JP7530909B2 - Methods, Devices, and Systems for Analyte Detection and Analysis - Patent application - Google Patents

Description

CROSS-REFERENCE This application claims priority to and the benefit of U.S. Provisional Application No. 62/818,549, filed March 14, 2019, U.S. Provisional Application No. 62/837,684, filed April 23, 2019, U.S. Provisional Application No. 62/914,293, filed October 11, 2019, U.S. Patent Application No. 16/445,798, filed June 19, 2019, U.S. Patent Application No. 16/677,067, filed November 7, 2019, and U.S. Patent Application No. 16/677,115, filed November 7, 2019, the entire contents of each of which are incorporated herein by reference.

Biological sample processing has a variety of applications in the fields of molecular biology and medicine (e.g., diagnostics). Nucleic acid sequencing can provide information that can be used to diagnose certain conditions in a subject and/or tailor treatment plans in some cases. Sequencing is used in a wide variety of molecular biology applications including vector design, gene therapy, vaccine design, industrial strain design, and validation. Biological sample processing can include fluidics and/or detection systems.

Despite the prevalence of biological sample processing systems and methods, such systems and methods may have low efficiency that can be time consuming and result in waste of valuable resources such as reagents. Recognized herein is the need for methods and systems for sample processing and/or analysis with high efficiency.

The present disclosure provides methods, devices, and systems for sample processing and/or analysis. The methods, devices, and systems described herein can include an open substrate or use thereof. The open substrate can include one or more analytes thereon. For example, one or more analytes can be bound, attached, immobilized, or otherwise associated with the open substrate directly or indirectly (e.g., via an intermediate entity such as a binder or linker). The open substrate can include an array. In some cases, the environment of the open substrate, such as the local environment around the open substrate, can be controlled, such as to facilitate one or more reactions or one or more detections. The methods, devices, and systems described herein can include an immersion optical system, or use thereof. The immersion optical system can be configured to detect an analyte or activity thereof on the open substrate. The methods, devices, and systems described herein can include spatial indexing of the open substrate or an array thereof, or use thereof.

In various aspects, the present disclosure provides a method for scanning a surface, the method including: (a) scanning a scan field including a portion of the surface using a scanning system, the scan field being directional relative to an axis of rotation of the surface; and (b) (i) rotating the surface about the axis of rotation of the surface and (ii) rotating the scan field about the axis of rotation of the scan field such that the scan field substantially maintains its directional orientation relative to the axis of rotation of the surface before, during, or after translation of the surface relative to the scan field.

In some embodiments, the scan field has a substantially linear shape. In some embodiments, the scan field has a major axis and the directionality includes a line coincident with the major axis of the scan field that passes through an axis of rotation of the surface. In some embodiments, the scan field follows an arc on the surface. In some embodiments, scanning the surface includes imaging the surface. In some embodiments, the scan field includes an imaging field. In some embodiments, the scan field follows a scan path on the surface and the scan path includes an imaging path. In some embodiments, the scanning system includes an imaging system.

In some embodiments, the directionality includes a major axis of the scan field, the major axis being parallel to (i) the axis of rotation of the surface and (ii) a radial line passing through the axis of rotation of the scan field. In some embodiments, the translation of the surface relative to the scan field includes translating in a direction that is not directly toward or away from the axis of rotation of the surface. In some embodiments, the translation of the surface relative to the scan field includes translating along a translation path, where a line that includes the net movement along the translation path does not intersect both the scan field and the axis of rotation of the surface.

In some embodiments, the scan field rotates relative to the surface about the axis of rotation of the scan field. In some embodiments, the axis of rotation of the scan field is approximately perpendicular to the surface. In some embodiments, the axis of rotation of the scan field is approximately parallel to the axis of rotation of the surface. In some embodiments, the axis of rotation of the scan field passes through the axis of symmetry of the scan field. In some embodiments, the scan field is rotated by rotating an objective lens. In some embodiments, the scan field is rotated by rotating a lens. In some embodiments, the scan field is rotated by rotating a prism. In some embodiments, the scan field is rotated by rotating a mirror. In some embodiments, the scan field is rotated by rotating a camera. In some embodiments, the scan field is rotated by rotating a diffractive optical element (DOE). In some embodiments, the scan field is rotated using a motor.

In some embodiments, the surface is generally circular and the scan field is translated along a chord of the surface. In some embodiments, the surface is generally circular and the axis of rotation of the scan field is translated along a chord of the surface. In some embodiments, the chord does not pass through the axis of rotation of the surface. In some embodiments, the scan field is translated by moving the surface. In some embodiments, the scan field is translated by moving the scanning system. In some embodiments, the scan field follows a circle on the surface. In some embodiments, the scan field follows a spiral on the surface. In some embodiments, the surface rotation and the surface translation are performed simultaneously. In some embodiments, the surface translation is linear with respect to the axis of rotation of the surface. In some embodiments, the surface translation is not generally circular with respect to the surface. In some embodiments, the surface translation increases or decreases the distance between the axis of rotation of the scan field and the axis of rotation of the surface.

In some embodiments, the scanning system comprises an objective lens in optical communication with the surface. In some embodiments, the scanning system comprises a camera. In some embodiments, the scan field is in optical communication with the camera. In some embodiments, the camera is a time delay integration (TDI) camera having a line rate. In some embodiments, the camera is a multi-line TDI camera. In some embodiments, the camera comprises an array of sensors, and the axis of rotation of the scan field passes through the center of the sensor array. In some embodiments, the line rate is set so that the camera captures images as the scan field advances along the surface from a first position to a second position, the second position adjacent to the first position. In some embodiments, the line rate is variable. In some embodiments, the line rate is higher when the objective lens is positioned further from the axis of rotation of the surface.

In some embodiments, the scanning system further comprises a tube lens. In some embodiments, the scanning system comprises two objective lenses in optical communication with the surface, namely, an objective lens and a second objective lens. In some embodiments, the two objective lenses are on the same side of the surface with respect to a plane perpendicular to the surface and intersecting the axis of rotation of the surface. In some embodiments, the two objective lenses are on opposite sides of the surface with respect to a plane perpendicular to the surface and intersecting the axis of rotation of the surface. In some embodiments, the two objective lenses follow a circular path on the surface. In some embodiments, the circular paths are concentric. In some embodiments, the objective lens and the second objective lens follow alternating circular paths. In some embodiments, the objective lens follows a circular path closer to the axis of rotation and the second objective lens follows a circular path further from the axis of rotation of the surface. In some embodiments, the two objective lenses follow individual spiral paths on the surface. In some embodiments, the spiral paths are alternated. In some embodiments, the helical paths are concentric, with the objective lens following a helical path closer to the axis of rotation of the surface and the second objective lens following a helical path further from the axis of rotation of the surface. In some embodiments, the objective lens follows a first path, the first path having a first width corresponding to a first width of the scan field, and the second objective lens follows a second path, the second path having a second path width corresponding to a second width of the second scan field. In some embodiments, the first path width and the second path width overlap by 30% or less, 20% or less, 10% or less, 5% or less, 1% or less.

In some embodiments, the scanning system includes four objective lenses in optical communication with the surface. In some embodiments, the four objective lenses are positioned on the same side of the surface relative to a plane perpendicular to the surface and intersecting the axis of rotation of the surface. In some embodiments, a first two of the four objective lenses are positioned on a first side of the surface and a second two of the four objective lenses are positioned on a second side of the surface opposite the first side relative to a plane perpendicular to the surface and intersecting the axis of rotation of the surface. In some embodiments, the scanning system includes 5, 6, 7, 8, 9, 10, 11, 12, 13 or more objective lenses in optical communication with the surface.

In some embodiments, the surface is rotated at a constant angular velocity. In some embodiments, the camera is configured to capture images at a predetermined frequency, and the surface is rotated relative to the objective lens at a variable angular velocity. In some embodiments, the angular velocity is varied such that, at the predetermined frequency, the camera captures images when the scan field is at a first position and when the scan field is at a second position, the second position being adjacent to the first position.

In some embodiments, the method further includes illuminating a portion of the surface defined by the illumination field. In some embodiments, the illumination field is illuminated using a laser. In some embodiments, the illumination field is illuminated using a light emitting diode (LED) or a lamp. In some embodiments, the power of the laser is adjusted to maintain a constant brightness on the surface and/or not to saturate the camera. In some embodiments, the illumination field at least partially overlaps with the scan field. In some embodiments, the scan field encompasses the illumination field. In some embodiments, the illumination field has a substantially similar shape to the scan field. In some embodiments, the illumination field is substantially rectilinear in shape. In some embodiments, the illumination field has a major axis.

In some embodiments, the scanning system further includes a plurality of illumination fields. In some embodiments, one or more of the plurality of illumination fields have a shape that is substantially linear. In some embodiments, the method further includes rotating the illumination field such that the illumination field maintains a predetermined orientation relative to an axis of rotation of the surface. In some embodiments, the illumination field maintains a predetermined orientation relative to the scan field. In some embodiments, the predetermined orientation includes a line that coincides with a major axis of the illumination field that passes through an axis of rotation of the surface. In some embodiments, the predetermined orientation includes a major axis of the illumination field that is parallel to a radial line, the radial line passing through the axis of rotation of the surface and the axis of rotation of the illumination field. In some embodiments, the scan field and the illumination field are rotated together. In some embodiments, the major axis of the illumination field is parallel to the major axis of the scan field. In some embodiments, the illumination field rotates about the axis of rotation of the illumination field. In some embodiments, the axis of rotation of the illumination field is substantially perpendicular to the surface. In some embodiments, the axis of rotation of the illumination field is substantially parallel to the axis of rotation of the surface. In some embodiments, the axis of rotation of the illumination field passes through an axis of symmetry of the illumination field. In some embodiments, the axis of rotation of the illumination field is the same as the axis of rotation of the scan field. In some embodiments, the illumination field is rotated by rotating a lens. In some embodiments, the illumination field is rotated by rotating a diffractive optical element (DOE). In some embodiments, the illumination field is rotated by rotating a prism. In some embodiments, the illumination field is rotated by rotating a mirror. In some embodiments, the illumination field is rotated by rotating a laser. In some embodiments, the illumination field is rotated using a motor.

In some embodiments, the method further includes scanning a second portion of the surface defined by a second scan field. In some embodiments, the second scan field is scanned using a second scanning system. In some embodiments, the second scanning system comprises a second objective lens in optical communication with the surface. In some embodiments, the second objective lens is focused independently of the first objective lens. In some embodiments, the second objective lens has a predetermined position relative to the first objective lens. In some embodiments, the second scan field is directional with respect to the axis of rotation of the surface. In some embodiments, the second scan field is radially adjacent to the scan field. In some embodiments, the scan field and the second scan field have the same orientation with respect to the axis of rotation of the surface. In some embodiments, the second scan field is rotated independently of the scan field such that the second scan field maintains its orientation with respect to the axis of rotation of the surface. In some embodiments, the second scan field is rotated in concert with the scan field.

In some embodiments, the first objective lens and the second objective lens are part of a scanning module, and the scanning module is translated relative to the surface along a line extending radially from the axis of rotation of the surface. In some embodiments, the surface is substantially circular, and at least one of the first objective lens or the second objective lens is not translated along a chord passing through the axis of rotation of the surface. In some embodiments, the first objective lens and the second objective lens are on the same side of the surface with respect to a plane perpendicular to the surface and intersecting the axis of rotation of the surface, and both the first objective lens and the second objective lens are translated together toward or away from the axis of rotation of the surface. In some embodiments, the first objective lens and the second objective lens are on opposite sides of the surface with respect to a plane perpendicular to the surface and intersecting the axis of rotation of the surface. In some embodiments, (i) the first objective lens is translated toward the axis of rotation of the surface when the second objective lens is translated away from the axis of rotation of the surface, or (ii) the first objective lens is translated away from the axis of rotation of the surface when the second objective lens is translated toward the axis of rotation of the surface.

In some embodiments, the surface is generally circular, and the first objective lens and the second objective lens are translated along parallel chords on either side of a plane perpendicular to the surface and intersecting the axis of rotation of the surface and equidistant from the axis of rotation of the surface. In some embodiments, the surface is mounted to a rotation module. In some embodiments, the rotation module is translated relative to the scanning system. In some embodiments, the rotation module is stationary and the scanning module is translatable. In some embodiments, the scanning module is stationary and the rotation module is translatable. In some embodiments, the rotation module is mounted to a track. In some embodiments, the scanning module is mounted to a scanning module track. In some embodiments, the scanning module track is linear. In some embodiments, multiple surfaces are mounted to multiple rotation modules, multiple rotation modules are mounted to a stage, and the stage is rotated to place each of the rotation modules in optical communication with the scanning module.

In some embodiments, after scanning the surface, the rotation module is moved to the chemistry module. In some embodiments, the method further includes translating the second rotation module such that the second surface is in optical communication with the scanning module. In some embodiments, the surface includes an array of nucleic acid colonies. In some aspects, the nucleic acid colonies are labeled with fluorophores. In some aspects, the intensity of the fluorophores indicates the sequence of the nucleic acid colonies. In some aspects, a laser excites the fluorophores at a first wavelength and a camera detects emission from the fluorophores at a second wavelength. In some embodiments, the laser illuminates an illumination field and the camera scans a scan field.

In some embodiments, two or more of the scanning, surface rotation, scanning field of view rotation, and translation are performed simultaneously. In some embodiments, three or more of the scanning, surface rotation, scanning field of view rotation, and translation are performed simultaneously. In some embodiments, the scanning, surface rotation, scanning field of view rotation, and translation are performed independently. In some embodiments, the method further includes repeating steps (a) and (b). In some embodiments, steps (a) and (b) are repeated for each base in the nucleic acid polymerization reaction, thereby sequencing the nucleic acid.

In various aspects, the present disclosure provides a scanning system comprising: a surface configured to rotate about an axis of rotation of the surface; a detector in optical communication with the surface, the detector having a scan field of view comprising a first portion of the surface; and an illumination source configured to illuminate an illumination field comprising a second portion of the surface, the illumination field and the scan field at least partially overlapping, the detector configured to maintain an orientation of the scan field relative to the axis of rotation of the surface during (i) rotation of the surface about the axis of rotation and (ii) translation of the surface relative to the scan field.

In some embodiments, the scan field of view follows an arc on the surface. In some embodiments, scanning the surface includes imaging the surface. In some embodiments, the scan field of view includes an imaging field of view. In some embodiments, the scan field of view follows a scan path along the surface, the scan path comprising an imaging path. In some embodiments, the scanning system comprises an imaging system. In some embodiments, the detector comprises a line scan camera. In some embodiments, the line scan camera comprises a TDI line scan camera. In some embodiments, the TDI line scan camera images the first scan field on a first camera region. In some embodiments, the TDI line scan camera images the second scan field on a second camera region. In some embodiments, the TDI line scan camera images the first scan field on the first camera region and images the first scan field on the second camera region. In some embodiments, the first camera region and the second camera region detect different wavelengths. In some embodiments, the first camera region and the second camera region detect different dynamic ranges.

In some embodiments, the surface is configured to translate along a translation axis relative to the scan field. In some embodiments, the translation axis intersects the rotation axis of the surface and a center point of the scan field. In some embodiments, the translation axis does not intersect the rotation axis of the surface and a center point of the scan field. In some embodiments, the directionality of the scan field changes from a first directionality to a second directionality relative to the rotation axis of the surface as the surface translates. In some embodiments, the scan field is configured to rotate about a rotation axis of the scan field relative to the rotation axis of the surface to correct the directionality of the scan field from the second directionality to the first directionality relative to the rotation axis of the surface. In some embodiments, the scan field is configured to rotate by rotating an objective lens. In some embodiments, the scan field is configured to rotate by rotating a lens. In some embodiments, the scan field is configured to rotate by rotating a prism. In some embodiments, the scan field is configured to rotate by rotating a mirror. In some embodiments, the scan field is configured to rotate by rotating a detector. In some embodiments, the scanning field is configured to rotate by rotating a diffractive optical element (DOE).

In some embodiments, the illumination source comprises a laser or a light emitting diode (LED). In some embodiments, the illumination source includes a substantially circular illumination profile. In some embodiments, the substantially circular illumination profile is expanded along a single axis. In some embodiments, the substantially circular illumination profile is expanded along a single axis using a cylindrical lens. In some embodiments, the scanning system further comprises a plurality of illumination sources having a substantially circular illumination profile, and the substantially circular illumination profile is expanded along a single axis. In some embodiments, the illumination source passes through a grating.

In some embodiments, a first portion of the surface is configured to move relative to the scan field. In some embodiments, a first region of the first portion of the surface is configured to move at a first speed relative to the scan field, and a second region of the first portion of the surface is configured to move at a second speed relative to the scan field. In some embodiments, the first region is closer to the axis of rotation of the surface than the second region, and the first speed is slower than the second speed. In some embodiments, an image of the first region is magnified on the detector by a first factor and an image of the second region is magnified on the detector by a second factor. In some embodiments, the first and second magnifications are different.

In some embodiments, the scanning system further comprises a lens having a lens axis positioned in an optical path between the scan field and the detector, where the lens axis is not perpendicular to the surface. In some embodiments, the scanning system further comprises an objective lens positioned in an optical path between the scan field and the detector. In some embodiments, the objective lens is in fluid contact with the surface. In some embodiments, the objective lens and the surface are at different temperatures.

In some embodiments, the scanning system further includes a temperature gradient across the fluid in contact with the surface and the objective lens. In some embodiments, the objective lens includes an insulating spacer in contact with the fluid. In some embodiments, the insulating spacer includes an air gap. In some embodiments, the objective lens is heated to reduce the temperature gradient. In some embodiments, the objective lens is cooled to increase the temperature gradient. In some embodiments, the fluid is configured to displace during rotation.

In some embodiments, the method further includes (i) scanning a focal region of the surface using an autofocus system to generate a focal map of the focal region, and (ii) adjusting the focus of the surface relative to the scanning system based on the focal map while scanning the scan field. In some embodiments, the surface rotates about an axis of rotation of the surface relative to the scan field while scanning a focal region of the surface using the autofocus system. In some embodiments, the focal region includes the scan field. In some embodiments, the focal region includes a field adjacent to the scan field. In some embodiments, the focal region does not include the scan field. In some embodiments, the focal region is scanned prior to scanning. In some embodiments, the focal region is scanned during scanning.

In some embodiments, the objective lens is configured to maintain fluid contact with the surface while the surface rotates about an axis of rotation of the surface relative to the objective lens. In some embodiments, the objective lens is configured to move in a direction substantially perpendicular to the surface, out of fluid contact with the surface and back into fluid contact. In some embodiments, the objective lens is configured to retain a droplet of fluid attached to the objective lens when the objective lens moves out of fluid contact with the surface. In some embodiments, the objective lens is configured to move an air bubble between the surface and the objective lens when the objective lens moves back into fluid contact with the surface. In some embodiments, the scanning system further comprises an adapter attached to the objective lens and configured to facilitate movement of the air bubble.

In some embodiments, the scanning system further comprises a chamber surrounding the surface and an objective lens configured to maintain a higher humidity within the chamber compared to outside the chamber. In some embodiments, the chamber comprises a reservoir below the surface configured to collect fluid. In some embodiments, the reservoir includes a fluid level, and the reservoir is configured to maintain a substantially constant fluid level. In some embodiments, the reservoir is configured to dispense an amount of fluid approximately equal to an amount of fluid collected by the reservoir. In some embodiments, the top of the chamber is held at a first temperature, the objective lens is held at a second temperature, the surface is held at a third temperature, and the reservoir is held at a fourth temperature. In some embodiments, the first temperature is greater than the second temperature. In some embodiments, the third temperature is less than the fourth temperature. In some embodiments, the second temperature is greater than the third temperature and less than the first temperature.

In various aspects, the disclosure provides a method for sequencing nucleic acid molecules, the method comprising: (i) providing an array of nucleic acid molecules on an uncoated surface; (ii) dispersing a layer of a solution over the uncoated surface at a rate of at least 1 nanoliter/second when measured at a temperature of 25 degrees Celsius, the solution comprising a reagent including at least one nucleotide that incorporates into a growing nucleic acid strand that is complementary to one of the nucleic acid molecules of the array of nucleic acid molecules; and (iii) detecting one or more signals indicative of the nucleotide that is incorporated into the growing nucleic acid strand.

In various aspects, the disclosure provides a method for processing a plurality of nucleic acid samples, comprising: (i) providing the plurality of nucleic acid samples, the plurality of nucleic acid samples including a first nucleic acid sample comprising a first set of nucleic acid molecules and a second nucleic acid sample comprising a second set of nucleic acid molecules, each sample of the plurality of nucleic acid samples having an identifiable sample source; (ii) loading the first nucleic acid sample as a first array of the first set of nucleic acid molecules into a first region of a substrate and loading the second nucleic acid sample as a second array of the second set of nucleic acid molecules into a second region of the substrate, the first region being different from the second region; and (iii) dispersing a solution throughout the substrate, the solution dispersing the solution throughout the first array or the second array. (iv) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecules of the first array or the second array; and (v) analyzing the first and second nucleic acid samples based, at least in part, on (a) the one or more signals and (b) the locations from the first and second regions from which the one or more signals are detected, and (1) determining a first subset of the nucleic acid molecules of the first or second array as derived from the first nucleic acid sample, and (2) determining a second subset of the nucleic acid molecules of the first or second array as derived from the second nucleic acid sample.

In various aspects, the disclosure provides a method for processing a plurality of nucleic acid samples, the method comprising: (i) providing the plurality of nucleic acid samples, the plurality of nucleic acid samples including a first nucleic acid sample comprising a first set of nucleic acid molecules and a second nucleic acid sample comprising a second set of nucleic acid molecules; (ii) loading the first nucleic acid sample onto a substrate to associate the first set of nucleic acid molecules with a first array of individually addressable locations; (iii) imaging the substrate to identify the first array of individually addressable locations; (iv) loading the second nucleic acid sample onto a substrate to associate the second set of nucleic acid molecules with a second array of individually addressable locations; (v) imaging the substrate to identify the second array of individually addressable locations; and (vi) dispersing the solution across the substrate. The method includes the steps of: (i) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecules of the first array or the second array; and (ii) analyzing the first and second nucleic acid samples based at least in part on (a) the one or more signals and (b) the location from the first array of individually addressable locations and the second array of individually addressable locations where the one or more signals are detected; and (1) determining a first subset of the nucleic acid molecules of the first or second array as derived from the first nucleic acid sample, and (20) determining a second subset of the nucleic acid molecules of the first or second array as derived from the second nucleic acid sample.

In various aspects, the disclosure provides a method for processing a plurality of nucleic acid samples, each of the plurality of nucleic acid samples comprising a fluorescent dye, comprising the steps of: (i) providing the plurality of nucleic acid samples, each of the plurality of nucleic acid samples comprising a fluorescent dye; (ii) separating the plurality of nucleic acid samples into a first set of one or more samples and a second set of one or more samples; (iii) loading the first set of one or more samples onto a first set of regions on a substrate, with one sample per region in the first set of regions; (iv) imaging the substrate to identify (a) locations within the first set of regions and (b) locations within the second set of regions on the substrate, wherein the second set of regions is different from the first set of regions with which the first set of one or more samples are associated; and (v) imaging the second set of one or more samples. (vi) imaging the substrate to identify (a) locations within the first set of regions and (b) locations within the second set of regions where the second set of one or more samples are associated; (vii) dispersing the solution across the substrate, the solution including sufficient reagents to react with nucleic acid molecules of the first set of one or more samples or the second set of one or more samples; (viii) detecting one or more signals indicative of a reaction between the reagents and the nucleic acid molecules; and (ix) analyzing each of the plurality of nucleic acid samples based at least in part on (a) the one or more signals and (b) the location from the first set of regions and the second set of regions where the one or more signals are detected.

In various aspects, the present disclosure provides a method for processing a biological sample, the method comprising: (i) moving a substrate through or along a reel, the surface of the substrate comprising an array having the biological sample fixed thereto; (ii) contacting the surface of the substrate with a reservoir comprising a solution, the solution comprising a plurality of probes; (iii) subjecting the biological sample to conditions sufficient to effect a reaction between one of the plurality of probes and the biological sample, thereby binding the probe to the biological sample; and (iv) detecting one or more signals from the probe bound to the biological sample, thereby analyzing the biological sample, the substrate being moved through or along the reel in the same direction for at least two successive cycles of (ii)-(iv).

In various aspects, the present disclosure provides a system for analyzing a biological specimen, the system comprising: a substrate containing the biological specimen, the substrate being maintained at or above a first temperature that is higher than an ambient temperature of an environment to which the substrate is exposed; and an optical imaging objective in optical communication with the substrate and exposed to the environment, the optical imaging objective being exposed to a temperature gradient between the first temperature of the substrate and the ambient temperature of the environment, the optical imaging objective comprising a first optical element and a second optical element adjacent to the first optical element, the second optical element being positioned farther from the substrate than the first optical element, the first optical element being configured to be at least partially immersed in an immersion fluid in contact with the substrate, the second optical element being in optical communication with the substrate through the first optical element, and the first optical element being configured such that a second temperature of the second optical element is maintained at or below a predetermined threshold.

In various aspects, the disclosure provides a method for analyzing a biological specimen, comprising the steps of: (i) providing a substrate containing the biological specimen, the substrate being at a first temperature greater than an ambient temperature of an environment to which the substrate is exposed; and (ii) providing an optical imaging objective lens in optical communication with the substrate and exposed to an environment, the optical imaging objective lens being exposed to a temperature gradient between the first temperature of the substrate and the ambient temperature of the environment, the optical imaging objective lens comprising a first optical element and a second optical element adjacent to the first optical element, the second optical element being adapted to selectively change the temperature of the substrate. (iii) controlling or maintaining a second temperature of the first optical element to adjust a magnitude or position of the temperature gradient through the optical imaging objective lens such that a third temperature gradient of the optical element is maintained below a predetermined threshold; and (iv) detecting one or more signals from the biological analyte using the optical imaging objective lens during movement of the substrate relative to the optical imaging objective lens.

In various aspects, the disclosure provides a method for storing a substrate having a surface coated with nucleic acid molecules, the method comprising: (i) providing the substrate having a surface on which a first set of nucleic acid molecules is immobilized, the nucleic acid molecules of the first set of nucleic acid molecules being configured to capture sample nucleic acid molecules from one or more nucleic acid samples; (ii) contacting the substrate having the surface comprising the first set of nucleic acid molecules with a second set of nucleic acid molecules under conditions sufficient to provide a treated surface in which at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized to nucleic acid molecules of the second set of nucleic acid molecules, the second set of nucleic acid molecules being not the sample nucleic acid molecules; and (iii) storing the substrate having the treated surface for a period of at least one hour.

In various aspects, the disclosure provides a method for nucleic acid processing, comprising: (i) providing a substrate having a processing surface having a first set of nucleic acid molecules immobilized thereon, where at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized to nucleic acid molecules of a second set of nucleic acid molecules, where the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules from one or more nucleic acid samples, where the second set of nucleic acid molecules are not the sample nucleic acid molecules, and where the substrate with the processed substrate is stored for a period of at least one hour; and (ii) removing the nucleic acid molecules of the second set of nucleic acid molecules from the processing surface.

In various aspects, the disclosure provides a kit comprising a substrate comprising a treated surface, the treated surface comprising a plurality of pairs of bound nucleic acid molecules, each pair of the plurality of pairs comprising a first nucleic acid molecule of a first set of nucleic acid molecules at least partially hybridized to a second nucleic acid molecule of a second set of nucleic acid molecules, the first set of nucleic acid molecules being immobilized on the surface, at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules being paired with a nucleic acid molecule of the second set of nucleic acid molecules, and the nucleic acid molecules of the first set of nucleic acid molecules being configured to capture a sample nucleic acid molecule from one or more nucleic acid samples when a nucleic acid molecule of the first set of nucleic acid molecules is not paired with a nucleic acid molecule of the second set of nucleic acid molecules.

In various aspects, the disclosure provides a kit comprising: a substrate having a surface on which a first set of nucleic acid molecules is immobilized, the first set of nucleic acid molecules comprising one or more first nucleic acid molecules, the one or more first nucleic acid molecules configured to capture sample nucleic acid molecules from one or more nucleic acid samples; and a solution comprising a second set of nucleic acid molecules, the second set of nucleic acid molecules comprising one or more second nucleic acid molecules, the one or more second nucleic acid molecules not being the sample nucleic acid molecules, the second set of nucleic acid molecules being selected such that upon contacting the solution with the surface, at least 70% of the one or more first nucleic acid molecules bind to a second nucleic acid molecule of the second set of nucleic acid molecules to generate one or more pairs of bound nucleic acid molecules, each pair of the one or more pairs comprising (i) a first nucleic acid molecule of the first set of nucleic acid molecules and a second nucleic acid molecule of the second set of nucleic acid molecules, and (ii) a section of substantially complementary sequence.

In various aspects, the disclosure provides a method for storing a substrate having a surface coated with nucleic acid molecules, the method comprising the steps of: (i) providing a substrate having a surface having a first set of nucleic acid molecules immobilized thereon, the nucleic acid molecules of the first set of nucleic acid molecules being configured to capture sample nucleic acid molecules from one or more nucleic acid samples, each nucleic acid molecule of the first set of nucleic acid molecules having a first nucleic acid sequence and a second nucleic acid sequence, the second nucleic acid sequence being substantially complementary to the first nucleic acid sequence; (ii) generating a treated surface by subjecting the surface to conditions sufficient to bind the first nucleic acid sequence of a nucleic acid molecule of the first set of nucleic acid molecules to the second nucleic acid sequence of the nucleic acid molecule to provide an immobilized hairpin molecule; and (iii) storing the substrate having the treated surface for a period of at least one hour.

In various aspects, the disclosure provides a method for storing a substrate having a surface coated with nucleic acid molecules, the method comprising the steps of: (i) providing a substrate having a surface having a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules derived from one or more nucleic acid samples, each nucleic acid molecule of the nucleic acid molecules of the first set of nucleic acid molecules comprising a first nucleic acid sequence; (ii) providing a second set of nucleic acid molecules, wherein each nucleic acid molecule of the second set of nucleic acid molecules comprises a second nucleic acid sequence substantially complementary to the first nucleic acid sequence, and wherein the second set of nucleic acid molecules are not the sample nucleic acid molecules; and (ii) i) contacting the surface comprising the first set of nucleic acid molecules with the second set of nucleic acid molecules to produce a treated surface on which at least 70% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized to a nucleic acid molecule of the second set of nucleic acid molecules; and (iv) storing the treated surface for at least 1 hour, wherein for each nucleic acid molecule of the first set of nucleic acid molecules hybridized to one nucleic acid molecule of the second set of nucleic acid molecules, the first nucleic acid sequence is hybridized to the second nucleic acid sequence and the first nucleic acid sequence hybridized to the second nucleic acid sequence is at least partially denatured between about 40°C and 60°C.

In various aspects, the present disclosure provides a method for detecting or analyzing an analyte, comprising: (i) providing an open substrate having a central axis, the open substrate comprising an array of analytes fixed adjacent to the open substrate, at least one analyte of the array of analytes being bound to a probe; and (ii) performing a nonlinear scan of the open substrate using a detector system to detect at least one signal or signal change from the bound probe, the detector system comprising a line scan camera and an illumination source, the illumination source configured to generate an illumination region on the open substrate, the open substrate comprising a first region and a second region, the first region and the second region (a) comprising different subsets of the array of analytes, (b) being at different radial positions of the open substrate relative to the central axis, and (c) being spatially resolved by the detector system, the bound probe being disposed in the first region of the open substrate, and the nonlinear scan being performed during a relative nonlinear motion between the open substrate and one or both of (1) the line scan camera and (2) the illumination region.

In various aspects, the present disclosure provides an apparatus for analyte detection or analysis, comprising: a housing configured to receive an open substrate having an array of analytes fixed adjacent to it, at least one analyte of the array of analytes being bound to a probe; and a detector system, the detector system comprising a line scan camera and an illumination source, the illumination source configured to generate an illumination area on the open substrate, the open substrate comprising a first region and a second region, the first region and the second region (a) comprising a subset of the array of fixed analytes, (b) at different radial positions of the open substrate relative to the central axis, and (c) spatially resolved by the detector system, the binding probe being disposed in the first region of the open substrate, the detector system being programmed to perform a nonlinear scan of the open substrate and detect at least one signal or signal change from the binding probe in the first region of the open substrate, the nonlinear scan being performed during a relative nonlinear movement between the open substrate and one or both of (1) the line scan camera and (2) the illumination area.

In various aspects, the present disclosure provides a computer readable medium having stored thereon persistent instructions that, when executed, cause one or more computer processors to perform a method for detecting or analyzing analytes, the method including the steps of providing an open substrate about a central axis, the open substrate comprising an array of analytes fixed adjacent to the open substrate, at least one analyte of the array of analytes being bound to a probe, and performing a non-linear scan of the open substrate using a detector system to detect at least one signal or signal change from the bound probe, the detector system including a line scan camera and an illuminator. A computer-readable medium is provided, the computer-readable medium comprising: an illumination source configured to generate an illumination region on the open substrate, the open substrate comprising a first region and a second region, the first region and the second region (a) comprising different subsets of the array of analytes, (b) being at different radial positions of the open substrate relative to the central axis, and (c) being spatially resolved by the detector system, the coupling probe being positioned in the first region of the open substrate, and the nonlinear scanning being performed during a relative nonlinear motion between the open substrate and one or both of (1) the line scan camera and (2) the illumination region.

In various aspects, the disclosure provides a method for processing a nucleic acid sample, comprising: (i) providing a first source including a first set of nucleic acid molecules and a second source including a second set of nucleic acid molecules, the first source being different from the second source; (ii) directing the first set of nucleic acid molecules from the first source to a substrate to result in the first set of immobilized nucleic acid molecules in a first array adjacent to the substrate; (iii) imaging the substrate to identify a first set of locations on the substrate with the first array adjacent to the substrate; and (iv) directing the second set of nucleic acid molecules from the second source to the substrate to result in the first set of immobilized nucleic acid molecules in a second array adjacent to the substrate. (v) imaging the substrate to identify a second set of locations on the substrate with the second array adjacent to the substrate, the second array being different from the first array; and (vi) (a) using signals detected from the first array and the second array, and (b) the locations at which the signals are detected, (1) identifying the first set of nucleic acid molecules or sequences thereof using the first source, and (2) identifying the second set of nucleic acid molecules or sequences thereof using the second source, wherein the first set of locations and the second set of locations each comprise at least 1,000,000 locations.

In various aspects, the present disclosure provides a method for scanning a surface, the method comprising: (i) scanning a scan field including a portion of the surface using a scanning system, the scan field being directional with respect to an axis of rotation of the surface; and (ii) (a) rotating the surface about the axis of rotation of the surface and (b) rotating the scan field about the axis of rotation of the scan field such that the scan field substantially maintains its directional orientation with respect to the axis of rotation of the surface before, during, or after translation of the surface relative to the scan field.

In various aspects, the present disclosure provides a system comprising: a scanner configured to scan a scan field that includes a portion of a surface, the scan field being directional relative to an axis of rotation of the surface; and a controller configured to direct (i) a rotation of the surface about the axis of rotation of the surface, and (ii) a rotation of the scan field about the axis of rotation of the scan field, to substantially maintain the orientation of the scan field relative to the axis of rotation of the surface before, during, or after translation of the surface and the scan field relative to one another.

In various aspects, the present disclosure provides a method for analyzing biological material, the method comprising: (i) operating an apparatus comprising: (a) a substrate having a surface bearing the biological material, the surface being at a first temperature higher than an ambient temperature; and (b) an optical imaging objective in optical communication with the surface, the optical imaging objective comprising: (1) a first optical element at least partially immersed in an immersion fluid in contact with the surface; and (2) a second optical element in optical communication with the surface through at least the first optical element, the second optical element being maintained at or below a second temperature different from the first temperature; and (ii) collecting a signal from the surface bearing the biological material using the optical imaging objective.

In various aspects, the present disclosure provides a system for analyzing biological material, comprising: a platform configured to support a substrate having a surface having the biological material, the surface being configured to have a first temperature higher than an ambient temperature when the substrate is supported by the platform; an optical imaging objective configured to be in optical communication with the surface when the substrate is supported by the platform, the optical imaging objective configured to include a temperature gradient between the first temperature and the ambient temperature, the optical imaging objective comprising (1) a first optical element configured to be at least partially immersed in an immersion fluid in contact with the surface, and (2) a second optical element in optical communication with the surface through at least the first optical element, the second optical element configured to be maintained at or below a second temperature different from the first temperature; and one or more computer processors individually or jointly programmed to direct collection of a signal from the surface having the biological material using at least the optical imaging objective.

Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, performs any of the preceding methods or methods elsewhere herein.

Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled to the processors. The computer memory comprises machine executable code that, when executed by the one or more computer processors, performs any of the preceding methods or methods elsewhere herein.

Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which merely exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with a disclosure contained in the specification, the specification is intended to take precedence and/or supersede any such conflicting material.

The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings (also referred to herein as "FIGS" and "FIGS"), in which:

1 illustrates a computer controlled system that is programmed or otherwise configured to carry out the methods provided herein. 1 shows a flow chart of an example of a method for sequencing a nucleic acid molecule. 1 shows a system for sequencing a nucleic acid molecule. 1 shows a system for sequencing a nucleic acid molecule at a first vertical level. 1 shows a system for sequencing nucleic acid molecules at a second vertical level. 1 shows a first example of a system for sequencing nucleic acid molecules using an array of fluidic channels. 1 shows a second example of a system for sequencing nucleic acid molecules using an array of fluidic channels. FIG. 1 illustrates a computerized system for sequencing nucleic acid molecules. 1 illustrates a system with different environmental conditions in an open substrate system. 1 shows a scheme for a line scan camera, and illustrates rows of pixels in a time delay integration (TDI) line scan camera. 1 shows a scheme for a line scan camera, and shows a trilinear pixel scheme for a color line scan camera including red (R), green (G), and blue (B) pixels. 1 shows a scheme for a line scan camera, and a bilinear pixel scheme for a color line scan camera including red, green, and blue pixels. 1 shows a scheme for a line scan camera, and a bilinear pixel scheme for a color line scan camera including red, green, and blue pixels. FIG. 2 shows an optical system for scanning successive areas of a substrate during rotational movement of the substrate. 1 shows an optical system for imaging a substrate during a rotational movement of the substrate using tailored optical distortion. 1 shows an optical system for imaging a substrate during a rotational movement of the substrate using tailored optical distortion. 1 shows an example of tailored optical distortion induced using a cylindrical lens. 1 illustrates diagrammatically a scheme for expanding a laser beam to provide a laser line; 1 illustrates diagrammatically a scheme for expanding a laser beam to provide a laser line; 1 shows an optical system for shaping a laser beam. 1 illustrates a scheme for detecting a signal emitted by a material bound to an open substrate, where the open substrate rotates and the detector system remains stationary during detection. 1 illustrates a scheme for detecting a signal emitted by a material bound to an open substrate, where the open substrate remains stationary and the detector system rotates during detection. 1 shows a scheme for detecting a signal emitted by a material bound to an open substrate, in which the open substrate rotates during delivery and dispersion of a solution onto the open substrate (left panel) and remains stationary during detection by a rotating detector system (right panel). 1 shows a first example of an interleaved spiral imaging scan. 13 shows a second example of interleaved imaging scans. 1 illustrates an example of a nested imaging scan. 1 illustrates a nested circular imaging scan configuration. 1 shows a cross-sectional view of an immersion optical system. 1 illustrates a schematic of a typical temperature gradient during optical imaging. 1 illustrates a schematic of an exemplary method for adjusting the temperature of a substrate. 1 illustrates a schematic of an exemplary method for adjusting the temperature of a substrate. 1 illustrates a schematic of an exemplary method for adjusting the temperature of a substrate. 1 illustrates a schematic of an exemplary method for adjusting the temperature of a substrate. 1 illustrates a schematic of an exemplary method for adjusting the temperature of a substrate. 1 illustrates a schematic of bubble formation in a fluid; 1 illustrates a schematic representation of an adapter for an optical imaging system. 1A-1C show schematic diagrams of an exemplary method of displacing a gas bubble, showing a substrate with fluid dispensed thereon; 1 illustrates a schematic of an exemplary method for displacing a gas bubble, showing an optical imaging objective in contact with a fluid. 1 illustrates generally a method for dispensing and removing immersion fluid onto a substrate. 1 illustrates a schematic representation of a method for trapping air bubbles and a typical adapter for an optical imaging objective. 1 illustrates a schematic representation of a method for trapping air bubbles and a typical adapter for an optical imaging objective. 1 shows the architecture for a system with a fixed axis substrate and moving fluid and optical elements. 1 shows the architecture for a system with a translational axis substrate and stationary fluidic and optical elements. 1 shows an architecture for a system with multiple stationary substrates and moving fluidic and optical elements. 1 shows the architecture for a system with multiple moving substrates on a rotating stage and stationary fluidic and optical elements. 1 shows an architecture for a system with multiple fixed substrates and moving optical elements. 1 shows an architecture for a system with multiple moving substrates and stationary fluidic and optical elements. 1 shows an architecture for a system with multiple moving substrates and stationary fluidic and optical elements. 1 illustrates an architecture for a system with multiple substrates being moved between multiple processing bays. 1 shows an architecture for a system with multiple imaging heads that share translation and rotation axes for scanning and independently rotate the field of view. 1 shows an architecture for a system with multiple imaging heads that share translation and rotation axes for scanning and independently rotate the field of view. 1 shows the architecture for a system with multiple spindles scanned by a shared optical detection system. 1 shows an architecture for a system with multiple rotating spindles. 1 shows a flow chart of an example method for processing a specimen. FIG. 1 shows a first example of a system for isolating an analyte; 2 shows a second example of a system for isolating an analyte. 1 shows an example of a control system for compensating for velocity gradients during scanning. 1 shows the movement of a substrate relative to two imaging heads positioned on the same side of the substrate's axis of rotation. 1 shows the movement of a substrate relative to two imaging heads positioned on either side of the substrate's axis of rotation. The movement of the substrate relative to the three imaging heads is shown. The movement of the substrate relative to the four imaging heads is shown. The movement of the substrate relative to the four imaging heads is shown. The movement of the substrate relative to the four imaging heads is shown. The movement of the substrate relative to the four imaging heads is shown. 1 shows successive ring paths of two imaging heads positioned on the same side of the rotation axis of the substrate. 1 shows a successive ring path of two imaging heads positioned on either side of the axis of rotation of the substrate. 1 shows staggered ring paths for two imaging heads positioned on the same side of the substrate's axis of rotation. 1 shows a staggered ring path of two imaging heads positioned on either side of the axis of rotation of the substrate. 13 illustrates the rotational scan direction of the imaging head resulting from non-radial motion of the substrate. 13 illustrates the rotational scan direction of the imaging head resulting from non-radial motion of the substrate. 1 shows a flow chart of an example method for analyte detection or analysis. The axes of rotation and relative translation of the surface and the imaging field of view are shown diagrammatically. 2 shows a schematic of an optical system for rotating the imaging field of view. 1 shows a schematic of an optical system for rotating the imaging field of view. 1 shows a schematic of an optical system for rotating the imaging field of view. 1 illustrates an example of imaging head positioning for optimal scanning efficiency. 1 illustrates an example of imaging head positioning for optimal scanning efficiency. 1 illustrates an example of imaging head positioning for optimal scanning efficiency. 1 illustrates a schematic of a method for processing a biological sample. 1 illustrates a schematic of a method for processing a biological sample. 3 shows different examples of cross-sectional surface profiles of a substrate. 3 shows different examples of cross-sectional surface profiles of a substrate. 3 shows different examples of cross-sectional surface profiles of a substrate. 3 shows different examples of cross-sectional surface profiles of a substrate. 3 shows different examples of cross-sectional surface profiles of a substrate. 3 shows different examples of cross-sectional surface profiles of a substrate. 3 shows different examples of cross-sectional surface profiles of a substrate. A method is presented for rendering oligonucleotide-coated surfaces resistant to nucleic acid contaminants. A method is presented for rendering oligonucleotide-coated surfaces resistant to nucleic acid contaminants. A method is presented for rendering oligonucleotide-coated surfaces resistant to nucleic acid contaminants. A method is presented for rendering oligonucleotide-coated surfaces resistant to nucleic acid contaminants. An example of a space loading method is shown below. Another example of the space loading method is shown. A multiple sample processing method is shown. 1 shows a schematic diagram of a typical optical layout. 1 shows an example of an image generated by imaging a substrate on which an analyte is immobilized. 1 shows an example of data obtained from a diagnostic procedure. Example data from a diagnostic procedure is shown: Panels A-F show spatial plots of diagnostic metrics calculated for images scanned at different individually addressable locations. 1 shows an example of flow-based sequencing data. Representative data from a processed image is shown. Representative data from a processed image is shown. A plot of aligned genomic reads is shown. The aligned coverage distribution across the reference genome is shown. 1 shows a partial cross-sectional view of a barrier system that maintains a fluid barrier. FIG. 47B shows a perspective view of a chamber of the barrier system of FIG. 47A. SUMMARY OF THE DISCLOSURE A system and method for spirally loading a sample or reagent onto a substrate is presented. 1 shows a typical coating of a substrate with a hexagonal lattice of beads. A method for loading beads onto a substrate is provided, and a method for loading beads into specific areas of a substrate is provided. Methods are provided for loading beads onto a substrate, and methods are provided for loading subsets of beads onto specific regions of a substrate. 1 shows a method for spirally loading a sample or reagent onto an open substrate.

While various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

The term "processing an analyte" as used herein generally refers to one or more stages of interaction with one or more sample substances. Processing an analyte may include performing a chemical reaction, a biochemical reaction, an enzymatic reaction, a hybridization reaction, a polymerization reaction, a physical reaction, any other reaction, or a combination thereof, in the presence or on the analyte. Processing an analyte may include physical and/or chemical manipulation of the analyte. For example, processing an analyte may include detecting a chemical or physical change, adding or subtracting a material, atom or molecule, identifying a molecule, detecting the presence of a fluorescent label, detecting a Forster Resonance Energy Transfer (FRET) interaction, or inferring the absence of fluorescence. The term "analyte" may refer to a molecule, a cell, a biological particle, or an organism. In some examples, a molecule may be a nucleic acid molecule, an antibody, an antigen, a peptide, a protein, or other biological molecule obtained or derived from a biological sample. An analyte may be derived from and/or from a biological sample, such as a cell or organism. An analyte may be synthetic.

As used herein, the term "sequencing" generally refers to the process of generating or identifying a sequence of a biomolecule, such as a nucleic acid molecule. Such a sequence may be a nucleic acid sequence, which may include a sequence of nucleobases (e.g., nucleobases). Sequencing may be, for example, single molecule sequencing or sequencing by synthesis. Sequencing may be performed using a template nucleic acid molecule immobilized on a support, such as a flow cell or one or more beads.

The term "biological sample" as used herein generally refers to any sample from a subject or specimen. A biological sample can be a fluid or tissue from a subject or specimen. A fluid can be blood (e.g., whole blood), saliva, urine, or sweat. A tissue can be from an organ (e.g., liver, lung, or thyroid), or a mass of cellular material, such as a tumor. A biological sample can be a fecal sample, a collection of cells (e.g., a buccal swab), or a hair sample. A biological sample can be acellular or cellular sample. Examples of biological samples include nucleic acid molecules, amino acids, polypeptides, proteins, carbohydrates, fats, or viruses. In one example, a biological sample is a nucleic acid sample that includes one or more nucleic acid molecules, such as deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). The nucleic acid molecule can be acellular or acellular nucleic acid molecule, such as cell-free DNA or cell-free RNA. The nucleic acid molecule can be from a variety of sources, including human, mammalian, non-human mammalian, ape, monkey, chimpanzee, reptile, amphibian, avian, or plant sources. Additionally, samples may be extracted from a variety of animal bodily fluids containing cell-free sequences, including, but not limited to, blood, serum, plasma, vitreous, sputum, urine, tears, sweat, saliva, semen, mucosal excretions, mucus, spinal fluid, amniotic fluid, lymphatic fluid, etc. Cell-free polynucleotides may be fetal in origin (via fluids taken from a pregnant subject) or derived from the subject's own tissues.

The term "subject" as used herein generally refers to an individual from whom a biological sample is obtained. The subject may be a mammal or a non-mammal. The subject may be an animal, such as a monkey, dog, cat, bird, or rodent. The subject may be a human. The subject may be a patient. The subject may be exhibiting symptoms of a disease. The subject may be symptomatic. The subject may be undergoing treatment. The subject may not be undergoing treatment. The subject may have or be suspected of having a disease or disorder, such as cancer (e.g., breast cancer, colorectal cancer, brain cancer, leukemia, lung cancer, skin cancer, liver cancer, pancreatic cancer, lymphoma, esophageal cancer, or cervical cancer) or an infectious disease. Subjects may have a medical condition such as achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth, Cri du Chat, Crohn's disease, cystic fibrosis, Delcam's disease, Down's syndrome, Duane's syndrome, Duchenne muscular dystrophy, factor V Leiden thrombocytosis, familial hypercholesterolemia, familial Mediterranean fever, fragile x syndrome, Gaucher's disease, hemochromatosis, hemophilia, holoprosencephalomyelopathy, Huntington's disease, Chlamydia trachomatis ... The patient may have or be suspected of having a genetic disorder such as Reinfelter's syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, muscular dystrophy, thalassemia, trimethylaminuria, Turner syndrome, velar cardiofacial syndrome, WAGR syndrome, or Wilson's disease.

As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleic acid fragment," "oligonucleotide," and "polynucleotide" generally refer to polynucleotides that may be of various lengths, such as deoxyribonucleotides or deoxyribonucleic acid (DNA) or ribonucleotides or ribonucleic acid (RNA), or analogs thereof. Non-limiting examples of nucleic acids include DNA, RNA, genomic or synthetic DNA/RNA, or coding or non-coding regions of a gene or gene fragment, multiple loci (single locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence. A nucleic acid molecule can have a length of at least about 10 nucleobases ("bases"), 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 1 megabase (Mb), or more. A nucleic acid molecule (e.g., a polynucleotide) can include a sequence of the four naturally occurring nucleotide bases, namely, adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) if the polynucleotide is RNA). A nucleic acid molecule may contain one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotide(s).

Examples of nucleotide analogs include inosine, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, deazaxanthine, deazaguanine, isocytosine, isoguanine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylketosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylketosine, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, ethynyl nucleotide bases, 1-propynyl nucleotide bases, azido nucleotide bases, phosphoroselenoate nucleic acids, and modified versions thereof (e.g., by oxidation, reduction, and/or alkyl, hydroxyalkyl, hydroxyl, or halogen moieties). In some cases, a nucleotide may include modifications at its phosphate moiety, including modifications to the triphosphate moiety. Further non-limiting examples of modifications include longer phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties), modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphate), and modifications with selenium moieties (e.g., phosphoroselenoate nucleic acids). Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms typically available to form hydrogen bonds with a complementary nucleotide and/or at one or more atoms typically not available to form hydrogen bonds with a complementary nucleotide), the sugar moiety, or the phosphate backbone. Nucleic acid molecules may also contain amine-modifying groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexylamide-dCTP (aha-dCTP), to allow for the covalent attachment of amine-reactive moieties, such as N-hydroxysuccinimide ester (NHS). Substitution of standard DNA or RNA base pairs in the oligonucleotides of the present disclosure may provide, for example, higher density in bits/cubic mm, greater safety (resistance to accidental or deliberate synthesis of natural toxins), easier discrimination in light-programmed polymerases, and/or less secondary structure. Nucleotide analogs can react or be conjugated with detectable moieties for nucleotide detection.

The term "nucleotide" as used herein generally refers to any nucleotide or nucleotide analog. A nucleotide may be naturally occurring or non-naturally occurring. A nucleotide analog may be a modified, synthetic, or engineered nucleotide. A nucleotide analog may be non-naturally occurring or may include a non-standard base. A naturally occurring nucleotide may include a canonical base. A nucleotide analog may include a modified polyphosphate chain (e.g., a triphosphate coupled to a fluorophore). A nucleotide analog may include a label. A nucleotide analog may be terminated (e.g., reversibly terminated). A nucleotide analog may include an alternative base.

The terms "amplify," "amplification," and "nucleic acid amplification" are used interchangeably and generally refer to generating one or more copies of a nucleic acid or template. For example, "amplification" of DNA generally refers to generating one or more copies of a DNA molecule. Nucleic acid amplification may be linear, exponential, or a combination thereof. Amplification may be emulsion-based or non-emulsion-based. Non-limiting examples of nucleic acid amplification reactions that may be used in combination with the methods disclosed herein include reverse transcription, primer extension, polymerase chain reaction (e.g., PCR), ligase chain reaction, helicase-dependent amplification, asymmetric amplification, rolling circle amplification, and multiple displacement amplification (MDA). When PCR is used, any form of PCR may be used, with non-limiting examples including real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot-start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap extension PCR, thermal asymmetric interlaced PCR, and touch-down PCR. Furthermore, amplification can be performed in a reaction mixture that includes various components that participate in or facilitate amplification (e.g., primer(s), template, nucleotides, polymerase, buffer components, cofactors, etc.). In some cases, the reaction mixture includes a buffer that allows for incorporation of context-independent nucleotides. Non-limiting examples include magnesium ions, manganese ions, and isocitrate buffers. Further examples of such buffers are described in Tabor, S. et al. C.C. PNAS, 1989, 86, 4076-4080, and U.S. Patent Nos. 5,409,811 and 5,674,716, each of which is incorporated herein by reference in its entirety.

The terms "dispense" and "disperse" may be used interchangeably herein. In some cases, dispensing may include dispersion and/or dispersion may include dispensing. Dispensing generally refers to dispensing, depositing, providing, or delivering, such as a reagent, solution, or other object. Dispensing may include dispersion, which may generally refer to spreading.

Useful methods for clonal amplification from a single molecule include rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), incorporated herein by reference), bridge PCR (Adams and Kron, Methods for Amplification of Nucleic Acids with Two Primers Attached to a Single Solid Support, Mosaic Technologies, Inc. (Winter Hill, MA); Whitehead Institute for Biomedical Research, Cambridge, MA, (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000); Pemov et al., Nucl. Acids Res. Res. 33:e11 (2005); or US Pat. No. 5,641,658, each of which is incorporated herein by reference), polony generation (Mitra et al., Proc. Natl. Acad. Sci. USA 100:5926-5931 (2003); Mitra et al., Anal. Biochem. 320:55-65 (2003), each of which is incorporated herein by reference), emulsion (Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), each of which is incorporated herein by reference), or ligation to bead-based adapter libraries (Brenner et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), each of which is incorporated herein by reference), or bead-based adapter libraries (Brenner et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), each of which is incorporated herein by reference). al., Nat. Biotechnol. 18:630-634 (2000); Brenner et al., Proc. Natl. Acad. Sci. USA 97:1665-1670 (2000); Reinartz, et al., Brief Funct. Genomic Proteomic 1:95-104 (2002), each of which is incorporated herein by reference.

The term "detector" as used herein generally refers to a device capable of detecting a signal, including a signal indicative of the presence or absence of one or more incorporated nucleotides or fluorescent labels. A detector can detect multiple signals. The signal or signals can be detected in real time during, substantially during, or after a biological reaction, such as a sequencing reaction (e.g., sequencing during a primer extension reaction). In some cases, a detector can include optical and/or electronic components capable of detecting a signal. The term "detector" can be used in detection methods. Non-limiting examples of detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, acoustic detection, magnetic detection, and the like. Optical detection methods include, but are not limited to, light absorption, ultraviolet-visible (UV-vis) light absorption, infrared light absorption, light scattering, Rayleigh scattering, Raman scattering, surface-enhanced Raman scattering, Mie scattering, fluorescence, luminescence, and phosphorescence. Spectroscopic detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques such as gel electrophoresis. Electrochemical detection methods include, but are not limited to, electrochemical detection of the amplification products after high performance liquid chromatography separation of the amplification products.

The term "continuous area scanning" as used herein generally refers to ring, spiral, or arc area scanning on a rotating substrate using an optical imaging system and a detector. Continuous area scanning can scan a substrate or array along a non-linear path. Alternatively or additionally, continuous area scanning can scan a substrate or array along a linear or substantially linear path. The detector may be a continuous area scanning detector. The scan direction may be substantially θ in an (R, θ) coordinate system, with the object rotation motion being in the θ direction. Over any field on an object (substrate) imaged by a scanning system, the apparent velocity is the velocity of a field point on the object.

tangential velocity blur may vary with the radial position (R) of the rotating substrate. A continuous area scanning detector may scan at the same speed for all image locations and may therefore not be able to operate at the correct scan speed for all imaged points in a curved (or arcuate or non-linear) scan. Thus, the scan may be perturbed by velocity blur of imaged field points moving at a speed different from the scan speed. A continuous rotating area scan may include an optical detection system or method that performs algorithmic, optical, and/or electronic correction to substantially compensate for this tangential velocity blur, thereby reducing this scanning aberration. For example, compensation is accomplished algorithmically by using an image processing algorithm that deconvolves the differential velocity blur at various image locations corresponding to different radii on the rotating substrate to compensate for the differential velocity blur. In some cases, the camera or scanner may apply or use blur to compensate for the differential velocity blur.

In another example, compensation is achieved by using an anamorphic magnification gradient. This can serve to magnify the substrate in one axis (anamorphic magnification) by different amounts at two or more substrate locations across the scan direction. The anamorphic magnification gradient can change the imaging speed of two or more locations to be substantially equal, thereby compensating for tangential velocity differences at two locations on the substrate. This compensation can be adjustable to account for different velocity gradients across the field of view at different radii on the substrate.

The imaging field of view can be divided into two or more regions, each of which can be electronically controlled to scan at a different speed. These speeds can be adjusted to the average projected object speed within each region. The regions may be optically defined using one or more beam splitters or one or more mirrors. The two or more regions may be directed to two or more detectors. A region can be defined as a segment of a single detector.

As used herein, the term "continuous area scanning detector" generally refers to an imaging array sensor capable of continuously integrating over a scan area where the scan is electronically synchronized to an image of a relatively moving object. A continuous area scanning detector may include a time delay integration (TDI) charge coupled device (CCD), a hybrid TDI, or a complementary metal oxide semiconductor (CMOS) pseudo-TDI device. For example, a continuous area scanning detector may comprise a TDI line scan camera.

As used herein, the term "open substrate" generally refers to a substantially flat substrate in which a single active surface is physically accessible at any point from the substrate normal. Substantially flat may refer to a micrometer or nanometer level of planarity. Alternatively, substantially flat may refer to a flatness that is less than the nanometer level or greater than the micrometer level (e.g., millimeter level).

As used herein, the term "anamorphic magnification" generally refers to the differential magnification between two axes of an image. The anamorphic magnification gradient may include the difference in the anamorphic magnification of a first axis across the displacement of a second axis. The magnification in the second axis may be 1, or any other value that is substantially constant across the field.

As used herein, the term "field of view" generally refers to the area on a sample or substrate that is optically mapped to the active area of a detector.

Analyte Processing Using Open Substrates Conventional microfluidic systems have utilized substrates that contain a large number of elongated channels. Typical flow cell geometries in such substrates introduce the need to compromise between two competing requirements: 1) minimize volume to minimize reagent usage, and 2) maximize effective hydraulic diameter to minimize flow time. This trade-off may be particularly important for wash operations that may require large wash volumes and therefore long times to complete. The trade-off is dictated by the Poiseuille equation, which governs flow in laminar regions, and is therefore inherent to microfluidic systems that utilize such flow cell geometries. Also, such flow cell geometries may be susceptible to contamination. Because such flow cell geometries allow for a finite, limited number of channels in a microfluidic system, such a finite number of channels may be shared between multiple different mixtures containing different analytes, reagents, drugs, and/or buffers. Fluid contents flowing through the same channel may become contaminated.

Described herein are devices, systems, and methods for processing analytes using open substrate or flow cell geometries that can address at least the problems discussed above. The devices, systems, and methods can be used to facilitate any application or process that involves a reaction or interaction between an analyte and a fluid (e.g., a fluid containing reagents, drugs, buffers, other analytes, etc.). Such a reaction or interaction can be chemical (e.g., a polymerase reaction) or physical (e.g., a displacement). The systems and methods described herein can benefit from higher efficiency, such as faster reagent delivery and less volume of reagent required per surface area. The systems and methods described herein can avoid contamination issues common to microfluidic channel flow cells that are fed from multiport valves that can be a source of carryover from one reagent to the next. The devices, systems, and methods can benefit from faster completion times, use of fewer resources (e.g., various reagents), and/or reduced system costs. The open substrate or flow cell geometries, as described herein, can be used to process any analyte, such as, but not limited to, nucleic acid molecules, protein molecules, antibodies, antigens, cells, and/or organisms. The open substrate or flow cell configuration can be used for any application or process, such as, but not limited to, sequencing by synthesis, sequencing by ligation, amplification, proteomics, single cell processing, barcoding, and sample preparation, as described herein.

The systems and methods may utilize a substrate that includes an array (e.g., a planar array) of individually addressable locations. Each location, or a subset of such locations, may have an analyte (e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.) immobilized thereon. For example, the analytes may be immobilized to the individually addressable locations via a support, such as a bead. The multiple analytes immobilized to the substrate may be copies of a template analyte. For example, the multiple analytes may have sequence homology. In other examples, the multiple analytes immobilized to the substrate may be different. The multiple analytes may be the same type of analyte (e.g., a nucleic acid molecule) or may be a combination of different types of analytes (e.g., a nucleic acid molecule, a protein molecule, etc.). One or more surfaces of the substrate may be exposed to and accessible from a surrounding open environment. For example, the array may be exposed and accessible from such a surrounding open environment. In some cases, the surrounding open environment may be controlled and/or restricted in a larger controlled environment, as described elsewhere herein.

A reagent may be dispensed onto a substrate at multiple locations, and/or multiple reagents may be dispensed onto a substrate at a single location via different mechanisms. In some cases, dispensing (of multiple reagents to multiple locations and/or to a single location) may be accomplished by relative motion between the substrate and a dispenser (e.g., a nozzle). For example, a reagent may be dispensed onto a substrate at a first location and then moved to a second location that is different from the first location due to forces (e.g., centrifugal force, centripetal force, inertial force, etc.) caused by the motion of the substrate. In another example, a reagent may be dispensed onto a reference location, and the substrate may be moved relative to the reference location such that the reagent is dispensed onto multiple locations on the substrate. In some cases, dispensing (of multiple reagents to multiple locations and/or to a single location) may be accomplished without relative motion between the substrate and a dispenser. For example, multiple dispensers may be used to dispense reagents to different locations and/or multiple reagents to a single location, or combinations thereof (e.g., multiple reagents to multiple locations). In another example, an external force such as wind (e.g., involving a pressure differential) can be applied to one or more surfaces of the substrate to direct the reagents to different locations across the substrate. In another example, a method for dispensing reagents (e.g., to multiple locations and/or multiple reagents to a single location) can include vibration. In such an example, the reagents can be distributed or dispensed to a single area or multiple areas of the substrate (or surface of the substrate). The substrate (or its surface) can then be subjected to vibration, which can spread the reagents to different locations across the substrate (or surface). Alternatively, or in addition, the method can include dispensing the reagents to the substrate using mechanical, electrical, physical, or other means. For example, a solution can be dispensed onto the substrate, and a physical scraper (e.g., a squeegee) can be used to spread the dispensed material or spread the reagents to different locations and/or obtain a desired thickness or uniformity across the substrate. Beneficially, such flexible dispensing can be achieved without contamination of the reagents. In some cases, when a volume of reagent is dispensed onto a substrate at a first location and then moved to a second location different from the first location, the volume of reagent can travel one or more paths such that one or more paths of travel are coated with the reagent. In some cases, such one or more paths of travel can include a desired surface area of the substrate (e.g., the entire surface area, one or more partial surface areas, etc.).

The reagent may be dispensed at a desired flow rate onto the uncoated surface or substrate. The flow rate of the fluid dispense may be about (e.g., at ambient temperature, or about 25° C.) 1 picoliters/minute, 10 picoliters/minute, 100 picoliters/minute, 1 nanoliters/minute, 10 nanoliters/minute, 100 nanoliters/minute, 1 microliters/minute, 10 microliters/minute, 100 microliters/minute, 1 milliliters/minute, 10 milliliters/minute, 100 milliliters/minute, up to 1 liter/minute. The flow rate of the fluid dispense may be between any of these flow rates. The flow rate of the fluid dispense may be at least any of these flow rates. Alternatively, the flow rate of the fluid dispense may be at most any of these flow rates. The flow rate may be adjusted according to the desired properties (e.g., thickness) of the reagent or solution layer.

The solution may include a reagent, a sample, or any useful substance. The solution may include a fluid having desirable flow characteristics. For example, the fluid may have a temperature variable viscosity. The solution may include a non-Newtonian fluid. The solution may include a power law fluid, such as a shear thinning (thixotropic) fluid or a shear thickening fluid. The solution may include a Newtonian fluid.

In some cases, the substrate may be rotatable about an axis. The analyte may be immobilized on the substrate during rotation. Reagents (e.g., nucleotides, antibodies, washing reagents, enzymes, etc.) may be dispensed onto the substrate before or during rotation of the substrate (e.g., spinning at a high rotational speed) to coat the array with the reagent and allow the analyte to interact with the reagent. For example, if the analyte is a nucleic acid molecule and the reagent includes nucleotides, the nucleic acid molecule may incorporate or otherwise react (e.g., transiently bind) one or more nucleotides. In another example, if the analyte is a protein molecule and the reagent includes antibodies, the protein molecule may bind or otherwise react with one or more antibodies. In another example, if the reagent includes washing reagents, the substrate (and/or the analyte on the substrate) may be washed of any unreacted (and/or unbound) reagents, drugs, buffers, and/or other particles.

In some cases, the substrate may be movable in any vector or direction, as described elsewhere herein. For example, such motion may be non-linear (e.g., in rotation about an axis). In another example, such motion may be linear. In other examples, motion may be a hybrid of linear and non-linear motion. Analytes may be immobilized on the substrate during any such motion. Reagents (e.g., nucleotides, antibodies, washing reagents, enzymes, etc.) may be dispensed onto the substrate before or during substrate motion to facilitate coating of the array with reagents and to allow analytes to interact with the reagents.

In some cases, when the substrate is rotatable, high speed coating across the substrate may be achieved by tangential inertia directing the spinning reagents partially radially (i.e., away from the axis of rotation) unconstrained during rotation, a phenomenon commonly referred to as centrifugal force. High speed rotation may include rotational speeds of at least 1 revolution per minute (rpm), at least 2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least 50 rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, at least 10,000 rpm, or more. This mode of directing reagents across the substrate may be referred to herein as centrifugal or inertial pumping. The inertial force can direct the unbound reagent across the substrate in any direction during any type of motion of the substrate (e.g., rotational, non-rotational, linear, non-linear, accelerated, etc.).

One or more signals (e.g., optical signals) can be detected from detection regions on the substrate before, during, or after dispensing of a reagent to generate an output. For example, the output can be an intermediate or final result obtained from processing the specimen. The signal can be detected on multiple occasions. The dispensing, rotation (or other movement), and/or detection operations can be repeated any number of times to process the specimen, in any order (independently or simultaneously). In some examples, the substrate can be washed (e.g., by dispensing a wash reagent) between successive dispenses of reagents. One or more detection operations can be performed within a desired time frame. For example, the detection operations can be performed within about 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds. In some cases, at least two detection operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds, etc. In some cases, at least three detection operations may be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds.

Provided herein is a method for processing a biological analyte, the method including providing a substrate including an array having biological analytes immobilized thereon, the substrate being rotatable about a central axis. In some cases, the array can be a planar array. In some examples, the array can be an array of wells. In some cases, the substrate can be textured and/or patterned. The method can include directing a solution across the substrate and contacting the solution with the biological analyte while rotating the substrate. The solution can be directed radially (e.g., outwardly) relative to the substrate to coat the substrate and contact the biological analyte immobilized in the array. In some examples, the solution can include a plurality of probes. In some cases, the solution can be a wash solution. The method can include subjecting the biological analyte to conditions sufficient to effect a reaction between at least one probe of the plurality of probes and the biological analyte. The reaction can generate one or more signals from at least one probe bound to the biological analyte. The method can include detecting the one or more signals, thereby analyzing the biological analyte.

In other cases, provided herein is a method for processing biological specimens that includes providing a substrate including an array having biological specimens fixed thereto, the substrate being movable relative to a reference axis. The method can include directing a solution across the substrate and contacting the solution with the biological specimens during the substrate movement. In some cases, the movement can be linear. In some cases, the movement can be non-linear. In some cases, the motion can be a hybrid of linear and non-linear motion.

In other cases, provided herein are methods of processing biological specimens, the methods including providing a substrate including an array having biological specimens fixed thereto. In some examples, the methods can include dispensing solutions at two different locations on the substrate and/or array. In some cases, the methods can include dispensing multiple solutions at a single location on the substrate and/or array, such as by using multiple dispensers. In some cases, the methods can include dispensing multiple solutions at multiple locations on the substrate and/or array. In some cases, the methods can include dispensing a single solution at a single location. The substrate can move relative to one or more dispensers. The substrate can be stationary relative to one or more dispensers. One or more dispense operations can be performed within a desired time frame. For example, the dispense operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds. In some cases, at least two dispense operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds, etc. In some cases, at least three dispense operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds.

Any operation or process of one or more of the methods disclosed herein may be performed within a desired time frame. In some cases, a combination of two or more operations or processes disclosed herein may be performed within a desired time frame. For example, both the dispense operation and the detection method may be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds. In some cases, at least two dispense and detection operations may be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds, etc. In some cases, at least three dispense and detection operations may be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds.

One or more methods disclosed herein can eliminate the need for barcoding of analytes (e.g., nucleic acid molecules), which can be time-consuming and expensive. For example, as described elsewhere herein, instead of or in addition to barcoding, the substrate and/or array may be spatially indexed to identify analytes. One or more methods disclosed herein can eliminate the need for unique barcoding of each individual analyte (e.g., each individual nucleic acid molecule).

The biological analyte can be any analyte derived from a sample. For example, the biological analyte can be a macromolecule, such as a nucleic acid molecule, a carbohydrate, a protein, a lipid, etc. The biological analyte can include multiple macromolecular groups, such as glycoproteins, proteoglycans, ribozymes, liposomes, etc. The biological analyte can be an antibody, an antibody fragment, or an engineered variant thereof, an antigen, a cell, a peptide, a polypeptide, etc. In some cases, the biological analyte includes a nucleic acid molecule. The nucleic acid molecule can include at least about 10, 100, 1000, 10, ... N-mers may contain 1, 2, 3, 4, 5 or 6 nucleotides and may bind to a common sequence. In some cases, the nucleic acid molecules may be amplified to generate a colony of nucleic acid molecules bound to a substrate or bound to beads that may be associated with or immobilized to a substrate. In some examples, the nucleic acid molecules may be attached to beads and subjected to a nucleic acid reaction, e.g., amplification, to generate a clonal population of nucleic acid molecules attached to beads.

Each nucleic acid molecule in any given nucleic acid sample may include a key sequence. The key sequence may be a synthetic sequence. In some instances, the key sequence may be up to about 6, 5, 4, 3, 2, or 1 base in length. Alternatively, the key sequence may be longer than 6 bases. The key sequence may be indicative of the originating sample. For example, the key sequence may be unique to a sample such that each sample of a plurality of samples has a unique key sequence. Individual analytes in a single sample may share the same key sequence. Alternatively, each sample may have a unique key sequence between its immediate neighbors when loaded onto the substrate. Advantageously, when two samples containing different key sequences are loaded into adjacent or otherwise proximate regions on the substrate, the nucleic acid molecules from the different samples may be easily distinguished based on the different key sequences, even if there is cross-contamination between regions (e.g., an outer nucleic acid molecule that is inadvertently loaded into an adjacent region due to spillover, etc.) with relatively short reads (e.g., much shorter than the reads of a unique barcode sequence configured to differentiate individual molecules).

The substrate may be a solid substrate. The substrate may comprise, in whole or in part, one or more of rubber, glass, metals such as silicon, aluminum, copper, titanium, chromium, or steel, ceramics such as titanium oxide or silicon nitride, plastics such as polyethylene (PE), low density polyethylene (LDPE), high density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadiene styrene (ABS), polyacetylene, polyamide, polycarbonate, polyester, polyurethane, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamine formaldehyde (MF), urea formaldehyde (UF), polyether ether ketone (PEEK), polyetherimide (PEI), polyimide, polylactic acid (PLA), furan, silicone, polysulfone, any mixture of any of the foregoing materials, or any other suitable material. The substrate may be fully or partially coated with one or more layers of a metal such as aluminum, copper, silver, or gold, an oxide such as silicon oxide (Si _x O _y , where x, y can take any possible value), a photoresist such as SU8, a surface coating such as an aminosilane or a hydrogel, polyacrylic acid, polyacrylamide dextran, polyethylene glycol (PEG), or any combination of any of the aforementioned materials, or any other suitable coating. The substrate may be completely or partially opaque to visible light. In some cases, the substrate may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% opaque to visible light. The substrate may have an opacity within a range defined by any two of the foregoing values. The substrate may be fully or partially transparent to visible light. In some cases, the substrate may transmit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% visible light. The substrate may have a transmittance within a range defined by any two of the aforementioned values. In some cases, the illumination output (e.g., laser output) may be adjusted based on the opacity or transmittance of the substrate. The one or more layers can have a thickness of at least 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, or at least 1 millimeter (mm). The one or more layers can have a thickness within a range defined by any two of the foregoing values. The surface of the substrate may be modified to include any of the binders or linkers described herein. The surface of the substrate may be modified to include active chemical groups such as amines, esters, hydroxyls, epoxides, etc., or combinations thereof. In some examples, such binders, linkers, active chemical groups, etc. may be added to the substrate as an additional layer or coating.

The substrate may have the general form of a cylinder, a cylindrical shell or disk, a rectangular prism, or any other geometric form. The substrate may have a thickness (e.g., smallest dimension) of at least 100 μm, at least 200 μm, at least 500 μm, at least 1 mm, at least 2 mm, at least 5 mm, or at least 10 mm. The substrate may have a thickness within a range defined by any two of the aforementioned values. The substrate may have a first lateral dimension (such as the width of a substrate having the general form of a rectangular prism or the radius of a substrate having the general form of a cylinder) of at least 1 mm, at least 2 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, at least 200 mm, at least 500 mm, or at least 1,000 mm. The substrate may have a first lateral dimension within a range defined by any two of the aforementioned values. The substrate can have a second lateral dimension (such as the length of a substrate having the general form of a rectangular prism) of at least 1 mm, at least 2 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, at least 200 mm, at least 500 mm, or at least 1,000 mm. The substrate can have a second lateral dimension within a range defined by any two of the preceding values.

The surface of the substrate may be planar. The surface of the substrate may be uncovered and exposed to the atmosphere. Alternatively or additionally, the surface of the substrate may be textured or patterned. For example, the substrate may comprise grooves, troughs, hills, and/or pillars. The substrate may define one or more cavities (e.g., microscale or nanoscale cavities). The substrate may define one or more channels. The substrate may have a regular texture and/or pattern across the surface of the substrate. For example, the substrate may have a regular geometric structure (e.g., a wedge, a cuboid, a cylinder, a sphere, a hemisphere, etc.) above or below a reference level of the surface. Alternatively, the substrate may have an irregular texture and/or pattern across the surface of the substrate. For example, the substrate may have any structure above or below a reference level of the substrate. In some examples, the texture of the substrate may include structures having a maximum dimension of up to about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% or less of the total thickness of the substrate or a layer of the substrate. In some cases, the texture and/or pattern of the substrate may define at least a portion of the individually addressable locations on the substrate. The textured and/or patterned substrate may be substantially planar.

For example, Figures 37A-37G show different examples of cross-sectional surface profiles of substrates. Figure 37A shows a cross-sectional surface profile of a substrate with a completely flat surface. Figure 37B shows a cross-sectional surface profile of a substrate with hemispherical troughs or grooves. Figure 37C shows a cross-sectional surface profile of a substrate with pillars, or alternatively or in combination with wells. Figure 37D shows a cross-sectional surface profile of a substrate with a coating. Figure 37E shows a cross-sectional surface profile of a substrate with spherical particles. Figure 37F shows the cross-sectional surface profile of Figure 37B with a first type of binder seeded or associated with each groove. Figure 37G shows the cross-sectional surface profile of Figure 37B with a second type of binder seeded or associated with each groove.

The substrate may comprise an array. For example, the array may be disposed on a side of the substrate. The array may be a planar array. The array may have the general shape of a circle, ring, rectangle, or any other shape. The array may include linear and/or non-linear rows. The array may be evenly spaced or distributed. The array may be arbitrarily spaced or distributed. The array may have regular spacing. The array may have irregular spacing. The array may be a textured array. The array may be a patterned array. The array may include a plurality of individually addressable locations. The individually addressable locations may be arranged in any convenient pattern. For example, the individually addressable locations may be randomly oriented on the array. The plurality of individually addressable locations may form distinct radial regions around a disk-shaped substrate. The plurality of individually addressable locations may form a square, rectangle, disk, circle, ring, pentagon, hexagon, heptagon, octagon, array, or any other pattern. One or more types of individually addressable locations may be generated. One or more types of individually addressable locations can form alternating regions of different types of individually addressable locations. One or more types of individually addressable locations can form blocked regions of different types of individually addressable locations. For example, if two types (A and B) of individually addressable locations are desired, the individually addressable locations can be arranged as alternating ABABAB, blocked AAABBB, or random, e.g., ABBAAB, AABBBA, BABBAA, etc. The types of individually addressable locations can be arranged in any useful pattern, such as squares, rectangles, disks, rings, pentagons, hexagons, radial patterns, etc. In some cases, the two types of individually addressable locations can have different chemical, physical, and/or biological properties (e.g., hydrophobicity, charge, color, topography, size, dimensions, geometric shape, etc.). For example, a first type of individually addressable location binds to a first type of biological analyte and does not bind to a second type of biological analyte, and a second type of individually addressable location binds to a second type of biological analyte and does not bind to the first type of biological analyte.

The analyte to be processed may be immobilized on the array. The array may include one or more binders, e.g., one or more physical or chemical linkers or adaptors, as described herein, bound to the biological analyte. For example, the array may include linkers or adaptors bound to the nucleic acid molecule. Alternatively or additionally, the biological analyte may be bound to a bead, which may be immobilized on the array. In some cases, a subset of the array may not be bound to the sample or analyte. For example, in a substrate configured to rotate about a central axis, the sample may not be bound to a plurality of individually addressable locations of the array located near the central axis. In some cases, the array may be bound to the sample or analyte, but not all of the array may be processed. For example, the substrate may be bound to the sample or analyte (e.g., including a nucleic acid molecule), but regions of the array adjacent to the border of the array may not undergo further processing (e.g., detection).

An individually addressable location may include a location of an analyte or a group of analytes that are accessible for manipulation. Manipulation may include placement, extraction, reagent dispensing, seeding, heating, cooling, or agitation. Extraction may include extracting an individual analyte or a group of analytes. For example, extraction may include extracting at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1,000 analytes or groups of analytes. Alternatively or additionally, extraction may include extracting up to 1,000, up to 500, up to 200, up to 100, up to 50, up to 20, up to 10, up to 5, or up to 2 analytes or groups of analytes. Manipulation may be accomplished, for example, by localized microfluidic, pipette, optical, laser, acoustic, magnetic, and/or electromagnetic interactions with the analyte or its surroundings.

In some cases, the individually addressable locations may be indexed, for example, spatially, such that an analyte fixed or bound to each individually addressable location may be identified. In some embodiments, the individually addressable locations are indexed by defining a portion of a substrate. In some embodiments, the surface of the substrate is defined using etching. In some embodiments, the surface of the substrate is defined using a notch in the surface. In some embodiments, the surface of the substrate is defined using a dye or ink. In some embodiments, the surface of the substrate is defined by depositing a topographical mark on the surface. In some embodiments, a sample, such as a control nucleic acid sample, may be used to define the surface of the substrate. As will be appreciated, a combination of positive and negative boundaries (or the absence thereof) may be used to index the individually addressable locations. In some cases, a single reference point or axis (e.g., a single boundary) may be used to index all of the individually addressable locations. In some embodiments, each of the individually addressable locations is indexed. In some embodiments, a subset of the individually addressable locations is indexed. In some embodiments, the individually addressable locations are not indexed, but different regions of the substrate are indexed.

The individually addressable locations, or individual regions that comprise the individually addressable locations, may be indexed or otherwise distinguished. In some cases, the individually addressable locations, or individual regions, may be distinguished only by sample loading (e.g., no physical boundaries). In some cases, a single region may be distinguished from other regions. In some cases, a single type of region may be distinguished from other types of regions. For example, different types of regions may contain different types of analytes or different sets of samples. For example, a first type of region ("A") may contain a first set of samples (or samples of a first type) and a second type of region ("B") may contain a second set of samples (or samples of a second type). A substrate may include a set of multiple regions A and a set of multiple regions B, where the multiple regions A are distinguishable from the multiple regions B. To enable such distinction, different samples may be loaded into the different types of regions in a predetermined spatial configuration.

In some cases, a key or barcode sequence on a sample can be used to distinguish and/or index a spatial location, an originating sample, or a combination thereof. For example, each nucleic acid molecule in any given nucleic acid sample can include a key sequence. The key sequence can be a synthetic sequence. The key sequence can be up to about 6 bases long, 5 bases long, 4 bases long, 3 bases long, 2 bases long, or 1 base long. Alternatively, the key sequence can be longer than 6 bases long. The key sequence can indicate the originating sample. For example, the key sequence can be unique to a sample such that each sample of a plurality of samples has a unique key sequence. Individual analytes of a single sample can share a common key sequence. Alternatively, each sample can have a unique key sequence between its immediate neighbors when loaded onto the substrate. Advantageously, when two samples containing different key sequences are loaded into adjacent or otherwise proximate regions on a substrate, even if there is cross-contamination between regions (e.g., an outer nucleic acid molecule that is inadvertently loaded into an adjacent region due to spillover, etc.) with relatively short reads (e.g., much shorter than the reads of a barcode sequence configured to distinguish individual molecules), the nucleic acid molecules from the different samples can be readily distinguished based on the different key sequences.

In some cases, spatial separation of analytes can be used to augment or replace the use of key or barcode sequences. For example, FIG. 40 shows a scheme for analysis of analytes in a single region or in multiple regions, including 7, 15, and 96 regions. For example, as shown in FIG. 40, analytes can be distributed across the entire surface (top left, "1-plex") distributed in separate regions, including 7 regions (top right, "7-plex"), 15 regions (bottom left, "15plex"), or 96 regions (bottom right, "96plex"). Analytes can be distributed in about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, or about 500 regions. The analytes may be distributed in regions of 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 200, 200 to 300, 300 to 400, or 400 to 500. In some cases, each region may contain a different analyte.

In some cases, different types of regions can be used for sample processing. A first type of region ("A") may include a first set of samples (or a first type of samples), and a second type of region ("B") may include a second set of samples (or a second type of samples). The first type of region and the second type of region may be regularly spaced apart from each other, as described elsewhere herein. In some cases, the first type of region and the second type of region may be spaced apart from a reference axis of the substrate. For example, the first type of region may be spaced apart from a reference axis of the substrate at least 1 micrometer, 10 micrometers, 100 micrometers, 1 millimeter, 10 millimeters, 100 millimeters, 1 centimeter, 10 centimeters, 100 centimeters, or more. Similarly, the second type of region may be spaced apart from a reference axis of the substrate. For example, the first type of region may be located at least 1 micrometer, 10 micrometers, 100 micrometers, 1 millimeter, 10 millimeters, 100 millimeters, 1 centimeter, 10 centimeters, 100 centimeters, or more from the reference axis of the substrate. Both types of regions may be located at least 1 micrometer, 10 micrometers, 100 micrometers, 1 millimeter, 10 millimeters, 100 millimeters, 1 centimeter, 10 centimeters, 100 centimeters, or more from the reference axis of the substrate.

For example, Figures 39A-39B show two examples of spatial loading schemes. In Figure 39A, the substrate includes two types of regions "A" and "B" arranged in a radial alternating manner relative to the central axis of the substrate. In Figure 39B, the substrate includes two types of regions "A" and "B" arranged in a triangular alternating manner across the substrate. Sample locations can be determined by loading a first set of samples into the A region, the first set of samples including a plurality of beads bound to the analytes of the first set of samples, detecting the plurality of beads and/or analytes and their locations on the substrate, and then loading a second set of samples into the B region, the second set of samples including a plurality of beads bound to the analytes of the second set of samples, detecting the plurality of beads and/or analytes and their locations on the substrate. Each sample in the first set of samples and the second set of samples can be associated with a label (e.g., a fluorescent dye). Even if the first set of samples is loaded primarily into the A region, there can be some crossovers where stray beads from the first set of samples are anchored in the B region. Even if the second set of samples is loaded mainly in the B region, there may be some crossovers where floating beads from the second set of samples are anchored in the A region. The location of the analytes of the first set of samples including crossover beads can be determined from the first image. The location of the analytes of the second set of samples including crossover beads can be determined from the second image. Advantageously, if the same type of fluorescent dye distinguishes analytes of two different samples ("P" and "Q"), with "P" deposited in the A region and "Q" deposited in the B region, it is possible to identify whether the analyte is from the "P" sample or the "Q" sample based on the type of region where the fluorescent signal is detected. The different regions may be alternating. The multiple regions may form any pattern, such as a triangle, square, rectangle, disk, circle, ring, pentagon, hexagon, heptagon, octagon, array, or any other pattern. The multiple regions may form an irregular pattern. The multiple regions may be discrete regions that are not patterned. The regions may be interleaved, interspersed, discontinuous, and/or of different sizes.

Although the examples herein describe two types of regions, there may be any number of regions (e.g., alternating regions) to achieve the alternating spatial distinctions described herein. For example, there may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 regions.

In some cases, the individually addressable locations may include distinct surface chemistries. The distinct surface chemistries may distinguish between different addressable locations. The distinct surface chemistries may distinguish between different regions. For example, a first location may have a first affinity for an object (e.g., a bead containing a nucleic acid molecule, e.g., an amplicon, immobilized thereon) and a second location may have a second different affinity for the object due to the different surface chemistry. The first location and the second location may or may not be located in the same region. The first location and the second location may or may not be arranged on the surface in alternating fashion. In another example, a first region (e.g., including a plurality of individually addressable locations) may have a first affinity for an object and a second region may have a second different affinity for the object due to the different surface chemistry. The first location type or region type may include a first surface chemistry and the second location type or region type may include a second surface chemistry. In some cases, a third location type or region type may include a third surface chemistry. For example, as shown in FIG. 50A, a first location type or region type may include a positively charged surface chemistry and/or a hydrophobic surface chemistry, and a second location type or region type may include a negatively charged surface chemistry and/or a hydrophilic surface chemistry. The same object (e.g., a bead with a nucleic acid molecule, e.g., an amplicon, immobilized thereon) may have a higher affinity for the first location type or region type compared to the second location type or region type. The same object may be attracted toward the first location type or region type and repelled from the second location type or region type. In another example, as shown in FIG. 50B, a first location type or region type (e.g., a positively charged surface chemistry or a negatively charged surface chemistry) that includes a first surface chemistry may interact with (e.g., have an affinity toward) a first sample type (e.g., a bead with a nucleic acid molecule, e.g., an amplicon, immobilized thereon) and exclude a second sample type (e.g., a bead that lacks, e.g., completely or in a substantial volume, a nucleic acid molecule, e.g., an amplicon, immobilized thereon). In some cases, the surface chemistry may include amines. In some cases, the surface chemistry may include silanes (e.g., tetramethylsilane). In some cases, the surface chemistry may include hexamethyldisilazane (HMDS). In some cases, the surface chemistry may include (3-aminopropyl)triethoxysilane (APTMS). In some cases, the surface chemistry may include any oligonucleotide molecule that has any degree of affinity for a surface primer molecule or another molecule.

The individually addressable locations of the plurality of locations (eg, alternating locations) can have regions. In some cases, the location is about 0.1 square microns (μm ² ), about 0.2 μm ² , about 0.25 μm ² , about 0.3 μm ² , about 0.4 μm ² , about 0.5 μm ² , about 0.6 μm ² , about 0.7 μm ^{2 , about 0.8 μm 2 ,} about 0.9 μm ² , about 1 μm ² , about 1.1 μm ² , about 1.2 μm ² , about 1.25 μm ² , about 1.3 μm ² , about 1.4 μm ² , about 1.5 μm ² , about 1.6 μm ^{2 , about 1.7 μm 2 ,} about 1.75 μm ² , about 1.8 μm ² , about 1.9 μm ² , about 2 μm ² , about 2.25 μm ² , about 2.5 μm ² , about 2.75 μm ² , about 3 μm ² , about 3.25 μm ² , about 3.5 μm ² , about 3.75 μm ² , about 4 μm ² , about 4.25 μm ² , about 4.5 μm ² , about 4.75 μm ² , about 5 μm ² , about 5.5 μm ² , or about 6 μm ^2. The locations can have an area within a range defined by any two of the foregoing values. The locations can have an area less than about 0.1 μm ² or greater than about 6 μm ² . In some cases, the location is about 0.1 microns (μm), about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 1.1 μm, about 1.2 μm, about 1.25 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.8 μm, about 1.9 μm, about 2.0 μm, about 2.1 μm, about 2.2 μm, about 2.3 μm, about 2.4 μm, about 2.5 μm, about 2.6 μm, about 2.7 μm, about 2.8 μm, about 2.9 μm, about 3.0 μm, about 3.1 μm, about 3.2 μm, about 3.3 μm, about 3.4 μm, about 3.5 μm, about 3.6 μm, about 3.7 μm, about 3.8 μm, about 3.9 ... The positions may have a width of about 1.7 μm, about 1.75 μm, about 1.8 μm, about 1.9 μm, about 2 μm, about 2.25 μm, about 2.5 μm, about 2.75 μm, about 3 μm, about 3.25 μm, about 3.5 μm, about 3.75 μm, about 4 μm, about 4.25 μm, about 4.5 μm, about 4.75 μm, about 5 μm, about 5.5 μm, or about 6 μm. In some cases, the positions may have a width within a range defined by any two of the aforementioned values. The positions may have a width less than about 0.1 μm or greater than about 6 μm. The positions (e.g., of the same type) may be distributed on the substrate with a pitch determined by the distance between the center of a first position and the center of a nearest or adjacent position (e.g., of the same type). The positions are about 0.1 microns (μm), about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 1.1 μm, about 1.2 μm, about 1.25 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.75 μm, about 1.8 μm, about 1.9 μm, The positions may be spaced apart at a pitch of about 2 μm, about 2.25 μm, about 2.5 μm, about 2.75 μm, about 3 μm, about 3.25 μm, about 3.5 μm, about 3.75 μm, about 4 μm, about 4.25 μm, about 4.5 μm, about 4.75 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some cases, the positions may be positioned at a pitch that is within a range defined by any two of the aforementioned values. The positions may be arranged at a pitch of less than about 0.1 μm or more than about 10 μm. In some cases, the pitch between any two positions of the same type may be determined as a function of the size of the loaded specimens (e.g., beads). For example, if the loaded object is a bead having a maximum diameter, the pitch may be at least as large as the maximum diameter of the loaded object.

Although the examples herein generally describe loading two samples or two sets of samples, any number of samples or sets of samples can be immobilized on the substrate. For example, the substrate may have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten samples or sets of samples immobilized. In some cases, at least about 10, 100, 1000, 10,000, 10 ... Where the samples are nucleic acid samples, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 nucleic acid samples may be immobilized on the substrate. In some cases, at least about 10, 100, 1000, 10, ...

Indexing may be performed using a detection method and may be performed in any convenient or useful step. The indexed, e.g., defined, substrate may be subjected to detection, such as optical imaging, to locate the indexed locations, the individually addressable locations, and/or the biological analyte. The imaging may be performed using a detection unit. The imaging may be performed using one or more sensors. The imaging does not have to be performed with the naked eye. The indexed substrate may be imaged prior to loading of the biological analyte. After loading of the biological analyte into the individually addressable locations, the substrate may be imaged again, e.g., to determine occupancy or to determine the location of the biological analyte relative to the substrate. In some cases, the substrate may be imaged after repeated cycles of nucleotide addition (or other probes or other reagents), as described elsewhere herein. Indexing of the substrate and known initial locations (individually addressable locations) of the biological analyte may allow for analysis and identification of the individually addressable locations and/or sequence information per location. Furthermore, spatial indexing may allow for identification of possible errors, e.g., sample contamination, sample loss, etc.

In some cases, indexing can be performed to identify, process, and/or analyze more than one type of biological analyte, as described above. For example, a first type of biological analyte, which may be labeled, may be loaded into a first set of locations in the substrate. The substrate may be imaged for a first indexing step of the first type of biological analyte. A second type of biological analyte may be loaded into a second set of locations in the substrate and imaged for a second indexing step of the second type of biological analyte. In some cases, the second type of biological analyte may be labeled such that the second type of biological analyte is distinguishable from the first type of biological analyte. Alternatively, the first type of biological analyte and the second type of biological analyte may be labeled in substantially the same detectable manner (e.g., the same dye), and the first and second images may be processed to generate a difference image, where the overlapping signals are due to the locations of the first type of biological analyte and the different signals are due to the locations of the second type of biological analyte. Alternatively, the first type of biological analyte and the second type of biological analyte may be labeled with a cleavable (or otherwise removable) label or tag (e.g., a fluorescent tag), and the label is cleaved after each imaging operation so that only the relevant analyte locations are imaged in each imaging operation. The substrate may then be analyzed and all of the locations containing the first biological analyte may be attributed to the first biological analyte and all of the locations containing the second biological analyte may be attributed to the second analyte. In some cases, labeling of the first and second analytes may not be necessary and attribution of locations to either the first or second analyte may be performed based on spatial location alone. This process may be repeated for any number or type of biological analyte.

The array may be coated with a binder. For example, the array may be randomly coated with the binder. Alternatively, the array may be coated with a binder arranged in a regular pattern (e.g., linear array, radial array, hexagonal array, etc.). The array may be coated with at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of individually addressable locations or the surface area of the substrate. The array may be coated with the binder on a portion of the individually addressable locations or a portion of the surface area of the substrate that is within a range defined by any two of the aforementioned values. The binder may be integral with the array. The binder may be added to the array. For example, the binder may be added to the array as one or more coating layers on the array.

The binder can immobilize the biological analyte through non-specific interactions, such as one or more of hydrophilic interactions, hydrophobic interactions, electrostatic interactions, physical interactions (e.g., adhesion to pillars or sedimentation in wells), and the like. The binder can immobilize the biological analyte through specific interactions. For example, if the biological analyte is a nucleic acid molecule, the binder can include an oligonucleotide adaptor configured to bind to the nucleic acid molecule. Alternatively or additionally, for example, to bind to other types of analytes, the binder can include one or more of an antibody, an oligonucleotide, a nucleic acid molecule, an aptamer, an affinity binding protein, a lipid, a carbohydrate, and the like. The binder can immobilize the biological analyte through any possible combination of interactions. For example, the binder can immobilize the nucleic acid molecule through a combination of physical and chemical interactions, a combination of protein and nucleic acid interactions, and the like. The array can include at least about 10, 100, 1000, 10,000, 100,000, 1 million, 10 ... Alternatively or additionally, the array may include up to about 100 million, 10 million, 1 million, 100,000, 10,000, 100, 10 or fewer binders. The array may have some binders within a range defined by any two of the preceding values. In some cases, a single binder may bind to a single biological analyte (e.g., a nucleic acid molecule). In some examples, a single binder may bind to multiple biological analytes (e.g., multiple nucleic acid molecules). In some examples, multiple binders may bind to a single biological analyte. Although the examples herein describe interactions between binders and nucleic acid molecules, binders may immobilize other molecules (such as proteins), other particles, cells, viruses, other organisms, etc.

In some cases, each location or a subset of such locations may have an analyte immobilized thereon (e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.). In other examples, a portion of the multiple individually addressable locations may have an analyte immobilized thereon. The multiple analytes immobilized on the substrate may be copies of a template analyte. For example, the multiple analytes (e.g., nucleic acid molecules) may have sequence homology. In other examples, the multiple analytes immobilized on the substrate may not be copies. The multiple analytes may be the same type of analyte (e.g., nucleic acid molecules) or may be a combination of different types of analytes (e.g., nucleic acid molecules, protein molecules, etc.).

In some examples, the array can include multiple types of binders. For example, the array can include different types of binders for binding different types of analytes. For example, the array can include a first type of binder (e.g., oligonucleotides) configured to bind a first type of analyte (e.g., nucleic acid molecules), and a second type of binder (e.g., antibodies) configured to bind a second type of analyte (e.g., proteins), etc. In another example, the array can include a first type of binder (e.g., a first type of oligonucleotide molecule) for binding a first type of nucleic acid molecule, and a second type of binder (e.g., a second type of oligonucleotide molecule) for binding a second type of nucleic acid molecule, etc. For example, the substrate can be configured to bind different types of analytes to specific fractions or specific locations on the substrate by having different types of binders at specific fractions or specific locations on the substrate.

The biological analyte may be fixed to the array at a predetermined individually addressable location of the plurality of individually addressable locations. The array can have any number of individually addressable locations. For example, the array can have at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10 million, at least 20 million, at least 50 million, at least 100 million, at least 200 million, at least 500 million, at least 100 million, at least 200 million, at least 500 million, at least 1 billion, at least 2 billion, at least 5 billion, at least 10 billion, at least 20 billion, at least 50 billion, or at least 100 billion individually addressable locations. An array can have several individually addressable locations within a range defined by any two of the aforementioned values. Each individually addressable location can be individually digitally and/or physically accessible (from the plurality of individually addressable locations). For example, each individually addressable location can be electronically or digitally located, identified, and/or accessed for mapping, sensing, association with a device (e.g., a detector, processor, dispenser, etc.), or other processing. Each individually addressable location can be indexed, as described elsewhere herein. Alternatively, the substrate can be indexed such that each individually addressable location can be identified during at least one step of a process. Alternatively or additionally, each individually addressable location can be physically located, identified, and/or accessed, for example, for physical manipulation or extraction of an analyte, reagent, particle, or other component located at the individually addressable location.

A plurality of biological analytes may be immobilized on the array at spatially distinct locations. As described elsewhere herein, a mask or barrier may be used to obtain spatial separation of the biological analytes. Alternatively or in combination, the biological analytes may be separated using different fluid compositions. In some cases, the fluid compositions may be immiscible. For example, a first solution (e.g., an oil, organic solution, or other hydrophobic or lipophilic solution) may contain a first biological analyte, and a second solution (e.g., a hydrophilic, aqueous, polar, or ionic solution) may contain a second biological analyte. The first and second solutions may be immiscible. The substrate may be exposed to the first solution in a defined area, for example, using a mask (e.g., covering or masking other areas of the substrate). In some cases, the first biological analyte may be associated with the defined area (e.g., an individually addressable location), and the first solution may be removed from the substrate. The substrate may then be exposed to a second solution. The second biological analyte may then be associated with the unoccupied sites of the substrate. Alternatively, the substrate may be pretreated so that biological analytes can be loaded into separate locations. In one non-limiting example, the substrate may be patterned with distinct hydrophobic and hydrophilic regions (e.g., using photolithography, soft lithography, etching, etc.) that can attract or repel subsets of biological analytes. In another non-limiting example, an inert polymer such as polyethylene glycol (PEG) may be patterned into separate regions to prevent attachment or biological analytes to the substrate in the separate regions.

Each individually addressable location may have a general shape or form of a circle, a pit, a bump, a rectangle, or any other shape or form. Each individually addressable location may have a first lateral dimension (such as a radius of an individually addressable location having the general shape of a circle, or a width of an individually addressable location having the general shape of a rectangle). The first lateral dimension may be at least 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or at least 10,000 nm. The first lateral dimension may be within a range defined by any two of the aforementioned values. Each individually addressable location may have a second lateral dimension (such as a length of an individually addressable location having the general shape of a rectangle). The second lateral dimension may be at least 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or at least 10,000 nm. The second lateral dimension may be within a range defined by any two of the foregoing values. In some examples, each individually addressable location may have or be associated with a binder to immobilize the analyte to the binder, as described herein. In some examples, only a portion of the individually addressable locations may have or be associated with a binder. In some examples, an individually addressable location may have or be associated with multiple binders to immobilize the analyte thereto.

The individually addressable locations can be generated using a variety of methods. In one embodiment, the method can include generating the individually addressable locations using one or more barriers. In some embodiments, the barriers may be removed during any convenient operation. For example, the barriers can be removed before or after binding the analyte to the individually addressable locations. The barriers can be removed before or after loading a solution containing multiple probes. The barriers can be removed before or after subjecting the analyte to conditions sufficient to cause a reaction between the probes and the analyte. The barriers can be removed before or after detection of one or more signals from the bound probes and analytes. The barriers may be removed before or after detection of the bound probes and analytes. The barriers can be removed before or after repeating any of the above processes. In some cases, the barriers may not be removed.

The barrier may include a physical, chemical, biological, or any other type of obstacle. In some embodiments, the barrier includes a physical obstacle. In one such example, a mold may be used, and a portion of the mold may prevent the movement of the fluid to a particular area. The mold may be produced using a variety of means, such as injection molding, machining, heat treatment, fiber spinning, bonding and joining, casting, rolling, forging, 3D printing, etc. In some embodiments, the barrier may be configured to dissolve at any convenient step. The barrier may be configured to dissolve, evaporate, or sublime. In some cases, the barrier may be melted and removed. In some cases, removal of the barrier or a portion of the barrier may be accomplished using an air knife. In some cases, the barrier includes a chemical obstacle. In some cases, the barrier includes a polymer. The barrier may include polyethylene glycol (PEG). In some cases, the barrier may include a solution. The solution may be viscous. The solution may have a temperature variable viscosity. The solution may be a non-Newtonian fluid. The solution may be a power law fluid, such as a shear thinning fluid (e.g., thixotropic) or a shear thickening fluid. The solution may be a Newtonian fluid. In some embodiments, the barrier comprises a fluid that is immiscible with the loading solution. In some cases, the barrier is a hydrophobic region on the substrate.

A mask may additionally or alternatively be used to prevent binding of the sample and/or biological analyte to regions of the substrate. Alternatively or in combination, a subset of the individually addressable locations containing the biological analyte may be masked, for example, to prevent binding of the probe to the biological analyte. The mask may include a barrier, such as a physical, chemical or biological barrier. The mask may include a film having a removed portion. In some cases, the mask may be interfaced with the substrate prior to introduction of the biological analyte. In such cases, introduction of the biological analyte may allow the biological analyte to bind to the exposed regions of the mask-substrate interface, while the unexposed regions may remain free of the biological analyte. In any convenient process, the substrate may be unmasked. Any combination of masks may be used. For example, a first mask may be used to load a first biological analyte into the desired regions. The first mask may then be removed and a second mask may be used to load a second biological analyte into the desired regions. The first and second regions may have overlapping regions or may remain spatially distinct. The barriers and masks may be used in combination or separately.

The analytes bound to the individually addressable locations may include, but are not limited to, molecules, cells, organisms, nucleic acid molecules, nucleic acid colonies, beads, clusters, polonies, DNA nanoballs, or any combination thereof (e.g., beads having one or more nucleic acid molecules, e.g., one or more clonal populations of nucleic acid molecules attached thereto). The bound analytes may be immobilized in an array in a regular, patterned, periodic, random, or pseudorandom configuration, or any other spatial arrangement. In some embodiments, the analyte is bound to a bead(s), which may then be associated with or immobilized to a substrate or region of a substrate (e.g., an individually addressable location). In some embodiments, the analyte comprises a bead or multiple beads. In some cases, the bead or multiple beads may comprise another analyte (e.g., a nucleic acid molecule) or a clonal population of other analytes (e.g., nucleic acid molecules amplified on the beads). Such other analytes may be attached or otherwise bound to the beads. For example, the analyte may comprise multiple beads, each bead having a clonal population of nucleic acid molecules bound thereto. In some cases, the beads are magnetic and the application of a magnetic field or the use of a magnet can be used to direct the analyte or beads containing the analyte to the individually addressable locations. In some cases, a fluid can be used to direct the analyte to the individually addressable locations. The fluid can be a ferrofluid and a magnet can be used to direct the fluid to the individually addressable locations. The individually addressable locations may alternatively or in combination include materials that are sensitive to stimuli, such as thermal, chemical, or electrical or magnetic stimuli. For example, the individually addressable locations may include photosensitive polymers or reagents that are activated upon exposure to electromagnetic radiation. In some cases, caged molecules can be used to reveal binding (e.g., biotin) moieties on the substrate. Subsequent exposure to light of a particular wavelength can result in uncaging of the binding moieties. Beads containing the analyte, e.g., containing streptavidin, can then associate with the uncaged binding moieties. In some cases, a subset of the individually addressable locations may not include beads. In such cases, blank beads can be added to the substrate. The blank beads can then occupy areas not occupied by the analyte. In some cases, the blank beads have a higher binding affinity or activity for the individually addressable locations than the beads containing the analyte. In some cases, the unoccupied locations may be destroyed. In some cases, the unoccupied locations may be subjected to a process to remove unbound analytes, such as aspiration, washing, air blasting, etc. In some cases, a sample containing a biological analyte may be loaded onto a substrate using a device, such as a microfluidic device, a closed flow cell, etc. The loaded biological analyte may then associate or be immobilized with the substrate or an individually addressable location of the substrate. In such cases, the device may be removed after loading of the sample.

The biological analyte may be bound to any number of beads. Different biological analytes may be bound to any number of beads. The beads may be unique (i.e., different from one another). Any number of unique beads may be used. For example, at least about 10, 100, 1000, 10,000, 10,000, 1 million, 10 million, 100 million, 100 million, or more different beads may be used. Alternatively or additionally, up to about 100 million, 10 million, 1 million, 100,000, 10,000, 100, 10, or fewer different beads may be used. The number of different beads may be within a range defined by any two of the aforementioned values. The beads may be distinguished from one another using a property of the beads, such as color, reflectance, anisotropy, brightness, fluorescence, etc.

The sample can be diluted so that the approximate occupancy of the individually addressable locations is controlled. The sample can be diluted to at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:10,000, 1:10,000, 1:100,000, 1:100,000, 1:10 million, 1:10 million, 1:10 billion. Alternatively, it may be diluted up to the dilution of the A sample, and may be diluted to a dilution of at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:10,000, 1:10 ...

In some examples, the sample may include beads. The beads may be distributed on the surface in any pattern or randomly. The beads may be distributed in one or more regions of the surface (e.g., regions having a particular surface chemistry). In some cases, the beads may be distributed on a hexagonal lattice surface or regions of the surface, as shown in FIG. 49, which shows a zoomed-out image of a portion of the surface in the right panel and a zoomed-in image of a cross section of a portion of the surface in the left panel. In some examples, the sample including beads may be distributed on a surface that includes distinct locations/regions that are distinguished by surface chemistry (e.g., as shown in FIGS. 50A and 50B). For example, the sample including beads may be dispensed on a surface that includes positively charged locations/regions and/or hydrophobic locations/regions. The beads may have a high affinity for a first location type or region type (e.g., positively charged). The beads may have a low affinity for a second location type or region type (e.g., hydrophobic). The locations may contain 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less beads per location. In some embodiments, the beads may be substantially centered within the individually addressable locations. The locations may have a width of up to about 0.5, up to about 0.6, up to about 0.7, up to about 0.8, up to about 0.9, up to about 1, up to about 1.1, up to about 1.2, up to about 1.3, up to about 1.4, up to about 1.5, up to about 1.6, up to about 1.7, up to about 1.8, up to about 1.9, up to about 2, up to about 2.1, up to about 2.2, up to about 2.3, up to about 2.4, up to about 2.5, up to about 2.6, up to about 2.7, up to about 2.8, up to about 2.9, or up to about 3 times the diameter (e.g., maximum diameter) of the bead. In some embodiments, the regions may be spaced apart with a pitch determined by the distance between the center of a first location and the center of a nearest or adjacent location of the same type. The locations may be spaced apart with a pitch that is at least about 1, at least about 1.2, at least about 1.4, at least about 1.6, at least about 1.8, at least about 2, at least about 2.2, at least about 2.4, at least about 2.6, at least about 2.8, at least about 3, at least about 3.2, at least about 3.4, at least about 3.6, at least about 3.8, at least about 4, at least about 4.2, at least about 4.4, at least about 4.6, at least about 4.8, or at least about 5 times the diameter (e.g., maximum diameter) of the beads. In some cases, one or more of location size, location spacing, bead affinity, and location surface chemistry can be adjusted to reduce deviation of bead contact points from the center of the region.

A surface comprising a plurality of individually addressable locations may be loaded with beads. The beads may be loaded onto the surface with an occupancy rate determined by the number of locations of a given location type that contain at least one bead out of the total number of locations of the same location type. A surface comprising a plurality of locations may have an occupancy rate of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99.5%, or up to about 100%. For example, the surface may have at least about 90% of the locations of a given location type that are loaded with at least one bead. The beads may land on the surface with a landing efficiency determined by the number of beads that bind to the surface out of the total number of beads dispensed onto the surface. The beads may be dispensed onto the surface with a landing efficiency of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, up to about 100%. In some embodiments, one or more of the temperature, incubation time, surfactant, or salt concentration of the solution containing the beads may be adjusted to increase the bead occupancy. In some embodiments, one or more of the temperature, incubation time, surfactant, or salt concentration of the solution containing the beads may be adjusted to increase the bead loading efficiency.

In some cases, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the available surface area of the substrate may be configured to receive beads. If less than 100% of the available surface area carries beads (e.g., beads are immobilized), the negative space (e.g., positions where no beads are present) can be used as a reference to identify and/or index different individually addressable locations of the positive space (e.g., positions where beads are present). In one example, a single individually addressable location acting on the negative space is sufficient to index the entire substrate. In such an example, a single individually addressable location always remains "dark" during imaging, such as in an array, as opposed to other individually addressable locations in the positive space that light up (e.g., fluoresce) at different times, so that the single, always "dark" individually addressable location can serve as a reference for all other individually addressable locations. In another example, multiple individually addressable locations acting on the negative space can facilitate substrate indexing. Alternatively or additionally, a "bright" reference bead that is always (e.g., always fluoresces regardless of time) can be used as a reference to identify and/or index different individually addressable locations in the positive space. In such a case, different individually addressable locations can be identified and/or indexed even when 100% or substantially 100% of the available surface area is loaded with beads, including the reference beads.

A sample including beads can be dispensed onto a surface. In some cases, the beads can be dispensed onto a surface in a substantially helical pattern. For example, the system shown in FIG. 48 and/or the method shown in FIG. 51 can be used to dispense beads in a helical pattern. In some examples, the beads can be dispersed in a helical pattern moving radially inward toward the axis of rotation of the surface. In some examples, the beads can be dispersed in a helical pattern moving radially outward from the axis of rotation of the surface. As shown in FIG. 48, a sample (e.g., a sample including beads) can be dispensed from a dispensing probe 4801 (e.g., a nozzle) onto a substrate 4803 (e.g., a wafer) to form a layer 4805. The dispensing probe can be positioned at a fixed height ("Z") above the substrate. In the illustrated example, the beads can be held within layer 4805 and fixed to the substrate by electrostatic retention. Each set of beads can include a population of amplification products (e.g., nucleic acid molecules) fixed thereto, where the amplification products accumulate on the negative charges on the beads with an affinity for the positive charges. The substrate includes alternating surface chemistry between the distinguishable locations, with a first location type including APTMS carrying a positive charge that has an affinity for the negative charge of the amplified beads (e.g., beads that have an amplification product immobilized thereon and are distinguished from negative beads that do not), and a second location type including HMDS that has a lower affinity and/or is a repellent for the amplified beads. Within layer 4805 containing the dispensed sample, the amplified beads can land successfully at a first location of the first location type (such as 4807). In the illustrated example, the location size is 1 micron, the pitch between different locations of the same location type (e.g., the first location type) is 2 microns, and the layer has a depth of 15 microns. To obtain a substantially spiral pattern, the substrate and/or the dispense probe can have an angular and/or linear velocity relative to each other.

The sample may be dispensed as shown on an open surface as shown in FIG. 51. In some cases, the substrate may be rotating relative to the dispensing probe. In some cases, the dispensing probe may be moving radially relative to the substrate relative to the axis of rotation of the substrate. In some cases, the substrate may be moving linearly relative to the dispensing probe. In some cases, the substrate may be rotating relative to the dispensing probe while moving linearly relative to the dispensing probe, thereby dispensing the sample in a helical pattern. In some cases, the substrate may be rotating relative to the dispensing probe while the dispensing probe is moving radially relative to the substrate relative to the axis of rotation of the substrate, thereby dispensing the sample in a helical pattern. The substrate and the dispensing probe may be moved in any configuration relative to one another to achieve a substantially helical pattern. The substrate may be rotating at a rotational frequency of 60 rpm or less, 50 rpm or less, 40 rpm or less, 30 rpm or less, 25 rpm or less, 20 rpm or less, 15 rpm or less, 14 rpm or less, 13 rpm or less, 12 rpm or less, 11 rpm or less, 10 rpm or less, 9 rpm or less, 8 rpm or less, 7 rpm or less, 6 rpm or less, 5 rpm or less, 4 rpm or less, 3 rpm or less, 2 rpm or less, or 1 rpm or less. In some cases, the rotational frequency may be within a range defined by any two of the aforementioned values. In some cases, the substrate may be rotating at a rotational frequency of about 5 rpm.

The spiral dispense pattern can have a path width determined by the width of the area coated by the fluid dispensed during one rotation of the surface. The spiral dispense pattern can have a path pitch determined by the distance between the center of the fluid dispense path at a first position and the center of the fluid dispense path at a second position after one rotation of the substrate. In some cases, the path width may be greater than the path pitch. For example, fluid dispensed along the path during a substrate rotation may overlap with fluid dispensed along the path during a preceding substrate rotation. In some cases, the path pitch may be greater than the path width. For example, fluid dispensed along the path during a substrate rotation may be separated from fluid dispensed along the path during a preceding substrate rotation. In some cases, the path width may be similar to the path pitch. For example, fluid dispensed along the path during a substrate rotation may not be substantially separated from fluid dispensed along the path during a preceding substrate rotation, and fluid dispensed along the path during a substrate rotation may not substantially overlap with fluid dispensed along the path during a preceding substrate rotation.

The substrate may be configured to rotate about an axis. In some cases, the systems, devices, and apparatus described herein may further comprise a rotation unit configured to rotate the substrate. The rotation unit may comprise a motor and/or rotor for rotating the substrate. Such a motor and/or rotor may be directly or indirectly mechanically connected to the substrate via intermediate components (e.g., gears, stages, actuators, disks, pulleys, etc.). The rotation unit may be automated. Alternatively or additionally, the rotation unit may receive a manual input. The rotation axis may be an axis passing through the center of the substrate (e.g., as shown in FIG. 33). The axis may be an off-center axis. For example, the substrate may be affixed to a chuck (e.g., a vacuum chuck) of a spin-coating apparatus. The substrate may be configured to rotate at a rotational speed of at least 1 revolution per minute (rpm), at least 2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least 50 rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, or at least 10,000 rpm. The substrate may be configured to rotate at a rotational speed within a range defined by any two of the aforementioned values. The substrate may be configured to rotate at different rotational speeds during different operations described herein. The substrate may be configured to rotate at a rotational speed that varies according to a time-dependent function, such as a ramp, a sine wave, a pulse, or other function or combination of functions. The time-varying function may be periodic or aperiodic.

The substrate may be configured to move with any vector relative to a reference point. In some cases, the systems, devices, and apparatuses described herein may further comprise an operating unit configured to move the substrate. The operating unit may include any mechanical component, such as a motor, rotor, actuator, linear stage, drum, roller, pulley, etc., to move the substrate. Such components may be mechanically connected to the substrate directly or indirectly through intermediate components (e.g., gears, stages, actuators, disks, pulleys, etc.). The operating unit may be automated. Alternatively or additionally, the operating unit may receive manual input. The substrate may be configured to move with any velocity. In some cases, the substrate may be configured to move with different velocities during different operations described herein. The substrate may be configured to move with a velocity that varies according to a time-dependent function, such as a ramp, a sine wave, a pulse, or other function or combination of functions. The time-varying function may be periodic or non-periodic.

The solution may be supplied to the substrate before or during a rotation (or other motion) of the substrate to centrifugally (or otherwise inertially) direct the solution across the array. In some cases, the solution may be supplied to the planar array in pulses during a rotation of the substrate, thereby generating annular waves of solution moving radially outward. In some cases, the solution may be supplied to the planar array during other pulsed motions of the substrate, thereby generating waves of solution moving in a particular direction. The pulses may have periodic or non-periodic (e.g., arbitrary) intervals. The series of pulses may include a series of waves that generate a surface reagent exchange. The surface reagent exchange may include a wash with each subsequent pulse including a reduced concentration of the surface reagent. The solution may have a temperature different from that of the substrate, thereby providing a source or sink of thermal energy to the substrate or analytes located on the substrate. The thermal energy may provide a temperature change to the substrate or analytes. The temperature change may be transient. The temperature change may enable, disable, enhance, or inhibit a chemical reaction, such as a chemical reaction performed on the analyte. For example, the chemical reaction may include denaturation, hybridization, or annealing of nucleic acid molecules. The chemical reaction may include a step in a polymerase chain reaction (PCR), bridge amplification, or other nucleic acid amplification reaction. Temperature changes may modulate, increase, or decrease the signal detected from the analyte.

The array may be in fluid communication with at least one sample inlet (of a fluid channel). The array may be in fluid communication with the sample inlet through a non-solid gap, e.g., an air gap. In some cases, the array may further be in fluid communication with at least one sample outlet. The array may be in fluid communication with the sample outlet through an air gap. The sample inlet may be configured to direct a solution to the array. The sample outlet may be configured to receive a solution from the array. The solution may be directed to the array using one or more dispensing nozzles. For example, the solution may be directed to the array using at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 dispensing nozzles. The solution may be directed to the array using a number of nozzles that are within a range defined by any two of the aforementioned values. In some cases, different reagents (e.g., different types of nucleotide solutions, different probes, washing solutions, etc.) can be dispensed through different nozzles, such as to prevent contamination. Each nozzle can be connected to a dedicated fluid line or fluid valve, which can further prevent contamination. Certain reagents may be dispensed through one or more nozzles. One or more nozzles may be directed at the center of the substrate or in close proximity to the center of the substrate. Alternatively, one or more nozzles may be directed at or near a location on the substrate other than the center of the substrate. Alternatively or in combination, one or more nozzles may be directed closer to the center of the substrate than one or more of the other nozzles. For example, one or more nozzles used to dispense washing reagents may be directed closer to the center of the substrate than one or more nozzles used to dispense active reagents. One or more nozzles may be positioned at different radii from the center of the substrate. Two or more nozzles can be operated in combination to deliver fluid to the substrate more efficiently. One or more nozzles may be configured to deliver fluid to the substrate as a jet, a spray (or other dispersion fluid), and/or droplets. One or more nozzles may be operated to spray the fluid before delivery to the substrate. For example, the fluid may be delivered as aerosol particles.

The solution may be dispensed onto the substrate while the substrate is stationary. The substrate may then be rotated (or other motion) following dispensing of the solution. Alternatively, the substrate may be rotated (or other motion) prior to dispensing the solution. The solution may then be dispensed onto the substrate while the substrate is rotating (or otherwise moving).

Rotation of the substrate creates a centrifugal force (or inertial force directed away from the axis) on the solution, causing the solution to flow radially outward over the array. In this manner, rotation of the substrate can direct the solution across the array. Continued rotation of the substrate over a period of time can dispense a fluid film of approximately constant thickness across the array. The rotation speed of the substrate can be selected to achieve a desired thickness of the film of solution on the substrate. The film thickness can be related to the rotation speed by equation (1):

(1)

where h(t) is the thickness of the fluid film at time t, μ is the viscosity of the fluid, ω is the rotational speed, and C is a constant.

Alternatively or in combination, the viscosity of the solution may be selected to achieve a desired thickness of the film of the solution on the substrate. For example, the rotation speed of the substrate or the viscosity of the solution may be selected to achieve a film thickness of at least 10 nanometers (nm), at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, or at least 1 mm. The rotation speed of the substrate and/or the viscosity of the solution may be selected to achieve a film thickness within a range defined by any two of the aforementioned values. The viscosity of the solution may be controlled by controlling the temperature of the solution. The thickness of the film may be measured or monitored. The measurement or monitoring of the film thickness may be incorporated into a feedback system to better control the thickness of the film. The thickness of the film may be measured or monitored by various techniques. For example, the thickness of the film can be measured or monitored by thin film spectroscopy using a thin film spectrometer, such as a fiber spectrometer.

In some cases, one or more factors such as the rotational speed of the substrate, the acceleration of the substrate (e.g., rate of change of speed), the viscosity of the solution, the angle of dispensing the solution (e.g., contact angle of the reagent stream), the radial coordinate of dispensing the solution (e.g., on-center, off-center, etc.), the temperature of the substrate, the temperature of the solution, and other factors can be adjusted and/or otherwise optimized to achieve a desired wetting on the substrate and/or film thickness on the substrate, such as to facilitate uniform coating of the substrate. In some cases, a surfactant may be added to the solution, or a surfactant may be added to the surface to facilitate uniform coating or to facilitate sample loading efficiency. Such optimization can prevent the solution from exiting the substrate along a relatively concentrated flow or travel path such that the fluid contacts the substrate only over a partial surface area (as opposed to the entire surface area), in which case a significantly larger amount of reagent may have to be dispensed to achieve a uniform and complete coating of the substrate. Such optimization can also prevent the solution from scattering or otherwise reflecting or bouncing off the substrate upon contact and disturbance with the surface fluid. Alternatively or in combination, the thickness of the solution can be adjusted using mechanical, electrical, physical, or other mechanisms. For example, the solution can be dispensed onto a substrate and then leveled using a physical scraper, such as a squeegee, to obtain a desired thickness uniformity across the substrate.

In some cases, the rotation of the substrate may be slow enough so as not to generate substantial centrifugal forces (or inertial forces directed away from the axis) on the solution. Advantageously, one or more reagents dispensed onto the substrate may remain substantially local to the landing location, without significantly and/or preemptively moving outward relative to the axis of rotation, for example to invade another reaction space on the open substrate. The substrate may be rotating at a rotational frequency of 60 rpm or less, 50 rpm or less, 40 rpm or less, 30 rpm or less, 25 rpm or less, 20 rpm or less, 15 rpm or less, 14 rpm or less, 13 rpm or less, 12 rpm or less, 11 rpm or less, 10 rpm or less, 9 rpm or less, 8 rpm or less, 7 rpm or less, 6 rpm or less, 5 rpm or less, 4 rpm or less, 3 rpm or less, 2 rpm or less, or 1 rpm or less. In some cases, the rotation frequency may be within a range defined by any two of the aforementioned values. In some cases, the substrate may be rotating at a rotation frequency of about 5 rpm. In some cases, the solution may be dispensed onto the surface in a spiral pattern. For example, the solution may be dispensed into a spiral pattern using the system shown in FIG. 48 or FIG. 51. As shown in FIG. 48, a solution (e.g., a sample or a wash solution) may be dispensed from a dispensing probe (e.g., a nozzle). In some embodiments, the solution may be dispensed from multiple dispensing probes. For example, a first reagent in the solution may be dispensed from a first dispensing probe, a second reagent in the solution may be dispensed from a second dispensing probe, and a third reagent in the solution may be dispensed from a third dispensing probe. Reagents dispensed from different dispensing probes may combine on the substrate to form a homogenous solution. The dispensing probe may be positioned at a fixed height above the substrate (e.g., a wafer). Reagents may be dispensed onto an open surface as shown in FIG. 51. In some cases, the substrate may be rotating relative to the dispensing probes. In some cases, the dispensing probe may move radially relative to the substrate relative to the substrate's axis of rotation. In some cases, the substrate may move linearly relative to the dispensing probe. In some cases, the substrate may rotate relative to the dispensing probe while moving linearly relative to the dispensing probe, thereby dispensing the sample in a spiral pattern. In some cases, the substrate may rotate relative to the dispensing probe while the dispensing probe moves radially relative to the substrate relative to the substrate's axis of rotation, thereby dispensing the sample in a spiral pattern. The rotation speed of the substrate, the flow rate of the solution, or the viscosity of the solution may be selected to achieve a film thickness of at least 10 nanometers (nm), at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, or at least 1 mm.

In some cases, the solution may be heated before being dispensed onto the substrate. The solution may be at a temperature higher than ambient temperature. The solution may be heated to about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C before being dispensed. In some cases, the solution may be heated to a temperature within a range defined by any two of the aforementioned values.

In some cases, the substrate may be rotated at a variable angular velocity. As the radial distance of the dispensing probe from the axis of rotation of the substrate changes, the angular velocity of the substrate may be varied such that the linear velocity of the substrate relative to the dispensing probe is substantially maintained. For example, the angular velocity of the substrate may decrease as the dispensing probe dispenses fluid in a spiral path that progresses outward relative to the axis of rotation of the substrate. In another example, the angular velocity of the substrate may increase as the dispensing probe dispenses fluid in a spiral path that progresses inward relative to the axis of rotation of the substrate. In some examples, the dispensing probe may dispense fluid at a variable flow rate. The flow rate of the dispensing probe may be varied such that the amount of fluid dispensed per unit area of the substrate is substantially maintained. For example, the flow rate of the dispensing probe may increase as the dispensing probe dispenses fluid in a spiral path that progresses outward relative to the axis of rotation of the substrate. In another example, the flow rate of the dispensing probe may decrease as the dispensing probe dispenses fluid in a spiral path that progresses inward relative to the axis of rotation of the substrate.

One or more solutions dispensed onto the surface may undergo a reaction on the surface. For example, a first solution (e.g., containing a reactant) dispensed onto the surface may react with a second solution (e.g., containing an enzyme) dispensed onto the surface above the first solution. One or more solutions dispensed onto the surface may inactivate or quench a chemical reaction. For example, a quenching solution (e.g., containing EDTA or an acid) may be added to the substrate above the reaction to quench the reaction. A solution (e.g., a solution containing reactants, a solution containing enzymes, or a quenching solution) may be dispensed onto the surface in a pattern (e.g., a spiral pattern). In some embodiments, the quenching solution is dispensed onto the surface in the same pattern as the solution containing reactants, thereby maintaining a substantially constant reaction time at each point on the surface where the solution is dispensed. In some embodiments, the quenching solution is dispensed onto the surface in the same pattern as the solution containing enzymes, thereby maintaining a substantially constant reaction time at each point on the surface where the solution is dispensed. For example, a solution containing reactants may be dispensed onto the surface in a spiral path directed inward toward the axis of rotation of the substrate. The solution containing reactants may be dispensed at a constant speed and the substrate may be rotated at a variable speed such that the volume dispensed per unit area is substantially constant. The solution containing enzymes may be dispensed along the same spiral path as the solution containing reagents. The solution containing enzymes may be dispensed at a constant speed and the substrate may be rotated at a variable speed such that the time between dispensing the solution containing reactants and dispensing the solution containing enzymes is substantially the same at any point along the spiral path. A quenching solution may be dispensed along the same spiral path as the solution containing enzymes. The quenching solution may be dispensed at a constant speed and the substrate may be rotated at a variable speed such that the time between dispensing the solution containing enzymes and the quenching solution is substantially the same at any point along the spiral path. Alternatively or additionally, one or more solutions dispensed onto the surface may similarly activate or catalyze a chemical reaction. For example, an activating solution (e.g., containing a catalyst) can be added to the substrate above the reaction (e.g., in the same dispense pattern) to activate or catalyze the reaction.

Various methods can be used to dispense one or more solutions onto a substrate to ensure substantially similar reaction times across the areas of the substrate contacted with the one or more solutions. In some embodiments, the solutions can be spin-coated onto the surface by dispensing the solutions at or near the axis of rotation of the rotating substrate such that the centrifugal force of the rotating substrate promotes outward spreading of the solutions away from the axis of rotation. Spin-coating may be well suited to dispense one or more solutions that initiate or quench a reaction that occurs on a time scale that is slow relative to the dispense time. In some embodiments, one or more solutions can be delivered directly to a reaction site without substantially displacing the one or more solutions from the delivery point. Methods of direct delivery of a solution to a reaction site may include aerosol delivery of the solution, applying the solution using an applicator, curtain coating the solution, slot die coating the solution, dispensing the solution from a translating dispensing probe, dispensing the solution from an array of dispensing probes, immersing the substrate in the solution, or contacting the substrate with a sheet containing the solution.

Aerosol delivery may include delivering the solution to the substrate in an aerosol form by directing the solution to the substrate using a pressure nozzle or ultrasonic nozzle. Applying the solution using an applicator may include contacting the substrate with an applicator containing the solution and translating the applicator relative to the substrate. For example, applying the solution using an applicator may include painting the substrate. The solution may be applied in a pattern by translating the applicator, rotating the substrate, translating the substrate, or a combination thereof. The pattern may be a spiral pattern. The pattern may be a circular pattern. Curing coating may include dispensing the solution from a dispensing probe in a continuous stream (e.g., a curtain or a flat sheet) to the substrate and translating the dispensing probe relative to the substrate. The solution may be curtain coated in a pattern by translating the dispensing probe, rotating the substrate, translating the substrate, or a combination thereof. The pattern may be a spiral pattern. The pattern may be a circular pattern. Slot die coating may include dispensing the solution from a dispensing probe positioned near the substrate such that the solution forms a meniscus between the substrate and the dispensing probe and translating the dispensing probe relative to the substrate. The solution may be slot die coated in a pattern by translating the dispensing probe, rotating the substrate, translating the substrate, or a combination thereof. The pattern may be a spiral pattern. The pattern may be a circular pattern. Dispensing the solution from a translating dispensing probe may include translating the dispensing probe relative to the substrate in a pattern (e.g., a spiral pattern, a circular pattern, a linear pattern, a striped pattern, a cross-hatched pattern, or a diagonal pattern). Dispensing the solution from an array of dispensing probes may include dispensing the solution from an array of nozzles (e.g., a showerhead) positioned above the substrate such that the solution is dispensed substantially simultaneously across an area of the substrate. Immersing the substrate in the solution may include immersing the substrate in a reservoir containing the solution. In some embodiments, the reservoir may be a shallow reservoir to reduce the volume of solution required to coat the substrate. Contacting the substrate with the sheet containing the solution may include contacting the substrate with a sheet of material (e.g., a porous or fibrous sheet) that is permeated with the solution. The solution may be transferred to the substrate. In some embodiments, the sheet of material may be a disposable sheet. In some embodiments, the sheet of material may be a reusable sheet. In some embodiments, the solution may be dispensed onto the substrate using the method shown in FIG. 51. As shown in FIG. 51, a jet of solution may be dispensed from a nozzle onto a rotating substrate. The nozzle may move radially relative to the rotating substrate, thereby dispensing the solution in a spiral pattern onto the substrate.

One or more solutions or reagents may be delivered to the substrate by any of the delivery methods disclosed herein. In some embodiments, two or more solutions or reagents are delivered to the substrate using the same delivery method. In some embodiments, two or more solutions are delivered to the substrate such that the time between contact of a solution or reagent with a subsequent solution or reagent is substantially similar for each area of the substrate contacted with the one or more solutions or reagents. In some embodiments, the solutions or reagents may be delivered as a single mixture. In some embodiments, the solutions or reagents may be dispensed into two or more component solutions. For example, each component of the two or more component solutions may be dispensed from a separate nozzle. The separate nozzles may dispense the two or more component solutions substantially simultaneously to substantially the same area of the substrate such that a homogenous solution is formed on the substrate. In some embodiments, the dispensing of each component of the two or more components may be separated in time. The dispensing of each component may be performed using the same method. For example, the first and second components are dispensed onto the substrate using the same method at substantially the same rate and in substantially the same pattern such that the time between contact of the first and second components is substantially similar for each area of the substrate contacted by the first and second components. In some embodiments, the first solution may initiate a reaction on the substrate (e.g., a solution including magnesium). In some embodiments, the second solution may stop a reaction on the substrate (e.g., a solution including ethylenediaminetetraacetic acid (EDTA)). In some embodiments, the time between the start of a reaction and the stop of a reaction may be substantially the same for each area of the substrate contacted by the first and second solutions. The first solution may form a substantially uniform film upon delivery to the substrate. The second solution may include a fast-diffusing component that may diffuse rapidly upon contact with the first solution. In some embodiments, the fast-diffusing component may initiate a reaction or the fast-diffusing component may stop a reaction.

In some embodiments, direct delivery of solutions or reagents may be combined with spin coating. For example, a first solution may be directly delivered in a spiral pattern using any of the direct delivery methods disclosed herein. The spiral pattern may be oriented inward toward the axis of rotation of the substrate. The first solution may initiate a reaction. The second solution may be delivered in the same pattern as the first solution. The second solution may stop the reaction. The second solution may wash away the first solution. The first and second solutions may be dispensed such that the reaction proceeds for a substantially constant time in each spatial region of the substrate.

The solution may be incubated on the substrate. In some aspects, the solution may be incubated on the substrate under conditions that maintain a layer of fluid on the surface. The solution may be incubated for at least about 5 minutes, up to about 10 minutes, up to about 15 minutes, up to about 20 minutes, up to about 25 minutes, up to about 30 minutes, up to about 35 minutes, up to about 40 minutes, up to about 45 minutes, up to about 50 minutes, up to about 55 minutes, up to about 60 minutes, up to about 65 minutes, up to about 70 minutes, up to about 75 minutes, up to about 80 minutes, up to about 85 minutes, or up to about 90 minutes. In some cases, the incubation time may be within a range defined by any two of the aforementioned values. In some cases, the incubation may exceed 90 minutes. In some examples, the layer of fluid can maintain a film thickness of at least 10 nanometers (nm), at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, or at least 1 mm during incubation. One or more of the chamber temperature, chamber humidity, substrate rotation, or fluid composition can be adjusted such that the layer of fluid is maintained during incubation. In some examples, the substrate may be rotated at a rotational frequency of 60 rpm or less, 50 rpm or less, 40 rpm or less, 30 rpm or less, 25 rpm or less, 20 rpm or less, 15 rpm or less, 14 rpm or less, 13 rpm or less, 12 rpm or less, 11 rpm or less, 10 rpm or less, 9 rpm or less, 8 rpm or less, 7 rpm or less, 6 rpm or less, 5 rpm or less, 4 rpm or less, 3 rpm or less, 2 rpm or less, or 1 rpm or less. In some cases, the rotational frequency may be within a range defined by any two of the aforementioned values. In some cases, the substrate may be rotating at a rotational frequency of about 5 rpm.

The substrate or its surface may include other features that aid in retention of the solution or reagent on the substrate or uniformity of the thickness of the solution or reagent on the substrate. In some cases, the surface may include a raised edge (e.g., a rim) that may be used to retain the solution on the surface. The surface may include a rim near the outer edge of the surface, thereby reducing the amount of solution that flows over the outer edge.

The solution may be a reaction mixture that includes various components. For example, the solution may include a plurality of probes configured to interact with the analyte. For example, the probes may have binding specificity for the analyte. In another example, the probes may not have binding specificity for the analyte. The probes may be configured to permanently bind to the analyte. The probes may be configured to temporarily bind to the analyte. For example, a nucleotide probe may be permanently incorporated into a growing strand hybridized to a nucleic acid molecule analyte. Alternatively, the nucleotide probe may temporarily bind to a nucleic acid molecule analyte. The temporarily coupled probe may then be removed from the analyte. Subsequent removal of the temporarily coupled probe from the analyte may or may not leave a residue (e.g., chemical residue) on the analyte. The type of probe in the solution may depend on the type of analyte. The probe may include a functional group or moiety configured to perform a specific function. For example, the probe may include a label (e.g., a dye). The probe may be configured to generate a detectable signal (e.g., an optical signal) upon coupling or otherwise interacting with the analyte, such as via the label. In some examples, the probes can be configured to generate a detectable signal upon activation (e.g., application of a stimulus). In another example, the nucleotide probes can include a reversible terminator (e.g., a blocking group) configured to terminate the polymerase reaction (until unblocked). The solution can include other components to aid, accelerate, or slow down the reaction between the probes and the analyte (e.g., enzymes, catalysts, buffers, saline, chelators, reducing agents, other agents, etc.). In some cases, the solution can be a wash solution. In some examples, after a reaction or interaction between the reagents (e.g., probes) in the reaction mixture with the analyte immobilized on the array, the wash solution can be directed to the substrate to contact the array. The wash solution can wash away free reagents from the previous reaction mixture solution.

Upon reaction between the probe and the analyte in the solution, a detectable signal, such as an optical signal (e.g., a fluorescent signal), may be generated. For example, the signal may originate from the probe and/or the analyte. The detectable signal may indicate a reaction or interaction between the probe and the analyte. The detectable signal may be a non-optical signal. For example, the detectable signal may be an electronic signal. The detectable signal may be detected by one or more sensors. For example, an optical signal may be detected via one or more photodetectors in the optical detection schemes described elsewhere herein. The signal may be detected during rotation of the substrate. The signal may be detected after the rotation has ended. The signal may be detected while the analyte is in fluid contact with the solution. The signal may be detected after washing of the solution. In some examples, after detection, the signal may be muted, such as by cleaving a label from the probe and/or analyte and/or modifying the probe and/or analyte. Such cleavage and/or modification may be affected by one or more stimuli, such as exposure to a chemical, an enzyme, light (e.g., ultraviolet light) or a temperature change (e.g., heat). In some cases, the signal may be rendered undetectable by stopping or changing the mode (e.g., detection wavelength) of one or more sensors, or by terminating or reversing the excitation of the signal. In some cases, detection of the signal may include capturing an image or generating a digital output (e.g., between different images).

The act of directing the solution to the substrate and detecting one or more signals indicative of a reaction between the probes in the solution and the analytes in the array may be repeated one or more times. Such acts may be repeated iteratively. For example, the same analyte immobilized at a predetermined location in the array may interact with multiple solutions in multiple repeated cycles. With each iteration, the additional signal detected may provide incremental or final data regarding the analyte during the process. For example, if the analyte is a nucleic acid molecule and the process is sequencing, the additional signal detected with each iteration may indicate a base in the nucleic acid sequence of the nucleic acid molecule. This operation may be repeated for at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10 million, at least 20 million, at least 50 million, at least 100 million, at least 20 ...0 million, at least 500 million, at least 100 million, at least 200 million, at least 500 million, or at least 1 billion cycles to process the analytes. In some examples, a different solution may be directed to the substrate for each cycle. For example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10,000,000, at least 20 million, at least 50 million, at least 100 million, at least 200 million, at least 500 million, or at least 1 billion solutions may be directed to the substrate.

In some cases, a cleaning solution may be directed to the substrate during each cycle (or at least once during each cycle). For example, a cleaning solution may be directed to the substrate after each type of reaction mixture solution is directed to the substrate. The cleaning solutions may be different. The cleaning solutions may be the same. The cleaning solution may be dispensed in pulses during rotation to generate annular waves as described herein. The cleaning solution may be dispensed in a continuous flow while the flow moves radially relative to the axis of rotation of the substrate during rotation, thereby dispensing the cleaning solution in a helical pattern. In some examples, the cleaning solution may be dispensed in a helical pattern progressing outwardly relative to the axis of rotation of the substrate. In some examples, the cleaning solution may be dispensed in a helical pattern progressing inwardly relative to the axis of rotation of the substrate. For example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10,000,000, at least 20 million, at least 50 million, at least 100 million, at least 200 million, at least 500 million, or at least 1 billion cleaning solutions may be directed to the substrate.

In some examples, a subset or the entire solution(s) may be recycled after the solution(s) contact the substrate. Recycling may include collecting, filtering, and reusing the subset or the entire solution. Filtering may be molecular filtering.

Nucleic acid sequencing using a rotating array In some examples, the method for sequencing may use sequencing by synthesis, in which the nucleic acid molecules are sequenced base by base by a primer extension reaction. For example, the method for sequencing a nucleic acid molecule may include providing a substrate including an array on which the nucleic acid molecules are fixed. The array may be a planar array. The substrate may be configured to rotate about an axis. The method may include directing a solution including a plurality of nucleotides across the array before or during rotation of the substrate. Rotation of the substrate may facilitate coating of the substrate surface with the solution. The nucleic acid molecule may be subjected to a primer extension reaction under conditions sufficient to incorporate or specifically bind at least one nucleotide from the plurality of nucleotides into a growing strand complementary to the nucleic acid molecule. A signal indicative of the incorporation or binding of the at least one nucleotide may be detected, thereby sequencing the nucleic acid molecule.

In some examples, the method may include immobilizing the nucleic acid molecules to the substrate prior to providing the substrate having the nucleic acid molecules immobilized thereon. For example, a solution containing a plurality of nucleic acid molecules may be directed to the substrate before, during, or after rotation of the substrate, and the substrate may be subjected to conditions sufficient to immobilize at least a subset of the plurality of nucleic acid molecules as an array on the substrate.

Figure 2 shows a flow chart of an example of a method 200 for sequencing nucleic acid molecules. In a first step 210, the method may include providing a substrate, as described elsewhere herein. The substrate may comprise an array of a plurality of individually addressable locations. The array may be a planar array. The array may be a textured array. The array may be a patterned array. For example, the array may define individually addressable locations with wells and/or pillars. A plurality of nucleic acid molecules, which may or may not be copies of the same nucleic acid molecule, may be fixed to the array. Each nucleic acid molecule from the plurality of nucleic acid molecules may be fixed to the array at a predetermined individually addressable location of the plurality of individually addressable locations.

The substrate may be configured to rotate about an axis. The axis may be an axis that passes through the center or substantially the center of the substrate. The axis may be an off-center axis. For example, the substrate may be affixed to a chuck (such as a vacuum chuck) of a spin-coating apparatus. The substrate may be configured to rotate at a rotational speed of at least 1 revolution per minute (rpm), at least 2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least 50 rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, or at least 10,000 rpm. The substrate may be configured to rotate at a rotational speed within a range defined by any two of the aforementioned values. The substrate may be configured to rotate at different rotational speeds during different operations described herein. The substrate may be configured to rotate at a rotational speed that varies according to a time-dependent function, such as a ramp, a sine wave, a pulse, or other function or combination of functions. The time-varying function can be periodic or aperiodic.

In a second step 220, the method may include directing the solution across the array before or during rotation of the substrate. The solution may be directed centrifugally across the array. In some cases, the solution may be directed to the array in pulses during rotation of the substrate, thereby generating annular waves of solution moving radially outward. In some examples, the solution may be directed to the array during rotation of the substrate in a continuous flow while the flow is moving radially relative to the axis of rotation of the substrate, thereby directing the solution to the array in a spiral pattern. In some cases, the substrate may be configured to rotate at a speed of 60 rpm or less, 50 rpm or less, 40 rpm or less, 30 rpm or less, 25 rpm or less, 20 rpm or less, 15 rpm or less, 14 rpm or less, 13 rpm or less, 12 rpm or less, 11 rpm or less, 10 rpm or less, 9 rpm or less, 8 rpm or less, 7 rpm or less, 6 rpm or less, 5 rpm or less, 4 rpm or less, 3 rpm or less, 2 rpm or less, or 1 rpm or less. In some cases, the rotation frequency may be within a range defined by any two of the aforementioned values. The solution may have a temperature different from the temperature of the substrate, thereby providing a source or sink of thermal energy to the substrate or nucleic acid molecules located on the substrate. The thermal energy may provide a temperature change to the substrate or nucleic acid molecules. The temperature change may be transient. Temperature changes can enable, disable, enhance, or inhibit chemical reactions, such as those performed on nucleic acid molecules. Chemical reactions can include denaturation, hybridization, or annealing of multiple nucleic acid molecules. Chemical reactions can include steps in polymerase chain reaction (PCR), bridge amplification, or other nucleic acid amplification reactions. Temperature changes can modulate, increase, or decrease the signal detected from the nucleic acid molecules (or from a probe in solution).

In some cases, the solution may include beads. The beads may be coated with the nucleic acid molecules to be sequenced. The solution including the beads may be dispensed onto a substrate using methods described herein. For example, the solution including the beads may be dispensed onto a substrate in a spiral pattern, as shown in FIG. 48 or FIG. 51. In some cases, the beads may preferentially interact with a first region type (e.g., positively charged regions) of the substrate, as shown in FIG. 50A. In some cases, the beads may not interact with a second region type (e.g., hydrophobic regions) of the substrate. In some cases, the beads coated with the nucleic acid molecules may interact with the first region type (e.g., positively charged regions) of the substrate, and the beads not coated with the nucleic acid molecules may not interact with the first region type of the substrate, as shown in FIG. 50B.

In some examples, the solution may include a probe configured to interact with a nucleic acid molecule. For example, in some examples, the solution may include a plurality of nucleotides (single bases), e.g., for performing sequencing by synthesis. The plurality of nucleotides may include nucleotide analogs, naturally occurring nucleotides, and/or non-naturally occurring nucleotides, collectively referred to herein as "nucleotides." The plurality of nucleotides may or may not be of the same type of base (e.g., A, T, G, C, etc.). For example, the solution may or may not include only one type of base. The solution may include at least one type of base of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 types. For example, the solution may include any possible mixture of A, T, C, and G. In some examples, the solution may include a plurality of natural and non-natural nucleotides. The plurality of natural and non-natural nucleotides may or may not be of the same type of base (e.g., A, T, G, C). In some cases, the solution may include a probe (e.g., an oligonucleotide primer) that is an oligomer. For example, in some instances, for example, to perform sequencing by synthesis, a solution may include a plurality of nucleic acid molecules, e.g., primers, that include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotide bases. The plurality of nucleic acid molecules may include nucleotide analogs, naturally occurring nucleotides, and/or non-naturally occurring nucleotides, collectively referred to herein as "nucleotides." The plurality of nucleotides may or may not include the same type of base (e.g., A, T, G, C, etc.). For example, the solution may or may not include only one type of base. The solution may include at least one type of base of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 types. For example, the solution may include any possible mixture of A, T, C, and G. In some instances, the solution may include a plurality of natural and non-natural nucleotides. The multiple natural and non-natural nucleotides may or may not be of the same type of base (e.g., A, T, G, C).

One or more nucleotides of the plurality of nucleotides may be terminated (e.g., reversibly terminated). For example, the nucleotides may comprise a reversible terminator or a moiety capable of reversibly terminating primer extension. Nucleotides comprising a reversible terminator may be accepted by a polymerase and incorporated into a growing nucleic acid sequence in the same manner as non-reversibly terminated nucleotides. The polymerase may be any naturally occurring mutant (i.e., native or wild type) or an engineered mutant of a polymerase (e.g., DNA polymerase, Taq polymerase, etc.). After incorporation of a nucleotide analog comprising a reversible terminator into a nucleic acid chain, the reversible terminator may be removed to allow further extension of the nucleic acid chain. A reversible terminator may comprise a blocking or capping group attached to the 3'-oxygen atom of the nucleotide or sugar moiety (e.g., pentose) of the nucleotide or nucleotide analog. Such a moiety is referred to as a 3'-O-blocked reversible terminator. Examples of 3'-O-blocked reversible terminators include, for example, 3'- _ONH2 reversible terminators, 3'-O-allyl reversible terminators, and 3'-O-aziomethyl reversible terminators. Alternatively, the reversible terminators may include blocking groups at the linker (e.g., cleavable linker) and/or dye portion of the nucleotide analog. The 3' unblocked reversible terminators may be attached to both the base of the nucleotide analog and a fluorescent group (e.g., a label as described herein). Examples of 3' unblocked reversible terminators include, for example, the "virtual terminator" developed by Helicos BioSciences Corp. and the "lightning terminator" developed by Michael L. Metzker et al. Cleavage of the reversible terminator may be achieved, for example, by irradiating the nucleic acid molecule containing the reversible terminator. In some instances, the plurality of nucleotides may not include a terminal nucleotide.

One or more of the nucleotides of the plurality of nucleotides can be labeled with a dye, fluorophore, or quantum dot. For example, the solution can include labeled nucleotides. In another example, the solution can include unlabeled nucleotides. In another example, the solution can include a mixture of labeled and unlabeled nucleotides. Non-limiting examples of dyes include SYBR Green, SYBR Blue, DAPI, propidium iodine, Hoechst, SYBR Gold, ethidium bromide, acridine, proflavine, acridine orange, acriflavine, fluorocoumanine, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine and acridine, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOX blue, SYTOX green, SYTOX orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BO BO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red) , fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Far Red, allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes such as europium and terbium, carboxytetrachlorofluorescein, 5 and/or 6-carboxyfluorescein (FAM), VIC, 5-(or 6-)iodoacetamidofluorescein, 5-{[2(and 3)-5-(acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein), Lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxyrhodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate-4-amino-naphthalimide, phycobiliproteins, Atto 390, 425, 465, 488, 495, 532, 565, 594, 633, 647, 647N, 665, 680 and 700 dyes, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750 and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755 and 800 dyes, or other fluorophores, black hole quencher dyes such as BH1-0, BHQ (Biosearch QSY dye fluorescence quenchers such as QSY7, QSY9, QSY21, QSY35 (Molecular Probes/Invitrogen) and other quenchers such as Dabcyl, Dabsyl, Cy5Q and Cy7Q and dark cyanine dyes (GE Healthcare); Dy-Quenchers such as DYQ-660 and DYQ-661 (Dyomics), and ATTO fluorescence quenchers such as ATTO540Q, 580Q, 612Q (ATTO-TEC GmbH). In some cases, the label may have a linker. For example, the label may have a disulfide linker attached to the label. Non-limiting examples of such labels include Cy5-azide, Cy-2-azide, Cy-3-azide, Cy-3.5-azide, Cy5.5-azide, and Cy-7-azide. In some cases, the linker can be a cleavable linker. In some cases, the label can be of a type that is not self-quenching or exhibits proximity quenching. Non-limiting examples of label types that do not exhibit self-quenching or proximity quenching include bimane derivatives, such as monobromobimane. Alternatively, the label can be of a type that exhibits self-quenching or proximity quenching. Non-limiting examples of such labels include Cy5-azide, Cy-2-azide, Cy-3-azide, Cy-3.5-azide, Cy5.5-azide, and Cy-7-azide. In some cases, the blocking group of the reversible terminator can include a dye.

Solutions may be directed to the array using one or more nozzles. In some cases, different reagents (e.g., different types of nucleotide solutions, wash solutions, etc.) may be dispensed through different nozzles, such as to prevent contamination. Each nozzle may be connected to a dedicated fluid line or fluid valve, which may further prevent contamination. Certain reagents may be dispensed through one or more nozzles. One or more nozzles may be directed at or proximate to the center of the substrate. Alternatively, one or more nozzles may be directed at or near a location on the substrate other than the center of the substrate. Two or more nozzles may be operated in combination to more efficiently deliver fluid to the substrate.

The solution may be dispensed onto the substrate while the substrate is stationary. The substrate may then be rotated following dispensing of the solution. Alternatively, the substrate may be rotated prior to dispensing of the solution. The solution may then be dispensed onto the substrate while the substrate is rotating. The rotation of the substrate may create a centrifugal force (or an inertial force directed away from the axis) on the solution, causing the solution to flow radially outward onto the array.

In a third step 230, the method may include subjecting the nucleic acid molecule to a primer extension reaction. The primer extension reaction may be performed under conditions sufficient to incorporate at least one nucleotide from the plurality of nucleotides into a growing strand complementary to the nucleic acid molecule. The incorporated nucleotide may or may not be labeled.

In some cases, step 230 may further include modifying at least one nucleotide. Modifying the nucleotide may include labeling the nucleotide. For example, the nucleotide may be labeled with a dye, a fluorophore, a quantum dot, or the like. The nucleotide may be cleavably labeled. In some examples, modifying the nucleotide may include activating (e.g., stimulating) the label of the nucleotide.

In a fourth step 240, the method may include detecting a signal indicative of the incorporation of at least one nucleotide. The signal may be an optical signal. The signal may be a fluorescent signal. The signal may be detected during rotation of the substrate. The signal may be detected after completion of rotation. The signal may be detected while the nucleic acid molecule to be sequenced is in fluid contact with the solution. The signal may be detected after fluid contact of the nucleic acid molecule with the solution. Step 240 may further include modifying the label of at least one nucleotide. For example, step 240 may further include cleaving the label of the nucleotide (e.g., after detection). The nucleotide may be cleaved by one or more stimuli, such as exposure to a chemical, an enzyme, light (e.g., ultraviolet light), or heat. Once the label is cleaved, the signal indicative of the incorporated nucleotide may not be detectable by one or more detectors.

Method 200 may further include repeating steps 220, 230, and/or 240 one or more times to identify one or more additional signals indicative of incorporation of one or more additional nucleotides, thereby sequencing the nucleic acid molecule. Method 200 may include iteratively repeating steps 220, 230, and/or 240. With each iteration, the additional signals may indicate incorporation of an additional nucleotide. The additional nucleotide may be the same nucleotide detected in the previous iteration. The additional nucleotide may be a different nucleotide than the nucleotide detected in the previous iteration. In some examples, at least one nucleotide may be modified (e.g., labeled and/or cleaved) between each iteration of steps 220, 230, or 240. For example, the method may include repeating steps 220, 230, and/or 240 at least once, at least twice, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, at least 1,000 times, at least 2,000 times, at least 5,000 times, at least 10,000 times, at least 200,000 times, at least 500,000 times, at least 1 million times, at least 2 million times, at least 5 million times, at least 10 million times, at least 20 million times, at least 50 million times, at least 100 million times, at least 200 million times, at least 500 million times, at least 100 million times, at least 200 million times, at least 500 million times, or at least 1 billion times. The method may include repeating steps 220, 230, and/or 240 a number of times that are within a range defined by any two of the aforementioned values. Thus, method 200 may result in the sequencing of nucleic acid molecules of any size.

The method can include periodically introducing different solutions to the array during rotation of the substrate. For example, the method can include introducing a first solution including a first type of nucleotide (e.g., in a plurality of nucleotides of the first type) to the array, followed by a second solution including a second type of nucleotide, followed by a third type of nucleotide, followed by a fourth type of nucleotide, etc. In another example, the different solutions can include combinations of different types of nucleotides. For example, the first solution can include a first canonical type of nucleotide (e.g., A) and a second canonical type of nucleotide (e.g., C), the second solution can include a first canonical type of nucleotide (e.g., A) and a third canonical type of nucleotide (e.g., T), and the third solution can include a first canonical type, a second canonical type, a third canonical type, and a fourth canonical type (e.g., G). In another example, the first solution may include labeled nucleotides, the second solution may include unlabeled nucleotides, and the third solution may include a mixture of labeled and unlabeled nucleotides. The method may include directing at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10,000,000, at least 20 million, at least 50 million, at least 100 million, at least 200 million, at least 50 million, at least 500 million, or at least 1 billion solutions to the array. The method may include directing some solutions to the array that are within a range defined by any two of the aforementioned values. The solutions may be different. The solutions may be the same.

The method may include directing at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10,000,000, at least 20 million, at least 50 million, at least 100 million, at least 200 million, at least 500 million, at least 500 million, or at least 1 billion wash solutions to the substrate. For example, wash solutions may be directed to the substrate after each type of nucleotide is directed to the substrate. The wash solutions may be different. The wash solutions may be the same. The wash solutions may be dispensed in pulses during rotation to generate the annular waves described herein. The cleaning solution is dispensed in a continuous stream while the stream moves radially relative to the axis of rotation of the substrate during rotation, thereby allowing the cleaning solution to be dispensed in a spiral pattern.

The method may further include recycling a subset or all of the solution(s) after the solution(s) contact the substrate. Recycling may include collecting, filtering, and reusing the subset or all of the solution. Filtering may be molecular filtering.

Steps 220 and 230 may be performed at a first location, and step 240 may be performed at a second location. The first and second locations may comprise first and second processing bays, respectively, as described herein (e.g., with respect to FIG. 23H). The first and second locations may comprise first and second rotating spindles, respectively, as described herein (e.g., with respect to FIG. 24). The first rotating spindle may be external or internal to the second rotating spindle. The first and second rotating spindles may be configured to rotate at different angular velocities. Alternatively, step 220 may be performed at a first location, and steps 230 and 240 may be performed at a second location.

The method may further include transferring the substrate between the first and second positions. Steps 220 and 230 may be performed while the substrate is rotating at a first angular velocity, and step 240 may be performed while the substrate is rotating at a second angular velocity. The first angular velocity may be less than the second angular velocity. The first angular velocity may be between about 0 rpm and about 100 rpm. The second angular velocity may be between about 100 rpm and about 1,000 rpm. Alternatively, step 220 may be performed while the substrate is rotating at the first angular velocity, and steps 230 and 240 may be performed while the substrate is rotating at the second angular velocity.

Many variations, modifications, and adaptations based on the method 200 provided herein are possible. For example, the order of steps of the method 200 may be changed, some steps may be deleted, some steps may be duplicated, and additional steps may be added as appropriate. Some steps may be performed sequentially. Some of these steps may be performed in parallel. Some steps may be performed once. Some steps may be performed multiple times. Some of the steps may include sub-steps. Some of the steps may be automated. Some steps may be manual. Some of the steps may be performed separately, for example, at different locations or during different steps and/or processes. For example, directing a solution containing multiple probes to a substrate may be performed separately from the reaction and detection process.

For example, in some cases, in the third step 230, instead of promoting a primer extension reaction, the nucleic acid molecule may be subjected to conditions that allow for transient binding of nucleotides from the plurality of nucleotides to the nucleic acid molecule. The transiently bound nucleotides may be labeled. The transiently bound nucleotides may be removed, such as after detection (see, e.g., step 240). A second solution may then be directed to the substrate, this time under conditions that promote a primer extension reaction, such that the nucleotides of the second solution become incorporated (e.g., into a growing strand hybridized to the nucleic acid molecule). The incorporated nucleotides may be unlabeled. After washing, another solution of labeled nucleotides may be directed to the substrate, e.g., for another cycle of transient binding, without detection.

In some cases, the solution may include different probes, for example, for sequencing by ligation. For example, the solution may include multiple oligonucleotide molecules. For example, the oligonucleotide molecules may have a length of about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases. The oligonucleotide molecules may be labeled with dyes (e.g., fluorescent dyes) as described elsewhere herein. In some examples, to detect repeat sequences in nucleic acid molecules, such as homopolymer repeat sequences, dinucleotide repeat sequences, and trinucleotide repeat sequences, the solution may include a targeting probe (e.g., homopolymer probe) configured to bind to the repeat sequence. The solution may include one type of probe (e.g., nucleotides). The solution may include different types of probes (e.g., nucleotides, oligonucleotide molecules, etc.). The solution may include different types of probes (e.g., oligonucleotide molecules, antibodies, etc.) for interacting with different types of analytes (e.g., nucleic acid molecules, proteins, etc.). Different solutions containing different types of probes can be directed to the substrate multiple times, with or without detection during successive cycles (e.g., detection may be performed during some successive cycles but not during some other cycles), to sequence or otherwise process nucleic acid molecules, depending on the type of processing.

FIG. 3 shows a system 300 for sequencing nucleic acid molecules or processing specimens. The system may be configured to perform methods 200 or 1400. Although the systems (e.g., 300, 400, 500a, 500b, etc.) are described with respect to processing nucleic acid molecules, the systems may be used to process any other type of biological specimen described herein.

The system may include a substrate 310. The substrate may include any substrate described herein, such as any substrate described herein with respect to FIG. 2. The substrate may include an array. The substrate may be open. The array may include one or more locations 320 configured to immobilize one or more nucleic acid molecules or analytes. The array may include any array described herein, such as any array described herein with respect to method 200. For example, the array may include a plurality of individually addressable locations. The array may include a linker (e.g., any binder described herein) attached to the nucleic acid molecule to be sequenced. Alternatively, or in combination, the nucleic acid molecule to be sequenced may be coupled to a bead. The bead may be immobilized on the array. The array may be textured. The array may be a patterned array. The array may be planar.

The substrate may be configured to rotate about axis 305. The axis may be an axis that passes through the center of the substrate. The axis may be an off-center axis. The substrate may be configured to rotate at any rotational speed described herein, such as any rotational speed described herein with respect to methods 200 or 1400.

The substrate may be configured to undergo a change in relative position with respect to the first or second longitudinal axis 315 and 325. For example, the substrate may be translatable along the first and/or second longitudinal axis (as shown in FIG. 3). Alternatively, the substrate may be stationary along the first and/or second longitudinal axis. Alternatively or in combination, the substrate may be translatable along the axis (as shown in FIG. 4). Alternatively or in combination, the substrate may be stationary along the axis. The relative position of the substrate may be configured to alternate between positions. The relative position of the substrate may be configured to alternate between positions with respect to the longitudinal axis or one or more of the axes. The relative position of the substrate may be configured to alternate between positions with respect to any of the fluidic channels described herein. For example, the relative position of the substrate may be configured to alternate between a first position and a second position. The relative positions of the substrates may be configured to alternate between at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positions. The relative positions of the substrates may be configured to alternate between several positions within a range defined by any two of the aforementioned values. The first or second longitudinal axis may be substantially perpendicular to the axis. The first or second longitudinal axis may be substantially parallel to the axis. The first or second longitudinal axis may be coincident with the axis.

The system may include a first fluid channel 330. The first fluid channel may include a first fluid outlet port 335. The first fluid outlet port may be configured to dispense a first fluid to the array. The first fluid outlet port may be configured to dispense any fluid described herein, such as any solution described herein. The first fluid outlet port may be external to the substrate. The first fluid outlet port may not contact the substrate. The first fluid outlet port may be a nozzle. The first fluid outlet port may have an axis substantially coincident with the axis. The first fluid outlet port may have an axis substantially parallel to the axis.

The system may include a second fluid channel 340. The second fluid channel may include a second fluid outlet port 345. The second fluid outlet port may be configured to dispense a second fluid to the array. The second fluid outlet port may be configured to dispense any fluid described herein, such as any solution described herein. The second fluid outlet port may be external to the substrate. The second fluid outlet port may not contact the substrate. The second fluid outlet port may be a nozzle. The second fluid outlet port may have an axis substantially coincident with the axis. The second fluid outlet port may have an axis substantially parallel to the axis.

The first and second fluids may include different types of reagents. For example, the first fluid may include a first type of nucleotide, such as any nucleotide described herein, or a nucleotide mixture. The second fluid may include a second type of nucleotide, such as any nucleotide described herein, or a nucleotide mixture. Alternatively, the first and second fluids may include the same type of reagent (e.g., the same type of fluid is dispensed through multiple fluid outlet ports (e.g., nozzles) to increase the coating speed). Alternatively or in combination, the first or second fluid may include a wash reagent. The first fluid channel 330 and the second fluid channel 340 may be fluidically isolated. Advantageously, when the first and second fluids include different types of reagents, each of the different reagents may remain free of contamination from the other reagent during dispensing.

The first fluid outlet port may be configured to dispense the first fluid during rotation of the substrate. The second fluid outlet port may be configured to dispense the second fluid during rotation of the substrate. The first and second fluid outlet ports may be configured to dispense at non-overlapping times. Alternatively, the first and second fluid outlet ports may be configured to dispense at overlapping times, such as when the first and second fluids contain the same type of reagent. The substrate may be configured to rotate at different speeds or different rotational frequencies when the first and second outlet ports dispense. Alternatively, the substrate may be configured to rotate at the same speed and rotational frequency when the first and second outlet ports dispense. During rotation, the array may be configured to direct the first fluid in a substantially radial direction away from the axis. The first fluid outlet port may be configured to direct the first fluid to the array during at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, or at least 1 million complete rotations of the substrate. The first fluid outlet port may be configured to direct the first fluid to the array during a number of complete rotations within a range defined by any two of the aforementioned values.

The system can include a third fluid channel 350 with a third fluid outlet port 355 configured to dispense a third fluid. The system can include a fourth fluid channel 360 with a fourth fluid outlet port 365 configured to dispense a fourth fluid. The third and fourth fluid channels can be similar to the first and second fluid channels described herein. The third and fourth fluids can be the same or different fluids as the first and/or second fluids. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more fluids (or reagents) can be used. For example, 5-10 fluids (or reagents) can be used.

3 illustrates a change in the position of the substrate, alternatively or in addition, one or more of the first, second, third, and fourth fluid channels may be configured to undergo a change in position. For example, any of the first, second, third, or fourth fluid channels may be translatable along the first and/or second longitudinal axes. Alternatively, any of the first, second, third, or fourth fluid channels may be stationary along the first and/or second longitudinal axes. Alternatively or in addition, any of the first, second, third, or fourth fluid channels may be translatable along an axis. Alternatively or in addition, any of the first, second, third, or fourth fluid channels may be stationary along an axis.

The relative positions of one or more of the first, second, third, and fourth fluid channels may be configured to alternate positions with respect to one or more of the longitudinal axes or axes. For example, the relative positions of any of the first, second, third, or fourth fluid channels may be configured to alternate between a first position and a second position (e.g., by moving such channels, by moving the substrate, or by moving the channels and the substrate). The relative positions of any of the first, second, third, or fourth fluid channels may be configured to alternate between at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty or more positions. The relative positions of any of the first, second, third, or fourth fluid channels may be configured to alternate between several positions that are within a range defined by any two of the aforementioned values. The first or second longitudinal axis may be substantially perpendicular to the axis. The first or second longitudinal axis may be substantially parallel to the axis. The first or second longitudinal axis may be coincident with the axis.

In some cases, the system may include one or more fluid channels for receiving fluid from a substrate (not shown in FIG. 3). With reference to FIGS. 4A-4B, the fifth fluid channel 430 may include a first fluid inlet port 435. The first fluid inlet port may be located at a first level of the axis (as shown in FIG. 4). In some examples, the first fluid inlet port may surround the periphery of the substrate 310 (e.g., circular). The first fluid inlet port may be downstream of the substrate 310 and in fluid communication with the substrate when the substrate is in a first position, such as relative to the axis. The fifth fluid channel may be in fluid communication with the first fluid channel 330 (as shown in FIG. 3). For example, the first fluid inlet port may be configured to receive a solution that passes from the first fluid outlet port to the substrate and then exits the substrate (e.g., due to inertial forces during rotation of the substrate). For example, the first fluid inlet port may be configured to receive a solution in a recycling process, such as the recycling processes described herein with respect to methods 200 or 1400. In some examples, the solution received by the fifth fluid channel through the first fluid inlet port may be fed back (e.g., after filtering) to the first fluid channel to be dispensed to the substrate through the first fluid outlet port. The fifth fluid channel and the first fluid channel may define at least a portion of a first periodic fluid flow path. The first periodic fluid flow path may include a filter, such as a filter described herein with respect to method 200 or 1400. The filter may be a molecular filter. In other examples, the solution received by the fifth fluid channel may be fed back (e.g., after filtering) to a different fluid channel (other than the first fluid channel) that is dispensed through a different fluid outlet port.

The system may include a sixth fluid channel 440. The sixth fluid channel may include a second fluid inlet port 445. The second fluid inlet port may be located at a second level of the axis (as shown in FIG. 4). In some cases, the second fluid inlet port may surround the periphery of the substrate 310. The second fluid inlet port may be downstream of the substrate 310 and in fluid communication with the substrate when the substrate is in a second position, such as relative to the axis. The sixth fluid flow path may be in fluid communication with the second fluid flow path 340. For example, the second fluid inlet port may be configured to receive a solution that passes from the second fluid outlet port to the substrate and then exits the substrate. For example, the second fluid inlet port may be configured to receive a solution in a recycling process, such as the recycling process described herein with respect to methods 200 or 1400. In some examples, the solution received by the sixth fluid channel through the second fluid inlet port can be fed back (e.g., after filtering) to the second fluid channel for dispensing to the substrate through the second fluid outlet port. The sixth fluid channel and the second fluid channel can define at least a portion of a second periodic fluid flow path. The second periodic fluid flow path can include a filter, such as the filters described herein with respect to methods 200 or 1400. The filter can be a molecular filter.

The system may include a shield (not shown) that prevents fluid communication between the substrate and the second fluid inlet port when the substrate is in the first position, and between the substrate and the first fluid inlet port when the substrate is in the second position.

The system may further include one or more detectors 370. The detector may be a photodetector such as one or more photodetectors, one or more photodiodes, one or more avalanche photodiodes, one or more photomultiplier tubes, one or more photodiode arrays, one or more avalanche photodiode arrays, one or more cameras, one or more charge-coupled device (CCD) cameras, or one or more complementary metal-oxide semiconductor (CMOS) cameras. The camera may be a TDI or other continuous area scanning detector as described herein, including, for example, a TDI line scan camera. The detector may be a fluorescent detector. The detector may be in sensing communication with the array. For example, the detector may be configured to detect a signal from the array. The signal may be an optical signal. The signal may be a fluorescent signal. The detector may be configured to detect a signal from the substrate during rotation of the substrate. The detector may be configured to detect a signal from the substrate when the substrate is not rotating. The detector may be configured to detect a signal from the substrate after the substrate rotation has ended. FIG. 3 shows an exemplary area 375 on a substrate optically mapped to a detector.

The system may include one or more sources (not shown in FIG. 3 ) configured to deliver electromagnetic radiation to the substrate. The light source may include one or more light sources (e.g., illumination sources). The light source may include one or more incoherent or coherent light sources. The light source may include one or more narrowband or broadband light sources. The light source may be configured to emit optical radiation having a bandwidth of up to 1 Hertz (Hz), up to 2 Hz, up to 5 Hz, up to 10 Hz, up to 20 Hz, up to 50 Hz, up to 100 Hz, up to 200 Hz, up to 500 Hz, up to 1 kilohertz (kHz), up to 2 kHz, up to 5 kHz, up to 10 kHz, up to 20 kHz, up to 50 kHz, up to 100 kHz, up to 200 kHz, up to 500 kHz, up to 1 megahertz (MHz), up to 2 MHz, up to 5 MHz, up to 10 MHz, up to 20 MHz, up to 50 MHz, up to 100 MHz, up to 200 MHz, up to 500 MHz, up to 1 gigahertz (GHz), up to 2 GHz, up to 5 GHz, up to 10 GHz, up to 20 GHz, up to 50 GHz, up to 100 GHz, or a bandwidth within a range defined by any two of the aforesaid values. The light source may comprise one or more light emitting diodes (LEDs). The light source may include one or more lasers. The light source may include one or more single mode laser sources. The light source may include one or more multimode laser sources. The light source may include one or more laser diodes. The laser may be a continuous wave laser or a pulsed laser. The light beam emitted by the laser may be a Gaussian or nearly Gaussian beam, and the beam may be manipulated using one or more optical elements (e.g., mirrors, lenses, prisms, wave plates, etc.). For example, the beam may be collimated. In some cases, the beam may be manipulated to provide a laser line (e.g., using one or more Powell lenses or cylindrical lenses). FIG. 11A shows an example of beam shaping using a cylindrical lens to provide a laser line. A collimated beam with a radius r ₀ is incident on a cylindrical plano-concave lens with a focal length −f. The beam expands with a half angle θ corresponding to r ₀ /f. The laser line has a thickness of about 2r ₀ and a length L of about 2(r ₀ /f)(z+f) at a distance z from the lens. In some embodiments, the beam thickness may be expanded along a single axis, e.g., the y-axis, and the beam thickness remains substantially unchanged along a second axis, e.g., the x-axis, as shown in FIG. 11B. Expansion along a single axis can be achieved using a cylindrical lens, e.g., a plano-concave cylindrical lens with a focal length of −f along the expansion axis. The beam shaping lens may be part of a line shaper element, as shown in FIG. 11C. The line shaper element may comprise one or more optical elements configured to expand the beam along a single axis. The line shaper element may further comprise one or more optical elements, e.g., a second cylindrical lens, for collimating the expanded beam. In some embodiments, the second cylindrical lens is a plano-convex cylindrical lens. The expanded beam can result in a laser line, as shown in FIG. 11B. The laser line may impinge directly on the substrate or may be projected onto the substrate so that it is approximately perpendicular to a central axis about which the open substrate may rotate.

The source (e.g., optical or illumination source) of the system may be configured to emit light including one or more wavelengths in the ultraviolet (about 100 nm to about 400 nm), visible (about 400 nm to about 700 nm), or infrared (about 700 nm to about 10,000 nm) regions of the electromagnetic spectrum, or any combination therefor. For example, the light source may emit radiation including one or more wavelengths in the range of 600 nm to 700 nm. The light sources, individually or in combination, may emit radiation having an optical power of at least 0.05 watts (W), at least 0.1 W, at least 0.2 W, at least 0.5 W, at least 1 W, at least 2 W, at least 5 W, at least 10 W, or an optical power within a range defined by any two of the foregoing values. The light source may be configured to interact with the molecules on the substrate to generate a detectable optical signal that may be detected by a photodetector. For example, the light source may be configured to generate optical absorption, optical reflectance, scattering, phosphorescence, fluorescence, or any other optical signal described herein.

The system may include a seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth fluid channel. Each fluid channel may include a fluid outlet port or a fluid inlet port in fluid communication with the substrate. For example, the ninth, tenth, thirteenth, fourteenth, seventeenth, or eighteenth fluid channel may include a fluid outlet port. The seventh, eighth, eleventh, twelfth, fifteenth, sixteenth, nineteenth, or twentieth fluid channel may include a fluid inlet port. Alternatively, the system may include more than twenty fluid channels including fluid outlet ports or fluid inlet ports.

Thus, the system may comprise a fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port may be configured to dispense a fifth, sixth, seventh, eighth, ninth, or tenth fluid to the array. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port may be configured to dispense any fluid described herein, such as any solution described herein. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port may be similar to the first, second, third, or fourth fluid outlet port described herein. Alternatively, the system may comprise more than ten fluid outlet ports.

The fluid channels may be fluidically isolated from one another. For example, the fluid channels may be fluidically isolated upstream of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port may be external to the substrate. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port may not contact the substrate. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port may be a nozzle.

The system may include a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet port. The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet port may be in fluid communication with the substrate when the substrate is in the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth position (e.g., relative to an axis), respectively. Alternatively, the system may include more than ten fluid inlet ports.

The ninth, tenth, thirteenth, fourteenth, seventeenth, or eighteenth fluid channels may be in fluid communication with the seventh, eighth, eleventh, twelfth, fifteenth, or sixteenth fluid channels, respectively. Each pair of fluid channels may define at least a portion of a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth annular fluid flow path, respectively. Each annular fluid flow path may be configured similarly to the first or second annular fluid flow paths described herein, with the fluid inlet port of the annular fluid flow path configured to receive the solution passing from the fluid outlet port of the annular fluid flow path to the substrate. Each circulating fluid flow path may be configured to receive the solution in a recycling process described herein. Each annular fluid flow path may include a filter as described herein.

The fifth, sixth, seventh, eighth, ninth, or tenth fluid may comprise a different type of reagent. For example, the fifth, sixth, seventh, eighth, ninth, or tenth fluid may comprise a fifth, sixth, seventh, eighth, ninth, or tenth type of nucleotide, such as any nucleotide described herein, respectively. Alternatively or in combination, the fifth, sixth, seventh, eighth, ninth, or tenth fluid may comprise a wash reagent.

The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports may be configured to dispense the fifth, sixth, seventh, eighth, ninth, or tenth fluids, respectively, during rotation of the substrate. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports may be configured to dispense at overlapping or non-overlapping times.

4A shows a system 400 for sequencing nucleic acid molecules at a first vertical level. The system may be substantially similar to system 300 described herein or may differ from system 300 in the arrangement of one or more of its elements. System 400 may include a substrate 310 as described herein. System 400 may utilize a vertical motion parallel to axis 305 to expose (e.g., make available in fluid communication) substrate 310 to different fluid channels. The system may include a first fluid channel 330 and a first fluid outlet port 335 as described herein. The system may include a second fluid channel 340 and a second fluid outlet port 345 as described herein. The system may include a third fluid channel 350 and a third fluid outlet port 355 as described herein. The system may include a fourth fluid channel 360 and a fourth fluid outlet port 365 as described herein. The system may include a detector 370 as described herein. The detector may be in optical communication with the illustrated region. The system can include any light source (not shown in FIG. 4A) described herein.

The fifth fluid channel 430 and the first fluid inlet port 435 may be disposed at a first level along the vertical axis, as shown in FIGS. 4A and 4B. The sixth fluid channel 440 and the second fluid inlet port 445 may be disposed at a second level along the vertical axis. In this manner, the system may be considered to include a first and a second fluid flow path, each fluid flow path disposed at a different vertical level. The substrate 310 may be vertically movable between the first and second levels, from the first level to the second level, and from the second level to the first level. Alternatively, the substrate may be fixed vertically, but the level may be vertically movable relative to the substrate 310. As another alternative, the substrate and the level may be vertically movable.

The system 400 may include multiple levels. The levels may be oriented perpendicular to one another. The system may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more levels. Each level may include one or more sublevels (e.g., incremental levels between any two levels). Each level may be for dispensing and/or retrieving a different fluid (or reagent). Some levels may be for dispensing the same fluid (or reagent).

While at the first vertical level, the substrate may be in fluid communication with the fifth fluid channel and the first fluid inlet port, but not with the sixth fluid channel and the second fluid inlet port. The substrate may be isolated from the sixth fluid channel and the second fluid inlet port by a shield (not shown), as described herein. The first fluid or first solution described herein may be dispensed into the substrate while the substrate is at this first vertical level. For example, any excess of the first solution that spins out of the substrate may be received by the first fluid inlet port while the substrate is at the first vertical level. In another example, a cleaning solution (e.g., dispensed from a different fluid outlet port than the first fluid) that is ejected from the substrate along with a portion of the first fluid may be received by the first fluid inlet port while the substrate is at the first vertical level. The substrate may then be moved to a second vertical level by vertically moving the substrate. Alternatively, the fifth or sixth fluid channel may be moved vertically. Alternatively or additionally, the substrate and one or more fluid channels may be moved relative to one another (e.g., along an axis).

4B shows a system 400 for sequencing nucleic acid molecules at a second vertical level. While at the second vertical level, the substrate can be in fluid communication with the sixth fluid channel and the second fluid inlet port, but not with the fifth fluid channel and the first fluid inlet port. The substrate can be isolated from the fifth fluid channel and the first fluid inlet port by a shield (not shown), as described herein. A second fluid or a second solution, as described herein, can be dispensed onto the substrate while the substrate is in this second vertical position. Alternatively, the first solution can be removed while the substrate is in the second vertical position. In some cases, the first solution can be recycled while the substrate is in the second vertical position. The substrate can then be moved vertically to return the substrate to the first vertical level, or to another vertical level as described herein. Alternatively, the fifth or sixth fluid channel can be moved vertically. Alternatively or additionally, the substrate and one or more fluid channels can be moved relative to one another (e.g., along an axis).

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet ports may be located at the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth vertical levels, respectively. The substrate may be moved to the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth vertical levels by vertically moving the substrate or by vertically moving the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth fluid flow channels. Any of the fluid solutions described herein may be dispensed to the substrate at any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or higher vertical levels. Any of the fluid solutions described herein can be removed from the substrate at any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more vertical levels. Any of the fluid solutions described herein can be recycled from the substrate at any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more vertical levels.

5A shows a first example of a system 500a for sequencing nucleic acid molecules using an array of fluid channels. The system may be substantially similar to system 300 or 400 described herein, or may differ from system 300 or 400 in the arrangement of one or more of its elements. System 500a may utilize a geometric arrangement of multiple fluid channels to expose a substrate to different fluids. System 500a may include a substrate 310 as described herein. The system may include a first fluid channel 330 and a first fluid outlet port 335 as described herein. The system may include a second fluid channel 340 and a second fluid outlet port 345 as described herein. The system may include a fifth fluid channel 430 and a first fluid inlet port 435 (not shown in FIG. 5A) as described herein. The system may include a sixth fluid channel 440 and a second fluid inlet port 445 (not shown in FIG. 5A) as described herein. The system may include a detector 370 (not shown in FIG. 5A) as described herein. The system can include any illumination source (not shown in FIG. 5A) described herein.

The first fluid channel and the first fluid outlet port may be disposed in a first location, as shown in FIG. 5A. The second fluid channel and the second fluid outlet port may be disposed in a second location. The system may be configured to dispense the first fluid from the first fluid outlet port and the second fluid from the second fluid outlet port.

The system may include any of the third, fourth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth fluid channels described herein. The system may include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports described herein. The system may include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet ports described herein.

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports may be located at the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth locations, respectively. The system may be configured to dispense the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluids from the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports, respectively.

Any two or more of the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, or more fluid channels can form an array of fluid channels. The array of fluid channels can be movable. Alternatively, the array of fluid channels can be in a fixed position relative to the substrate. Each fluid channel of the array of fluid channels can be arranged such that the longitudinal axis of the fluid channel forms an angle with the axis of rotation of the substrate. The angle can have a value of at least 0 degrees, at least 5 degrees, at least 10 degrees, at least 15 degrees, at least 20 degrees, at least 25 degrees, at least 30 degrees, at least 35 degrees, at least 40 degrees, at least 45 degrees, at least 50 degrees, at least 55 degrees, at least 60 degrees, at least 65 degrees, at least 70 degrees, at least 75 degrees, at least 80 degrees, at least 85 degrees, or at least 90 degrees. The angle can have a value within a range defined by any two of the aforementioned values. Each fluid channel of the array of fluid channels can be at a similar angle with the substrate. Alternatively, one or more of the fluid channels can be at a different angle with the substrate.

Figure 5B shows a second example of a system 500b for sequencing nucleic acid molecules using an array of fluid channels.

The system may be substantially similar to system 300 or 400 described herein, or may differ from system 300 or 400 in the arrangement of one or more of its elements. System 500b may utilize multiple fluid flow paths configured to move relative to the substrate to expose the substrate to different fluids. System 500b may include substrate 310 as described herein. The system may include first fluid channel 330 and first fluid outlet port 335 as described herein. The system may include second fluid channel 340 and second fluid outlet port 345 as described herein. The system may include fifth fluid channel 430 and first fluid inlet port 435 (not shown in FIG. 5B) as described herein. The system may include sixth fluid channel 440 and second fluid inlet port 445 (not shown in FIG. 5B) as described herein. The system may include detector 370 (not shown in FIG. 5B) as described herein. The system may include any light source (not shown in FIG. 5B) as described herein.

The first fluid channel and the first fluid outlet port may be attached to a fluid dispenser 510. The fluid dispenser may be a movable fluid dispenser, such as one including a movable gantry arm, as shown in FIG. 5B. Alternatively, the fluid dispenser may be fixed or stationary. The fluid dispenser may be configured to move to a first position to dispense the first fluid from the first fluid outlet port. The second fluid channel and the second fluid outlet port may also be attached to the fluid dispenser. The fluid dispenser may be configured to move to a second position to dispense the second fluid from the second fluid outlet port.

The system may include any of the third, fourth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth fluid channels described herein. The system may include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports described herein. The system may include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet ports described herein.

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports may be attached to a fluid dispenser. The fluid dispenser may be configured to move to the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth position to dispense the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid from the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports, respectively. Alternatively, the fluid dispenser may be kept stationary and the substrate 310 moved to different positions to receive different fluids.

6 shows a computerized system 600 for sequencing nucleic acid molecules. The system can include a substrate 310, such as a substrate described herein with respect to methods 200 or 1400, or system 300. The system can further include a fluid flow unit 610. The fluid flow unit can include any element associated with fluid flow described herein, such as any or all of elements 330, 335, 340, 345, 350, 355, 360, 365, 430, 435, 440, 445, and 370 described herein with respect to systems 300, 400, 500a, or 500b. The fluid flow unit can be configured to direct a solution comprising a plurality of nucleotides as described herein to the array of substrates before or during rotation of the substrate. The fluid flow unit can be configured to direct a wash solution as described herein to the array of substrates before or during rotation of the substrate. In some cases, the fluid flow unit can include a pump, a compressor, and/or an actuator for directing the fluid flow from the first location to the second location. For method 1400, the fluid flow system can be configured to direct any solution to the substrate 310. For method 1400, the fluid flow system can be configured to collect any solution from the substrate 310. The system can further include a detector 370, such as any detector described herein with respect to systems 300 or 400. The detector can sense and communicate with the array of the substrate.

The system may further include one or more computer processors 620. The one or more processors may be individually or collectively programmed to perform any of the methods described herein. For example, the one or more processors may be individually or collectively programmed to perform any or all of the operations of the methods of the present disclosure, such as method 200 or 1400. In particular, the one or more processors may be individually or collectively programmed to (i) direct a fluid flow unit to direct a solution containing a plurality of nucleotides across the array during or before rotation of the substrate, (ii) expose the nucleic acid molecule to a primer extension reaction under conditions sufficient to incorporate at least one nucleotide from the plurality of nucleotides into a growing strand complementary to the nucleic acid molecule, and (iii) use a detector to detect a signal indicative of incorporation of at least one nucleotide, thereby sequencing the nucleic acid molecule.

Although the rotation system is described with respect to sequencing applications, such rotation methods may be used for other applications (e.g., pre-sequencing applications, sample preparation, etc.), such as template seeding and surface amplification processes. For example, the reagents dispensed during or before rotation of the substrate can be tailored for other applications. Although the reagents dispensed to the substrate in the rotation system are described with respect to nucleotides, any reagent that can react with nucleic acid molecules (or any other molecules or cells) immobilized on the substrate, such as probes, adapters, enzymes, and labeling reagents, can be dispensed to the substrate before, during, or after rotation to achieve rapid coating of the substrate with the dispensed reagents.

A system described herein (e.g., any of systems 300, 400, 500a, or 500b, or any other system described herein), or any element thereof, may be environmentally controlled. For example, the system may be maintained at a specified temperature or humidity. The system (or any element thereof) may be maintained at a temperature of at least 20 degrees Celsius (°C), at least 25°C, at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, at least 100°C, up to 100°C, up to 95°C, up to 90°C, up to 85°C, up to 80°C, up to 75°C, up to 70°C, up to 65°C, up to 60°C, up to 55°C, up to 50°C, up to 45°C, up to 40°C, up to 35°C, up to 30°C, up to 25°C, up to 20°C, or a temperature within a range defined by any two of the foregoing values. Different elements of the system may be maintained at different temperatures or within different temperature ranges, such as the temperatures or temperature ranges described herein. Elements of the system may be set at a temperature above the dew point to prevent condensation. Elements of the system may be set at a temperature below the dew point to collect the condensation.

7 is a diagram illustrating a system with different environmental conditions in an open substrate system. The open substrate system can include a substrate 3502 and a container 3504 surrounding the substrate. The substrate 3502 can be any substrate described herein. The container 3504 can define an ambient environment for the substrate 3502. In some cases, the ambient environment can be limited and/or closed. In some cases, the ambient environment can be sealed (e.g., hermetically sealed, frictionally sealed, air pressure, etc.). In some cases, the ambient environment can be sealed using a pressure differential (e.g., air pressure, mechanical pressure, etc.). The open substrate system can include at least two non-overlapping regions having different environmental conditions, a first region 3522 and a second region 3524. In some examples, the first region 3522 in contact with or in close proximity to a surface of the substrate 3502, such as a surface containing one or more analytes described herein, can be maintained at a first set of temperatures and a first set of humidity. In some cases, the second region 3524 in contact with or adjacent to the top (or also referred to herein as a lid or cover) of the container 3504 may be maintained at a second set of temperature and a second set of humidity. The first set of temperature and the first set of humidity may be controlled to prevent or minimize evaporation of one or more reagents on the surface of the substrate. For example, the first set of temperature and the first set of humidity may be configured to prevent evaporation of less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the volume of the solution layer dispensed on the uncovered surface. The second set of temperature and the second set of humidity may also be controlled to enhance or limit condensation. For example, the first set of temperature may be the minimum temperature in the ambient environment of the open substrate system. For example, the second set of temperature may be the maximum temperature in the ambient environment of the open substrate system. In some cases, the environmental conditions of the different regions may be achieved by controlling the temperature of the enclosure. In some examples, the environmental conditions in the different regions may be achieved by controlling the temperature of selected portions or the entire vessel. In some examples, the environmental conditions in the different regions may be achieved by controlling the temperature of selected portions or the entire substrate. In some examples, the environmental conditions in the different regions may be achieved by controlling the temperature of the reagent dispensed onto the substrate. Any combination of these may be used to control the environmental conditions in the different regions. Heat transfer may be achieved by any method including, for example, conductive, convective, and radiative methods. For example, the first region 3522 may be maintained at a lower temperature by controlling the temperature of the substrate 3502, and the second region 3524 may be maintained at a higher temperature by controlling the temperature of the top of the vessel 3504 by conduction.

The system may further include a reservoir below the substrate 3522 (not shown in FIG. 7). The reservoir may be configured to hold a fluid. The reservoir may be configured to collect fluid, precipitate, or condensate from another surface, such as the substrate 3522 or the top of the vessel 3504. Fluid may be removed from the reservoir. In some cases, fluid may be volumetrically removed from the reservoir. For example, fluid may be volumetrically removed from the reservoir to balance the amount of fluid added to the system. In some cases, fluid is continuously added to the system and fluid is continuously removed from the reservoir. The amount of fluid added may be equal to the amount of fluid removed. In some cases, the volume of fluid in the reservoir is held constant. The volume of fluid in the reservoir may be determined based on the relative humidity of the system. The relative humidity of the system may depend on the amount of fluid in the reservoir, the amount of fluid in the system, the temperature of the system, or any combination thereof.

The open substrate system of the present disclosure may include a barrier system configured to maintain a fluid barrier. FIG. 47A shows a partial cross-sectional view of a barrier system 4700 maintaining a fluid barrier 4713. FIG. 47B shows a perspective view of a chamber 4715 of the barrier system 4700. The barrier system 4700 and/or its respective components may correspond to the systems and/or their respective components having different environmental conditions shown in FIG. 7. For example, the substrate 310 shown in FIGS. 15-24 may be disposed within the barrier system 4700.

The barrier system 4700 includes a sample environment 4705 defined by a plate 4703, a chamber 4715, and a fluid barrier 4713. The chamber 4715 and the plate 4703 may be separated by a physical gap. The sample environment 4705 may be isolated (and/or insulated) from an external environment 4707.

The fluid barrier 4713 can act as a transition region between the sample environment 4705 and the external environment 4707. A substrate (e.g., substrate 310 shown in Figures 15-24) can be placed in the sample environment 4705. The fluid barrier 4713 can contain fluid (e.g., air) from the sample environment 4705, the external environment 4707, or both. The fluid barrier 4713 can be a low pressure region. The fluid barrier 4713 can have a lower pressure than the sample environment, the external environment, or both. The fluid barrier 4713 can be maintained via a fluid flow unit such as a pressure changer 4711. The fluid barrier 4713 can contain fluids of coherent or bulk operation.

The pressure change device 4711 may be integral with the chamber 4715. For example, as shown in FIG. 47A and FIG. 47B, the pressure change device may be integrated as a fluid channel 4720 in the wall of the chamber 4715. For example, suction may be applied through the fluid channel 4720 to draw fluid from the external environment 4707, or the sample environment 4705, or both, to generate a partial vacuum curtain (e.g., coherent operation, bulk operation, etc.), thereby creating the fluid barrier 4713. Alternatively, the fluid may be subjected to negative pressure. Fluid exhaust may be exhausted at the other end of the fluid channel. Alternatively or additionally, the device may not be integral with the chamber 4715. The fluid flow unit and/or the pressure change device 4711 may operate via one or more compressors (e.g., to generate negative pressure), pumps (e.g., to generate positive pressure), suction devices, and/or other devices to provide a lower pressure in the transition region. The chamber 4715 can include one or more fluid channels 4720 for implementing the fluid barriers of the present disclosure.

47A and 47B show two pressure change devices 4711, it will be understood that any number of such devices may be present. For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more such devices. Alternatively or additionally, there may be up to about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 such devices. In some cases, one or more pressure change devices 4711 may be implemented as an annular fluid channel surrounding the sample environment region, or other fluid channels along the perimeter or border of the sample environment region. In some examples, one or more additional fluid flow paths (e.g., 4733) may be provided near the bottom of the chamber to draw excess fluid (e.g., liquid, gas) from the sample environment region.

Beneficially, the fluid barrier 4713 can provide a low or zero friction seal between the sample environment 4705 and the external environment 4707. In some cases, the fluid flow rate through the fluid barrier 4713 can be at least about 5 liters per minute (L/min), 5.5 L/min, 6 L/min, 6.5 L/min, 7 L/min, 7.5 L/min, 8 L/min, 8.5 L/min, 9 L/min, 9.5 L/min, 10 L/min, 10.5 L/min, 11 L, 11.5 L/min, 12 L/min, 12.5 L/min, 13 L/min, 13.5 L/min, 14 L/min, 14.5 L/min, 15 L/min, or more. Alternatively or additionally, the fluid flow rate may be up to about 15 L/min, 14.5 L/min, 14 L/min, 13.5 L/min, 13 L/min, 12.5 L/min, 12 L/min, 11.5 L/min, 11 L/min, 10.5 L/min, 10 L/min, 9.5 L/min, 9 L/min, 8.5 L/min, 8 L/min, 7.5 L/min, 7 L/min, 6.5 L/min, 6 L/min, 5.5 L/min, 5 L/min, or less. As will be appreciated, the fluid flow rate may vary depending on different parameters (e.g., minimum distance between the plate and the chamber, pressure, temperature, etc.). In some examples, for a gap of about 500 microns between the plate 4703 and the chamber 4715, the fluid flow rate can be about 10 L/min or about 13 milliliters/minute (mL/min)/millimeter (mm) around the circumference at a velocity of about 0.42 meters/second (m/s). Barrier systems, methods, and apparatus that can be used in the open substrate systems of the present disclosure are described in U.S. Pat. No. 10,512,911 and International Patent Application No. PCT/US19/64916, filed December 6, 2019, each of which is incorporated herein by reference in its entirety.

The system may be temperature controlled. In some cases, elements of the system may be held at different temperatures. Temperature differences between individual elements in the system can control the accumulation of condensation or precipitation on the individual elements of the system. The top of the container 3504 may be held at a different temperature than the substrate 3502, the objective lens (e.g., as shown in FIG. 15), or the reservoir. Alternatively or additionally, the substrate may be held at a different temperature than the top of the container, the objective lens, or the reservoir. Alternatively or additionally, the reservoir may be held at a different temperature than the top of the container, the objective lens, or the substrate. Alternatively or additionally, the objective lens may be held at a different temperature than the top of the container, the reservoir, or the substrate. In some cases, the top of the container is held at a higher temperature than at least one other element in the system to prevent the accumulation of condensation on the top surface of the container. In an exemplary configuration, the top of the container is kept at the highest temperature, the substrate is kept at the lowest temperature, and the reservoir and objective lens are kept at an intermediate temperature, thereby preventing condensation from forming on the top of the container or from forming or dripping on the objective lens. In another example, the objective lens is kept at the highest temperature, the top of the container is kept at an intermediate temperature, and the substrate and reservoir are kept at a lower temperature than the top of the container, thereby preventing condensation from forming on the top of the container or from forming or dripping on the objective lens. In some cases, the objective lens may be fully or partially surrounded by a seal. The seal may be configured to prevent moisture from the container surrounding the substrate (e.g., as shown in FIG. 7 ) from reaching other optical components in the system (e.g., as described with respect to FIG. 41 ). The seal may include a flexible material. The flexible seal may be configured to allow relative movement of individual elements of the system while maintaining a seal. In some embodiments, the flexible seal may stretch, expand, or contract. For example, the flexible seal may be configured to allow independent movement of two or more imaging heads, as described with respect to FIGS. 29F-29G. Alternatively or additionally, the seal may include a waterproof material. For example, the seal may be rubber, silicone, latex, plastic, Teflon, nitrile, elastin, elastomer, or polymer. The seal may surround the objective lens and contact the top of the container. In some cases, a portion of the objective lens, including the front lens, is not covered by the seal. The front lens of the objective lens may be exposed to the container surrounding the substrate. In some cases, the front lens of the objective lens may be in fluid contact with the substrate.

The system (or any element thereof) is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at most The system (or any element thereof) may be configured to maintain a relative humidity of at most 100%, at most 95%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, or at most 5%, or within a range defined by any two of the foregoing values. The system (or any element thereof) may be configured to evaporate less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the volume of the solution layer dispensed on the uncovered surface.

The system (or any element thereof) may be contained within a sealed container, housing, or chamber that insulates the system (or any element thereof) from the external environment or atmosphere, allowing for control of temperature or humidity. The environmental units (e.g., humidifiers, heaters, heat exchangers, compressors, etc.) may be configured to adjust one or more operating conditions in each environment. In some cases, each environment may be regulated by an independent environmental unit. In some cases, a single environmental unit may regulate multiple environments. In some examples, multiple environmental units may adjust different environments, individually or collectively. The environmental units may use active or passive methods to adjust the operating conditions. For example, temperature may be controlled using heating or cooling elements. Humidity may be controlled using humidifiers or dehumidifiers. In some cases, some of the internal environments within the container or chamber may be further controlled from other parts of the internal environment. Different parts may have different local temperatures, pressures, and/or humidity. For example, the internal environment may include a first internal environment and a second internal environment separated by a seal.

Alternatively or in addition, the systems or methods described herein may include a solution that includes an agent that may reduce evaporation. For example, the solution may include glycerol, which may prevent evaporation of the solution.

In some cases, the seal may include an immersion objective, which is described in more detail elsewhere herein. For example, the immersion objective may be part of a seal that separates an internal environment within the container into a first internal environment having 100% (or substantially 100%) humidity and a second environment having one or more of ambient temperature, pressure, or humidity. The immersion objective may be in contact with one or more of the detector and the imaging lens.

Substrate Preparation and Contaminant-Resistant Substrates As described above, a substrate can include a surface that includes a plurality of binders bound thereto. In some cases, the plurality of binders can include a plurality of nucleic acid molecules (e.g., a plurality of nucleic acid molecules that are directly coupled to the surface or indirectly coupled to the surface via a plurality of linkers, as described herein). Surfaces (e.g., substantially planar substrates and/or particles including a substrate having a plurality of particles immobilized thereon) coated with oligonucleotides (e.g., nucleic acid molecules) can be used for a variety of applications, including, for example, gene expression analysis by hybridization capture (gene arrays), single nucleotide polymorphism (SNP) genotyping, capture of a subset of a sequencing library (e.g., target capture or exome sequencing), synthesis of cDNA from mRNA by oligo-dT capture, and capture of specific sequences of nucleic acid molecules for on-surface amplification of the nucleic acid molecules for downstream analysis, such as next-generation sequencing. The oligonucleotide-coated surface may be prepared prior to its use in any such application and may be stored between its generation and its ultimate use (e.g., during transport from manufacturing site to surgical site, sample processing and preparation, etc.). The oligonucleotide-coated surface may be stored for at least one hour, and in some cases may be stored for months or years. During storage, the oligonucleotide-coated surface may come into contact with one or more solutions or other materials that may contain nucleic acid molecules that may be considered contaminants. Contaminating nucleic acid molecules may hybridize to the oligonucleotides coupled to the surface, resulting in reduced efficiency and/or erroneous results in downstream analysis (e.g., during use in applications such as those described herein). For example, an oligonucleotide-coated surface prepared for use in sequencing analysis may be contaminated with unrelated sequencing libraries during handling of the surface prior to use in sequencing analysis (e.g., before placing the substrate containing the surface in a sequencing instrument, or before starting an amplification process such as a clonal amplification process).

Non-associated interactions of oligonucleotides (e.g., binders) bound to the surface of a substrate can be reduced by blocking the surface-bound oligonucleotides (e.g., binding oligonucleotides) with oligonucleotides that contain sequences that are fully or partially complementary to the sequences of the surface-bound oligonucleotides. The blocking oligonucleotides may be provided in solution and may be considered "free" oligonucleotides. For example, the blocking oligonucleotides may fully or partially hybridize to all or a subset of the oligonucleotides coupled to the surface of the substrate, thereby providing a partially double-stranded nucleic acid molecule that includes the binding oligonucleotides and the blocking oligonucleotides. Such partially double-stranded nucleic acid molecules may be resistant to hybridization to nucleic acid molecules that the surface may come into contact with, including potential contaminating nucleic acid molecules that may not be relevant to the ultimate analysis, such as ultimate nucleic acid sequencing. The blocking oligonucleotides can be removed from the oligonucleotide-coated surface (e.g., via application of a suitable stimulus, such as a chemical or thermal stimulus, or via enzymatic degradation) to provide an oligonucleotide-coated surface that is ready for use in an analysis process. The surface may be subjected to one or more washing processes (e.g., one or more wash streams) to remove the blocking oligonucleotides (e.g., as described herein). Removal of the blocking oligonucleotides can provide the surface-coupled oligonucleotides as free oligonucleotides that can participate in a variety of reactions, including capture of a complementary or partially complementary nucleic acid molecule of interest.

The oligonucleotide-coated surface may be stored for any useful time. For example, the oligonucleotide-coated surface may be stored for at least 1 hour, e.g., at least 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or more. The oligonucleotide-coated surface may be stored under any useful conditions. For example, the oligonucleotide-coated surface may be stored under standard temperature and pressure conditions (e.g., room temperature), e.g., about 18°C to about 30°C, e.g., about 20°C to about 25°C and about 1 atmosphere. The oligonucleotide-coated surface may be stored in a dry environment (e.g., in air or in a nitrogen- or argon-rich environment) or in a solution (e.g., a buffer solution such as saline sodium citrate).

The oligonucleotide-coated surfaces may be stored in a package or container, which may include one or more such oligonucleotide-coated surfaces. For example, multiple oligonucleotide-coated surfaces may be provided in a given package or container. The package or container containing one or more oligonucleotide-coated surfaces may be a rigid package or container, or a flexible package or container. For example, one or more oligonucleotide-coated surfaces, e.g., one or more substantially flat substrates containing one or more oligonucleotide-coated surfaces, may be provided in a flexible package. The package or container may include or be formed of, for example, glass, a plastic polymer, a metal (e.g., a metal foil), or any other material. The package or container containing one or more oligonucleotide-coated surfaces may be sealed (e.g., hermetically sealed). The package or container containing one or more oligonucleotide-coated surfaces may be resealable upon opening. For example, a first oligonucleotide-coated surface may be removed from the package or container, and a second oligonucleotide-coated surface may be retained within the package or container. The oligonucleotide-coated surface may also be configured to be stored outside the package or container for a period of time, e.g., at least about 1 hour, 2 hours, 6 hours, or more (e.g., as described herein).

The oligonucleotide-coated surface may be prepared at the manufacturing and/or shipping site. Alternatively, the oligonucleotide-coated surface may be prepared by a user, such as a user of a sequencing device. In some cases, an oligonucleotide-coated surface may be prepared at the manufacturing and/or shipping site, comprising a plurality of blocking oligonucleotides coupled (e.g., hybridized) to a plurality of oligonucleotides coupled to the oligonucleotide-coated surface. Alternatively, an oligonucleotide-coated surface may be prepared by a user, such as a user of a sequencing device. The plurality of blocking oligonucleotides coupled to the plurality of oligonucleotides coupled to the oligonucleotide-coated surface may be removed by a user, such as a user of a sequencing device. For example, the plurality of blocking oligonucleotides coupled to the plurality of oligonucleotides coupled to the oligonucleotide-coated surface may be removed by a user immediately prior to the user using the oligonucleotide-coated surface (e.g., for sequencing applications, as described herein).

An oligonucleotide-coated surface may be used one or more times for one or more applications. For example, a surface coated with oligonucleotides may be configured for a single use. Alternatively, an oligonucleotide-coated surface may be configured to be used multiple times for the same and/or different applications. For example, oligonucleotides coupled to a surface may be "reloaded" for use in a subsequent application, or the surface may be washed clean and new oligonucleotides may be coupled to the surface for use in a subsequent application. In another example, an oligonucleotide-coated surface may include one or more different regions (e.g., as described herein) that include one or more different oligonucleotides (e.g., binders) bound thereto. The one or more different oligonucleotides may be configured for use in one or more different applications. In one example, an oligonucleotide-coated surface includes a first plurality of oligonucleotides bound to a first region and a second plurality of oligonucleotides bound to a second region, the first plurality of oligonucleotides and the second plurality of oligonucleotides having different nucleic acid sequences. The first plurality of oligonucleotides may be configured to at least partially hybridize to the first plurality of blocking oligonucleotides, and the second plurality of oligonucleotides may be configured to at least partially hybridize to the second plurality of blocking oligonucleotides, where the first plurality of blocking oligonucleotides and the second plurality of blocking oligonucleotides have different nucleic acid sequences. The first plurality of blocking oligonucleotides hybridized to the first plurality of oligonucleotides coupled to the surface may be removable upon application of a first stimulus (e.g., as described herein), and the second plurality of blocking oligonucleotides hybridized to the second plurality of oligonucleotides coupled to the surface may be removable upon application of a second stimulus, where the second stimulus is different from the first stimulus. Thus, the first and second plurality of blocking oligonucleotides can be provided to an oligonucleotide-coated surface (e.g., at the same time or at different times) to provide a dual-treated surface. The first plurality of blocking oligonucleotides hybridized to the oligonucleotides of the first plurality of oligonucleotides coupled to the surface may be removed by application of a first stimulus (e.g., after a first storage period) so that the first plurality of oligonucleotides coupled to the first region are free to participate in a first application, such as a first sequencing assay. Application of the first stimulus may not affect the second plurality of blocking oligonucleotides coupled to the second plurality of oligonucleotides coupled to the second region. Thus, the second plurality of blocking oligonucleotides hybridized to the oligonucleotides of the second plurality of oligonucleotides coupled to the surface may be retained during the first application. The second plurality of blocking oligonucleotides hybridized to the oligonucleotides of the second plurality of oligonucleotides coupled to the surface may be removed by application of a second stimulus (e.g., after a second storage period) so that the second plurality of oligonucleotides coupled to the second region are free to participate in a second application, such as a second sequencing assay.

Oligonucleotides can be coupled to the oligonucleotide-coated surface via any useful mechanism, including, for example, nonspecific interactions (e.g., one or more of hydrophilic interactions, hydrophobic interactions, electrostatic interactions, physical interactions (e.g., adhesion to pillars or sedimentation in a well), etc.) or specific interactions (e.g., as described herein).

The oligonucleotides may be coupled to the oligonucleotide-coated surface in a random or semi-random manner. Alternatively, the oligonucleotides may be coupled to the oligonucleotide-coated surface in a predetermined pattern (e.g., as described herein). In some cases, the substrate comprising the oligonucleotide-coated surface may include one or more different binders (e.g., dispersed in a plurality of oligonucleotides or disposed in different regions of the substrate). For example, the substrate comprising the oligonucleotide-coated surface may include a first set of oligonucleotides coupled to the surface and a second set of oligonucleotides coupled to the surface, where the oligonucleotides of the first set of oligonucleotides have a nucleic acid sequence that is different from the nucleic acid sequence of the oligonucleotides of the second set of oligonucleotides. In one example, the oligonucleotides of the first set of oligonucleotides may include a first nucleic acid sequence, and the oligonucleotides of the second set of oligonucleotides may include a second nucleic acid sequence that is different from the first nucleic acid sequence. In some cases, the oligonucleotides of the first set of oligonucleotides and the oligonucleotides of the second set of oligonucleotides may include a common third nucleic acid sequence, such as a poly(T) sequence.

The oligonucleotides may be coupled to one or more particles immobilized on the surface of the substrate. For example, the surface of the substrate may include a plurality of particles (e.g., beads) immobilized thereon (e.g., as described herein), the plurality of particles including a plurality of oligonucleotides bound thereto. Optionally, each particle includes a different plurality of oligonucleotides coupled thereto (e.g., a plurality of oligonucleotides including a nucleic acid sequence that differs from the nucleic acid sequence of another plurality of oligonucleotides coupled to a different particle). For example, each particle of the plurality of particles on the surface of the substrate may include a plurality of oligonucleotides bound thereto, with all of the oligonucleotides bound to a given particle including a common barcode sequence, and each plurality of oligonucleotides bound to each different particle of the plurality of particles including a different barcode sequence (e.g., as described herein).

The oligonucleotide-coated surface may comprise any useful number of oligonucleotides coupled thereto (e.g., as described herein). For example, the oligonucleotide-coated surface may comprise at least 10, 100, 1,000, 10,000, 10,000, 1 million, 10 million, 100 million, 100 million, or more oligonucleotides. In some cases, the oligonucleotide-coated surface may comprise a plurality of regions comprising a plurality of different oligonucleotides, where these different oligonucleotides may have the same or different nucleic acid sequences and may comprise the same or different numbers of oligonucleotides. For example, the oligonucleotide-coated surface may comprise a first region comprising a first plurality of oligonucleotides and a second region comprising a second plurality of oligonucleotides, where the first plurality of oligonucleotides and/or the second plurality of oligonucleotides comprise at least 10, 100, 1,000, 10,000, 10,000, 1 million, 10 million, 100 million, 100 million, or more oligonucleotides. The density of oligonucleotides bound to a region of the surface can be, for example, at least about 1,000 molecules/ ^mm2 , such as at least about 10,000 molecules/ ^mm2 , 100,000 molecules/ ^mm2 , 1 million molecules/ ^mm2 , ¹⁰ million molecules/mm2 or more. The density of oligonucleotides bound to the surface can vary from region to region. For example, a surface can include a first region having a first density of oligonucleotides bound thereto and a second region having a second density of oligonucleotides bound thereto, the first density being greater than the second density.

The oligonucleotides coupled to the surface of the substrate may include one or more different nucleic acid sequences. For example, the oligonucleotides coupled to the surface of the substrate may include a barcode sequence, an adapter sequence, a primer sequence (e.g., a universal primer sequence), a poly(T) sequence, a random N-mer sequence, a flow cell adapter sequence, a sequencing primer, a unique molecular identifier, a key sequence, an index sequence, or any other useful sequence. The one or more sequences of the oligonucleotides coupled to the surface may be configured to capture a specific sample molecule or population thereof. In some cases, the oligonucleotide-coated surface may include multiple oligonucleotides coupled thereto, each oligonucleotide of the multiple oligonucleotides including at least one common or shared sequence. For example, each oligonucleotide of the multiple oligonucleotides coupled to the oligonucleotide-coated surface or a predetermined region thereof may include a common barcode sequence. Alternatively or additionally, each oligonucleotide of the multiple oligonucleotides coupled to the oligonucleotide-coated surface or a predetermined region thereof may include a poly(T) sequence (e.g., for capture of sample nucleic acid molecules including poly(A) sequences, such as mRNA molecules) or another specific capture sequence. In some cases, each oligonucleotide of a plurality of oligonucleotides coupled to an oligonucleotide-coated surface or region thereof may contain one or more common sequences (e.g., as described herein) and a different unique molecular identifier or key sequence.

The oligonucleotides attached to the surface of the substrate can have any useful length. For example, the oligonucleotides coupled to the surface of the substrate can include at least 6 bases, such as 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35 or more bases. In some cases, only a portion of the bases of the oligonucleotides coupled to the surface of the substrate can be accessible to blocking or other oligonucleotides. For example, one or more nucleotides of the oligonucleotides coupled to the surface of the substrate can include a blocking moiety and/or can be coupled to other moieties, such as a moiety that anchors the oligonucleotide to the surface. In some cases, the oligonucleotides coupled to the surface of the substrate can include one or more reversible terminators.

Similarly, the blocking oligonucleotide may have any useful length. For example, the blocking oligonucleotide may contain at least 6 bases, e.g., 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35 or more bases. In some cases, only a portion of the bases of the blocking oligonucleotide may be available to hybridize to an oligonucleotide bound to an oligonucleotide coated surface. For example, one or more nucleotides of the blocking oligonucleotide may comprise a blocking moiety and/or may be coupled to another moiety. In some cases, the blocking oligonucleotide may contain one or more groups that may be substantially inert or non-reactive (e.g., in a buffer solution). In some cases, the blocking oligonucleotide may contain one or more reversible terminators.

The oligonucleotides coupled to the surface of the substrate can have any useful composition. The oligonucleotides coupled to the surface can include nucleotides, nucleotide analogs, non-standard nucleotides, and/or modified analogs (e.g., as described herein). For example, the oligonucleotides coupled to the surface can include DNA nucleotides, RNA nucleotides, and/or mixtures thereof. Similarly, the blocking oligonucleotides coupled to the surface can have any useful composition, so long as they include a nucleic acid sequence that is fully or partially complementary to the oligonucleotides coupled to the surface of the substrate. The blocking oligonucleotides can include DNA nucleotides, RNA nucleotides, and/or mixtures thereof. In one example, the oligonucleotides coupled to the surface include DNA nucleotides, and the blocking oligonucleotides configured to hybridize partially or completely to the oligonucleotides coupled to the surface include DNA nucleotides. In another example, the oligonucleotides coupled to the surface include RNA nucleotides, and the blocking oligonucleotides configured to hybridize partially or completely to the oligonucleotides coupled to the surface include RNA nucleotides.

The oligonucleotide coupled to the surface may include an adaptor or its complement. For example, the oligonucleotide may include a sequence that is complementary to the sequence of an adaptor bound to a sample nucleic acid molecule (e.g., a single-stranded sample nucleic acid molecule, such as a single-stranded sample RNA molecule). The adaptor may be a single-stranded adaptor and may have any useful composition. For example, the adaptor may include DNA nucleotides, RNA nucleotides, or a combination thereof. The adaptor may have any useful length and other properties. The adaptor may be located at the end of the oligonucleotide that is distal from the surface to which the oligonucleotide is bound. The adaptor may include a barcode sequence (e.g., as described herein).

The oligonucleotides coupled to the surface and/or blocking oligonucleotides may include functional features such as terminators (e.g., reversible terminators), blocking moieties, or label or reporter moieties. For example, the blocking oligonucleotides may include a label moiety such as a fluorescent label (e.g., a dye as described herein). The label moiety or other functional feature may be linked to the nucleotide of the oligonucleotide through a linker moiety. For example, the nucleotide of the blocking oligonucleotide may include a label moiety (e.g., a dye) linked to the base of the nucleotide through a linker moiety. The nucleotide may be located at the end of the blocking oligonucleotide. Alternatively, or in addition, the nucleotide of the blocking oligonucleotide may include a terminator (e.g., a reversible terminator). The terminator may be linked to the sugar of the nucleotide through a linker moiety. The nucleotide may be located at the end of the blocking oligonucleotide. Such functional features may facilitate control of the interaction between the blocking oligonucleotide and the oligonucleotide coupled to the surface of the substrate and/or provide a mechanism for identifying where the blocking oligonucleotide has hybridized to the oligonucleotide coupled to the surface. Alternatively or in addition, the oligonucleotides coupled to the surface may contain a label or reporter moiety that emits a first signal when the oligonucleotide is not coupled and a second signal when the oligonucleotide is coupled to a blocking oligonucleotide. For example, the second signal may be attenuated, reduced, quenched, or amplified relative to the first signal. In some cases, when the oligonucleotide coupled to the surface hybridizes to the blocking oligonucleotide, no detectable signal may be emitted by the label or reporter moiety. In this way, the coupling between the oligonucleotide coupled to the surface and the blocking oligonucleotide may be monitored (e.g., to measure the blocking efficiency of the blocking oligonucleotide). For example, each of the oligonucleotides coupled to the surface may contain a dye that emits a signal when the oligonucleotide is "free", and this signal is severely attenuated when the oligonucleotide is "blocked" (e.g., hybridized to the blocking oligonucleotide). The blocking efficiency of the blocking oligonucleotide (and therefore the resistance to contamination of the treated surface) can be measured by optically examining the surface before and after providing the blocking oligonucleotide. In some cases, different fluorescent dyes can be used on different regions of the surface (e.g., in the case of oligonucleotides with different nucleic acid sequences that can be coupled to different regions of the surface).

A treated surface comprising a plurality of oligonucleotides immobilized thereon and a plurality of blocking oligonucleotides coupled to the oligonucleotides of the plurality of oligonucleotides may have any degree of "resistance to contamination." The percentage of the oligonucleotides of the plurality of oligonucleotides coupled to the blocking oligonucleotides of the plurality of blocking oligonucleotides may indicate the resistance of the treated surface to contamination. In some cases, at least 50% of the oligonucleotides of the plurality of oligonucleotides may be coupled to the blocking oligonucleotides. For example, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the oligonucleotides of the plurality of oligonucleotides may be coupled to the blocking oligonucleotides. Coupling between the oligonucleotides coupled to the surface and the blocking oligonucleotides may be monitored by optical detection (e.g., as described herein) or any other useful method.

Figures 38A-D show a blocking oligonucleotide approach. In Figure 38A, a substrate is provided that includes a binding oligonucleotide. In Figure 38B, the bound oligonucleotide is blocked using a blocking oligonucleotide (e.g., as described herein). As shown in Figure 38C, while the blocking oligonucleotide is coupled to the binding oligonucleotide, contaminating nucleic acid molecules cannot bind to the binding oligonucleotide. Figure 38D shows the removal of the blocking oligonucleotide using various mechanisms, including thermal denaturation, chemical denaturation, chemical degradation, and enzymatic degradation. After the blocking oligonucleotide is removed, relevant target nucleic acid molecules (e.g., from samples for various applications such as sequencing) can bind to the substrate-bound oligonucleotides (e.g., substrate-bound oligonucleotides that include sequences at least partially complementary to the target nucleic acid molecules described herein).

In one aspect, the disclosure provides a method of storing a substrate comprising a surface coated with nucleic acid molecules. A substrate having a surface comprising a first set of immobilized nucleic acid molecules may be provided. The nucleic acid molecules of the first set of nucleic acid molecules may be configured to capture sample nucleic acid molecules from one or more nucleic acid samples (e.g., nucleic acid samples for sequencing). The substrate comprising a surface comprising the first set of nucleic acid molecules may be contacted with a second set of nucleic acid molecules under conditions sufficient to obtain a treated surface in which at least 70% (e.g., at least 75%, 80%, 85%, 90% or more) of the nucleic acid molecules of the first set of nucleic acid molecules may hybridize to the nucleic acid molecules of the second set of nucleic acid molecules, and the second set of nucleic acid molecules are not sample nucleic acid molecules. Excess nucleic acid molecules of the second set of nucleic acid molecules may be washed away. The substrate having the treated surface may be stored for a period of at least 1 hour, 6 hours, 12 hours, 24 hours, 2 days or more, etc. The treated surface may be stored under any useful conditions (e.g., as described herein). During storage of the treated surface, each nucleic acid molecule of the first set of nucleic acid molecules that hybridizes to a nucleic acid molecule of the second set of nucleic acid molecules may not hybridize to another nucleic acid molecule.

The second set of nucleic acid molecules may be provided on the surface of the substrate in solution. Each nucleic acid molecule of the second set of nucleic acid molecules may comprise a sequence substantially complementary to the sequence of the first set of nucleic acid molecules. The sequence of the first set of nucleic acid molecules may comprise at least 6 bases, e.g., at least 10 bases, 20 bases, or more. Each nucleic acid molecule of the first set of nucleic acid molecules may comprise at least 6 bases, e.g., at least 10 bases, 20 bases, or more. The first set of nucleic acid molecules and/or the second set of nucleic acid molecules may comprise DNA nucleotides, RNA nucleotides, or a combination thereof. Each nucleic acid molecule of the first set of nucleic acid molecules may comprise the same nucleic acid sequence. In some cases, the first set of nucleic acid molecules may comprise one or more different nucleic acid sequences. The first set of nucleic acid molecules may comprise a first subset of nucleic acid molecules comprising a first nucleic acid sequence and a second subset of nucleic acid molecules comprising a second nucleic acid sequence, where the first and second nucleic acid sequences are different. Both the first subset of nucleic acid molecules and the second subset of nucleic acid molecules may comprise a third nucleic acid sequence. The third nucleic acid sequence may include a poly(T) sequence.

The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface at independently addressable locations. The independently addressable locations may be substantially planar and may comprise one or more wells. The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface of a substrate according to a predetermined pattern. The density of the first set of nucleic acid molecules on the surface may be at least 10,000 molecules/ ^mm2 , such as at least 100,000, 1 million, 10 million, or more molecules/ ^mm2 . The surface of the substrate may be substantially planar. The substrate may comprise one or more particles immobilized thereon.

The method may further include removing the nucleic acid molecules of the second set of nucleic acid molecules from the treated surface after a storage period of the treated surface. The nucleic acid molecules may be removed, for example, by enzymatic degradation or by denaturation with a chemical or thermal stimulus (e.g., application of a chemical stimulus such as sodium hydroxide). After removing these nucleic acid molecules, the first set of nucleic acid molecules immobilized on the surface may be used, for example, for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of the nucleic acid molecules or derivatives thereof, or a combination thereof.

In another aspect, the disclosure provides a method of preparing a substrate having a treated surface for use in nucleic acid processing. A substrate having a treated surface may be provided, the substrate comprising a first set of nucleic acid molecules immobilized thereon. At least 70% (e.g., at least 80%, 85%, 90%, 95%, or more) of the nucleic acid molecules of the first set of nucleic acid molecules may hybridize to nucleic acid molecules of the second set of nucleic acid molecules. The nucleic acid molecules of the first set of nucleic acid molecules may be configured to capture sample nucleic acid molecules from one or more nucleic acid samples. The second set of nucleic acid molecules are different from the sample nucleic acid molecules. The substrate having the treated substrate may be stored for a period of at least 1 hour, e.g., at least 6 hours, 12 hours, 24 hours, 2 days, or more. The treated surface may be stored under any useful conditions (e.g., as described herein). During storage of the treated surface, each nucleic acid molecule of the first set of nucleic acid molecules that hybridizes to a nucleic acid molecule of the second set of nucleic acid molecules may not hybridize to another nucleic acid molecule.

The nucleic acid molecules of the second set of nucleic acid molecules from the treated surface can be removed (e.g., as described herein). For example, the nucleic acid molecules can be removed from the treated surface by enzymatic degradation or by denaturation with a chemical or thermal stimulus (e.g., application of a chemical stimulus such as sodium hydroxide). After removing these nucleic acid molecules, the first set of nucleic acid molecules immobilized on the surface can be used, for example, for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of the nucleic acid molecules or derivatives thereof, or a combination thereof.

Each nucleic acid molecule of the second set of nucleic acid molecules may comprise a sequence substantially complementary to a sequence of the first set of nucleic acid molecules. The sequence of the first set of nucleic acid molecules may comprise at least 6 bases, e.g., at least 10 bases, 20 bases, or more. Each nucleic acid molecule of the first set of nucleic acid molecules may comprise at least 6 bases, e.g., at least 10 bases, 20 bases, or more. The first set of nucleic acid molecules and/or the second set of nucleic acid molecules may comprise DNA nucleotides, RNA nucleotides, or a combination thereof. Each nucleic acid molecule of the first set of nucleic acid molecules may comprise the same nucleic acid sequence. In some cases, the first set of nucleic acid molecules may comprise one or more different nucleic acid sequences. The first set of nucleic acid molecules may comprise a first subset of nucleic acid molecules comprising the first nucleic acid sequence and a second subset of nucleic acid molecules comprising the second nucleic acid sequence, where the first and second nucleic acid sequences are different. Both the first subset of nucleic acid molecules and the second subset of nucleic acid molecules may comprise a third nucleic acid sequence. The third nucleic acid sequence may comprise a poly(T) sequence.

The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface at independently addressable locations. The independently addressable locations may be substantially planar and may comprise one or more wells. The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface of a substrate according to a predetermined pattern. The density of the first set of nucleic acid molecules on the surface may be at least 10,000 molecules/ ^mm2 , such as at least 100,000, 1 million, 10 million, or more molecules/ ^mm2 . The surface of the substrate may be substantially planar. The substrate may comprise one or more particles immobilized thereon.

In another aspect, the disclosure provides a method of storing a substrate comprising a surface coated with nucleic acid molecules, the method comprising providing a substrate having a surface comprising a first set of immobilized nucleic acid molecules. The nucleic acid molecules of the first set of nucleic acid molecules may be configured to capture sample nucleic acid molecules from one or more nucleic acid samples. Each nucleic acid molecule of the first set of nucleic acid molecules may comprise a first nucleic acid sequence. A second set of nucleic acid molecules may also be provided, each nucleic acid molecule of the second set of nucleic acid molecules comprising a second nucleic acid sequence that may be substantially complementary to the first nucleic acid sequence. The second set of nucleic acid molecules may be different from the sample nucleic acid molecules. The surface comprising the first set of nucleic acid molecules may be contacted with the second set of nucleic acid molecules to generate a treated surface in which at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or more) of the nucleic acid molecules of the first set of nucleic acid molecules may hybridize to the nucleic acid molecules of the second set of nucleic acid molecules. For each nucleic acid molecule of the first set of nucleic acid molecules that hybridizes to a nucleic acid molecule of the second set of nucleic acid molecules, the first nucleic acid sequence can be hybridized to the second nucleic acid sequence. The first nucleic acid sequence hybridized to the second nucleic acid sequence can be at least partially denatured at about 40°C to 60°C, for example, about 50°C to 60°C. The treated surface can then be stored for a period of time, for example at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days or more. The treated surface can be stored under any useful conditions (e.g., as described herein). During storage of the treated surface, each nucleic acid molecule of the first set of nucleic acid molecules that hybridizes to a nucleic acid molecule of the second set of nucleic acid molecules can be non-hybridized to another nucleic acid molecule.

The second set of nucleic acid molecules may be provided on the surface of the substrate in solution. The first and second nucleic acid sequences may each comprise at least 6 bases, e.g., at least 10 bases, 20 bases, or more. Each nucleic acid molecule of the second set of nucleic acid molecules may comprise at least 6 bases, e.g., at least 10 bases, 20 bases, or more. Similarly, each nucleic acid molecule of the first set of nucleic acid molecules may comprise at least 6 bases, e.g., at least 10 bases, 20 bases, or more. A given nucleic acid molecule of the first set of nucleic acid molecules and a given nucleic acid molecule of the second set of nucleic acid molecules may comprise the same number of nucleotides. Alternatively, a given nucleic acid molecule of the first set of nucleic acid molecules and a given nucleic acid molecule of the second set of nucleic acid molecules may comprise a different number of nucleotides. The first set of nucleic acid molecules and/or the second set of nucleic acid molecules may comprise DNA nucleotides, RNA nucleotides, or a combination thereof. In some cases, the first set of nucleic acid molecules may comprise one or more different nucleic acid sequences. The first set of nucleic acid molecules can include a first subset of nucleic acid molecules that include a first nucleic acid sequence and a second subset of nucleic acid molecules that include a third nucleic acid sequence, where the first and third nucleic acid sequences are different. The first subset of nucleic acid molecules and the second subset of nucleic acid molecules can both include a fourth nucleic acid sequence. The fourth nucleic acid sequence can include a poly(T) sequence.

The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface at independently addressable locations. The independently addressable locations may be substantially planar and may comprise one or more wells. The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface of a substrate according to a predetermined pattern. The density of the first set of nucleic acid molecules on the surface may be at least 10,000 molecules/ ^mm2 , such as at least 100,000, 1 million, 10 million, or more molecules/ ^mm2 . The surface of the substrate may be substantially planar and may comprise a plurality of wells. The substrate may comprise one or more particles immobilized thereon.

The method may further include removing the nucleic acid molecules of the second set of nucleic acid molecules from the treated surface after a storage period of the treated surface. The nucleic acid molecules may be removed, for example, by enzymatic degradation or by denaturation with a chemical or thermal stimulus (e.g., application of a chemical stimulus such as sodium hydroxide). The nucleic acid molecules of the second set of nucleic acid molecules may be removed from the treated surface by denaturing the first nucleic acid sequence hybridized to the second nucleic acid sequence, for example, by heating the treated surface or a solution in contact with the treated surface to about 40°C to 60°C. After removing these nucleic acid molecules, the first set of nucleic acid molecules immobilized on the surface may be used, for example, for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of the nucleic acid molecules or derivatives thereof, or a combination thereof.

In some cases, a single nucleic acid molecule may serve as both the surface-bound nucleic acid molecule and the blocking nucleic acid molecule. For example, the surface-bound nucleic acid molecule may include a first sequence and a second sequence, where the second sequence may be complementary to the first sequence. The second sequence may hybridize to the first sequence to provide a hairpin molecule immobilized on the surface. Such a scheme may provide higher blocking efficiency and therefore higher resistance to contamination. The portion of the nucleic acid molecule that includes the second sequence may be separated from the immobilized portion of the nucleic acid molecule that includes the first sequence (e.g., by cleaving the molecule at a cleavage site located between the first and second sequences), and the portion of the nucleic acid molecule that includes the second sequence may be removed and washed away (e.g., via denaturation or enzymatic degradation).

Thus, in another aspect, the disclosure may provide a method of storing a substrate comprising a surface coated with nucleic acid molecules, comprising providing a substrate having a surface comprising a first set of immobilized nucleic acid molecules, the nucleic acid molecules of the first set of nucleic acid molecules may be configured to capture sample nucleic acid molecules from one or more nucleic acid samples. Each nucleic acid molecule of the first set of nucleic acid molecules may comprise a first nucleic acid sequence and a second nucleic acid sequence, the second nucleic acid sequence being substantially complementary to the first nucleic acid sequence. The first sequence and the second sequence may each comprise at least 6 bases, e.g., at least 10 bases, 12 bases, 15 bases, 20 bases or more. The treated surface may be generated by subjecting the surface to conditions sufficient to bind the first nucleic acid sequence of the nucleic acid molecules of the first set of nucleic acid molecules to the second nucleic acid sequence of the nucleic acid molecules to provide immobilized hairpin molecules. The substrate with the treated surface may then be stored for a period of at least 1 hour, e.g., at least 2 hours, 6 hours, 12 hours, 24 hours, 2 days or more. The engineered surface can be stored under any useful conditions (e.g., as described herein). During storage of the engineered surface, each nucleic acid molecule of the first set of nucleic acid molecules can be non-hybridized to another nucleic acid molecule. At least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or more) of the nucleic acid molecules of the first set of nucleic acid molecules can be present as fixed hairpin molecules during storage of the engineered surface.

The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface at independently addressable locations. The independently addressable locations may be substantially planar and may comprise one or more wells. The nucleic acid molecules of the first set of nucleic acid molecules may be immobilized on a surface of a substrate according to a predetermined pattern. The density of the first set of nucleic acid molecules on the surface may be at least 10,000 molecules/ ^mm2 , such as at least 100,000, 1 million, 10 million, or more molecules/ ^mm2 . The surface of the substrate may be substantially planar and/or may comprise a plurality of wells. The substrate may comprise one or more particles immobilized thereon.

The first set of nucleic acid molecules may include one or more different nucleic acid sequences. For example, the first set of nucleic acid molecules may include a first subset of nucleic acid molecules including a first nucleic acid sequence and a second nucleic acid sequence, and a second subset of nucleic acid molecules including a third nucleic acid sequence and a fourth nucleic acid sequence. The third nucleic acid sequence may be substantially complementary to the fourth nucleic acid sequence. The first nucleic acid sequence may be different from the third and fourth nucleic acid sequences. The first subset of nucleic acid molecules and the second subset of nucleic acid molecules may both include a fifth nucleic acid sequence, and the fifth nucleic acid sequence may include a poly(T) sequence.

The method may further include separating the second sequence from the first sequence of the immobilized hairpin molecules after storing the treated surface for a period of time. Separation of the first and second sequences may be accomplished by enzymatic degradation or denaturation using a chemical or thermal stimulus (e.g., a chemical stimulus such as sodium hydroxide). After separating these sequences, the first set of nucleic acid molecules immobilized on the surface may be used, for example, for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of the nucleic acid molecules or derivatives thereof, or combinations thereof. Each nucleic acid molecule of the first set of nucleic acid molecules may include a cleavable base. The cleavable base may be disposed between the first and second sequences of the nucleic acid molecule. After separating the first and second sequences of the immobilized hairpin molecules, the nucleic acid molecules may be cleaved at the cleavable base, thereby removing the second sequence of the nucleic acid molecule from the surface.

The present disclosure also provides kits including engineered surfaces and kits for preparing engineered surfaces. The kits may include a substrate including an engineered surface and one or more reagents for treating the engineered surface (e.g., for removing blocking oligonucleotides from the engineered surface and preparing the surface for use in a subsequent application). The kits may include a substrate including a surface and a plurality of oligonucleotides for coupling to the substrate. The kits may also include a plurality of blocking oligonucleotides configured to hybridize to the plurality of oligonucleotides, as well as reagents for removing the blocking oligonucleotides and/or preparing the surface for use in a subsequent application. The kits provided herein may also include reagents for use in subsequent applications and/or instructions for storing, preparing, unblocking, or otherwise utilizing the surface of the substrate.

In one aspect, the disclosure provides a kit including a substrate including an engineered surface, the engineered surface including a plurality of pairs of bound nucleic acid molecules, each pair of the plurality of pairs including a first nucleic acid molecule of a first set of nucleic acid molecules at least partially hybridized to a second nucleic acid molecule of a second set of nucleic acid molecules. The first set of nucleic acid molecules may be immobilized on the surface. At least 70% (e.g., 75%, 80%, 85%, 90%, 95% or more) of the nucleic acid molecules of the first set of nucleic acid molecules may be paired with a nucleic acid molecule of the second set of nucleic acid molecules. The nucleic acid molecules of the first set of nucleic acid molecules may be configured to capture a sample nucleic acid molecule from one or more nucleic acid samples if the nucleic acid molecule of the first set of nucleic acid molecules is not paired with a nucleic acid molecule of the second set of nucleic acid molecules.

The treated surface may be stored for at least 6 hours, 12 hours, 24 hours, 2 days, or more. The treated surface may be stored under any useful conditions (e.g., as described herein). During storage of the treated surface, each nucleic acid molecule of the first set of nucleic acid molecules in each pair of the plurality of pairs may not hybridize to another nucleic acid molecule (e.g., a sample nucleic acid molecule).

The kit may include one or more reagents for treating the nucleic acid molecule. For example, the kit may include a kit further including a chemical stimulus (e.g., sodium hydroxide) configured to remove the second nucleic acid molecule from the treatment surface.

The surface of the substrate may be substantially planar and/or may include a plurality of wells. In some cases, the substrate may include one or more particles (e.g., beads) fixed thereto. The nucleic acid molecules of the first set of nucleic acid molecules may be fixed to the surface at independently addressable locations. The independently addressable locations may be substantially planar and/or may include one or more wells. In some cases, the density of the first set of nucleic acid molecules on the surface may be at least 10,000 molecules/ ^mm2 , such as at least 100,000, 1 million, 10 million, or more molecules/ ^mm2 . The nucleic acid molecules of the first set of nucleic acid molecules may be fixed to the surface according to a predetermined pattern or may be randomly distributed on the surface.

The second nucleic acid molecule may comprise a sequence substantially complementary to the sequence of the first nucleic acid molecule. The sequence of the first nucleic acid molecule and/or the second nucleic acid molecule may comprise at least 6 bases, e.g., at least 10, 12, 16, 20 or more bases. In some cases, the first nucleic acid molecule and the second nucleic acid molecule may comprise the same number of nucleotides. Alternatively, the first nucleic acid molecule and the second nucleic acid molecule may comprise a different number of nucleotides. Each nucleic acid molecule of the second set of nucleic acid molecules may comprise at least 6 bases. The first and/or second set of nucleic acid molecules may comprise DNA nucleotides, RNA nucleotides, or a mixture thereof.

Each nucleic acid molecule of the first set of nucleic acid molecules may comprise the same nucleic acid sequence. Alternatively, the first set of nucleic acid molecules may comprise one or more different nucleic acid sequences. For example, the first set of nucleic acid molecules may comprise a first subset of nucleic acid molecules comprising a first nucleic acid sequence and a second subset of nucleic acid molecules comprising a second nucleic acid sequence. The first and second nucleic acid sequences may be different. Both the first and second subsets of nucleic acid molecules may comprise a third nucleic acid sequence, which may comprise a poly(T) sequence.

In another aspect, the disclosure provides a kit comprising a substrate comprising a surface comprising a first set of immobilized nucleic acid molecules, the first set of nucleic acid molecules comprising one or more first nucleic acid molecules. The one or more first nucleic acid molecules may be configured to capture sample nucleic acid molecules from one or more nucleic acid samples. The kit may also comprise a solution comprising a second set of nucleic acid molecules, the second set of nucleic acid molecules comprising one or more second nucleic acid molecules, the one or more second nucleic acid molecules being not said sample nucleic acid molecules. The second set of nucleic acid molecules may be selected such that upon contacting the solution with the surface, at least 70% of the one or more first nucleic acid molecules (e.g., at least 75%, 80%, 85%, 90%, 90%, or more) bind to a second nucleic acid molecule of the second set of nucleic acid molecules to generate one or more pairs of bound nucleic acid molecules. Each pair of the one or more pairs may comprise (i) a first nucleic acid molecule of the first set of nucleic acid molecules and a second nucleic acid molecule of the second set of nucleic acid molecules, and (ii) a portion of a substantially complementary sequence. Each nucleic acid molecule of the first set of nucleic acid molecules in each pair of one or more pairs may not hybridize to another nucleic acid molecule (e.g., during storage on the treatment surface). For example, a paired nucleic acid molecule may not hybridize to a sample nucleic acid molecule.

The treated surface may be stored for at least 6 hours, 12 hours, 24 hours, 2 days, or more. The treated surface may be stored under any useful conditions (e.g., as described herein).

The kit may include one or more reagents for treating the nucleic acid molecule. For example, the kit may include a kit further including a chemical stimulus (e.g., sodium hydroxide) configured to remove the second nucleic acid molecule from the treatment surface.

The surface of the substrate may be substantially planar and/or may include a plurality of wells. In some cases, the substrate may include one or more particles (e.g., beads) fixed thereto. The nucleic acid molecules of the first set of nucleic acid molecules may be fixed to the surface at independently addressable locations. The independently addressable locations may be substantially planar and/or may include one or more wells. In some cases, the density of the first set of nucleic acid molecules on the surface may be at least 10,000 molecules/ ^mm2 , such as at least 100,000, 1 million, 10 million, or more molecules/ ^mm2 . The nucleic acid molecules of the first set of nucleic acid molecules may be fixed to the surface according to a predetermined pattern or may be randomly distributed on the surface.

The substantially complementary sequence portion of each pair of the one or more pairs may include a first sequence of the first nucleic acid molecule of the one or more first nucleic acid molecules and a second sequence of the second nucleic acid molecule of the one or more second nucleic acid molecules. The first sequence may be substantially complementary to the second sequence. The first and second sequences may each include the same number of bases. In some cases, the first and second sequences may each include about 6-20 bases. The first nucleic acid molecule of the one or more first nucleic acid molecules and the second nucleic acid molecule of the one or more second nucleic acid molecules may include the same number of nucleotides. Alternatively, the first nucleic acid molecule of the one or more first nucleic acid molecules and the second nucleic acid molecule of the one or more second nucleic acid molecules may include a different number of nucleotides. Each nucleic acid molecule of the second set of nucleic acid molecules may include at least 6 bases. The first and/or second set of nucleic acid molecules may include DNA nucleotides, RNA nucleotides, or a mixture thereof. The sequence of the nucleic acid molecule of the first nucleic acid molecule and/or the nucleic acid molecule of the second nucleic acid molecule can include at least 6 bases, e.g., at least 10, 12, 16, 20 or more bases.

Each nucleic acid molecule of the first set of nucleic acid molecules may comprise the same nucleic acid sequence. Alternatively, the first set of nucleic acid molecules may comprise one or more different nucleic acid sequences. For example, the first set of nucleic acid molecules may comprise a first subset of nucleic acid molecules comprising a first nucleic acid sequence and a second subset of nucleic acid molecules comprising a second nucleic acid sequence. The first and second nucleic acid sequences may be different. Both the first and second subsets of nucleic acid molecules may comprise a third nucleic acid sequence, which may comprise a poly(T) sequence.

Optical system for imaging a rotating substrate For substrates that exhibit smooth and stable rotational motion, it may be easier or more cost-effective to image the substrate using a rotational motion system instead of a linear motion system. Rotational motion as used herein may generally refer to motion in a polar coordinate system that includes an angular component φ and a radial component r that is primarily in the angular direction φ. Conventional optical imaging systems have utilized time-delay and integration (TDI) cameras to achieve high duty cycles and maximum integration times per field point. TDI cameras (e.g., TDI line-scan cameras) can use detection principles similar to charge-coupled device (CCD) cameras. Compared to CCD cameras, TDI cameras can shift charge row-by-row across the sensor at the same rate that the image traverses the focal plane of the camera. In this way, TDI cameras can enable longer image integration times while reducing artifacts such as blurring that may otherwise be associated with long image exposure times. TDI cameras can perform integration while simultaneously reading out, and therefore can have a higher duty cycle than cameras that perform these functions in a serial manner. The use of a TDI camera to extend integration times can be important for high-throughput fluorescent samples where signal generation may be limited by fluorescence lifetimes. For example, alternative imaging techniques such as point scanning may be unable to acquire a sufficient number of photons from a point for the limited amount of integration time required for high speed due to limitations imposed by the fluorescence lifetimes of dye molecules, thus preventing their use in high-throughput systems.

8A-8D show an exemplary scheme for a line scan camera. As shown in FIG. 8A, a TDI line scan camera can include two or more vertically arranged rows of pixels (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 24, 36, 48, 50, 60, 72, 84, 96, 100, 108, 120, 128, 132, 150, 200, 256, 512, 1024, 2000, 2048, 4000, 4096, 8000, 8192, 12000, 16000, 16384, or more pixels). During operation of the camera (e.g., movement of the camera relative to an open substrate), photoelectrons from each pixel in a given row can be summed to the row below the given row (e.g., in the direction of relative object motion) by shifting the accumulated charge between pixel rows. 8B and 8C show pixel schemes for use in color line scan cameras. Such cameras can include rows of pixels with different color filters for detecting and/or blocking different wavelengths of light. For example, FIG. 8B shows a tri-line pixel scheme including rows of red, green, and blue filters. This tri-line pixel scheme can be replicated one or more times to facilitate TDI applications. FIG. 8C shows a bi-line pixel scheme including rows of alternating red and blue filters and rows of green filters. FIG. 8D shows an alternative bi-line pixel scheme including multiple Bayer patterns (e.g., a 2×2 pixel array including a first row of alternating blue and green pixels and a second row of alternating green and red pixels). As with the tri-line scheme, the bi-line pattern can be replicated one or more times to facilitate TDI applications. The color line scan schemes shown in FIGS. 8B-8D can be replaced with alternative color combinations including cyan, yellow, green, and magenta. Red, green, blue, and emerald; cyan, magenta, yellow, and white; or any other combination of colors, in any arrangement (e.g., alternating, non-alternating).

Conventional TDI detection schemes may have limited applicability to imaging of rotating systems, such as the rotating nucleic acid sequencing system described herein. When scanning a curved path, such as the curved path generated by the rotating system described herein, the TDI sensor can only shift charge at the correct rate for a single speed (commonly referred to as clocking or line triggering). For example, the TDI sensor can only clock at the correct rate along an arc located at a particular distance from the center of rotation. Locations that are closer to the center of rotation may be too fast, and locations that are closer to the center of rotation may be too slow. In either case, a mismatch between the rotation rate of the rotating system and the clock rate of the TDI sensor can cause blurring that varies with the distance of the location from the center of the rotating system. This effect is sometimes referred to as tangential velocity blurring. Tangential velocity blurring can produce an image distortion of magnitude σ defined by equation (2):

(2)

where h, w, and A are the effective height, width, and area of the TDI sensor projected onto the object plane, respectively. These values can be adjusted using one or more optical elements (e.g., lenses, prisms, mirrors, etc.). R is the distance of the center of the field from the center of the rotating system. The effective height, width, and area of the sensor are the height, width, and area that generate the signal, respectively. For fluorescence imaging, the effective height, width, and area of the sensor may be the height, width, and area that correspond to the illuminated area on the sample, respectively. In addition to the tangential velocity blur effect, equation (2) implies that an increase in sensor area, which may be a goal of many imaging systems, may result in imaging complexity for TDI imaging of rotating systems. As a result, conventional TDI systems may require small image sensors to image rotating systems and therefore may not be suitable for simultaneous high sensitivity and high throughput imaging of such systems.

Described herein are systems and methods for imaging a rotating system that can address at least the problems discussed above. The systems and methods described herein can benefit from greater efficiency, such as faster imaging times.

9 shows an optical system 700 for continuous area scanning of a substrate during rotational motion of the substrate. The term "continuous area scanning (CAS)" as used herein generally refers to a method in which a relative moving object is imaged by repeatedly and electronically or computationally advancing (clocking or triggering) an array sensor at a rate that compensates for the object's motion in the detection plane (focal plane). CAS can produce images with a scan dimension larger than the field of view of the optical system. TDI scanning can be an example of CAS, where clock control involves shifting the photocharge on the area sensor during signal integration. For a TDI sensor, at each clocking step, the charge can be shifted by one row, and the last row is read out and digitized. Other modalities can achieve similar functionality by high speed area imaging and simultaneous addition of digital data to synthesize continuous or stepwise continuous scans.

The optical system may include one or more sensors 710. As shown in FIG. 9, the sensor may detect an optically projected image from the sample. The optical system may include one or more optical elements, such as the optical element 810 described in the context of FIG. 8. The optical element may be, for example, a lens, a prism, a mirror, a wave plate, a filter, an attenuator, a grating, an aperture, a beam splitter, a diffuser, a polarizer, a polarizer remover, a retroreflector, a spatial light modulator, or any other optical element. The system may include a plurality of sensors, such as at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1,000 sensors. The system may include at least 2, at least 4, at least 8, at least 16, at least 32, at least 64, at least 128, at least 256, at least 512, or at least 1,024 sensors. The multiple sensors may be homogeneous or heterogeneous. Alternatively, the system may include up to about 1000, 500, 200, 100, 50, 20, 10, 5, 2, or fewer sensors. Alternatively, the system may include up to about 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, or fewer sensors. The system may include a number of sensors within a range defined by any two of the aforementioned values. The sensor may include an image sensor. The sensor may include a CCD camera. The sensor may include a CMOS camera. The sensor may include a TDI camera (e.g., a TDI line scan camera). The sensor may include a pseudo-TDI high frame rate sensor. The sensor may include a CMOS TDI or hybrid camera. The sensors may be integrated into a single package. The sensors may be integrated into a single semiconductor substrate. The system may further include any light source (not shown in FIG. 9) described herein.

The sensor may be configured to detect images from a substrate, such as substrate 310 described herein, during a rotational motion of the substrate. The rotational motion may be relative to an axis of the substrate. The axis may be an axis through the center of the substrate. The axis may be an off-center axis. The substrate may be configured to rotate at any rotational speed described herein. The rotational motion may include a compound motion. The compound motion may include additional components of rotation and radial motion. The compound motion may be helical (or substantially helical). The compound motion may be ring (or substantially ring-like).

Each sensor may be located in a conjugate focal plane with respect to the substrate. Each sensor may be in optical communication with the substrate. The conjugate focal plane may be an approximate plane in an imaging system (e.g., a CAS sensor) where an image of an area of the substrate is formed. The sensor may be located in a plane conjugate with a plane containing the substrate (e.g., an image plane). The conjugate focal plane may be segmented into a plurality of regions, such as at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 regions. The conjugate focal plane may be segmented into at least 2, at least 4, at least 8, at least 16, at least 32, at least 64, at least 128, at least 256, at least 512, or at least 1,024 regions. The conjugate focal plane may be segmented into several regions that are within a range defined by any two of the aforementioned values. The conjugate focal plane may be divided into a plurality of regions along an axis substantially perpendicular to the projection direction of the rotational motion. The angle between the axis of rotational motion and the projection direction may be 1 degree or less, 2 degrees or less, 3 degrees or less, 4 degrees or less, 5 degrees or less, 6 degrees or less, 7 degrees or less, 8 degrees or less, 9 degrees or less, 10 degrees or less, 11 degrees or less, 12 degrees or less, 13 degrees or less, 14 degrees or less, or 15 degrees or less from the normal, or an angle within a range defined by any two of the preceding values. The conjugate focal plane may be divided into multiple regions along an axis parallel to the projection direction of the rotational motion. The conjugate focal plane may be spatially segmented. For example, the conjugate focal plane may be segmented by abutting or otherwise placing multiple sensors in a single focal plane and clocking each sensor independently.

Alternatively or in combination, the conjugate focal plane can be segmented by optically splitting the conjugate focal plane into multiple separate focal paths, each of which can form a sub-image on an independent sensor of the multiple sensors and can be clocked independently. The focal paths may be optically split using one or more optical elements, such as a lens array, a mirror, or a prism. Each sensor of the multiple sensors may be in optical communication with a different region of the rotating substrate. For example, each sensor may image a different region of the rotating substrate. Each sensor of the multiple sensors may be clocked at a speed appropriate for the region of the rotating substrate imaged by the sensor, which may be based on the distance of the region from the center of the rotating substrate or the tangential velocity of the region. For example, a first sensor (e.g., a line scan camera) imaging a first region through a first objective lens positioned further from the axis of rotation of the rotating substrate may be clocked at a faster rate than a second sensor imaging a second region through a second objective lens positioned closer to the axis of rotation of the rotating substrate.

One or more sensors may be configured to be in optical communication with at least two of the multiple regions in the conjugate focal planes. One or more of the sensors may comprise multiple segments. Each segment of the multiple segments may be in optical communication with a region of the multiple regions. Each segment of the multiple segments may be independently clocked. The independent clocking of the segments may be linked to a rate of an image in an associated region of the focal plane. The independent clocking may include a TDI line rate or a pseudo-TDI frame rate.

The system may further include a controller (not shown). The controller may be operably coupled to one or more sensors. The controller may be programmed to process optical signals from each region of the rotating substrate. For example, the controller may be programmed to process optical signals from each region with independent clocking during the rotational motion. The independent clocking may be based at least in part on the distance of each region from the projection of the axis and/or the tangential velocity of the rotational motion. The independent clocking may be based at least in part on the angular velocity of the rotational motion. Although a single controller is described, multiple controllers may be configured, individually or collectively, to perform the operations described herein.

FIG. 10A illustrates an optical system 800 for imaging a substrate during a rotational movement of the substrate using tailored optical distortion. The optical system may include one or more sensors 710. The one or more sensors may include any sensor described herein. The optical system may include any light source described herein (not shown in FIG. 10A). FIG. 10B illustrates an optical system 801 for imaging a substrate during a rotational movement of the substrate using tailored optical distortion. The optical system may include one or more sensors 710. The one or more sensors may include any sensor described herein. The optical system may include any light source described herein (not shown in FIG. 10B). The optical system may include a lens 810, for example a plano-convex lens. In some embodiments, the substrate 310 is tilted relative to the lens 810 and the detector 710. In some embodiments, the lens 810 is tilted relative to the detector 710, thereby generating an anamorphic magnification of light (e.g., fluorescent or scattered light) from the substrate. Anamorphic magnification may result in a differential magnification of light from a first region of the substrate 820 and a second region of the substrate 830. The light from the first region of the substrate may be magnified by a first amount at a first location on the detector 825, and the light from the second region of the substrate may be magnified by a second amount at a second location on the detector 835. In some embodiments, anamorphic magnification may occur along a single axis. In some embodiments, a cylindrical lens may be used to generate anamorphic magnification along a single axis.

The sensor may be configured to detect images from a substrate, such as substrate 310 described herein, during a rotational movement of the substrate. The rotational movement may be about an axis of the substrate. The axis may be an axis through the center of the substrate. The axis may be an off-center axis. The substrate may be configured to rotate at any rotational speed described herein.

The system 800 may further comprise an optical element 810. The optical element may be in optical communication with the sensor. The optical element may be configured to direct an optical signal from the substrate to the sensor. The optical element may generate an optical magnification gradient across the sensor. At least one of the optical element and the sensor may be adjustable. For example, at least one of the optical element and the sensor may be adjustable to generate an optical magnification gradient across the sensor. The optical magnification gradient may be along a direction substantially perpendicular to the projection direction of the rotational motion of the substrate. The optical element may be configured to rotate, tilt, or be positioned to manipulate the optical magnification gradient. The optical element may generate a magnification approximately proportional to the inverse of the distance to the axis of the substrate. The magnification gradient may be generated by selecting the relative orientation of the substrate, the optical element, and the sensor. For example, the magnification gradient may be generated by tilting the object and the image plane as shown in Figures 10A and 10B. The magnification gradient may indicate a geometric characteristic. For example, the ratio of the first optical magnification of the first region 820 at the maximum distance from the center of the substrate to the second optical magnification of the second region 830 at the minimum distance from the center of the substrate may be substantially equal to the ratio of the maximum distance to the minimum distance. Thus, the first and second optical magnifications may be in the same ratio as the radii of the respective sample regions. Although the illustrated system 800 and system 801 include a single optical element 810, the system 800 or system 801 may include multiple optical elements, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more optical elements. Various arrangements or configurations of optical elements may be used. For example, the system 800 may include lenses and mirrors to direct light.

The optical element may be a lens. The lens may be a field lens. The lens may be a cylindrical lens (e.g., as shown in FIG. 10C). The cylindrical lens may be plano-cylindrical. The lens may be plano-concave or plano-convex. The cylindrical lens may have a positive or negative curvature. The curvature of the cylindrical lens may vary. The curvature of the cylindrical lens may vary in a direction perpendicular to the projected direction of rotational motion. The shape of the surface of the lens may be conical. The lens may be tilted with respect to the sensor, thereby generating an anamorphic magnification gradient. The tilt of the lens may be adjustable, thereby generating an adjustable anamorphic magnification gradient.

10C shows an example of tailored optical distortion induced using a cylindrical lens. As shown in FIG. 10C, the cylindrical lens can have a first side A and a second side B. The first side A can be located closer to the image sensor (such as the TDI camera sensor described herein) than the second side B. Such a configuration can be achieved by tilting the cylindrical lens with respect to the image sensor. In this way, the cylindrical lens can direct light to different locations on the image sensor, with light passing through side B being directed more divergently than light passing through side A. In this way, the cylindrical lens can provide an anamorphic magnification gradient across the image sensor, as shown in FIG. 10C.

The lens tilt may provide an anamorphic magnification gradient across the sensor. The tilt, and therefore the anamorphic gradient, may be in a direction substantially perpendicular to the motion of the image on the sensor. The lens tilt may be adjustable. The adjustment may be automatic by using a controller. The adjustment may be coupled to the radius of the scanning substrate area relative to the substrate rotation axis. The ratio of the minimum anamorphic magnification to the maximum anamorphic magnification may be exactly or approximately the ratio of the minimum projection radius to the maximum projection radius relative to the substrate rotation axis.

Alternatively or in combination, a gradient in the radius of curvature of the lens can provide an anamorphic magnification gradient across the sensor. The curvature gradient may be in a direction substantially perpendicular to the motion of the image on the sensor.

The system may further include a controller (not shown). The controller may be operably coupled to the sensor and the optical element. The controller may be programmed to direct adjustment of at least one of the sensor and the optical element to generate an optical magnification gradient across the sensor. The magnification gradient may be generated along a direction substantially perpendicular to the projection direction of the rotational motion. The controller may be programmed to direct adjustment of the sensor and/or the optical element to generate an anamorphic optical magnification gradient. The optical magnification gradient may be across the sensor in a direction substantially perpendicular to the projection direction of the rotational motion. The controller may be programmed to direct rotation or tilt of the optical element. The controller may be programmed to direct adjustment of the magnification gradient. For example, the controller may be programmed to direct adjustment of the magnification gradient at least in part to a radial extent of a field of view dimension for projection about an axis of the substrate. The controller may be programmed to apply a rotational motion to the substrate. Although a single controller is described, multiple controllers may be configured individually or collectively to perform the operations described herein.

The optical systems described herein may utilize multiple scan heads. The multiple scan heads may operate in parallel along different imaging paths. For example, the scan heads may be operated to generate an interleaved spiral scan, a nested spiral scan, an interleaved ring scan, a nested ring scan, or a combination thereof. The scan head may include one or more of a detection element, such as a camera (e.g., a TDI line scan camera), an illumination source (e.g., as described herein), and one or more optical elements (e.g., as described herein).

FIG. 13A shows a first example of an interleaved spiral imaging scan. A first region of the scan head may operate along a first spiral path 910a. A second region of the scan head may operate along a second spiral path 920a. A third region of the scan head may operate along a third spiral path 930a. Each of the first, second, and third regions may be independently clocked. The scan head may include any of the optical systems described herein. The use of multiple imaging scan paths may increase imaging throughput by increasing imaging speed.

13B shows a second example of interleaved helical imaging scanning. The first scan head may operate along a first helical path 910b. The second scan head may operate along a second helical path 920b. The third scan head may operate along a third helical path 930b. Each of the first scan head, the second scan head, and the third scan head may be clocked independently or in unison. Each of the first, second, and third scan heads may comprise any of the optical systems described herein. The use of multiple imaging scan paths can increase the imaging throughput by increasing the net imaging speed. Operating multiple scan heads of field width in parallel can increase the throughput of the optical system. For example, each scan head may be fixed at a different angle relative to the center of substrate rotation.

13C shows an example of a nested spiral imaging scan. The first scan head may operate along a first spiral path 910c. The second scan head may operate along a second spiral path 920c. The third scan head may operate along a third spiral path 930c. Each of the first, second, and third scan heads may be independently clocked. Each of the first, second, and third scan heads may comprise any of the optics described herein. The use of multiple imaging scan paths can increase imaging throughput by increasing imaging speed. The scan heads can move together in a radial direction. Multiple scan heads of a field width can be operated in parallel to increase the throughput of the optics. For example, each scan head may be fixed at a different angle. The scan may be a discrete ring rather than a spiral.

13A-13C show three imaging paths, there may be any number of imaging paths and any number of scan heads. For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more imaging paths or scan heads. Or, there may be up to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer imaging paths or scan heads. Each scan head may be configured to receive light having a wavelength within a predetermined wavelength range. For example, a first scan head may be configured to receive a first light having a wavelength within a first wavelength range. A second scan head may be configured to receive a second light having a wavelength within a second wavelength range. A third scan head may be configured to receive a third light having a wavelength within a third wavelength range. Similarly, the fourth, fifth, sixth, seventh, eighth, ninth, or tenth scan head may be configured to receive fourth, fifth, sixth, seventh, eighth, ninth, or tenth light having a wavelength within the fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength range, respectively. The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may be the same. The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may overlap. Any two, three, four, five, six, seven, eight, nine, or ten of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may be different. The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may be in the ultraviolet, visible, or near infrared regions of the electromagnetic spectrum. Each of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may include a wavelength emitted by a fluorophore, dye, or quantum dot described herein. In this manner, the system may be configured to detect optical signals from multiple fluorophores, dyes, or quantum dots.

Scanning the surface may include detecting the focus of the surface relative to the detector. In some embodiments, scanning the surface includes adjusting the focus of the surface relative to the detector. The optical system of the present disclosure may further include one or more autofocus systems for detecting the position of the surface relative to the objective lens, as described elsewhere herein. The autofocus system may include an autofocus illumination source. The autofocus system may detect when the surface is out of focus relative to the detector. The autofocus system may be configured to send a signal to the focusing system to adjust the position of the surface relative to the objective lens, thereby returning the surface to a focused position relative to the detector. In some embodiments, the autofocus system may map a portion or all of the surface before scanning the surface to generate an autofocus map of the surface. The autofocus map of the surface may include surface texture, irregularities, or tilt that may affect the focus. The autofocus map of the surface may be used to predict the focus position of the surface and adjust the position of the surface relative to the objective lens to compensate for the surface texture, irregularities, or tilt. In some embodiments, the autofocus system may map a first portion of the surface (e.g., a first ring) before scanning the first portion of the surface. The map of the first portion of the surface may be used to predict and adjust the focus of the surface while scanning the first portion of the surface. The map of the first portion of the surface may be used to predict the focus of the surface while scanning a second portion of the surface (e.g., a second ring). The second portion of the surface may be proximate to the first portion of the surface such that the map of the first portion of the surface may approximate the map of the second portion of the surface. The autofocus system may map the second portion of the surface while scanning the second portion of the surface. The map of the second portion of the surface may be used to predict and adjust the focus of the surface while scanning a third portion of the surface (e.g., a third ring). In some embodiments, maps generated while scanning a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or more portions of the surface may be used to predict and adjust the focus of the surface while scanning a fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirtieth, or more portions of the surface, respectively. Successive surface portions may be positioned close to each other such that a map of a previous portion of the surface may approximate a map of a subsequent portion of the surface. In some embodiments, the autofocus system may map the entire surface before scanning. In some embodiments, the autofocus system adjusts focus as it scans without generating a map.

14 illustrates a nested circular imaging scan. The first scan head 1005 may be operated along a first generally circular path 1010. The second scan head 1015 may be operated along a second generally circular path 1020. The third scan head 1025 may be operated along a third generally circular path 1030. The fourth scan head 1035 may be operated along a fourth generally circular path 1040. The fifth scan head 1045 may be operated along a fifth generally circular path 1050. The sixth scan head 1055 may be operated along a sixth generally circular path 1060. Each of the first, second, third, fourth, fifth, and sixth scan heads may be independently clocked. Each of the first, second, third, fourth, fifth, and sixth scan heads may comprise any optical system described herein. Each of the first, second, third, fourth, fifth, and sixth scan heads may be configured to remain at a fixed position during scanning of the substrate. Alternatively, one or more of the first, second, third, fourth, fifth, and sixth scan heads may be configured to move during scanning of the substrate. The use of multiple scan heads imaging along a substantially circular imaging path can significantly increase imaging throughput. For example, the configuration of scan heads shown in FIG. 14 can enable imaging of all addressable locations on a substrate during a single rotation of the substrate. Such a configuration can have the additional advantage of simplifying the mechanical complexity of the imaging system by requiring only one scanning motion (e.g., rotation of the substrate).

14 shows six imaging paths and six scan heads, there may be any number of imaging paths and any number of scan heads. For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more imaging paths or scan heads. Or, there may be up to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or less imaging paths or scan heads. Each scan head may be configured to receive light having a wavelength within a predetermined wavelength range. For example, a first scan head may be configured to receive a first light having a wavelength within a first wavelength range. A second scan head may be configured to receive a second light having a wavelength within a second wavelength range. A third scan head may be configured to receive a third light having a wavelength within a third wavelength range. A fourth scan head may be configured to receive a fourth light having a wavelength within a fourth wavelength range. A fifth scan head may be configured to receive a fifth light having a wavelength within a fifth wavelength range. The sixth scan head may be configured to receive a sixth light having a wavelength within the sixth wavelength range. Similarly, the seventh, eighth, ninth, or tenth scan head may be configured to receive a seventh, eighth, ninth, or tenth light having a wavelength within the seventh, eighth, ninth, or tenth wavelength range, respectively. The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may be identical. The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may overlap. Any two, three, four, five, six, seven, eight, nine, or ten of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may be different. The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may be in the ultraviolet, visible, or near infrared regions of the electromagnetic spectrum. Each of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may include a wavelength emitted by a fluorophore, dye, or quantum dot described herein. In this manner, the system may be configured to detect optical signals from multiple fluorophores, dyes, or quantum dots.

29A-29D, 30A-30D, and 31A-31B show additional examples of imaging schemes including multiple imaging heads. For example, FIG. 31B shows a rotational scanning direction of multiple imaging heads with non-radial motion of the substrate.

FIG. 15 shows a cross-sectional view of an immersion optical system 1100. The system 1100 may be used to optically image a substrate as described herein. The system 1100 may be integrated with any other optical system or system for nucleic acid sequencing as described herein (e.g., any of systems 300, 400, 500a, 500b, 700, or 800), or any element thereof. The system may include an optical imaging objective 1110. The optical imaging objective may be an immersion optical imaging objective. The optical imaging objective may be configured to be in optical communication with a substrate, such as substrate 310 as described herein. The optical imaging objective may be configured to be in optical communication with any other optical element as described herein. The optical imaging objective may be partially or completely surrounded by a housing 1120. The housing may partially or completely surround the sample-facing end of the optical imaging objective. The housing and fluid may include an interface between an atmosphere in contact with the substrate and an ambient atmosphere. The atmosphere in contact with the substrate and the surrounding atmosphere may differ in relative humidity, temperature, and/or pressure. The housing may have a generally cup-like shape or form. The housing may be any container. The housing may be configured to contain a fluid in which the optical imaging objective is immersed or an immersion fluid 1140 (such as water or an aqueous or organic solution). The housing may be configured to maintain a minimum distance 1150 between the substrate and the housing to avoid contact between the housing and the substrate during rotation of the substrate. In some cases, air or pressure differentials can be used to maintain the minimum distance. The minimum distance can be at least 100 nm, at least 200 nm, at least 500 nm, at least 1 μm, at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, at least 1 mm, or a distance within a range defined by any two of the aforementioned values. Even at a minimum distance, the housing can contain the fluid due to surface tension effects. The system can include a fluid flow tube 1130 configured to deliver fluid inside the housing. The fluid flow tube can be connected to the housing via an adapter 1135. The adapter can include a threaded adapter, a compression adapter, or any other adapter. An electric field application unit (not shown) can be configured to adjust the hydrophobicity of one or more surfaces of the container, for example by applying an electric field, to retain at least a portion of the fluid in contact with the immersion objective lens and the open substrate.

As used herein, the fluid in contact with the immersion objective may be referred to as an "immersion fluid" or "fluid." The immersion fluid may include any suitable immersion medium for imaging. For example, the immersion medium may include an aqueous solution. Non-limiting examples of aqueous immersion fluids include water. In some cases, the aqueous solution may include salts, surfactants, oils, and/or any other chemicals or reagents useful for imaging. In some cases, the immersion medium includes an organic solution. Non-limiting examples of organic immersion fluids include oils, perfluorinated polyethers, perfluorocarbons, and hydrofluorocarbons. In some cases, the immersion fluid may be substantially the same as the wash buffers described elsewhere herein, or any buffer used in the methods described herein. The immersion fluid may be tailored based on the optical requirements of the systems and methods described herein. For example, if a high numerical aperture (NA) is required, an appropriate immersion fluid (e.g., oil) may be used for imaging. In some cases, the immersion fluid may be selected to match the refractive index of a solution on a substrate (e.g., a buffer), a surface (e.g., a coverslip or substrate), or an optical component (e.g., an objective lens).

The optical imaging objective may be in fluid contact with the open substrate. The open substrate may comprise a layer of fluid covering a surface of the substrate. The optical imaging objective may be configured to scan the surface including the layer of fluid. The layer of fluid on the surface may comprise the same fluid as the immersion fluid. The layer of fluid on the surface may comprise a different fluid than the immersion fluid. The layer of fluid on the surface may be miscible with the immersion fluid or the layer of fluid on the surface may not be miscible with the immersion fluid. In some cases, the layer of fluid is deeper in places where it contacts the optical imaging objective than at other points on the surface. Part of the layer of fluid may adhere to the optical imaging objective. In some cases, part of the layer of fluid may move with the optical imaging objective relative to the substrate during scanning. The optical imaging objective may remain in fluid contact with the substrate during scanning. The optical imaging objective may be configured to have a long travel distance in a direction perpendicular to the substrate. In some cases, the optical imaging objective may be configured to be lifted away from the substrate such that the optical imaging objective is no longer in fluid contact with the substrate. For example, the optical imaging objective may be moved away from the substrate while fluid is being dispensed onto the substrate. A portion of the layer of fluid, the immersion fluid, or both may adhere to the optical imaging objective upon leaving fluid contact with the substrate. The portion of the layer of fluid adhered to the optical imaging objective may prevent air bubbles from forming or accumulating between the substrate and the optical imaging objective when the optical imaging objective re-enters fluid contact with the substrate.

The optical imaging objective may be configured to scan a side of the substrate that does not include a layer of fluid. For example, the optical imaging objective may be configured to scan a bottom side of the substrate. In some cases, the optical imaging objective may not be in fluid contact with the substrate. For example, the optical imaging objective may be an air objective.

The fluid may be in contact with the substrate. The optical imaging objective and the housing may be configured to provide a physical barrier between a first location where a chemical processing operation is performed and a second location where a detection operation is performed. In this way, the chemical processing operation and the detection operation may be performed in independent operating conditions, avoiding contamination of the detector. The first and second locations may have different humidity, temperature, pressure, or atmospheric mixture.

The system of the present disclosure may be contained in a container or other closed environment. For example, the container may isolate the internal environment 1160 from the external environment 1170. The internal environment 1160 may be controlled to localize temperature, pressure, and/or humidity as described elsewhere herein. In some cases, the external environment 1170 may be controlled. In some cases, the internal environment 1160 may be further compartmentalized to separately control portions of the internal environment (e.g., a first internal environment for chemical processing operations, a second internal environment for detection operations, etc.), such as through or with the housing 1120. Different portions of the internal environment may be isolated via seals. For example, the seals may include an immersion objective lens as described herein.

The disclosed system may be configured to analyze a dynamic (e.g., rotating or otherwise moving) open substrate (e.g., as described herein) using a stationary detector system. Alternatively or additionally, one or more components of the detector system may be moving. For example, the detector system may include a sensor (e.g., a camera) and an illumination source. The sensor may be moving while the optical element (e.g., a prism) is stationary. The illumination source may move in conjunction with the sensor. For example, the sensor may be a line scan camera (e.g., a TDI line scan camera), the illumination source may be an LED line light or a laser (e.g., a laser with a beam expanded into a line), and the illumination source may illuminate the area being detected by the sensor. The sensor (and may be the illumination source) may rotate at the same or different speed as the open substrate. In some cases, the sensor (which may be the illumination source) may translate across the open substrate in a predetermined pattern, such as a spiral pattern. Alternatively, the sensor (which may be the illumination source) may translate radially across the open substrate. In some cases, the sensor (which may be the illumination source) may remain in the same physical location, but may rotate around a central axis of the detector system or its component(s). In other cases, the illumination source may illuminate an area of the open substrate that may be larger than the area detectable by the sensor in a given scan or set of scans. However, illumination over a wide swath of the open substrate may promote bleaching of beads and/or fluorophores that may be disposed on the open substrate. Thus, the illumination source may be configured to illuminate only a limited area of the open substrate at a given time (e.g., an area that may be, at least partially overlap, or within the area detectable by the sensor).

In another example, the detector system may include a sensor (e.g., a camera), an illumination source, and one or more optical elements (e.g., a lens, a mirror, a prism, etc.), and the sensor and illumination source may remain stationary while the optical element (e.g., a prism) moves. For example, the optical element may rotate at the same speed as the open substrate, or the optical element may translate across the open substrate (e.g., radially or in a predetermined pattern, such as a spiral pattern). In some cases, the optical element may remain in the same physical location but rotate about a central axis (e.g., the optical element or the detector system). The movement of the optical elements of the detector system relative to the moving open substrate may have the effect of enabling detection at one or more different regions of the open substrate. For example, the movement of one or more optical elements of the detector system may result in illumination of different regions of the open substrate to enable detection of signals associated with different regions of the open substrate. Distortions in the illumination (e.g., laser light) and variations in detection sensitivity across different regions of the open substrate may be compensated for by subsequent processing (e.g., using a processor, as described herein).

Alternatively, the disclosed system may be configured to analyze a fixed open substrate using a detector system that includes one or more dynamic components. For example, the detector system may include a sensor (e.g., a camera) and an illumination source. The sensor may be moving while the optical element (e.g., a prism) is stationary. The illumination source may move in conjunction with the sensor. For example, the sensor may be a line scan camera (e.g., a TDI line scan camera), the illumination source may be an LED line light or a laser (e.g., a laser with a beam expanded into a line), and the illumination source may illuminate the area being detected by the sensor. The sensor (which may be the illumination source) may rotate (e.g., about a central axis of the open substrate). In some cases, the sensor (which may be the illumination source) may translate across the open substrate in a predetermined pattern, such as a spiral pattern. Alternatively, the sensor (which may be the illumination source) may translate radially across the open substrate. In some cases, the sensor (which may be the illumination source) may remain in the same physical location but may rotate around the central axis of the detector system or its component(s).

In another example, the detector system may comprise a sensor (e.g., a camera), an illumination source, and one or more optical elements (e.g., lenses, mirrors, prisms, etc.), and the sensor and illumination source may remain stationary while the optical elements (e.g., prisms) move. For example, the optical elements may rotate (e.g., around a central axis of the open substrate or around a central axis of the optical elements or detector system) or translate across the open substrate (e.g., radially or in a predetermined pattern such as a spiral pattern). Movement of the optical elements of the detector system relative to a fixed open substrate may have the effect of enabling detection at one or more different regions of the open substrate. For example, movement of one or more optical elements of the detector system may result in illumination of different regions of the open substrate to enable detection of signals associated with different regions of the open substrate. Distortions of the illumination (e.g., laser light) and variations in detection sensitivity across different regions of the open substrate may be compensated for by subsequent processing (e.g., using a processor as described herein).

The system may be calibrated (e.g., using an open substrate that contains no analyte or that contains a known analyte or collections thereof) to facilitate any of the detection schemes provided herein.

In any of the foregoing examples, multiple sensors and/or illumination sources may be used (e.g., to detect different areas of an open substrate, as described herein). The multiple sensors and/or illumination sources may all remain stationary or may all move during the detection process. In other cases, certain sensors and/or illumination sources may move while other sensors and/or illumination sources may be stationary during the detection process. Some or all of the sensors may analyze the substrate. For example, only the moving sensors or only the stationary sensors may detect signals from the open substrate.

The scan direction of one or more detector systems (e.g., imaging heads) can rotate due to non-radial motion of the detector systems relative to the substrate. For example, the detector systems can have different tangential velocity vectors relative to the substrate while tracing different imaging paths at different radial positions along the substrate, and the tangential velocity vectors can point in substantially different directions. Such effects can manifest as a rotation of the imaging field of view when a first detector system traces a first set of imaging paths or when a second detector system traces a second set of imaging paths (see, e.g., FIGS. 31A and 31B).

The present disclosure provides an apparatus in which processing of analytes on an open substrate and detection of signals associated with the analytes occur in the same environment. For example, the open substrate may be held in the same or approximately the same physical position during processing of the analytes and subsequent detection of signals associated with the processed analytes. For systems in which the detector system or a component thereof is moving during detection, the apparatus may include a mechanical component configured to affect the movement of the detector system of that component.

The present disclosure also provides an apparatus in which the processing of analytes on an open substrate and the detection of signals associated with the analytes occur in different environments. For example, the open substrate may be held at a first physical location during processing of the analytes and held at a second physical location during detection of signals associated with the processed analytes. The open substrate may be transferred between the various physical locations, for example, via a mechanical component. In some cases, the open substrate may be transferred between the various physical locations using a robotic arm, an elevator mechanism, or another mechanism. The first physical location may be located, for example, above, below, adjacent to, or across the second physical location. For example, the first physical location may be located above the second physical location, and the open substrate may be transferred between these locations between analyte processing and detection. In another example, the first physical location may be located adjacent to the second physical location, and the open substrate may be transferred between these locations between analyte processing and detection. The first and second physical locations may be separated by a barrier, such as a retractable barrier.

12A-12C show various detection schemes. FIG. 12A shows a scheme including a system 3900 in which an open substrate 3910 rotates and a detector system 3920 remains stationary during detection. The detector system 3920 can include a line scan camera (e.g., a TDI line scan camera) 3930 and an illumination source 3940. FIG. 12B shows an alternative scheme including a system 3900 in which an open substrate 3910 remains stationary and a detector system 3920 rotates during detection. FIG. 12C shows a scheme including an apparatus including a first system 3950 in which an open substrate 3910 is subjected to analyte processing. As shown in FIG. 3, the first system 3950 can include a plurality of fluidic channels 3960, 3970, 3980, and 3990, which can include a plurality of fluidic outlet ports 3965, 3975, 3985, and 3995. The apparatus may be configured to transfer the open substrate 3910 to a second system 3900, where the open substrate 3910 is configured to remain stationary and the detector system 3920 is configured to rotate during detection. Although the examples described herein provide for relative rotational motion of the substrate and/or detector system, the substrate and/or detector system may alternatively or additionally undergo relative non-rotational motion, such as relative linear motion, relative non-linear motion (e.g., curved, arcuate, angular, etc.), and any other type of relative motion.

In one aspect, the disclosure provides a method for analyte detection or analysis, comprising providing an open substrate comprising a central axis (e.g., as described herein). The open substrate may be, for example, a wafer or disk, such as a wafer or disk having one or more materials patterning its surface. The open substrate may be substantially planar. The open substrate may have an array of analytes immobilized thereon (e.g., as described herein). The immobilized analytes may be immobilized in the array via one or more binders. The array may include at least 100,000 such binders. In some cases, the immobilized analytes of the immobilized analytes may be bound to beads, and the beads may be immobilized in the array. The immobilized analytes may include nucleic acid molecules.

A solution having a plurality of probes may be delivered (e.g., as described herein) to a region proximal to the central axis to introduce the solution into the open substrate. The solution may be dispersed across the open substrate such that at least one probe of the plurality of probes may bind to at least one immobilized analyte of the immobilized analytes to form a bound probe. The plurality of probes may include a plurality of oligonucleotide molecules. Alternatively, the plurality of probes may include a plurality of nucleotides or nucleotide analogs. All or a subset of the plurality of nucleotides or nucleotide analogs may be fluorescently labeled. In one example, the immobilized analytes may include nucleic acid molecules, and the plurality of probes may include fluorescently labeled nucleotides such that at least one fluorescently labeled nucleotide of the fluorescently labeled nucleotides binds to at least one nucleic acid molecule of the nucleic acid molecule via nucleotide complementary bonds. All or a subset of the plurality of nucleotides or nucleotide analogs may include the same base (e.g., the same canonical nucleic acid base, e.g., A, T, C, or G). Similarly, all or a subset of the plurality of nucleotides or nucleotide analogs may be reversibly terminated. The reversible terminators and possibly fluorescent moieties such as dyes may be cleaved from the nucleotides (e.g., following their incorporation into a growing nucleic acid strand) using a cleaving agent, which may be included in a separate solution provided to the open substrate (e.g., as described herein). The open substrate may also be provided with a wash solution to remove excess probes and other reagents, which may be dispersed across the open substrate (e.g., while spinning the open substrate using at least centrifugal force, as described herein).

After generation of the binding probes, a detector system can be used to detect at least one signal from the binding probes. The detector system can include a line scan camera (e.g., a TDI line scan camera) and an illumination source (e.g., a laser, such as an LED line light or a continuous wave laser). In some cases, the illumination source can include a laser, and the detector system can include an optical element (e.g., a cylindrical lens) configured to change the shape of a beam (e.g., a Gaussian beam) emitted by the laser (e.g., as described herein). The open substrate can include a first region and a second region, the first region and the second region including a subset of the array of immobilized analytes, at different radial positions of the open substrate relative to a central axis, and spatially resolvable by the detector system. The binding probes can be disposed in the first region of the open substrate. The detector system can perform a nonlinear scan of the open substrate. The illumination source and the detector system are described in more detail with respect to FIG. 41.

During the dispersion and delivery processes, the open substrate may be rotating (e.g., at a first physical location). The detector system (e.g., sensor and illumination source) may be stationary during these processes.

During the detection process, the open substrate may be stationary. The sensor and/or illumination source of the detector system may be moving during detection. For example, the sensor and illumination source may be rotating at the same speed during detection. The sensor and/or illumination source may rotate around the central axis of the open substrate. Alternatively, the sensor and/or illumination source may rotate around the central axis of the detector system or a component thereof and remain in the same physical location. The sensor and/or illumination source may translate relative to the open substrate in a predetermined pattern, such as a helical pattern. Alternatively, the line scan camera and/or illumination source may translate (e.g., radially translate) across the open substrate. The detector system may further comprise a prism (e.g., a Dove prism) that may rotate during the detection process (e.g., around the central axis of the open substrate or around the central axis of the detector system or a component thereof while remaining in the same physical location). In one example, the prism may rotate or translate relative to the open substrate while the sensor and illumination source remain stationary. Such prisms can be used to disperse light to and from an open substrate, for example, to disperse light from an illumination source onto an open substrate and to detect optical signals from the open substrate, such as fluorescence.

The detector system may be configured to illuminate an area of the open substrate using an illumination source and subsequently detect a signal from the area using a sensor (e.g., a line scan camera). For example, the illumination source may illuminate an area of the open substrate (e.g., a fixed open substrate) prior to detection by the sensor. In such a situation, the sensor and illumination source may move in tandem relative to the open substrate. One or more optical elements, such as one or more lenses, mirrors, filters, or other optical elements, may move in conjunction with these other components of the detector system (e.g., to manipulate the light provided to or detected from the open substrate).

During the dispersion and/or delivery process, an additional probe may be formed, and this additional coupled probe may be located in a second region of the open substrate. During detection, at least one signal may be detected from the further coupled probe simultaneously with at least one signal from the coupled probe. These signals may be detected with different sensitivities.

The detector system can compensate for velocity differences at different radial positions of the array relative to the central axis within the scan region. The detector system can include an optical imaging system having an anamorphic magnification gradient substantially transverse to a scan direction along the open substrate, the anamorphic magnification gradient can at least partially compensate for tangential velocity differences substantially perpendicular to the scan direction. The detection can include reading two or more regions on the open substrate at two or more different scan velocities, respectively, to at least partially compensate for tangential velocity differences in the two or more regions. The detection can further include using an immersion objective lens in optical communication with the detector system and the open substrate to detect at least one signal (e.g., as described herein). The immersion objective lens can be in contact with a fluid that is in contact with the open substrate. The fluid can be in a container, and an electric field can be used to adjust the hydrophobicity of one or more surfaces of the container to retain at least a portion of the fluid in contact with the immersion objective lens and the open substrate.

The delivery and/or dispersion process may be performed in a first environment having a first operating condition, and the detection process may be performed in a second environment having a second operating condition different from the first operating condition. The first and second environments may be located at the same physical location. For example, the delivery and/or dispersion process may be performed under a first set of conditions while the open substrate is held at the first physical location, and the detection process may be performed under a second set of conditions while the open substrate is held at the same physical location. Alternatively, the first environment may include a first physical location at which the open substrate is accessible to a rotation unit configured to rotate the open substrate during the delivery and/or dispersion process. The second environment may include a second physical location at which the open substrate is accessible to the detector system. As described above, one or more components of the detector system and/or the open substrate may be moving during the detection process. The second physical location may include a mechanism for supporting the open substrate while holding it stationary, as well as a mechanism (e.g., a rotation unit) for moving the detector system or components thereof relative to the open substrate (e.g., as described herein). The first and second environments may be physically close to each other. In one example, the first environment may be located at a first physical location of the device that is located above a second physical location of the device that is part of the second environment. In another example, the first environment may be located at a first physical location of the device that is located adjacent or somewhat adjacent to a second physical location of the device that is part of the second environment. The first and second environments may be separable by one or more barriers. In one example, a retractable barrier, such as a sliding door, separates the first and second environments. The retractable barrier may remain closed during the sending and/or dispersion process and then retract to allow translation of the open substrate from the first environment to the second environment for subsequent detection. The retractable barrier may be held in a closed state during the detection process. The open substrate may be held in a container that is transported with the open substrate between the first and second environments.

The first and second environments can include one or more different operating conditions. For example, the first environment can include a first temperature, humidity, and pressure, and the second environment can include a second temperature, humidity, and pressure, where at least one of the temperature, humidity, and pressure differs between the first and second environments. A given environment can include multiple temperature, humidity, and/or pressure zones, where one or more such zones can differ in the first and second environments.

The present disclosure also provides an apparatus and computer readable medium for carrying out the methods provided herein. For example, the present disclosure provides a computer readable medium including stored persistent instructions that, when executed, cause one or more computer processors to carry out the methods provided herein. The present disclosure also provides an apparatus for analyte detection or analysis, including a housing configured to receive an open substrate (e.g., as described herein) having an array of analytes immobilized thereon. The apparatus may include one or more dispensers configured to dispense a solution having a plurality of probes to a region proximate a central axis of the open substrate. The apparatus may also include a rotation unit configured to rotate the open substrate about the central axis to distribute the solution across the open substrate by at least centrifugal force, such that at least one probe of the plurality of probes binds to at least one immobilized analyte of the immobilized analytes to form a bound probe. The rotation unit may be disposed in a first region of the apparatus, the first region being different from a second region of the apparatus. The apparatus may also include a detector system, the detector system may include a sensor (e.g., a line scan camera) and an illumination source (e.g., as described herein). The detector system may be disposed in the second region of the apparatus. Alternatively, the detector system may be located in a first region of the device. The open substrate may include a first region and a second region, the first region and the second region including a subset of the array of immobilized analytes, at different radial positions of the open substrate relative to the central axis, and spatially resolved by the detector system. The binding probes may be located in the first region of the open substrate, and the detector system may be programmed to perform a non-linear scan of the open substrate and detect at least one signal from the binding probes in the first region of the open substrate. The device may include one or more processors configured, for example, to direct the detector system to direct the dispersion and delivery of one or more solutions to the open substrate or to detect one or more signals from the open substrate. The processor may be programmed to direct the detector system to compensate for velocity differences at different radial positions of the array relative to the central axis of the open substrate within the scan region. For example, the processor may be programmed to direct the detector system to scan two or more regions of the open substrate with two or more different scan velocities, respectively, to at least partially compensate for tangential velocity differences in the two or more regions. The apparatus may further include one or more optical systems, such as, for example, one or more optical systems configured to generate an anamorphic magnification gradient substantially transverse to the scan direction along the open substrate (e.g., as described herein). The processor may be programmed to adjust the gradient to compensate for different imaged radial positions relative to the central axis of the open substrate.

System Architectures for High Throughput Processing The nucleic acid sequencing systems and optical systems described herein (or any elements thereof) can be combined in a variety of architectures.

FIG. 23A shows the architecture of a system 1200a with a stationary substrate and moving fluid and optical elements. The system 1200a can include a substrate 310 as described herein. The substrate can be configured to rotate as described herein. The substrate can be glued or otherwise secured to a chuck (not shown in FIG. 23A) as described herein. The system can further include a fluid channel 330 and a fluid outlet port 335 as described herein, and/or any other fluid channel and fluid outlet port as described herein. The fluid channel and fluid outlet port can be configured to dispense any solution as described herein. The fluid channel and fluid outlet port can be configured to move 1215a relative to the substrate. For example, the fluid channel and fluid outlet port can be configured to move to a position above (e.g., near the center of) the substrate during periods when the fluid channel and fluid outlet port are dispensing a solution. The fluid channel and fluid outlet port can be configured to move to a position away from the substrate during periods when the fluid channel and fluid outlet port are not dispensing a solution. Alternatively, the reverse can apply. The system may further include an optical imaging objective 1110 as described herein. The optical imaging objective may be configured to move 1210a relative to the substrate. For example, the optical imaging objective may be configured to move to a position above the substrate (e.g., near the center of the substrate) during periods when the substrate is being imaged. The optical imaging objective may be configured to move to a position away from the substrate during periods when the substrate is not being imaged. This system may alternate between dispensing solutions and imaging, allowing for rapid sequencing of nucleic acids attached to a substrate using the systems and methods described herein.

FIG. 23B shows the architecture of system 1200b including a moving substrate and stationary fluidic and optical elements. System 1200b can include substrate 310 as described herein. The substrate can be configured to rotate as described herein. The substrate can be glued or otherwise secured to a chuck (not shown in FIG. 23B) as described herein. The system can further include fluidic channels 330 and fluidic outlet ports 335 as described herein, or any other fluidic channels and fluidic outlet ports as described herein. The fluidic channels and fluidic outlet ports can be configured to dispense any solution as described herein. The system can further include optical imaging objective 1110 as described herein. The fluidic channels, fluidic outlet ports, and optical imaging objective can be stationary. The substrate can be configured to move 1210b relative to the fluidic channels, fluidic outlet ports, and optical imaging objective. For example, the substrate can be configured to move to a position such that the fluidic channels and fluidic outlet ports are above (e.g., near the center of) the substrate during periods when the fluidic channels and fluidic outlet ports are dispensing solutions. The substrate may be configured to move away from the fluidic channels and fluidic outlet ports during periods when the fluidic channels and fluidic outlet ports are not dispensing solutions. The substrate may be configured to radially scan the objective lens over the substrate during periods when the substrate is being imaged. The substrate may be configured to move away from the optical imaging objective lens during periods when the substrate is not being imaged. This system can alternate between dispensing solutions and imaging, allowing for rapid sequencing of nucleic acids attached to the substrate using the systems and methods described herein.

23C shows the architecture of system 1200c including multiple stationary substrates and moving fluidic and optical elements. System 1200c may include first and second substrates 310a and 310b. The first and second substrates may be similar to substrate 310 described herein. The first and second substrates may be configured to rotate as described herein. The first and second substrates may be glued or otherwise secured to first and second chucks (not shown in FIG. 23C) as described herein. The system may further include first fluid channel 330a and first fluid outlet port 335a. The first fluid channel 330a may be similar to fluid channel 330 described herein or any other fluid channel described herein. The first fluid outlet port 335a may be similar to fluid outlet port 335 described herein or any other fluid outlet port described herein. The system may further include second fluid channel 330b and second fluid outlet port 335b. The second fluid channel 330b may be similar to the fluid channel 330 described herein or any other fluid channel described herein. The second fluid outlet port 335a may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The first fluid channel and the first fluid outlet port may be configured to dispense any solution described herein. The second fluid channel and the second fluid outlet port may be configured to dispense any solution described herein.

The system may further include an optical imaging objective 1110. The optical imaging objective 1110 may be configured to move 1210c relative to the first and second substrates. For example, the optical imaging objective may be configured to move to a position above the first substrate (e.g., near the center or scanning in a radial direction) during a period when the first fluid channel and the first fluid outlet port are not dispensing solution to the second substrate (during which the first substrate is imaged). The optical imaging objective may be configured to move to a position away from the first substrate during a period when the first fluid channel and the first fluid outlet port are dispensing solution. The optical imaging objective may be configured to move to a position above the second substrate (e.g., near the center or scanning in a radial direction) during a period when the second fluid channel and the second fluid outlet port are not dispensing solution to the second substrate (during which the second substrate is imaged). The optical imaging objective may be configured to move to a position away from the second substrate during the period when the second fluid channel and the second fluid outlet port are dispensing the solution.

The timing of dispensing the solution and imaging the substrate may be synchronized. For example, the solution may be dispensed onto a first substrate while the second substrate is being imaged. Once the solution is dispensed onto the first substrate and the second substrate is imaged, the optical imaging objective may be moved from the second substrate to the first substrate. The solution may then be dispensed onto the second substrate while the first substrate is being imaged. Repeating this alternating pattern of dispensing and imaging allows for rapid sequencing of the nucleic acids bound to the first and second substrates using the systems and methods described herein. The alternating pattern of dispensing and imaging may speed up sequencing by increasing the duty cycle of the imaging process or the solution dispensing process.

23C as including two substrates, two fluid channels, two fluid outlet ports, and one optical imaging objective, system 1200c can include any number of each of substrates, fluid channels, fluid outlet ports, and optical imaging objectives. For example, the system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten substrates. Each substrate can be bonded or otherwise secured to a chuck as described herein. The system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid channels and/or at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid outlet ports. Each fluid channel and fluid outlet port may be configured to dispense a solution as described herein. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten optical imaging objectives. Each optical imaging objective may be moved between the substrates as described herein.

FIG. 23D illustrates the architecture of system 1200d, which includes multiple moving substrates on a rotation stage and stationary fluid and optical elements. System 1200d may include first and second substrates 310a and 310b. The first and second substrates may be similar to substrate 310 described herein. The first and second substrates may be configured to rotate as described herein. The first and second substrates may be glued or otherwise secured to first and second chucks (not shown in FIG. 23D) as described herein. The first and second substrates may be secured to rotation stage 1220d (e.g., approximately opposite ends of the rotation stage). The rotation stage may be configured to rotate about an axis. The axis may be an axis that passes through the center of the substrate. The axis may be an off-center axis. The rotation stage may scan approximately the radius of substrate 310b. The system may further include fluidic channels 330 and fluidic outlet ports 335. The fluidic channels and fluidic outlet ports may be configured to dispense any of the solutions described herein. The system may further include an optical imaging objective 1110. The longitudinal axis of the imaging objective 1110 may not coincide with the central axis of the second substrate 310b (although this is difficult to distinguish in FIG. 23D). The imaging objective 1110 may be located at a distance from the center of the second substrate 310b.

The rotation stage may be configured to change the relative positions of the first and second substrates to perform different sequencing operations. For example, the rotation stage may be configured to rotate such that the optical imaging objective is above or in optical communication with the first substrate during periods when the fluidic channels and fluidic outlet ports are not dispensing solution to the first substrate (and the first substrate is to be imaged). The rotation stage may be configured to rotate such that the optical imaging objective is away from the first substrate during periods when the fluidic channels and fluidic outlet ports are dispensing solution to the first substrate. The rotation stage may be configured to rotate such that the optical imaging objective is above or in optical communication with the second substrate during periods when the fluidic channels and fluidic outlet ports are not dispensing solution to the second substrate (and the second substrate is to be imaged). The rotation stage may be configured to rotate such that the optical imaging objective is away from the second substrate during periods when the fluidic channels and fluidic outlet ports are dispensing solution to the second substrate.

The timing of dispensing the solution and imaging the substrate may be synchronized. For example, the solution may be dispensed onto a first substrate while the second substrate is being imaged. Once the solution is dispensed onto the first substrate and the second substrate is imaged, the rotation stage may be rotated such that the solution may be dispensed onto the second substrate while the first substrate is being imaged. Repeating this alternating pattern of dispensing and imaging allows for rapid sequencing of the nucleic acids bound to the first and second substrates using the systems and methods described herein. The alternating pattern of dispensing and imaging may speed up sequencing by increasing the duty cycle of the imaging process or the solution dispensing process.

Although shown in FIG. 23D as including two substrates, one fluid channel, one fluid outlet port, and one optical imaging objective, system 1200d can include any number of each of substrates, fluid channels, fluid outlet ports, and optical imaging objectives. For example, the system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten substrates. Each substrate may be glued or otherwise secured to a chuck as described herein. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid channels, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid outlet ports. Each fluid channel and fluid outlet port may be configured to dispense a solution as described herein. The system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten optical imaging objectives. The rotation stage can be rotated to position any substrate under any fluid channel, fluid outlet port, or optical imaging objective at any time.

FIG. 23E illustrates the architecture of system 1200e, which includes multiple stationary substrates and moving optics. System 1200d may include first and second substrates 310a and 310b. The first and second substrates may be similar to substrate 310 described herein. The first and second substrates may be configured to rotate as described herein. The first and second substrates may be glued or otherwise secured to first and second chucks (not shown in FIG. 23E) as described herein. The system may further include a first fluid channel 330a and a first fluid outlet port 335a. The first fluid channel 330a may be similar to fluid channel 330 described herein or any other fluid channel described herein. The first fluid outlet port 335a may be similar to fluid outlet port 335 described herein or any other fluid outlet port described herein. The first fluid channel and the first fluid outlet port may be configured to dispense any solution described herein. The system may further include a second fluid channel 330b and a second fluid outlet port 335b. The second fluid channel 330b may be similar to the fluid channel 330 described herein or any other fluid channel described herein. The second fluid outlet port 335b may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The second fluid channel and the second fluid outlet port may be configured to dispense any solution described herein.

The system may further include an optical imaging objective 1110. The optical imaging objective may be attached to an imaging arm 1230e. The optical imaging objective may be configured to move 1220e along the optical imaging arm to image the entire area of the first or second substrate. The optical imaging arm may be configured to rotate 1210e. The optical imaging arm may be configured to rotate such that the optical imaging objective is in a position above or in optical communication with the first substrate during a period when the first fluid channel and the first fluid outlet port are not dispensing solution to the first substrate (and the first substrate should be imaged). The optical imaging arm may be configured to rotate such that the optical imaging objective is away from the first substrate during a period when the first fluid channel and the first fluid outlet port are dispensing solution to the first substrate. The optical imaging arm may be configured to rotate such that the optical imaging objective is above or in optical communication with the second substrate during periods when the second fluid channel and the second fluid outlet port are not dispensing solution to the second substrate (and the second substrate is to be imaged). The optical imaging arm may be configured to rotate such that the optical imaging objective is away from the second substrate during periods when the second fluid channel and the second fluid outlet port are dispensing solution to the second substrate.

The timing of dispensing the solution and imaging the substrate may be synchronized. For example, the solution may be dispensed onto a first substrate while the second substrate is being imaged. Once the solution is dispensed onto the first substrate and the second substrate is imaged, the optical imaging arm may be rotated such that the solution may be dispensed onto the second substrate while the first substrate is being imaged. Repeating this alternating pattern of dispensing and imaging allows for rapid sequencing of the nucleic acids bound to the first and second substrates using the systems and methods described herein. The alternating pattern of dispensing and imaging may speed up sequencing by increasing the duty cycle of the imaging process or the solution dispensing process.

23E as including two substrates, two fluid channels, two fluid outlet ports, and one optical imaging objective, system 1200e may include any number of each of substrates, fluid channels, fluid outlet ports, and optical imaging objectives. For example, the system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten substrates. Each substrate may be bonded or otherwise secured to a chuck as described herein. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid channels, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid outlet ports. Each fluid channel and fluid outlet port may be configured to dispense a solution as described herein. The system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten optical imaging objectives. The optical imaging arm can be rotated to position any substrate under any fluid channel, fluid exit port, or optical imaging objective at any time.

FIG. 23F illustrates the architecture of system 1200f, including multiple moving substrates and stationary fluidic and optical elements. System 1200f may include first and second substrates 310a and 310b. The first and second substrates may be similar to substrate 310 described herein. The first and second substrates may be configured to rotate as described herein. The first and second substrates may be glued or otherwise secured to first and second chucks (not shown in FIG. 23F) as described herein. The first and second substrates may be affixed to opposing ends of motion stage 1220f. The motion stage may be configured to move 1210f. The system may further include first fluidic channel 330a and first fluidic outlet port 335a. First fluidic channel 330a may be similar to fluidic channel 330 described herein or any other fluidic channel described herein. The first fluid outlet port 335a may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The first fluid channel and the first fluid outlet port may be configured to dispense any solution described herein. The system may further include a second fluid channel 330b and a second fluid outlet port 335b. The second fluid channel 330b may be similar to the fluid channel 330 described herein or any other fluid channel described herein. The second fluid outlet port 335b may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The second fluid channel and the second fluid outlet port may be configured to dispense any solution described herein. The system may further include an optical imaging objective 1110.

The translation stage may be configured to move the optical imaging objective lens to a position above or in optical communication with the first substrate during a period when the first fluid channel and the first fluid outlet port are not dispensing solution to the first substrate (and the first substrate is to be imaged). The translation stage may be configured to move the optical imaging objective lens away from the first substrate during a period when the first fluid channel and the first fluid outlet port are dispensing solution to the first substrate. The translation stage may be configured to move the optical imaging objective lens to a position above or in optical communication with the second substrate during a period when the second fluid channel and the second fluid outlet port are not dispensing solution to the second substrate (and the second substrate is to be imaged). The translation stage may be configured to move the optical imaging objective lens away from the second substrate during a period when the second fluid channel and the second fluid outlet port are dispensing solution to the second substrate.

The timing of dispensing the solution and imaging the substrate may be synchronized. For example, the solution may be dispensed onto a first substrate while the second substrate is being imaged. Once the solution is dispensed onto the first substrate and the second substrate is imaged, the motion stage may move such that the solution may be dispensed onto the second substrate while the first substrate is being imaged. Repeating this alternating pattern of dispensing and imaging allows for rapid sequencing of the nucleic acids bound to the first and second substrates using the systems and methods described herein. The alternating pattern of dispensing and imaging may speed up sequencing by increasing the duty cycle of the imaging process or the solution dispensing process.

Although shown in FIG. 23F as including two substrates, two fluid channels, two fluid outlet ports, and one optical imaging objective, system 1200f can include any number of each of substrates, fluid channels, fluid outlet ports, and optical imaging objectives. For example, the system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten substrates. Each substrate may be glued or otherwise secured to a chuck as described herein. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid channels, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid outlet ports. Each fluid channel and fluid outlet port may be configured to dispense a solution as described herein. The system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten optical imaging objectives. The motion stage can move to position any substrate under any fluid channel, fluid outlet port, or optical imaging objective at any time.

FIG. 23G shows the architecture of a system 2300g including multiple moving substrates and stationary fluidic and optical elements. The system 2300g may include first and second substrates 310a and 310b. The first and second substrates may be similar to the substrate 310 described herein. The first and second substrates may be configured to rotate as described herein. The first and second substrates may be glued or otherwise secured to first and second chucks (not shown in FIG. 23G) as described herein. The first and second substrates may be configured to translate along a fixed stage 2320g. The first and second substrates may be configured to move between a first fluid station including a first fluid channel 330a and a first fluid outlet port 335a, a second fluid station including a second fluid channel 330b and a second fluid outlet port 335b, and an imaging station including an optical imaging objective 1110. 23G shows a configuration in which the first substrate is placed at the first fluid station and the second substrate is placed at the imaging station. In another configuration, the first and second substrates can be translated relative to the optical head such that the first substrate is placed at the imaging station and the second substrate is placed at the second fluid station. The first and second translating substrates may be configured to move such that the optical imaging objective is in a position above or in optical communication with the first substrate during periods when the first fluid channel and the first fluid outlet port are not dispensing solution to the first substrate (and the first substrate should be imaged). The first and second translating substrates may be configured to move such that the optical imaging objective is away from the first substrate during periods when the first fluid channel and the first fluid outlet port are dispensing solution to the first substrate. The first and second translation substrates may be configured to move such that the optical imaging objective is above or in optical communication with the second substrate during periods when the second fluid channel and second fluid outlet port are not dispensing solution to the second substrate (and during which the second substrate is to be imaged). The first and second translation substrates may be configured to move such that the optical imaging objective is away from the second substrate during periods when the second fluid channel and second fluid outlet port are dispensing solution to the second substrate.

The timing of dispensing the solution and imaging the substrate may be synchronized. For example, the solution may be dispensed onto a first substrate while the second substrate is being imaged. Once the solution is dispensed onto the first substrate and the second substrate is imaged, the motion stage may move such that the solution may be dispensed onto the second substrate while the first substrate is being imaged. Repeating this alternating pattern of dispensing and imaging allows for rapid sequencing of the nucleic acids bound to the first and second substrates using the systems and methods described herein. The alternating pattern of dispensing and imaging may speed up sequencing by increasing the duty cycle of the imaging process or the solution dispensing process.

Although shown in FIG. 23G as including two substrates, two fluid channels, two fluid outlet ports, and one optical imaging objective, system 2300g can include any number of each of substrates, fluid channels, fluid outlet ports, and optical imaging objectives. For example, the system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten substrates. Each substrate may be glued or otherwise secured to a chuck as described herein. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid channels, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid outlet ports. Each fluid channel and fluid outlet port may be configured to dispense a solution as described herein. The system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten optical imaging objectives. The translating substrate can be moved to position any substrate under any fluid channel, fluid outlet port, or optical imaging objective at any time.

Figure 23H shows the architecture of system 1200g with multiple substrates moved between multiple processing bays. System 1200g may include first, second, third and fourth substrates 310a, 310b, 310c, 310d and 310e, respectively. The first, second, third, fourth and fifth substrates may be similar to substrate 310 described herein. The first, second, third, fourth and fifth substrates may be configured to rotate as described herein. The first, second, third, fourth and fifth substrates may be glued or otherwise secured to first, second, third, fourth and fifth chucks (not shown in Figure 23H), respectively, as described herein.

The system may further include a first fluid channel 330a and a first fluid outlet port 335a. The first fluid channel 330a may be similar to the fluid channel 330 described herein or any other fluid channel described herein. The first fluid outlet port 335a may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The first fluid channel and the first fluid outlet port may be configured to dispense any solution described herein. The first fluid channel and the first fluid outlet port may be considered a first processing bay. The first processing bay may be configured to perform a first processing step, such as dispensing a first solution to any of the first, second, third, fourth, or fifth substrates.

The system may further include a second fluid channel 330b and a second fluid outlet port 335b. The second fluid channel 330b may be similar to the fluid channel 330 described herein or any other fluid channel described herein. The second fluid outlet port 335b may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The second fluid channel and the second fluid outlet port may be configured to dispense any solution described herein. The second fluid channel and the second fluid outlet port may be considered a second processing bay or processing station. The second processing bay may be configured to perform a second processing step, such as dispensing a second solution to any of the first, second, third, fourth, or fifth substrates.

The system may further include a third fluid channel 330c and a third fluid outlet port 335c. The third fluid channel 330c may be similar to the fluid channel 330 described herein or any other fluid channel described herein. The third fluid outlet port 335c may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The third fluid channel and the third fluid outlet port may be configured to dispense any solution described herein. The third fluid channel and the third fluid outlet port may be considered a third processing bay or processing station. The third processing bay may be configured to perform a third processing step, such as dispensing a third solution to any of the first, second, third, fourth, or fifth substrates.

The system may further include a fourth fluid channel 330d and a fourth fluid outlet port 335d. The fourth fluid channel 330d may be similar to the fluid channel 330 described herein or any other fluid channel described herein. The fourth fluid outlet port 335d may be similar to the fluid outlet port 335 described herein or any other fluid outlet port described herein. The fourth fluid channel and the fourth fluid outlet port may be configured to dispense any solution described herein. The fourth fluid channel and the fourth fluid outlet port may be considered a fourth processing bay or processing station. The fourth processing bay may be configured to perform a fourth processing step, such as dispensing a fourth solution to any of the first, second, third, fourth, or fifth substrates.

The system may further include a scanning optical imaging objective 1110. The optical imaging objective may be considered a fifth processing bay or station.

The system may further include a transfer arm 1220g. The transfer arm may be configured to move laterally 1210g or rotate 1215g. The transfer arm may be configured to move any of the first, second, third, fourth, or fifth substrates between different processing stations (e.g., by picking up the substrates and moving them to a new location). For example, at a first time point, the first substrate may undergo a first step (e.g., dispensing a first solution) in the first processing bay, the second substrate may undergo a second step (e.g., dispensing a second solution) in the second processing bay, the third substrate may undergo a third step (e.g., dispensing a third solution) in the first processing bay, the fourth substrate may undergo a fourth step (e.g., dispensing a fourth solution) in the fourth processing bay, and the fifth substrate may be imaged in the fifth processing bay. Once one or more of the first, second, third, or fourth steps or imaging is completed, the movement arm can move one or more of the first, second, third, fourth, or fifth substrates to one or more of the first, second, third, fourth, or fifth processing bays to complete another step. The pattern of completing one or more operations and moving one or more substrates to another processing bay to complete another operation can be repeated, allowing rapid sequencing of nucleic acids bound to the first, second, third, fourth, and fifth substrates using the systems and methods described herein. The alternating pattern of dispensing and imaging can speed up sequencing by increasing the duty cycle of the imaging process or solution dispensing process.

23H shows five substrates, four fluid channels, four fluid outlet ports, and one optical imaging objective, system 1200g may include any number of each of substrates, fluid channels, fluid outlet ports, and optical imaging objectives. For example, system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten substrates. Each substrate may be bonded or otherwise secured to a chuck as described herein. System may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid channels and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fluid outlet ports. Each fluid channel and fluid outlet port may be configured to dispense a solution as described herein. The system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten optical imaging objectives. The movement arm can move to position any substrate under any fluid channel, fluid outlet port, or optical imaging objective at any time.

23I shows the architecture of a system 1200h including multiple imaging heads that share translation and rotation axes and scan independently rotating fields of view. The system may include first and second read heads 1005 and 1015, respectively, configured to image the substrate 310. The first and second read heads may be similar to any of the read heads described herein (e.g., with respect to FIG. 14). At a particular time, the first and second read heads may be configured to image first and second paths 1010, 1020, respectively. The first and second paths may be similar to any of the paths described herein (e.g., with respect to FIG. 14). The first and second read heads may be configured to move substantially radially 1210h over the spinning substrate, thereby scanning the substrate. If either the first or second read heads do not move exactly radially, the image field or sensor of the read heads may be rotated to maintain a substantially tangential scanning direction, as described with respect to FIG. 34. Field rotation may be achieved using a rotating prism. Alternatively or additionally, mirrors or other optical elements may be used.

23I as including two read heads and two imaging paths, system 1200h may include any number of read heads or imaging paths. For example, the system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten read heads. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten imaging paths.

23J shows the architecture of a system 2300j with multiple scanning imaging heads sharing translation and rotation axes and having independently rotating fields. The system may include first, second, third, and fourth read heads 1005, 1015, 1025, and 1035, respectively, configured to image the substrate 310. The first, second, third, and fourth read heads may be similar to any read heads described herein (such as with respect to FIG. 14). At a particular time, the first, second, third, and fourth read heads may be configured to image first, second, third, and fourth paths 1010, 1020, 1030, and 1040, respectively. The first, second, third, and fourth paths may be similar to any paths described herein (such as with respect to FIG. 14). The first, second, third, and fourth read heads may be configured to move 1210h in a substantially radial direction over the spinning substrate, thereby scanning the substrate. If the first, second, third, and fourth readheads do not move precisely radially, the image fields or sensors of the readheads can be rotated to maintain a substantially tangential scanning direction. Field rotation can be accomplished using a rotating prism. Alternatively or additionally, mirrors or other optical elements may be used.

23J as having four read heads and four imaging paths, system 2300j can have any number of read heads or imaging paths. For example, the system can have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten read heads. The system can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten imaging paths.

23K shows the architecture of system 1200i including multiple spindles scanning with a shared optical detection system. The system may include first and second substrates 310a and 310b, respectively. The first and second substrates may be similar to substrate 310 described herein. The first and second substrates may be mounted on first and second spindles, respectively. The first and second spindles may impart rotational motion to the first and second substrates, respectively. The system may include first and second optical imaging objectives 1110a and 1110b, respectively. The first and second optical imaging objectives may be similar to optical imaging objective 1110 described herein. The first and second optical imaging objectives may be configured to collect light from the first and second substrates, respectively. The first and second optical imaging objectives may send the light collected from the first and second substrates, respectively, to first and second mirrors 1280a and 1280b, respectively. In some cases, only one of the first and second optical imaging objective lenses collects light at a particular moment.

The first and second mirrors may pass light to a shared movable mirror. In a first configuration 1285a, the shared movable mirror may direct light from the first substrate to a beam splitter 1295. The beam splitter may comprise a dichroic mirror. For example, as shown in FIG. 23K, the beam splitter may be configured to reflect excitation light from the excitation light source 1290 toward the substrate and transmit light from the substrate toward the detector 370. In an alternative configuration (not shown in FIG. 23K), the beam splitter may be configured to transmit excitation light from the excitation light source 1290 toward the substrate and reflect light from the substrate toward the detector 370. The beam splitter may pass or reflect light to the detector 370, allowing the first substrate to be imaged. The first substrate may be configured to be translated 1210i, allowing different positions on the first substrate to be imaged.

For the second configuration 1285b, the shared movable mirror may direct light from the second substrate to a beam splitter 1295. The beam splitter may pass or reflect the light to a detector 370, allowing the second substrate to be imaged. The second substrate may be configured to be translated 1210i, allowing different locations on the second substrate to be imaged. In this manner, by moving the movable mirror, the first and second substrates can be imaged with a common optical system.

The system may further include an excitation light source 1290. The light source may be configured to provide excitation light (e.g., for fluorescence imaging) to the first or second substrate. The excitation light may be selectively delivered to the first or second substrate using a moveable mirror in a manner similar to the detection described herein.

23K as including two substrates, two imaging optical objectives, and two mirrors, system 1200i may include any number of substrates, imaging optical objectives, or mirrors. For example, the system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten substrates. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten imaging optical objectives. The system may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mirrors.

Figure 23I shows the architecture of a system with multiple imaging heads that share translation and rotation axes and scan independently rotating fields of view.

Figure 23K shows the architecture of a system that includes multiple spindles scanning with a shared optical detection system.

24 shows the architecture of a system 1300 with multiple rotating spindles. The system 1300 can include a substrate 310 as described herein. The substrate can be configured to rotate as described herein. The system can further include a fluid channel 330 and a fluid outlet port 335 as described herein, or any other fluid channel and fluid outlet port as described herein. The fluid channel and fluid outlet port can be configured to dispense any solution as described herein. The fluid channel and fluid outlet port can be configured to move 1315a relative to the substrate. For example, the fluid channel and fluid outlet port can be configured to move to a position above (e.g., near the center of) the substrate during periods when the fluid channel and fluid outlet port are dispensing a solution. The fluid channel and fluid outlet port can be configured to move to a position away from the substrate during periods when the fluid channel and fluid outlet port are not dispensing a solution. The system can further include an optical imaging objective 1110 as described herein. The optical imaging objective can be configured to move 1310a relative to the substrate. For example, the optical imaging objective lens may be configured to move to a position above the substrate (e.g., near the center or scanning radially) during periods when the substrate is being imaged. The optical imaging objective lens may be configured to move to a position away from the substrate during periods when the substrate is not being imaged.

The system may further include a first spindle 1305a and a second spindle 1305b. The first spindle may be internal to the second spindle. The first spindle may be external to the second spindle. The second spindle may be internal to the first spindle. The second spindle may be external to the first spindle. The first and second spindles may each be configured to rotate independently of each other. The first and second spindles may be configured to rotate at different angular velocities. For example, the first spindle may be configured to rotate at a first angular velocity and the second spindle may be configured to rotate at a second angular velocity. The first angular velocity may be less than the second angular velocity. The first spindle may be configured to rotate at a relatively low angular velocity (e.g., an angular velocity of about 0 rpm to about 100 rpm) during the period when the solution is dispensed onto the substrate. The second spindle may be configured to rotate at a relatively high angular velocity (such as an angular velocity of about 100 rpm to about 1,000 rpm) during the period in which the substrate is being imaged. Alternatively, the reverse may apply. The substrate may be transferred between the first spindle and the second spindle to complete each of the dispense and imaging operations.

The system may include any number of spindles. For example, the system may include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more spindles. Alternatively or additionally, the system may include up to about 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 spindle. A given spindle may be inboard or outboard relative to one or more other spindles in the system. In some cases, each of the spindles may rotate independently of one another. In some cases, at least a subset of the spindles may rotate independently of one another. In some cases, at least a subset of the spindles may rotate dependently on one another (e.g., at the same time and at the same angular velocity). The spindles may rotate about the same axis or different axes. In some cases, each spindle may rotate at a different angular velocity. In some cases, at least a subset of the spindles may rotate at a different angular velocity.

Although shown in FIG. 24 as utilizing a moving fluid channel and optical imaging objective, system 1300 may be configured in other ways as described herein. For example, the system may be configured such that the fluid channel and optical imaging objective are stationary and the substrate is configured to move. The system may be configured in any other way as described herein.

Nucleic Acid Amplification and Sequencing Applications The methods and systems herein may be applied to a variety of sequencing and application techniques and methods. The open substrate system, solution dispensing method, rotating array system, substrate system, substrate preparation method, optical system, scanning system, or scanning method, or any combination thereof, disclosed herein may be applied to a variety of sequencing methods, including, for example, non-terminal sequencing, reversible terminator sequencing, rolling circle amplification sequencing, DNA nanoball sequencing, and massively parallel sequencing. The substrates disclosed herein may include one or more sequencing components suitable for binding or amplifying nucleic acids (e.g., adapters, primers, beads, antibodies, DNA nanoballs, nucleic acid templates, polymerases, nucleotides, fluorescent nucleotides, terminating nucleotides, or reversibly terminating nucleotides). The sequencing components may be immobilized on the substrate. In some examples, the sequencing components may be patterned on the substrate. In some examples, the sequencing components may be immobilized on the substrate without patterning. The substrate may include a pattern including discrete regions distinguished by surface chemistry. For example, a substrate may include a pattern that includes one or more regions that recruit one or more sequencing components and one or more regions that exclude one or more sequencing components. In some instances, a first sequencing component may be recruited to the substrate and a second sequencing component may be recruited to the first sequencing component. Additional sequencing components may be recruited, thereby sequencing the nucleic acid.

The solution dispensing methods disclosed herein can be used to dispense one or more sequencing components onto a substrate. For example, the sequencing components can include a sample, a processed sample, a support or particles (e.g., beads, etc.), amplification and/or sequencing reagents (e.g., wash solutions, buffers, primers, enzymes, catalysts, quenchers, nucleotides or analogs thereof, dyes, probes, tags, labels, etc.), fluidic components (e.g., surfactants, buffers, etc.), and/or optical components (e.g., reference beads, dyes, etc.). For example, a solution containing the sequencing components can be dispensed onto the substrate in a spiral pattern. In some examples, a solution containing the sequencing components can be dispensed in a circular, elliptical, linear, or non-linear path. The sequencing components can be dispensed onto a rotating substrate. As disclosed herein, the sequencing components can be dispensed onto the substrate in a pattern to ensure consistent reaction times in each region of the substrate contacted with the sequencing components. Alternatively, the sequencing components can be dispensed in any manner, including random or semi-random dispensing. Substrates containing sequencing components can be scanned using the scanning systems disclosed herein. Substrates containing sequencing components, such as fluorescent components (e.g., fluorescent nucleotides or fluorescent antibodies), can be imaged using the optical systems disclosed herein. Substrates containing sequencing components can be scanned while the substrate is rotating. In some examples, the substrate can be scanned using an optical system that includes one or more objective lenses. The one or more objective lenses can be configured as disclosed herein to enable efficient scanning of the substrate.

Reversible Terminator Sequencing The systems and methods disclosed herein may be compatible with reversible terminator sequencing methods. In some examples, the reversible terminator sequencing methods may include attaching a plurality of adaptors to a substrate. The adaptors may be bound to DNA templates or DNA template fragments. The adaptors may be fixed to a patterned substrate. The adaptors may be fixed to a substrate without patterning. The patterned substrate may include one or more regions that recruit adaptors and one or more regions that exclude adaptors. The adaptors may be delivered to a substrate with or without a DNA template or DNA template fragment. For example, an adaptor that includes a DNA template or fragment thereof may be delivered to a patterned substrate or a substrate lacking a pattern. In another example, an adaptor that includes a DNA template or fragment thereof may be delivered to a patterned substrate or a substrate lacking a pattern. A solution including a DNA template or DNA template fragment may be dispensed onto a substrate including adaptors, and the DNA template or DNA template fragment may be attached to the adaptors. The solution containing the DNA template or DNA template fragments can be dispensed onto the substrate using any of the dispensing methods or patterns disclosed herein. For example, the solution containing the DNA template or DNA fragments can be dispensed locally onto a target area of the substrate. In another example, the solution containing the DNA template or DNA fragments can be dispensed locally and distributed broadly across the substrate (e.g., spin-coated). In another example, the solution containing the DNA template or DNA fragments can be dispensed onto the substrate in a pattern (e.g., a helical pattern, a circular pattern, an elliptical pattern, a linear pattern, or a non-linear pattern). The solution containing the DNA template or DNA fragments can be dispensed while the substrate is rotating.

In some examples, the reversible terminator sequencing method may include attaching a plurality of primers to a substrate. In some examples, the primers may be attached to an adaptor. The primers may bind to the DNA template or DNA template fragments. The primers may be immobilized on a patterned substrate. The primers may be immobilized on a substrate without patterning. The patterned substrate may include one or more regions that recruit primers and one or more regions that exclude primers. A solution containing the DNA template or DNA template fragments may be dispensed onto a substrate containing primers using any of the dispensing methods or patterns disclosed herein. The DNA template or DNA template fragments may be mobilized to the substrate (e.g., by binding to the primers or adaptors). The DNA template or DNA template fragments may be amplified on the substrate. In some examples, the DNA template or DNA template fragments may be dispensed onto the substrate such that the DNA binding rate to a region is slower than the amplification fold rate in that region. For example, the amplification fold rate of the DNA template or DNA template fragment can be about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times the rate at which the DNA template or DNA template fragment reaches a region of the substrate. This can ensure that the DNA template or DNA template fragment is sufficiently amplified before another DNA template or DNA template fragment reaches the same region.

A solution containing DNA molecules can be dispensed onto a substrate (e.g., any of the substrates disclosed herein or patterned substrates) with a seeding efficiency determined by the fraction of DNA molecules dispensed onto the substrate that adhere to the substrate. In some examples, the solution containing DNA molecules can be dispensed with a seeding efficiency of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, or about 25%. In some examples, the DNA molecules are about 10,000 DNA molecules/ ^mm2 , about 20,000 DNA molecules/ ^mm2 , about 30,000 DNA molecules/ ^mm2 , about 40,000 DNA molecules/mm2, about 50,000 DNA molecules/ ^mm2 , about 100,000 DNA molecules/ ^mm2 , about 200,000 DNA molecules/ ^mm2 , about 300,000 DNA molecules/ ^mm2 , about 400,000 DNA molecules/ ^mm2 , about 500,000 DNA molecules/ ^mm2 , about 1 million DNA molecules/ ^mm2 , about 2 million DNA molecules/ ^mm2 , about 3 million DNA molecules/ ^mm2 , about 4 million DNA molecules/ ^mm2 , about 5 million DNA molecules/ ^mm2 , about 6 ^million DNA molecules/ ^mm2 , about 7 million DNA molecules/ ^mm2 , about 8 million DNA molecules/ ^mm2 , or about 9 million DNA molecules/ ^mm2. , or a density of about 10 million DNA molecules/ ^mm2 on the substrate.

The DNA molecules attached to the substrate may be monoclonally amplified. In some cases, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the DNA molecules attached to the substrate may be amplified.

One or more DNA molecules (e.g., one or more DNA templates or one or more DNA template fragments) can be amplified. Amplification can occur while the DNA molecules are attached to the substrate. Amplification can occur while the DNA molecules are dispensed onto the substrate. The DNA molecules can be amplified using a variety of amplification means, including, but not limited to, polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), bridge amplification, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), or multiple displacement amplification (MDA). The DNA molecules can be amplified using a nucleic acid that includes a reversible terminator. In some examples, the nucleic acid can include a base, a cleavable linker covalently attached to the base, and a fluorescent molecule covalently attached to the base via the cleavable linker. In some examples, the nucleic acid can include a reversible terminating group (e.g., at the 3' hydroxyl group) covalently attached to the nucleic acid. The reversible terminator may include a 3'-O-azidomy reversible terminator, a 3'-ONH ₂ reversible terminator, a 3'-ONH ₂ reversible terminator or a 3'-OH non-blocking reversible terminator (eg, a virtual terminator or a Lightning terminator).

The amplified DNA molecules (e.g., including fluorescent molecules) can be imaged using the optical systems or scanning methods disclosed herein. In some cases, imaging can include imaging a plurality of optically resolvable spots. The spots can include DNA molecules (e.g., DNA molecules including fluorescent molecules). In some cases, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the spots that include DNA molecules include a single DNA species (e.g., are monoclonal).

Massively Parallel Sequencing The systems and methods disclosed herein may be compatible with massively parallel sequencing methods. One or more DNA molecules (e.g., one or more DNA templates or one or more DNA template fragments) may be amplified. Amplification may occur while the DNA molecules are attached to the substrate. Amplification may occur while the DNA molecules are dispensed onto the substrate. The DNA molecules may be amplified using a variety of amplification means, including, but not limited to, polymerase chain reaction (PCR), recombinase polymerase amplification (RCA), bridge amplification, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RPA), or multiple displacement amplification (MDA). In some examples, massively parallel sequencing may include labeling the amplified DNA molecules (e.g., DNA templates or DNA template fragments) with an antibody (e.g., a fluorescently labeled antibody). The antibody may bind to a terminal nucleotide. For example, the antibody may bind to any of the reversibly terminated nucleotides disclosed herein. The antibody may selectively bind to a terminal nucleotide sequence, such as a terminal A, a terminal T, a terminal C, or a terminal G. In some instances, the antibody may include multiple fluorescent molecules.

The DNA molecules may be dispensed onto a substrate using any of the methods or systems disclosed herein. The DNA molecules may be dispensed onto any of the substrates disclosed herein (e.g., a patterned substrate or a substrate lacking a pattern). The antibodies may be dispensed onto a substrate in any of the patterns disclosed herein (e.g., a helical pattern, a circular pattern, an elliptical pattern, a linear pattern, or a non-linear pattern). The antibodies may be dispensed onto a substrate using any of the methods or systems disclosed herein. The antibodies may be dispensed onto any of the substrates disclosed herein (e.g., a patterned substrate or a substrate lacking a pattern). The antibodies may be dispensed onto a substrate in any of the patterns disclosed herein (e.g., a helical pattern, a circular pattern, an elliptical pattern, a linear pattern, or a non-linear pattern).

The substrate containing the DNA molecules and the antibodies can be imaged using the optical system or scanning methods disclosed herein. In some cases, imaging can include imaging a plurality of optically resolvable spots. The spots can include DNA molecules. The spots can include antibodies (e.g., antibodies that include fluorescent molecules). In some cases, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the spots that include DNA molecules include a single fluorescent antibody.

DNA Nanoball Sequencing The systems and methods disclosed herein may be compatible with DNA nanoball sequencing methods. DNA nanoball sequencing methods may include amplifying a DNA template or DNA template fragment using rolling circle replication. A DNA template fragment (e.g., a fragment comprising about 100 base pairs to about 350 base pairs) may be ligated to an adapter sequence. The adapter sequence may be ligated to the fragment, thereby circularizing the fragment. The circular fragment may be amplified using rolling circle amplification. In some aspects, rolling circle amplification of the ligated fragments may generate single-stranded copies of the fragments. The amplified nucleic acid molecule comprising the ligated amplified fragments may be compacted into a DNA nanoball.

The DNA fragments may be dispensed onto a substrate using any of the methods or systems disclosed herein. The DNA fragments may be dispensed onto any of the substrates disclosed herein (e.g., a patterned substrate or a substrate lacking a pattern). The DNA nanoballs may be dispensed onto a substrate using any of the methods or systems disclosed herein. The DNA nanoballs may be dispensed onto any of the substrates disclosed herein (e.g., a patterned substrate or a substrate lacking a pattern). In some examples, the DNA fragments may be amplified while attached to the substrate.

A substrate containing DNA fragments or DNA nanoballs may be imaged using the optical system or scanning methods disclosed herein. In some cases, imaging may include imaging a plurality of optically resolvable spots. The spots may include DNA nanoballs. The spots may include DNA fragments. In some cases, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the spots containing DNA nanoballs or DNA fragments may include a single fluorescent species of DNA nanoball or DNA fragment.

The systems and methods provided herein may be applicable to any sequencing or amplification scheme, such as those described herein. For any sequencing scheme or amplification, one or more operations may be performed from the substrate and/or one or more operations may be performed on the substrate. For example, in a sequencing scheme, amplification may be performed from the substrate and the amplification products (e.g., attached to a support) are then deposited on the substrate for sequencing. For example, in an amplification scheme, library preparation may be performed from the substrate and a library of template nucleic acid molecules may be deposited on the substrate for amplification. In another example, both amplification and subsequent sequencing are performed on the substrate. As described elsewhere herein, any sequencing components may be loaded onto the substrate, immobilized on the substrate, and/or dispensed onto objects immobilized on the substrate.

Applications to Other Specimens Although described herein as being useful for nucleic acid sequencing, the systems and methods herein may be applied to other specimens and/or other uses for processing such specimens. Figure 25 shows a flow chart of one example of a method 1400 for processing a specimen.

In a first step 1410, the method may include providing a substrate including a planar array having analytes immobilized thereon, the substrate configured to rotate about an axis. The axis may be an axis through the center of the substrate. The axis may be an axis off-center. The substrate may be any substrate herein. In some examples, the planar array may include a single type of analyte. In other examples, the planar array may include two or more types of analytes. The two or more types of analytes may be arranged randomly. The two or more types of analytes may be arranged in a regular pattern. For example, the two types of analytes may be arranged in a radial alternating manner. The analyte may be any biological sample herein or a derivative thereof. For example, the analyte may be a single cell analyte. The analyte may be a nucleic acid molecule. The analyte may be a protein molecule. The analyte may be a single cell. The analyte may be a particle. The analyte may be an organism. The analyte may be part of a colony. In some cases, the analyte may be a non-biological sample or may be derived from a non-biological sample. The analyte may be immobilized at an individually addressable location on the planar array. The analyte may be immobilized to the substrate via a linker configured to bind to the analyte. For example, the linker may include a carbohydrate molecule. The linker may include an affinity binding protein. The linker may be hydrophilic. The linker may be hydrophobic. The linker may be electrostatic. The linker may be labeled. The linker may be integral to the substrate. The linker may be a separate layer on the substrate.

In a second step 1420, the method may include directing a solution including a plurality of reactants across the planar array during rotation of the substrate. The solution may include any solution or reagent herein. The plurality of reactants may be configured to interact with an analyte immobilized on the planar array. For example, when the analyte is a nucleic acid molecule, the plurality of reactants may include a plurality of probes. A given probe of the plurality of probes may include a random sequence or a target sequence, such as a homopolymer sequence or a di- or tri-base repeat sequence. In some examples, the probe may be a dibase probe. In some examples, the probe may be about 1-10 bases long. In some examples, the probe may be about 10-20 bases long. In some examples, the probe may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or more bases. Alternatively or in combination, the probes can be up to about 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 bases. In another example, when the analyte is a protein molecule, the plurality of reactants can include a plurality of antibodies. A given antibody of the plurality of antibodies can have binding specificity for one or more types of proteins. In other examples, the plurality of reactants can include any combination of a plurality of oligonucleotide molecules, carbohydrate molecules, lipid molecules, affinity binding proteins, aptamers, antibodies, enzymes, or other reagents. The plurality of reactants can be hydrophilic. The plurality of reactants can be hydrophobic. The plurality of reactants can be electrostatic. The plurality of reactants can be labeled. The plurality of reactants can include a mixture of labeled and unlabeled components. In some examples, the plurality of reactants can be unlabeled.

In step 1430, the method may include subjecting the analyte to conditions sufficient to cause a reaction or interaction between the analyte and a plurality of reactants. In step 1440, the method may include detecting a signal indicative of a reaction between the analyte and a plurality of reactants, thereby analyzing the analyte. In some cases, the reactant may react with the analyte. Alternatively or additionally, the reactant may bind to or interact with the analyte. The analyte or one or more of the reactants may undergo a conformational change, a chemical change, a change in state, or any combination thereof, upon interaction with the analyte.

The method may further include directing the analyte across a substrate that includes a linker prior to step 1410. For example, the substrate may be rotated before or during dispensing of the analyte to coat the substrate surface and/or the planar array with the analyte. In some examples, the analyte may be bound to beads, which are immobilized on the planar array.

The method may further include recycling a subset of the solution contacted with the substrate, as described elsewhere herein. Recycling may include collecting, filtering, and reusing the subset of the solution. Filtering may include molecular filtering. Molecular filtering may include specific nucleic acid filtering (i.e., filtering of specific nucleic acids). Nucleic acid filtering may include exposure of the solution to an array of oligonucleotide extension compounds capable of specifically binding to contaminating nucleotides or nucleic acids.

The signal may be an optical signal. The signal may be a fluorescent signal. The signal may be a light absorption signal. The signal may be a light scattering signal. The signal may be a luminescence signal. The signal may be a phosphorescence signal. The signal may be an electrical signal. The signal may be an acoustic signal. The signal may be a magnetic signal. The signal may be any detectable signal. Alternatively or in addition to the optical sensors herein, the system may include one or more other detectors (e.g., acoustic detectors, etc.) configured to detect detectable signals.

In some examples, the method may further include rotating the substrate about a central axis prior to step 1420.

In some cases, the method may further include terminating rotation of the substrate before detecting the signal in step 1440. In other examples, the signal may be detected in step 1440 while the substrate is rotating.

The signal may be generated by binding of the label to the analyte. The label may be bound to a molecule, particle, cell, or organism. The label may be bound to a molecule, particle, cell, or organism prior to step 1410. The label may be bound to a molecule, particle, cell, or organism following step 1410. The signal may be generated by formation of a detectable product by a chemical reaction. The reaction may include an enzymatic reaction. The signal may be generated by formation of a detectable product by physical association. The signal may be generated by formation of a detectable product by proximity association. The signal generated by proximity association may include Forster resonance energy transfer (FRET). The proximity association may include association with a complementary enzyme. The signal may be generated by a single reaction. The signal may be generated by multiple reactions. The multiple reactions may be performed in series. The multiple reactions may be performed in parallel. The multiple reactions may include one or more repetitions of the reaction. For example, the reaction may include a hybridization reaction or a ligation reaction. The reactions may include hybridization reactions and ligation reactions.

The method may further include repeating steps 1420, 1430, and 1440 one or more times. Different solutions may be directed to the planar array during rotation of the substrate for successive cycles.

Many variations, modifications, and adaptations based on the method 1400 provided herein are possible. For example, the order of steps of the method 1400 may be changed, some steps may be removed, some steps may be duplicated, and additional steps may be added as appropriate. Some steps may be performed sequentially. Some of these steps may be performed in parallel. Some steps may be performed once. Some steps may be performed multiple times. Some of the steps may include sub-steps. Some of the steps may be automated. Some steps may be manual.

26 shows a first example of a system 1500 for isolating an analyte. The system can include a plurality of linkers 1510a, 1510b, 1510c, and 1510d. The plurality of linkers can be attached or otherwise fixed to the substrate 310 of the present specification. For example, each linker can be attached to a particular individually addressable location of the plurality of individually addressable locations of the present specification. The linkers 1510a, 1510b, 1510c, and 1510d can include any linker of the present specification. Some or all of the linkers 1510a, 1510b, 1510c, and 1510d can be the same. Some or all of the linkers 1510a, 1510b, 1510c, and 1510d can be different. The linkers can be configured to interact with the analytes 1520a and 1520b. For example, the linker may be configured to bind to the analytes 1520a and 1520b via any interaction herein. The analytes 1520a and 1520b may include any analyte herein. The analytes 1520a and 1520b may be the same. The analytes 1520a and 1520b may be different. The linker may be configured to specifically interact with a particular analyte and/or type thereof. For example, the linker 1510b may be configured to specifically interact with the analyte 1520a. The linker 1510d may be configured to specifically interact with the analyte 1520b. Any linker may be configured to interact with any analyte. In this manner, a particular analyte may be bound to a particular location on the substrate. Although shown in FIG. 26 as including four linkers and two analytes, the system 1500 may include any number of linkers and analytes. For example, system 1500 may include at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10 million, at least 20 million, at least 50 million, at least 100 million, at least 200 million, at least 500 million, at least 1 billion linkers, or a number of linkers within a range defined by any two of the foregoing values. The system 1500 may include at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10 million, at least 20 million, at least 50 million, at least 100 million, at least 200 million, at least 500 million, at least 1 billion analytes, or a number of analytes within a range defined by any two of the foregoing values.

27 illustrates a second example of a system 1600 for isolating an analyte. The system may include a well configured to physically capture a particle. The well may include an individually addressable location of a plurality of individually addressable locations herein. The well may be configured to capture an analyte. For example, the well may be configured to capture a droplet of blood 1630. For example, the droplet of blood may include white blood cells 1640, red blood cells 1650, and circulating tumor cells 1660. The well may be configured to capture any other analyte herein. The well may be layered using microfabrication materials and techniques. For example, the well may include a base layer 1605. The base layer may include silicon. The well may include an oxide layer 1610. The oxide layer may include silicon oxide. The well may include a metal layer 1615. The metal may include nickel or aluminum. The well may include a nanotube layer 1620. The nanotube layer may include one or more carbon nanotubes. The well can include a containment layer 1625. The containment layer can include photoresist. The photoresist can include SU-8. The nanotube layer and the containment layer can be configured to trap the cells together.

Figure 28 shows an example of a control system for compensating for velocity gradients during scanning. Such a control system can algorithmically compensate for velocity gradients. The control system can predictively or adaptively compensate for tangential velocity gradients. In the first control system shown on the left side of Figure 28, the control system can measure the residual (uncorrected) velocity error during scanning based on the scanning of a rotating substrate, calculate a compensation correction factor, and use the compensation correction factor to set (or adjust) the compensation factor to reduce the velocity error in the subsequent scan results. The first control system may be a closed loop control system that eliminates (or reduces) the velocity error. In the second control system shown on the right side of Figure 28, the control system can directly calculate (or predict) the expected velocity gradient based on knowledge of the geometry and relative position of the scan with respect to the substrate, and set (or adjust) the system to eliminate the expected gradient.

Multi-Head Imaging Using a Common Linear Motion The systems and methods herein may utilize multiple imaging heads (e.g., detector systems, such detector systems including sensors and illumination sources), each imaging head responsible for imaging a different location on the substrate herein. For example, as described herein, a first imaging head may image the substrate along a first imaging path. The first imaging path may include a first series of rings (one or more), a first series of spirals (one or more), or a different first imaging path. A second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth imaging head may image the substrate along the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth imaging path. The second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth imaging path may comprise a second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth series of rings, a second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth spiral, or a different second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth imaging path. The imaging path or scanning path may be an imaging path or scanning path over a substrate or a sample.

Such multi-head imaging systems and methods can increase the imaging speed of a substrate and/or reduce the amount of time that may be required to image a substrate. In some cases, multiple imaging heads can move independently relative to the substrate, such as by independently controlling the movement of each of the imaging heads. In some cases, a first sensor can image a first region of the substrate at a first speed, and a second sensor can image a second region of the substrate at a second speed. The imaging speed of the sensor can be determined based on the linear velocity of the region being imaged relative to the imaging head that comprises the sensor. For example, a first sensor can image a first region farther from the axis of rotation of the substrate at a faster speed than a second sensor that images a second region closer to the axis of rotation of the substrate.

As described herein, during detection (e.g., imaging) of the substrate or a region thereof, the substrate may be stationary and one or more detector systems or components thereof may be moving (e.g., rotating). For example, the substrate may be stationary and both the sensor (e.g., line scan camera) and the illumination source of the detector system may be moving (e.g., rotating) during detection. Alternatively, the substrate may be moving (e.g., rotating) and one or more detector systems or components thereof may be stationary. In some cases, the substrate and the detector system or components thereof may be moving. For example, the substrate may be rotating and the sensor and illumination source of the detector system may be moving. For example, the sensor and illumination source may be translated (e.g., translated radially) across the substrate, or the sensor and illumination source may remain located at the same physical location but rotate around a central axis of the detector system.

The required motion of the imaging heads can be reduced by moving the substrate relative to each of the imaging heads such that each of the imaging heads shares a single linear motion relative to the substrate. Such an improvement can be achieved by positioning each scan head at a different initial distance (e.g., radial distance) from the center of the substrate and operating each scan head at a different scan speed that depends on the initial distance of the scan head from the center of the substrate. The single shared linear motion may be along a linear vector. For example, the single shared linear motion may result in radial motion (e.g., directed through the axis of rotation) or non-radial motion (e.g., not directed through the axis of rotation) of one or more scan heads. In some cases, the imaging heads may be configured to move in a radial direction r relative to the substrate in a polar coordinate system that includes a radial component r and an angular component φ. In some cases, the imaging heads may be configured to move in a linear direction relative to the substrate that is not completely radial, e.g., in a direction that includes both an r component and a φ component. The imaging heads can operate on the same side of the axis of rotation of the substrate or on opposite sides of the axis of rotation of the substrate. In the case of non-radial linear motion of one or more heads, the scanning direction of each imaging head may rotate due to a change in angle relative to the axis of rotation. Such rotation may be compensated for by a counter rotation (e.g., using a prism) to allow for a fixed scanning direction for each imaging head.

29A illustrates the motion of a substrate relative to two imaging heads disposed on the same side of the substrate's axis of rotation. The substrate 310 may be any substrate herein. The first imaging head 1005 may be similar to any first imaging head herein. The second imaging head 1015 may be similar to any second imaging head herein. At a first time, the first imaging head 1005 and the second imaging head 1015 may be disposed on the same side of the substrate's axis of rotation 305, with the first imaging head 1005 following a first imaging path 1010 during the substrate rotation and the second imaging head 1015 taddles a second imaging path 1020 during the substrate rotation. The substrate may be configured to move in a linear radial direction 1810 relative to the first and second imaging heads. For example, the substrate may be configured to move in a radial direction r in a polar coordinate system including a radial component r and an angular component φ. In some cases, the substrate may be configured to move in a linear direction that is not entirely radial, for example, in a direction that includes both r and φ components. Thus, the first and second imaging paths may change position relative to the substrate over time. Each imaging head may be in optical communication with an imaging field. For example, the first and second imaging heads may be in optical communication with the first and second imaging fields, respectively. As described elsewhere herein, each of the first and second imaging fields may be configured to rotate relative to the substrate. The rotation of the first and second imaging fields may be independent, or the rotation of the first, second, third, or fourth imaging fields may be coordinated.

FIG. 29B illustrates the movement of the substrate relative to two imaging heads located on either side of the substrate's axis of rotation. Compared to FIG. 29A, at a first time, the first imaging head 1005 and the second imaging head 1015 may be located on either side of the substrate's axis of rotation 305 such that the first imaging head 1005 follows a first imaging path 1010 during the substrate rotation, and the second imaging head 1015 follows a second imaging path 1020 during the substrate rotation. The substrate may be configured to move in a linear radial direction 1810 relative to the first and second imaging heads. Thus, the first and second imaging paths may change position relative to the substrate over time. Each imaging head may be in optical communication with an imaging field. For example, the first and second imaging heads may be in optical communication with a first and second imaging field, respectively. As described elsewhere herein, each of the first and second imaging fields may be configured to rotate relative to the substrate. The rotation of the first and second imaging fields may be independent, or the rotation of the first, second, third, or fourth imaging fields may be coordinated.

29C illustrates the movement of the substrate relative to the three imaging heads. The third imaging head 1025 may be similar to any third imaging head herein. At a first time, the first imaging head 1005 may be disposed on one side of the substrate's axis of rotation 305, relative to a plane that includes the axis of rotation, and the second imaging head 1015 and the third imaging head 1025 may be disposed on opposite sides of the substrate's axis of rotation, such that the first imaging head 1005 follows a first imaging path 1010 during the substrate rotation, the second imaging head 1015 follows a second imaging path 1020 during the substrate rotation, and the third imaging head 1025 follows a third imaging path 1030 during the substrate rotation. The substrate may be configured to move in a linear radial direction 1810 relative to the first, second, and third imaging heads. Thus, the first, second, and third imaging paths may change position relative to the substrate over time. Each imaging head may be in optical communication with an imaging field. For example, the first, second, and third imaging heads may be in optical communication with the first, second, and third imaging fields, respectively. As described elsewhere herein, each of the first, second, and third imaging fields may be configured to rotate relative to the substrate. The rotation of the first, second, and third imaging fields may be independent, or the rotation of the first, second, and third imaging fields may be coordinated.

29D illustrates the movement of the substrate relative to the four imaging heads. The fourth imaging head 1035 may be similar to any fourth imaging head herein. At a first time, the first imaging head 1005 and the fourth imaging head 1035 may be located on one side of the substrate's axis of rotation 305 relative to a plane that includes the axis of rotation, and the second imaging head 1015 and the third imaging head 1025 may be located on the opposite side of the substrate's axis of rotation, such that the first imaging head 1005 follows a first imaging path 1010 during the substrate rotation, the second imaging head 1015 follows a second imaging path 1020 during the substrate rotation, the third imaging head 1025 follows a third imaging path 1030, and the fourth imaging head 1025 follows a fourth imaging path 1030 during the substrate rotation. The substrate may be configured to move in a linear radial direction 1810 relative to the first, second, third, and fourth imaging heads. Thus, the first, second, third, and fourth imaging paths may change position relative to the substrate over time. Each imaging head may be in optical communication with an imaging field. For example, the first, second, third, and fourth imaging heads may be in optical communication with the first, second, third, and fourth imaging fields, respectively. Each of the first, second, third, and fourth imaging fields may be configured to rotate relative to the substrate, as described elsewhere herein. The rotation of the first, second, third, or fourth imaging fields may be independent, or the rotation of the first, second, third, or fourth imaging fields may be coordinated.

29E illustrates a further embodiment of the movement of the substrate relative to the four imaging heads. The first, second, third, and fourth imaging heads 1005, 1015, 1025, and 1035 may each be similar to any imaging head herein. At a first time, the first imaging head 1005 and the second imaging head 1015 may be located on one side of the substrate's axis of rotation 305, and the third imaging head 1025 and the fourth imaging head 1035 may be located on the opposite side of the substrate's axis of rotation, relative to a plane that includes the axis of rotation, such that during rotation of the substrate 310, the first imaging head 1015 and the fourth imaging head 1035 follow the first and second halves of the first imaging path 1010, respectively, and the second imaging head 1015 and the third imaging head 1025 follow the first and second halves of the second imaging path 1020, respectively. The substrate may be configured to move in a linear radial direction 1810 relative to the first, second, third, and fourth imaging heads. Thus, the first, second, third, and fourth imaging heads may subsequently follow the first and second halves of the third imaging path 1030 and the fourth imaging path 1040. The first, second, third, and fourth imaging paths may change position relative to the substrate over time. Each imaging head may be in optical communication with an imaging field. For example, the first, second, third, and fourth imaging heads may be in optical communication with the first, second, third, and fourth imaging fields, respectively. Each of the first, second, third, and fourth imaging fields may be configured to rotate relative to the substrate, as described elsewhere herein. The rotation of the first, second, third, or fourth imaging fields may be independent, or the rotation of the first, second, third, or fourth imaging fields may be coordinated.

29F illustrates a further embodiment of the movement of the substrate relative to the four imaging heads. Each of the first, second, third, and fourth imaging heads 1005, 1015, 1025, and 1035 may be similar to any imaging head herein. At a first time, the first imaging head 1005 and the second imaging head 1015 may be located on one side of the substrate's axis of rotation 305 with respect to a plane that includes the axis of rotation, and the third imaging head 1025 and the fourth imaging head 1035 may be located on the opposite side of the substrate's axis of rotation such that the first imaging head 1005 follows a first imaging path 1010, the second imaging head 1015 follows a second imaging path 1020, the third imaging head 1025 follows a third imaging path 1030, and the fourth imaging head 1035 follows a fourth imaging path 1040 during the rotation of the substrate. The heads may be configured to translate in a linear direction. The translation may be radial or the translation may not be radial. The translation of one or more of the first, second, third, or fourth imaging heads may be coupled. Alternatively or additionally, the translation of the first, second, third, or fourth imaging heads may be independent. In some embodiments, the translation of the first and second imaging heads may be coupled, and the translation of the third and fourth imaging heads may be coupled. Thus, the first, second, third, and fourth imaging paths may change position relative to the substrate over time. Each imaging head may be in optical communication with an imaging field. For example, the first, second, third, and fourth imaging heads may be in optical communication with the first, second, third, and fourth imaging fields, respectively. Each of the first, second, third, and fourth imaging fields may be configured to rotate relative to the substrate, as described elsewhere herein. The rotation of the first, second, third, or fourth imaging field of view may be independent, or the rotation of the first, second, third, or fourth imaging field of view may be coordinated.

29G shows a further embodiment of the movement of the substrate relative to the four imaging heads. The first, second, third, and fourth imaging heads 1005, 1015, 1025, and 1035 may each be similar to any imaging head herein. At a first moment, the first imaging head 1005, the second imaging head 1015, the third imaging head 1025, and the fourth imaging head 1035 may be located on the same side of the substrate's axis of rotation 305 with respect to a plane that includes the axis of rotation. During the rotation of the substrate, the first imaging head 1005 may follow a first imaging path 1010, the second imaging head 1015 may follow a second imaging path 1020, the third imaging head 1025 may follow a third imaging path 1030, and the fourth imaging head 1035 may follow a fourth imaging path 1040. The head may be configured to translate in a linear direction. The translation may be radial, or the translation may not be radial. The translation of the first, second, third, or fourth imaging head may be independent. Thus, the first, second, third, and fourth imaging paths may change position relative to the substrate over time. Each imaging head may be in optical communication with an imaging field. For example, the first, second, third, and fourth imaging heads may be in optical communication with the first, second, third, and fourth imaging fields, respectively. Each of the first, second, third, and fourth imaging fields may be configured to rotate relative to the substrate, as described elsewhere herein. The rotation of the first, second, third, or fourth imaging fields may be independent, or the rotation of the first, second, third, or fourth imaging fields may be coordinated.

30A shows a succession of ring paths of two imaging heads located on the same side of the axis of rotation of the substrate. At a first time, a first imaging head (not shown in FIG. 30A) and a second imaging head (not shown in FIG. 30A) may be positioned on the same side of the axis of rotation 305 of the substrate 310, such that the first imaging head follows a first imaging path 1010a at a first time during the rotation of the substrate, and the second imaging head follows a second imaging path 1020a at a first time during the rotation of the substrate. For example, the two imaging heads may be positioned and configured as in FIG. 29A. As the substrate moves in a linear radial direction 1810 relative to the first and second imaging heads, the first and second imaging heads may follow a series of imaging paths during the rotation of the substrate. For example, if the first and second imaging heads are located on the same side of the rotation axis of the substrate, the first imaging head may follow imaging path 1010b at a second time, imaging path 1010c at a third time, and imaging path 1010d at a fourth time, while the second imaging path may follow imaging path 1020b at a second time, imaging path 1020c at a third time, and imaging path 1020d at a fourth time. When the first and second imaging heads are located on the same side of the rotation axis, the series of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} may proceed in the same direction relative to the substrate. For example, as shown in FIG. 30A, the series of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} can both proceed in a direction toward the center of the substrate.

30B shows a succession of ring paths of two imaging heads located on either side of the rotation axis of the substrate. Compared to FIG. 30A, at a first time, a first imaging head (not shown in FIG. 30B) and a second imaging head (not shown in FIG. 30B) may be positioned on either side of the rotation axis 305 of the substrate such that the first imaging head follows a first imaging path 1010a at a first time during the rotation of the substrate, and the second imaging head follows a second imaging path 1020a at a first time during the rotation of the substrate. For example, the two imaging heads may be positioned and configured as in FIG. 29B. As the substrate moves in a linear radial direction 1810 relative to the first and second imaging heads, one head moves toward the central axis and the other head moves away from the central axis, and the first and second imaging heads each follow a series of imaging paths during the rotation of the substrate. For example, when the first and second imaging heads are located on opposite sides of the axis of rotation of the substrate, the first imaging head may follow imaging path 1010b at a second time, imaging path 1010c at a third time, and imaging path 1010d at a fourth time, while the second imaging path may follow imaging path 1020b at a second time, imaging path 1020c at a third time, and imaging path 1020d at a fourth time. When the first and second imaging heads are located on opposite sides of the axis of rotation, the series of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} may proceed in opposite directions relative to the substrate. For example, as shown in FIG. 30B, a series of imaging paths {1010a, 1010b, 1010c, 1010d} can travel in a direction toward the center of the substrate, and a series of imaging paths {1020a, 1020b, 1020c, 1020d} can travel in a direction away from the center of the substrate.

30C illustrates alternating ring paths for two imaging heads located on the same side of the substrate's axis of rotation. At a first time, a first imaging head (not shown in FIG. 30C) and a second imaging head (not shown in FIG. 30C) may be positioned on the same side of the substrate's axis of rotation 305, such that the first imaging head follows a first imaging path 1010a at a first time during the substrate's rotation, and the second imaging head follows a second imaging path 1020a at a first time during the substrate's rotation. As the substrate moves in a linear radial direction 1810 relative to the first and second imaging heads, the first and second imaging heads may follow a series of imaging paths during the substrate's rotation. For example, if the first and second imaging heads are located on the same side of the rotation axis of the substrate, the first imaging head may follow imaging path 1010b at a second time, imaging path 1010c at a third time, and imaging path 1010d at a fourth time, while the second imaging path may follow imaging path 1020b at a second time, imaging path 1020c at a third time, and imaging path 1020d at a fourth time. The sequence of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} may be staggered such that successive imaging paths toward or away from the center of the substrate are followed by alternating imaging heads. When the first and second imaging heads are located on the same side of the rotation axis, the series of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} can proceed in the same direction relative to the substrate. For example, as shown in FIG. 30C, the series of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} can both proceed in a direction toward the center of the substrate.

30D shows the alternating ring paths of two imaging heads located on either side of the axis of rotation of the substrate. At a first time, a first imaging head (not shown in FIG. 30D) and a second imaging head (not shown in FIG. 30D) may be positioned on opposite sides of the axis of rotation 305 of the substrate 310, with the first imaging head following a first imaging path 1010a at a first time during the rotation of the substrate and the second imaging head following a second imaging path 1020a at a first time during the rotation of the substrate. As the substrate moves in a linear radial direction 1810 relative to the first and second imaging heads, one head moves toward the central axis and the other head moves away from the central axis, and the first and second imaging heads each follow a series of imaging paths during the rotation of the substrate. For example, if a first and second imaging head are located on either side of an axis of rotation of the substrate, the first imaging head may follow imaging path 1010b at a second time, imaging path 1010c at a third time, and imaging path 1010d at a fourth time, while the second imaging path may follow imaging path 1020b at a second time, imaging path 1020c at a third time, and imaging path 1020d at a fourth time. The sequence of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} may be staggered such that successive imaging paths toward or away from the center of the substrate are followed by alternating imaging heads. When the first and second imaging heads are located on opposite sides of the rotation axis, the series of imaging paths {1010a, 1010b, 1010c, 1010d} and {1020a, 1020b, 1020c, 1020d} can proceed in opposite directions relative to the substrate. For example, as shown in FIG. 30D, the series of imaging paths {1010a, 1010b, 1010c, 1010d} can proceed in a direction toward the center of the substrate, and the series of imaging paths {1020a, 1020b, 1020c, 1020d} can proceed in a direction away from the center of the substrate.

31A-31B illustrate rotational scanning directions of the imaging head with non-radial motion of the head relative to the substrate (e.g., motion including both r and φ components in a polar coordinate system). For example, as shown in FIG. 31A, the head may move along a direction 316 relative to the substrate that does not pass through the central axis. At a first time, the first imaging head (not shown in FIG. 31A) or the second imaging head (not shown in FIG. 31A) may be positioned off-axis from the longitudinal axis 315 of the substrate 310. In such a case, the first or second imaging head may have a tangential velocity relative to the substrate that changes direction as the substrate moves relative to the first or second imaging head. For example, as shown in FIG. 31A, the second imaging head may have a tangential velocity vector 2020a relative to the substrate while following imaging path 1020a and a tangential velocity vector 2020b relative to the substrate while following imaging path 1020c. As shown in Figure 31, the tangential velocity vectors 2020a and 2020b can point in substantially different directions. Such an effect can manifest as a rotation of the imaging field when the first imaging head follows the sequence of imaging paths {1010a, 1010b, 1010c, 1010d} or when the second imaging head follows the sequence of imaging paths {1020a, 1020b, 1020c, 1020d}.

31B illustrates the rotational scanning direction of the imaging field of view due to the non-radial motion of the imaging head relative to the substrate. For example, a first imaging head (not shown in FIG. 31B) imaging a first field of view 3101 and a third imaging head (not shown in FIG. 31B) imaging a third field of view 3103 can be translated relative to the substrate 310 in directions 3111 and 3113 that do not pass through the central axis. At a first time, the first imaging field of view 3101 or the third imaging field of view 3103 may be located off-axis from the longitudinal axis 315 of the substrate 310. In such a case, the first or third imaging field of view can have a tangential velocity relative to the substrate that changes direction as the substrate moves relative to the first or second imaging head. Following the non-radial translation, the first and third imaging fields of view may no longer be positioned perpendicular to the tangential motion of the substrate (shown by the grey rectangles). In some embodiments, the first and third imaging fields of view can undergo a counter rotation with respect to the substrate after the non-radial translation (shown by the dashed rectangle) such that the first and third imaging fields of view can be positioned perpendicular to the tangential motion of the substrate. The counter rotation can be accomplished using any of the methods disclosed herein, such as those described with respect to FIGS. 34A-34C.

34A-34C show an exemplary optical system for rotating the imaging field. Such a rotation of the imaging field can be compensated for by derotating the imaging field. For example, the imaging field may be derotated using a prism system such as a delta rotator prism, a Schmidt rotator, or a Dove prism. An exemplary optical system for inverting the imaging field using a Dove prism is shown in FIG. 34B. Alternatively or additionally, compensation may be achieved by using one or more mirrors or other optical elements (e.g., beam splitters (e.g., dichroic mirrors)) as described herein. Alternatively or additionally, compensation may be achieved by rotating one or more sensors in the optical head(s). For example, compensation can be achieved by rotating a detector (e.g., a line scan camera) and a line shaping element (e.g., a cylindrical lens). Exemplary optical systems for rotating a detector and a line shaping element are shown in FIGS. 34A and 34C. As shown in FIG. 33, the imaging field of view can be rotated about an axis of rotation that can be opposite to the axis of rotation of the surface to compensate for relative translational motion that may not intersect the axis of rotation of the surface.

35A-35C show exemplary optical path trajectories of an optical system 3500 with imaging heads. As shown in FIG. 35A, two imaging heads 3501 and 3502, each including an objective lens, can be positioned to image corresponding regions of a substrate 3503. The imaging heads may be positioned on either side of a radial line 3504. In some embodiments, the two imaging heads may be positioned at different distances from the radial line. The distance from the radial line may be determined by the diameter of the objective lens and the optical path trajectory of the imaging heads. The substrate may be configured to rotate about a rotation axis 3505 and translate along a translation axis 3506 relative to the imaging heads. The substrate may be rotated about the rotation axis such that the two imaging heads describe a circular optical path trajectory. The ideal optical path trajectory, in which the entire outer region of the surface is scanned without overlap, is outlined in solid lines in FIG. 35A-35C. The optical path trajectories resulting from coordinated operation of two imaging heads with partially overlapping optical path trajectories are outlined in dashed lines in Figures 35A-35C. For clarity, only the initial positions of imaging heads 3501 and 3502 are shown in Figures 35A-35C.

The first optical path 3511 of the first imaging head 3501 may be concentric with the first optical path 3521 of the second imaging head 3502, as shown in FIG. 35C. As the substrate translates along the translation axis, the first and second imaging heads may move to the second optical path 3512 and 3522, the third optical path 3513 and 3523, the third optical path, the fourth optical path 3514 and 3524, the fifth optical path 3515 and 3525, the sixth optical path 3516 and 3526, the seventh optical path 3517 and 3527, or more optical paths. In some embodiments, the optical path trajectories of the two imaging heads overlap, with the amount of overlap increasing for optical paths closer to the rotation axis of the substrate. The optical path trajectories and the distance of the imaging heads from the radial line may be optimized to minimize the overlap of the optical path trajectories of the two imaging heads, as shown in FIG. 35B. The light path trajectories may overlap by 0.10% or less, 0.20% or less, 0.50% or less, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, or 50% or less.

In some embodiments, the optical path trajectories of the two imaging heads do not substantially overlap. In some embodiments, the optical path trajectories of the two imaging heads are partially separated, with the amount of separation decreasing for optical paths closer to the rotation axis of the substrate. The optical path trajectories and the distance of the imaging heads from the radial lines can be optimized to reduce the amount of substrate that is not scanned without substantial overlap of the optical path trajectories of the two imaging heads (not shown in FIG. 32). In some examples, the unscanned portion of the substrate may comprise 0.10% or less, 0.20% or less, 0.50% or less, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, or 50% or less of the entire substrate surface. In some cases, the optical path trajectories of the tow imaging heads can be configured to reduce the amount of overlap to reduce the amount of photodamage to the substrate or reagents on the substrate.

Substrate movements herein, such as those described with respect to Figures 29-31, can be used to scan a surface containing analytes. In some cases, scanning the surface can include detecting analytes on the surface. Figure 32 shows a flow chart of an example of a method 2100 for analyte detection or analysis. In a first step 2110, the method 2100 can include rotating an open substrate about a central axis, the open substrate having an array of analytes fixed thereon.

In a second step 2120, the method 2100 may include delivering a solution having a plurality of probes to a region proximal to the central axis to introduce the solution into the open substrate.

In a third step 2130, the method 2100 may include dispersing the solution across the open substrate (e.g., by at least centrifugal force) such that at least one of the plurality of probes binds to at least one of the immobilized analytes to form a bound probe.

In a fourth step 2140, the method 2100 may include performing a first scan of the open substrate along a first set of one or more scan paths using a first detector while rotating the open substrate, and performing a second scan of the open substrate along a second set of one or more scan paths using a second detector. The first set of one or more scan paths and the second set of one or more scan paths may be different. The first detector or the second detector may detect at least one signal from the coupled probe. The first detector may be disposed at a first radial position relative to the central axis. The second detector is disposed at a second radial position relative to the central axis. The first detector and the second detector may undergo relative motion with respect to the central axis along the same linear vector to generate the first set of one or more scan paths and the second set of one or more scan paths, respectively.

The first and second detectors can operate at different scan rates. For example, the different scan rates of the first and second detectors can be a function of the first and second radial positions, respectively. Alternatively, the detectors can operate at a fixed line rate. For example, algorithmic processing can account for oversampling of the optical head located at the inner radial position.

The first set of one or more scan paths may include one or more circular scan paths having different radii. For example, the first set of one or more scan paths may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more circular scan paths, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 circular scan path, or a number of circular scan paths within a range defined by any two of the preceding values.

The second set of one or more scan paths may include one or more circular scan paths having different radii. For example, the second set of one or more scan paths may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more circular scan paths, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 circular scan path, or a number of circular scan paths within a range defined by any two of the preceding values.

The first set of one or more scan paths can include one or more spiral scan paths. For example, the first set of one or more scan paths can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more spiral scan paths, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 spiral scan path, or a number of spiral scan paths within a range defined by any two of the preceding values.

The second set of one or more scan paths can include one or more spiral scan paths. For example, the second set of one or more scan paths can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100 or more spiral scan paths, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 spiral scan path, or a number of spiral scan paths within a range defined by any two of the preceding values.

The same linear vector may be in a radial direction through the central axis. The same linear vector may not be in a radial direction (e.g., not through the central axis). The method may further include compensating for velocity differences (e.g., tangential velocity differences, etc., as described herein with respect to FIG. 31) of different regions at different radial positions relative to the central axis. The predetermined scan paths of the first set of one or more scan paths may include different regions. The predetermined scan paths of the second set of one or more scan paths may include different regions. The compensation may include using one or more prisms, such as one or more delta rotator prisms, Schmidt rotators, or Dove prisms.

The first detector and the second detector may be substantially stationary during the relative motion. The open substrate may undergo both rotational and translational motion during the relative motion. The first detector and the second detector may undergo motion during the relative motion. The open substrate may undergo rotational motion relative to the first detector and the second detector, and the first detector and the second detector may undergo linear motion about the central axis. The first detector may undergo relative motion during rotation of the open substrate. The second detector may undergo relative motion during rotation of the open substrate. The first detector may undergo relative motion when the open substrate is substantially stationary. The second detector may undergo relative motion when the open substrate is substantially stationary.

The predetermined scan paths of the first set of one or more scan paths may include an area scanned during relative motion. The predetermined scan paths of the second set of one or more scan paths may include an area scanned during relative motion. The predetermined scan paths of the first set of one or more scan paths may not include an area scanned during relative motion. The predetermined scan paths of the second set of one or more scan paths may not include an area scanned during relative motion.

The first detector and the second detector may have the same angular position relative to the central axis. The first detector and the second detector may have different angular positions relative to the central axis. The first detector and the second detector may have opposite angular positions (e.g., with a 180 degree separation) relative to the central axis.

The first detector is at an angle of at least about 1 degree, at least about 2 degrees, at least about 3 degrees, at least about 4 degrees, at least about 5 degrees, at least about 6 degrees, at least about 7 degrees, at least about 8 degrees, at least about 9 degrees, at least about 10 degrees, at least about 15 degrees, at least about 20 degrees, at least about 25 degrees, at least about 30 degrees, at least about 35 degrees, at least about 40 degrees, at least about 45 degrees, at least about 50 degrees, at least about 55 degrees, at least about 60 degrees, at least about 65 degrees, at least about 70 degrees, at least about 75 degrees, at least about 80 degrees, at least about 81 degrees, at least about 82 degrees, at least about 83 degrees, at least about 84 degrees, at least about 85 degrees, at least about 86 degrees, at least about 87 degrees, at least about 89 degrees, at least about 90 degrees, at least about 91 degrees, at least about 92 degrees, at least about 93 degrees, at least about 94 degrees, at least about 95 degrees, at least about 96 degrees, at least about 97 degrees, at least about 98 degrees, at least about 99 degrees, at least about 100 degrees, at least about 101 degrees, at least about 102 degrees, at least about 103 degrees, at least about 104 degrees, at least about 105 degrees, at least about 106 degrees, at least about 107 degrees, at least about 108 degrees, at least about 109 degrees, at least about 110 degrees, at least about 111 degrees, at least about 112 degrees, at least about 113 degrees, at least about 114 degrees, at least about 115 degrees, at least about 116 degrees, at least about 117 degrees, at least about 118 degrees, at least about 1 7 degrees, at least about 88 degrees, at least about 89 degrees, or more, up to about 89 degrees, up to about 88 degrees, up to about 87 degrees, up to about 86 degrees, up to about 85 degrees, up to about 84 degrees, up to about 83 degrees, up to about 82 degrees, up to about 81 degrees, up to about 80 degrees, up to about 75 degrees, up to about 70 degrees, up to about 65 degrees, up to about 60 degrees, up to about 55 degrees, up to about 50 degrees, up to about 45 degrees, up to about 40 degrees, up to about 35 degrees, up to about 30 degrees, up to about 25 degrees, up to about 20 degrees, up to about 15 degrees, up to about 10 degrees, up to about 9 degrees, up to about 8 degrees, up to about 7 degrees, up to about 6 degrees, up to about 5 degrees, up to about 4 degrees, up to about 3 degrees, up to about 2 degrees, up to about 1 degree, or less, or within a range defined by any two of the above values.

The second detector is at an angle of at least about 1 degree, at least about 2 degrees, at least about 3 degrees, at least about 4 degrees, at least about 5 degrees, at least about 6 degrees, at least about 7 degrees, at least about 8 degrees, at least about 9 degrees, at least about 10 degrees, at least about 15 degrees, at least about 20 degrees, at least about 25 degrees, at least about 30 degrees, at least about 35 degrees, at least about 40 degrees, at least about 45 degrees, at least about 50 degrees, at least about 55 degrees, at least about 60 degrees, at least about 65 degrees, at least about 70 degrees, at least about 75 degrees, at least about 80 degrees, at least about 81 degrees, at least about 82 degrees, at least about 83 degrees, at least about 84 degrees, at least about 85 degrees, at least about 86 degrees, at least about 87 degrees, at least about 89 degrees, at least about 90 degrees, at least about 91 degrees, at least about 92 degrees, at least about 93 degrees, at least about 94 degrees, at least about 95 degrees, at least about 96 degrees, at least about 97 degrees, at least about 98 degrees, at least about 99 degrees, at least about 100 degrees, at least about 101 degrees, at least about 102 degrees, at least about 103 degrees, at least about 104 degrees, at least about 105 degrees, at least about 106 degrees, at least about 107 degrees, at least about 108 degrees, at least about 109 degrees, at least about 110 degrees, at least about 111 degrees, at least about 112 degrees, at least about 113 degrees, at least about 114 degrees, at least about 115 degrees, at least about 116 degrees, at least about 117 degrees, at least about 118 degrees, at least about 1 7 degrees, at least about 88 degrees, at least about 89 degrees, or more, up to about 89 degrees, up to about 88 degrees, up to about 87 degrees, up to about 86 degrees, up to about 85 degrees, up to about 84 degrees, up to about 83 degrees, up to about 82 degrees, up to about 81 degrees, up to about 80 degrees, up to about 75 degrees, up to about 70 degrees, up to about 65 degrees, up to about 60 degrees, up to about 55 degrees, up to about 50 degrees, up to about 45 degrees, up to about 40 degrees, up to about 35 degrees, up to about 30 degrees, up to about 25 degrees, up to about 20 degrees, up to about 15 degrees, up to about 10 degrees, up to about 9 degrees, up to about 8 degrees, up to about 7 degrees, up to about 6 degrees, up to about 5 degrees, up to about 4 degrees, up to about 3 degrees, up to about 2 degrees, up to about 1 degree, or less, or within a range defined by any two of the above values.

A predetermined scan path of a first set of one or more scan paths may include a first region and a second region. The first region and the second region may be at different radial positions of the open substrate relative to the central axis. The first region and the second region may be spatially resolved by a first detector. A predetermined scan path of a second set of one or more scan paths may include a first region and a second region. The first region and the second region may be at different radial positions of the open substrate relative to the central axis. The first region and the second region may be spatially resolved by a second detector.

Reel-to-reel processing of biological specimens In some examples, the open substrate system of the present disclosure can include a substantially flexible substrate. For example, the substantially flexible substrate can include a film. The substantially flexible substrate can have any degree of deformability. In some examples, the open substrate system of the present disclosure can achieve dispensing via contact with a reagent reservoir or solution. In some examples, a substantially flexible substrate can be used with a reagent reservoir or solution. In some examples, a substantially rigid substrate can be used with a reagent reservoir or solution. In some cases, a substantially flexible substrate can be used with other dispensing mechanisms (e.g., nozzles) herein. In some cases, a substantially rigid substrate can be used with other dispensing mechanisms (e.g., nozzles) herein.

In one aspect, provided herein is a method for processing a biological sample, the method comprising: (a) providing a flexible substrate including an array on which the biological sample is fixed, the flexible substrate being movable via a reel; (b) contacting the flexible substrate with a reservoir including a solution including a plurality of probes; (c) subjecting the biological sample to conditions sufficient to effect a reaction between at least one of the plurality of probes and the biological sample, thereby binding at least one probe to the biological sample; and (d) detecting one or more signals from the at least one probe bound to the biological sample, thereby analyzing the biological sample.

In some embodiments, the method further includes using a recirculation tank.

In some cases, the dimension of the flexible substrate is the width of the field of view of the imaging method.

In some embodiments, the process of contacting the flexible substrate with the reservoir and/or subjecting the biological sample to conditions sufficient to effect a reaction occurs while the flexible substrate is moving through the reel.

In some embodiments, the flexible substrate is moved through a reel to contact the solution with the biological specimen. In some embodiments, the flexible substrate is further moved through a second reel to contact the flexible substrate with a second reservoir containing a second solution. In some cases, the second solution includes a wash buffer. In some cases, the second solution includes a plurality of probes, and the solution and the second solution are different.

In some embodiments, the process of contacting the flexible substrate with the reservoirs, subjecting the biological sample to conditions sufficient to effect a reaction, and detecting can be repeated any number of times, e.g., a sufficient number of times to complete an assay (e.g., determining the sequence of a nucleic acid molecule).

In some embodiments, the method further includes repeating the process of contacting the flexible substrate with the reservoir, subjecting the biological sample to conditions sufficient to effect a reaction, and detecting with an additional plurality of probes different from the plurality of probes. In some cases, the plurality of probes can include any probe described elsewhere herein. For example, the probes can include oligonucleotide molecules having any length. For example, the probes can include oligonucleotides between 1 and 10 bases in length. The given probe can be a dibase probe. The given probe can be between 10 and 20 bases in length. In some examples, the plurality of probes can be labeled.

In some embodiments, the biological specimen is a nucleic acid molecule, and analyzing the biological specimen includes identifying a sequence of the nucleic acid molecule. In some aspects, the plurality of probes is a plurality of nucleotides. In some embodiments, the plurality of probes is a plurality of oligonucleotide molecules. In some cases, subjecting the biological specimen to conditions sufficient to react includes subjecting the nucleic acid molecule to a primer extension reaction under conditions sufficient to incorporate at least one nucleotide from the plurality of nucleotides into a growing strand complementary to the nucleic acid molecule. In some aspects, the one or more signals indicate incorporation of at least one nucleotide. In some aspects, the plurality of nucleotides includes a nucleotide analog. In some aspects, the method further includes repeating the process of contacting the flexible substrate with the reservoir and subjecting the biological specimen to conditions sufficient to react with an additional plurality of nucleotides that are a second canonical base type, the second canonical base type being different from the first canonical base type. In some embodiments, the plurality of probes is a plurality of oligonucleotide molecules. In some embodiments, the biological specimen is a nucleic acid molecule, and the subjecting includes performing a complementary binding reaction between at least one probe and the nucleic acid molecule to identify the presence of homology between the at least one probe and the biological specimen upon detection.

In some embodiments, detection is performed using a sensor that continuously scans the array. In some embodiments, detection is performed using a sensor that linearly scans the array. In some cases, detection is performed using any sensor or sensing mechanism described herein.

In some embodiments, the method further includes using a tensioning mechanism to move the flexible substrate through the reel and into contact with the reservoir, thereby dispensing the solution onto the flexible substrate. Any other operating unit or mechanism can be used to actuate the flexible substrate.

In some embodiments, the fluid viscosity of the solution or the velocity of the flexible substrate is selected to provide a predetermined thickness of a layer of the solution adjacent to the array. In some embodiments, a squeegee near the substrate can be used to obtain the predetermined thickness of the layer. In some embodiments, the flexible substrate is textured or patterned. In some embodiments, the flexible substrate is substantially planar.

In some embodiments, the flexible substrate includes an array including a plurality of individually addressable locations, and the biological analyte is disposed at a predetermined individually addressable location of the plurality of individually addressable locations. In some embodiments, the array has one or more additional biological analytes immobilized thereon.

In some embodiments, contacting the flexible substrate with the reservoir includes achieving contact at a contact area between the flexible substrate and the reservoir. In some embodiments, contacting the flexible substrate with the reservoir includes achieving contact along a contact line between the substrate and the reservoir.

In some cases, the biological specimen may include any specimen described elsewhere herein. The specimen may be a single cell specimen. The specimen may be a nucleic acid molecule or a clonal population of nucleic acids. The specimen may be a protein molecule. The specimen may be a single cell. The specimen may be a particle. The specimen may be an organism. The specimen may be part of a colony. The specimen may be fixed to an individually addressable location on a planar array. The array on the flexible substrate may include two or more types of analytes. The two or more types of analytes may be randomly arranged. The two or more types of analytes may be arranged in a regular pattern.

In some examples, the analyte can be immobilized on the flexible substrate via a linker. The flexible substrate can include a linker bound to the analyte. The linker can be any linker herein. The linker can include a carbohydrate molecule. The linker can include an affinity binding protein. The linker can be hydrophilic. The linker can be hydrophobic. The linker can be electrostatic. The linker can be labeled. The linker can be integral with the substrate. The linker can be a separate layer on the substrate. In some embodiments, the biological analyte is bound to a bead, which is immobilized on the flexible substrate. The method can further include directing the biological analyte across the flexible substrate including the linker prior to providing the flexible substrate. The biological analyte can be bound to a bead, which is immobilized on the substrate. In some examples, for example, the flexible substrate including the linker can be contacted with a reservoir including a solution including the biological analyte. Alternatively or additionally, the biological analyte can be dispensed onto the flexible substrate according to any other dispensing mechanism herein.

The method may further include recycling a subset of the solution that contacted the substrate. Recycling may include collecting, filtering, and reusing the subset of the solution. The filtering may be molecular filtering. For example, the solution in the reservoir (after the substrate has passed through) may be recycled.

The signal may be an optical signal. The signal may be a fluorescent signal. The signal may be a light absorption signal. The signal may be a light scattering signal. The signal may be a luminescence signal. The signal may be a phosphorescence signal. The signal may be an electrical signal. The signal may be an acoustic signal. The signal may be a magnetic signal. The signal may be generated by binding of a label to an analyte. The label may be bound to a molecule, particle, cell or organism. The label may be bound to an analyte (e.g., a molecule, particle, cell or organism) prior to deposition on the substrate. The label may be bound to an analyte after deposition on the substrate. The signal may be generated by formation of a detectable product by a chemical reaction. The reaction may include an enzymatic reaction. The signal may be generated by formation of a detectable product by physical association. The signal may be generated by formation of a detectable product by proximity association. The signal generated by proximity association may include Förster resonance energy transfer (FRET). The proximity association may include association with a complementary enzyme. The signal may be generated by a single reaction. The signal may be generated by multiple reactions. The multiple reactions may be performed in series. The multiple reactions may be performed in parallel. The multiple reactions may include one or more repetitions of a reaction. The reaction may include a hybridization reaction or a ligation reaction. The reaction may include a hybridization reaction and a ligation reaction.

One or more processes of the methods herein can be repeated continuously. One or more methods herein can provide greater efficiency in reagent usage. One or more methods herein can allow for detection of one or more signals simultaneously at multiple locations along the array. In some cases, the throughput can be altered by changing the dimensions of the flexible substrate. For example, the flexible substrate can be a rectangular film, with wider films allowing for increased throughput. In another example, the length of the reel can be altered to match the detection method.

36A-36B, as shown in FIG. 36A and FIG. 36B, generally illustrate a method for processing a biological specimen. The biological specimen is immobilized on a flexible substrate, such as film 2710. In some cases, the biological specimen is immobilized on the film in an arrayed pattern at individually addressable locations. In other embodiments, the biological specimen is immobilized on the film in a random orientation. The film 2710 with the immobilized biological specimen can be moved through a reel or series of reels. In process 2712, the film 2710 with the immobilized biological specimen is moved through the reel and contacted with a reservoir 2730 containing a plurality of probes, such as a plurality of labeled probes. In some cases, the labeled probes are fluorescently labeled nucleotides. The labeled probes can bind to a subset of the individually addressable locations containing the biological specimen, for example, based on sequence complementarity. Then, in process 2714, the film is moved through a second reel and contacted with a reservoir 2740 containing a wash buffer. The wash buffer may allow removal of uncoupled probes, such as probes that are not bound or hybridized to the film. Detection of one or more signals from at least one probe bound to the biological specimen may be performed. In process 2715, detection may be performed using a sensor, such as an imager 2750, where an image of the film is taken. In some cases, the field of view of the image is one of the dimensions (e.g., width) of the film. In some cases, detection may be performed multiple times during processing. For example, as shown in FIG. 36A, detection may be performed after one or more wash steps after treatment with a probe (e.g., dATP, dCTP, dTTP, dGTP, or dUTP). In some cases, the surface may be imaged before treatment with a probe, as shown in FIG. 36B. In process 2716, the film 2710 is moved through a third reel and contacted with a reservoir 2760 containing multiple probes, such as multiple labeled probes. The labeled probes in reservoir 2760 may be different from the labeled probes in reservoir 2730. Similar to process 2712, the labeled probes in reservoir 2760 can bind, for example, based on sequence complementarity, to a subset of the individually addressable locations that contain the biological analyte. Processes 2714, 2715 can then be repeated. In some cases, one or more processes can be performed iteratively.

In some cases, the biological specimen is a nucleic acid molecule or a clonal population of nucleic acid molecules, and the film 2710 moves through a first reel to contact the film with a first reservoir containing a plurality of adenine (e.g., fluorescently labeled adenine) molecules. The adenine molecules can then hybridize with thymine molecules in the biological specimen. The film can then be passed through a reel to contact the film with a wash reservoir to remove unhybridized probe. Detection of the hybridized molecules can occur. Since the sequence of the probe molecules is known, detection of one or more signals can provide knowledge of the sequence of the biological specimen. The film can then be contacted with a reservoir containing labeled cytosine, labeled guanine, or labeled thymine, etc. Again, since the sequence of each of the probes is known, detection of one or more signals can provide knowledge of the sequence of the biological specimen. As will be appreciated, the specific nucleotides added to each reservoir can vary. For example, a first reservoir may contain adenine, cytosine, guanine, thymine, etc., and the next reservoir may contain adenine, cytosine, guanine, thymine, etc.

As will be appreciated, any process within the methods herein may be performed in any convenient step. For example, the flexible substrate may be contacted first with a first reservoir, followed by a wash reservoir, followed by a second reservoir, prior to detection. In other examples, the flexible substrate may be contacted with multiple reservoirs containing probes prior to detection. In other examples, the flexible substrate may be contacted with a detector before or after contacting the flexible substrate with any number of reservoirs. Additionally, any number of reels may be used. For example, it may be desirable to use a single reel for an operation. In some cases, two or more reels may be used. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more reels may be used.

In some cases, the detection method may include multi-channel imaging.

Immersion Optical Elements In certain embodiments, a system for using an optical sensor, such as an optical imaging objective, is disclosed herein. The present disclosure provides a system for temperature regulation and management for one or more of the systems or methods of the present disclosure. In some embodiments of one or more of the systems and methods of the present disclosure, an optical imaging objective is used during the detection method. In some cases, the optical imaging objective is immersed in a fluid that contacts the substrate, and the optical imaging objective is in optical communication with the detector. In some embodiments, the substrate functions optimally at a non-ambient temperature (e.g., about 50 degrees Celsius). In some cases, the optical imaging objective may be close to the ambient temperature. In such cases, a substrate operating at a higher temperature (e.g., about 50 degrees Celsius) can contact an objective operating at an ambient temperature (about 20° C.), thereby generating a temperature gradient between the substrate and the optical imaging objective. In some cases, it may be desirable to control the temperature gradient location and the magnitude of the temperature gradient. Thus, methods and systems for temperature regulation are provided herein.

16 is a schematic diagram of an exemplary temperature gradient that may occur between the optical imaging objective and the substrate. The optical imaging objective 1110 (e.g., as described with respect to FIG. 15) may comprise a first element 2810, a second element 2830, a third element 2840, and, optionally, one or more spacers 2820. For example, the first element 2810 may comprise a front lens or a meniscus lens, the second element 2830 may comprise a first lens group, such as a triplet lens group, and the third element 2840 may comprise a second lens group, such as a doublet lens group. Alternatively or additionally, the first element 2810 may comprise a plano-convex lens, the second element 2830 may comprise a meniscus lens, and the third element 2840 may comprise an achromatic lens. The optical imaging objective 1110 may be at ambient temperature. The substrate 310 may be a substrate herein and may include a biological specimen. In some cases, the substrate 310 is heated to a temperature higher than the ambient temperature. In some cases, the temperature difference between the substrate 310 and the optical imaging objective 1110 may generate a temperature gradient 2850. The temperature gradient 2850 may result in heat transfer between the substrate and the optical imaging objective 1110 and the ambient environment. In some cases, it may be desirable to regulate or adjust the temperature of the system or the substrate so that the substrate maintains a constant temperature.

17A-17E are schematic diagrams illustrating exemplary methods for adjusting the temperature of a substrate. FIG. 17A illustrates one embodiment of such a temperature adjustment method of a system. The system can include a substrate 310, which can be any substrate herein, an optical imaging objective 1110 herein, and an immersion fluid 1140. In some embodiments, it is desirable to maintain the substrate 310 at an elevated temperature (e.g., 50 degrees Celsius) while keeping other components of the system (e.g., 2830, 2840, 2820) at ambient temperature. In some cases, heat 2920 can be applied to the substrate 310. The heat can be transferred to other components of the system, such as the immersion fluid 1140 and a portion of the optical imaging objective 1110. In some cases, the first element 2810 of the optical imaging objective 1110 can be robust against large temperature gradients and not critical to the optical path or detection method. In one non-limiting example, the first element 2810 can be a substantially flat (e.g., planar) surface. In such a case, the first element 2810 may be robust to large temperature gradients and may not affect the optical path, detection, or magnification of the substrate or its disposed contents. In some cases, heat 2920 applied to the substrate 310 may be conductively transferred away from the optical imaging objective 1110. For example, heat 2920 applied to the substrate 310 may be transferred to the immersion fluid 1140, to the first element 2810, to one or more spacers 2820, and then toward the outer layer 2930 of the optical imaging objective. The transferred heat may then be transferred convectively away from the optical imaging objective 1110. In some cases, heat may be transferred away from the optical imaging objective, from the substrate 310 to the immersion fluid 1140, to the first element 2810. Heat may be convectively transferred to the second element 2830 and one or more spacers 2820, or may be convectively transferred away from the optical imaging objective 1110. In some embodiments, the thermal resistance of one or more components of the optical imaging objective lens 1110 can be modulated. For example, the outer layer 2930 of the imaging optical imaging objective lens 1110 may be configured to optimally distribute heat (e.g., using brass or low resistance materials, designing thin layers, etc.).

In some embodiments, the method may include heating the immersion fluid. In some cases, the immersion fluid 1140 may be preheated and applied to the substrate 310 so that the substrate maintains an elevated temperature (e.g., 50 degrees Celsius). The immersion fluid may be continuously replenished. For example, the system may include a fluid flow tube (e.g., 1130 in FIG. 15) configured to deliver the immersion fluid into the enclosed system. In such cases, heat may be transferred away from the optical imaging objective by convection and conduction. In some cases, a cooling element 2910a, such as a fan, may be used to move additional heat away from the optical imaging objective, which may move heat (e.g., convectively) away from the optical imaging objective 1110 and reduce the temperature of the components of the optical imaging objective 1110.

17B shows a schematic of another embodiment of a method for adjusting the temperature of the system. The system may include a substrate 310 of the present specification, an optical imaging objective lens 1110 of the present specification, and an immersion fluid 1140. In some embodiments, the immersion fluid 1140 may be heated. In some embodiments, heat 2920 is applied to the substrate 310. In some embodiments, the system includes an insulating spacer 2935 that may be configured to generate an insulating region 2940 including the second element 2830 and the third element 2840 that are insulated from the high temperature region (e.g., the first element 2810, the immersion fluid 1140, and the substrate 310). In such a case, the largest temperature gradient may occur in the space between the first element 2810 and the second element 2830. In some cases, the insulating spacer 2935 may have a higher thermal resistance than glass. In some embodiments, a cooling element 2910a may be used to further cool the optical imaging objective lens 1110. In some embodiments, the first element 2810 may be configured to dissipate heat (e.g., may be thin). In some embodiments, the insulating spacer 2935 may have a higher resistance than the first element 2810 and may reduce heat transfer to the second element 2830 and the third element 2840. Instead of or in addition to the insulating spacer, a gap (e.g., an air gap) may be disposed between the first element 2810 and the remainder of the objective lens 1110. In some embodiments, the first element 2810 may have optical properties that are insensitive to temperature. In some embodiments, the first element 2810 may have zero or very low optical power, for example, a window or a substantially flat (e.g., planar) element, thereby reducing the sensitivity of the first element 2810 to temperature or thermally induced dimensional variations.

17C shows a schematic of another embodiment of a method for adjusting the temperature of the system. The system can include a substrate 310 of the present specification, an optical imaging objective 1110 of the present specification, an immersion fluid 1140, and a heating element 2910b. In some embodiments, the optical imaging objective 1110 can be heated to a desired temperature (e.g., 50 degrees Celsius) or a temperature that matches the desired temperature of the substrate 310. In some cases, a resistive heater can be used for the optical imaging objective. Heating the optical imaging objective can result in heat transfer to the substrate 310. In some cases, heat 2920 can be applied to the substrate 310. In some embodiments, a heating element 2910b can be used to apply heat to the optical imaging objective, for example, by convection.

Figure 17D illustrates a schematic of another embodiment of a method for temperature regulation of a system. The system can include a substrate 310 as described herein, an optical imaging objective 1110 as described herein, and an immersion fluid 1140. In some embodiments, the optical imaging objective 1110 can be cooled. For example, a cooled immersion fluid 1140 can be continuously circulated between the optical imaging objective 1110 and the substrate 310. In some cases, the immersion fluid 1140 can be recycled to minimize reagent usage, as described elsewhere herein. In some embodiments, heat 2920 can be applied to the substrate 310.

17E shows a schematic of another embodiment of a method of temperature regulation of the system. The system may include a substrate 310 as described herein, an optical imaging objective 1110 as described herein, and an immersion fluid 1140. In some embodiments, the optical imaging objective 1110 may be cooled while the substrate 310 is heated. For example, the cooled immersion fluid 1140 may be continuously circulated between the optical imaging objective 1110 and the substrate 310. In some cases, the flow rate of the immersion fluid 1140 may be controlled such that a temperature gradient 2850 exists primarily in the immersion fluid 1140, with the immersion fluid 1140 closer to the substrate being hotter, while the immersion fluid 1140 closer to the optical imaging objective 1110 is cooled. In some cases, the immersion fluid 1140 may be reused to minimize reagent usage, as described elsewhere herein.

As will be appreciated, any combination of mechanisms for temperature regulation and/or adjustment can be used. For example, an optical imaging objective can include (i) an outer layer capable of carrying heat away from the optical imaging objective and (ii) a flat or planar first element having zero or low optical power that is robust to temperature. In some cases, the immersion fluid may be heated in addition to or instead of using an optical imaging objective having a conductive outer layer and/or a flat first element. Similarly, a cooling element can be implemented in any of the methods and systems described. Any suitable combination of temperature modulation methods can be used in conjunction with the systems and methods herein.

In certain embodiments, methods for fluid and bubble control in an optical detection system are also disclosed herein. In some embodiments, an optical imaging objective is used during the detection method. In some cases, the optical imaging objective is immersed in a fluid in contact with the substrate, and the optical imaging objective is in optical communication with a detector. In some cases, the optical imaging objective may include a camera or may be connected to a camera. In some cases, the camera or the optical imaging objective with a camera may be in fluid communication with the substrate. In some embodiments, the optical imaging objective or camera is positioned at a suitable working distance from the substrate. In some cases, the optical imaging objective may be immersed in the fluid. In some embodiments, the optical imaging objective or camera includes an adapter configured to maintain a fluid-filled cavity around the outlet of the optical imaging objective or camera. In some cases, the adapter can enable imaging of the substrate (or its uncoated surface) at a longer working distance. The adapter may be attached to or encase the optical imaging objective or camera. In some cases, the adapter includes a hydrophobic region, such as a region in contact with the immersion fluid. The hydrophobic region can allow fluid to be directed to or remain near the imaging region of the optical imaging objective. For example, the hydrophobic region can be configured to hold a volume of fluid between the optical imaging objective or camera and the imaging region of the substrate (or its uncoated surface). In some cases, the adapter includes a hydrophilic region, such as a region in contact with the immersion fluid. The hydrophilic region can allow fluid to be directed to or remain near the imaging region of the optical imaging objective. For example, the hydrophilic region can be configured to hold a volume of fluid between the optical imaging objective or camera and the imaging region of the substrate (or its uncoated surface). In some cases, the adapter includes both hydrophilic and hydrophobic regions, which can allow fluid to be directed to or remain near the imaging region of the optical imaging objective or camera.

FIG. 19 illustrates an exemplary adapter that may be attached to or accommodate an optical imaging objective. The adapter 3100 may enable imaging of a substrate at a larger working distance (e.g., greater than 500 microns). In some cases, the adapter simulates a shorter working distance by forming a fluid-filled cavity around the optical imaging objective 1110. In some embodiments, the adapter 3100 includes one or more inlet ports 3110 through which an immersion fluid may be dispensed. In some embodiments, the adapter 3100 may also include one or more other ports 3120 (e.g., an outlet port, additional inlet ports, etc.). The fluid may be directed to a cavity 3130 surrounding the optical imaging objective 1110. In some cases, the fluid may be an immersion fluid and may be dispensed onto the substrate 310. In some cases, the adapter 3100 may hold a volume of immersion fluid between the adapter and the substrate 310, for example, via surface tension. The use of an adapter can allow for a larger working distance while maintaining immersion of the optical imaging objective 1110 in the immersion fluid. In some cases, the adapter may include a hydrophobic region that allows the immersion fluid to remain or be directed into the imaging path of the optical imaging objective 1110.

A suitable working distance between the optical imaging objective lens and the substrate may be any suitable distance for imaging the substrate. In some cases, a working distance of 100 to 500 microns (μm) is suitable. For example, a suitable working distance may be 100, 150, 200, 250, 300, 350, 400, 450, 500 microns. In some cases, the working distance may be less than 100 microns. For example, the working distance may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 microns. In some cases, the working distance may be greater than 500 microns. For example, a suitable working distance may be 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microns or more. In some cases, a suitable working distance may be 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 microns or more. In some cases, the optical imaging objective may be a long working distance objective. For example, the optical imaging objective may have a working distance of more than 5, 6, 7, 8, 9, 10, 15, 20, 25 millimeters (mm).

In some cases, the working distance may be small enough so that the immersion fluid can be held (e.g., via surface tension) between the optical imaging objective and the substrate. In some cases, the working distance can be large so that the immersion fluid does not contact the optical imaging objective or the substrate. In some cases, an adapter may be added to the objective that can form a fluid-filled cavity around the objective so that the immersion fluid can be held (e.g., via surface tension) between the optical imaging objective and/or the adapter and the substrate.

In some embodiments, air bubbles may form in the immersion fluid, which may affect the optical and/or detection performance of the system. For example, air bubbles may form in the optical path of an optical imaging objective, which may degrade the imaging, focusing, and performance of the optical path (e.g., laser, LED, transmitted light, etc.). In some cases, it is desirable to prevent air bubble formation and/or remove air bubbles from the optical path. Accordingly, methods and systems are provided herein for preventing air bubble formation and removing air bubbles from the optical path.

Figure 18 shows a schematic of the formation of bubbles in an immersion fluid. The optical imaging objective 1110 of the present specification can be disposed on a substrate 310, such as a rotatable substrate, a planar substrate, and/or any substrate of the present specification. An immersion fluid 1140 is disposed between the optical imaging objective 1110 and the substrate 310, as described herein. In some cases, the immersion fluid may contain bubbles 3010. The bubbles 3010 may occur along the optical path of the optical imaging objective 1110 and may degrade the imaging performance of the detection method.

In some embodiments, the method may include substrate modification to prevent bubble formation. In some cases, the method includes degassing the immersion fluid prior to use for imaging. In some cases, the substrate modification may include immersion lithography. In some cases, a hydrophobic material, such as a resist, may be deposited on the surface of the substrate. Increasing the hydrophobicity of the substrate may increase the contact angle of the fluid on the surface of the substrate and reduce bubble formation.

In some cases, for example in immersion lithography, it may be desirable to minimize exposure of the immersion fluid to the substrate. Thus, the method may include a method for minimizing the area and duration of immersion fluid contact with the substrate. In some embodiments, the method includes a dispense port and a return port for dispensing the immersion fluid onto the substrate and removing the immersion fluid from the substrate, respectively. Return of the fluid may be obtained by various means, such as application of pressure or suction, gravity, centrifugal force, capillary force, electrical force, magnetic force, etc. In some cases, the dispense and return components may be used to minimize the use of reagents (e.g., immersion fluid). In such cases, the immersion fluid may be recycled as described elsewhere herein.

FIG. 21 illustrates a schematic of a method of dispensing and removing an immersion fluid onto a substrate. The substrate 310 may be any substrate herein. The immersion fluid 1140 may include an imaging buffer. In some cases, minimization of the amount of immersion fluid may be desired, or minimization of exposure of the substrate 310 to the immersion fluid 1140 is desired. In some embodiments, the method includes dispensing the immersion fluid 1140 through a dispense port 3210 and withdrawing the immersion fluid 1140 through a withdrawal port 3220. In some cases, the dispense port is located near the optical imaging objective 1110. In some cases, the withdrawal port is located outside, i.e., radially outward, of the optical imaging objective 1110 and the dispense port 3210. In some cases, multiple dispense and withdraw components may be used. As will be appreciated, any number of dispense and withdraw ports may be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more dispense or removal ports can be used. In some embodiments, the number of dispense ports used may not equal the number of removal ports used. In some cases, more dispense ports can be used than removal ports. In other cases, more removal ports are used than dispense ports. In some embodiments, the dispense and removal ports can be part of the adapter 3100 (see FIG. 19).

In some embodiments, the generation of air bubbles can be minimized by controlling the flow rate of the immersion fluid. In some cases, for example, in immersion lithography, the flow rate of the fluid dispense can be optimized. For example, the flow rate of the fluid dispense can be 1 picoliter/min, 10 picoliter/min, 100 picoliter/min, 1 nanoliter/min, 10 nanoliter/min, 100 nanoliter/min, 1 microliter/min, 10 microliter/min, 100 microliter/min, 1 milliliter/min, 10 milliliter/min, 100 milliliter/min, or up to 1 liter/min. The flow rate of the fluid dispense can be between any of these flow rates. Alternatively, the flow rate of the fluid dispense can be the maximum or any of these flow rates. The flow rate can be low enough to minimize the generation of air bubbles. In some embodiments, the flow rate can allow air or bubbles to rise above the objective lens and out of the optical path.

In some embodiments, the method can include dispensing a fluid onto a substrate and then using an optical imaging objective to displace the air bubble. FIGS. 20A-20B show a schematic of a method for displacing an air bubble. In FIG. 20A, an immersion fluid 1140 can be dispensed onto a substrate 310 as described herein. The immersion fluid 1140 can include an air bubble 3010. In FIG. 20B, the optical imaging objective 1110 can be contacted with the immersion fluid 1140 to displace the air bubble 3010. In some embodiments, the optical imaging objective 1110 can be fitted with an adaptor 3100 (not shown). In some cases, the adaptor 3100 can include multiple dispense and return ports. In such cases, the dispense or return ports can be used to draw fluid (e.g., via pressure differential, capillary forces, etc.) into the adaptor and thus away from the optical imaging objective.

In some embodiments, the method may include using an adapter to prevent bubble formation or to capture or trap bubbles. The adapter may be attached to an optical imaging objective lens as described herein. In some cases, the adapter may interface with an immersion fluid. In some cases, the adapter includes a dispensing port through which the immersion fluid may be dispensed onto the substrate. In some embodiments, the surface of the adapter that contacts the immersion fluid may be flat. In some cases, a thin layer of glass may be disposed between the optical imaging objective lens and the substrate to form a closed cavity to minimize bubble formation. In such an embodiment, a thin layer of glass may be disposed between the objective lens and the wafer to form a closed cavity. The closed cavity may be filled with bubble-free immersion fluid. At the other end of the thin layer of glass, a fluid may be introduced between the thin layer of glass and the substrate.

In some embodiments, an adapter can be used to remove air bubbles from the immersion fluid. In some cases, the adapter includes one or more dispense and/or return ports. In some embodiments, the dispense port can be used to rapidly flow the immersion fluid onto the substrate, thereby breaking or destroying larger air bubbles into smaller bubbles, which may be removed or destroyed by a separate mechanism. A fast flash can also push the air bubbles out of the adapter or out of the optical imaging objective.

In some embodiments, the adapter may include a port that can be used to remove air bubbles. For example, an aspiration (i.e., negative pressure) port can be located in the adapter that can be attached to an optical imaging objective. In some embodiments, the aspiration port can be used to remove nearby air bubbles. In other cases, the adapter may include a dispense port to quickly dispense fluid onto the substrate to move the air bubbles toward another area of the substrate. In some cases, the adapter may include an aspiration port to aspirate the air bubbles. As will be appreciated, any combination of adapter features (e.g., dispense ports, collection ports, aspiration ports) can be used.

In some embodiments, the adapter may be flat relative to the plane where the adapter contacts the immersion fluid, as shown, for example, in FIG. 22A. In some embodiments, the adapter may be convex along the plane or area where the adapter contacts the immersion fluid, as shown, for example, in FIG. 22B. In some cases, the bottom surface of the adapter may contact the immersion fluid and may be partially inclined, for example, in a conical shape. The inclined shape may reduce the contact area between the immersion fluid and the adapter. In some cases, the inclined shape may guide or direct the fluid into the optical path. In some cases, the optical imaging objective may be the portion closest to the substrate and/or immersion fluid. In some embodiments, the adapter may be asymmetric in shape to reduce the area of the adapter in contact with the immersion fluid.

In some embodiments where the adapter is tilted, the angle between the adapter and the immersion fluid may be any suitable angle. The angle may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 degrees. In some cases, the angle may be up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 degrees. In some cases, the angle may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 degrees. In some cases, the angle may be a non-integer angle.

In some embodiments, the adapter can include a trap that can capture and remove air bubbles from the optical path. For example, the adapter can include a cavity that can direct air bubbles into an interior region of the adapter. Alternatively, the cavity can be connected to an exit port that allows the air bubbles to break down or be removed.

22A-22B show a schematic of a method for trapping an air bubble. FIG. 22A shows an exemplary adapter 3100 of the present specification that accommodates an optical imaging objective lens 1110 of the present specification. The adapter 3100 may be flat or may be sloped (see FIG. 22B). The adapter 3100 may be in contact with the immersion fluid 1140 that may contain an air bubble 3010. The adapter 3100 may include a cavity that may trap the entrained air bubble 3010. In some cases, the air bubble may break, collapse, or burst within the cavity. In other cases, the cavity may be connected to a port (not shown). In FIG. 22B, the adapter may have a sloped bottom, which may reduce the contact area between the immersion fluid 1140 and the adapter 3100. The angle θ may be any suitable or useful angle.

In some cases, one or more components of the system may be moved (e.g., translated) to remove the air bubbles. In one non-limiting example, the optical imaging objective may be moved vertically away from the substrate and then repositioned in the imaging position, thereby allowing the entrained air bubbles to be displaced and/or broken up. In some cases, the entrained air bubbles may be displaced and/or broken up by moving the substrate relative to the objective. In another non-limiting example, the substrate may be moved in a plane, for example in a circular or linear motion (e.g., as shown in Figures 23A-23J). In some cases, the motion of the substrate may generate shear and velocity fields that displace and/or break up the air bubbles. In some cases, a combination of motion planes may be used. For example, either the optical imaging objective or the substrate, or both, may be moved in both vertical and planar directions. At any step of the motion, immersion fluid may be dispensed onto the substrate.

In some embodiments, the immersion fluid can be recovered and reused (or recirculated). In some cases, the immersion fluid may be treated prior to recycling or recirculation. Treatment may include removal of debris, removal of analytes (e.g., nucleotides, proteins, lipids, carbohydrates, etc.), removal of beads, or any other contaminants. Treatment may include degassing, debubbling, or removal of entrained air. As will be appreciated, any treatment may include any combination of these processes in any convenient order.

Optical Layout The present disclosure provides an optical system designed to implement the methods of the present disclosure. Figure 41 shows an exemplary optical system that can be used to scan a substrate, such as a rotating substrate, as disclosed herein. The optical system can include one or more separate optical paths. The one or more optical paths can include a mirrored optical layout. In some embodiments, the optical system can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 different optical paths. For example, the optical system can include two separate optical paths, as shown in Figure 41.

The optical path may include an excitation path and an emission path. The excitation path and the emission path may each comprise a plurality of optical elements in optical communication with the substrate. In some embodiments, the excitation path includes one or more of an excitation light source, a beam expander element, a line shaper element, a dichroic, and an objective lens. In some embodiments, the emission path may include one or more of an objective lens, a dichroic, a tube lens, and a detector. The objective lens in the excitation path may be the same as the objective lens in the emission path. The objective lens may be an immersion objective lens, or the objective lens may be an air objective lens. In some embodiments, the objective lens is immersed in water, a buffer, an aqueous solution, an oil, an organic solvent, a refractive index matching fluid, or other immersion fluid. The objective lens may be a 10x, 20x, 50x, or 100x objective lens.

The dichroic in the excitation path may be the same as the dichroic in the emission path. The dichroic may be a short-pass or long-pass dichroic. In some embodiments, the dichroic passes the excitation light and reflects the emission light. In other embodiments, the dichroic reflects the excitation light and passes the emission light. The dichroic may have a cutoff wavelength of about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1000 nm, about 1050 nm, or about 1100 nm. Dichroics are 250nm to 300nm, 300nm to 350nm, 350nm to 400nm, 400nm to 450nm, 450nm to 500nm, 500nm to 550nm, 550nm to 600nm, 600nm to 650nm, 650nm to 700nm, 700nm to 750nm, 750nm to 800nm, 800nm to 850nm, 850nm to 900nm, 9 It may have a cutoff wavelength of 00 nm to 950 nm, 950 nm to 1000 nm, 1000 nm to 1050 nm, 1050 nm to 1100 nm, 250 nm to 400 nm, 350 nm to 500 nm, 450 nm to 600 nm, 550 nm to 700 nm, 650 nm to 800 nm, 750 nm to 900 nm, 850 nm to 1000 nm, or 950 nm to 1100 nm.

The excitation light source may be configured to emit light, for example coherent light. The excitation light source may comprise one or more light emitting diodes (LEDs). The excitation light source may include one or more lasers. The excitation light source may comprise one or more single mode laser sources. The excitation light source may comprise one or more multimode laser sources. The excitation light source may comprise one or more laser diodes. The laser may be a continuous wave laser or a pulsed laser. The light beam emitted by the laser may be a Gaussian or nearly Gaussian beam, and the beam may be manipulated using one or more optical elements (e.g., mirrors, lenses, prisms, wave plates, etc.). For example, the beam may be collimated. In some cases, the beam may be manipulated to provide a laser line (e.g., using one or more Powell lenses or cylindrical lenses). The excitation light source may be coupled to an optical fiber.

The line shaper may be configured to expand the excitation light source along one axis, for example as shown in FIGS. 11A and 11B. The line shaper may comprise one or more lenses. In some embodiments, the line shaper includes one or more cylindrical lenses. The one or more cylindrical lenses may be convex cylindrical lenses, concave cylindrical lenses, or any combination thereof. In some embodiments, the line shaper is disposed on a rotation mount, for example a motorized rotation mount. The rotation mount may be configured to rotate the expanded excitation light source about a central axis without substantially shifting the center point of the excitation light source. In some embodiments, the line shaper element may be configured to rotate about the central axis in response to, simultaneously with, or in anticipation of translation of the substrate relative to the optical system. For example, the line shaper element may rotate about the central axis such that the axis of the expanded excitation light maintains a defined orientation relative to the axis of rotation of the substrate as the substrate translates relative to the optical axis in a direction neither directly toward nor away from the axis of rotation.

The beam expander can include one or more lenses. For example, the beam expander may comprise two lenses. The lenses may have different focal lengths. In some embodiments, a lens closer to the excitation light source may have a shorter focal length than a lens further from the excitation light source. The beam expander may be configured to expand the excitation light source by about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, or about 20 times. The beam expander may be configured to collimate the excitation light source. The beam expander may be configured to focus the excitation light source.

The tube lens may include one or more lenses. For example, the tube lens may include two lenses. The lenses may have different focal lengths, or the two lenses may have different focal lengths. The tube lens may be configured to expand the excitation light source by about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, or about 20 times. The tube lens may be configured to collimate the output light. The tube lens may be configured to focus the output light.

The detector may include any combination of a camera (e.g., CCD, CMOS, or line scan), a photodiode (e.g., avalanche photodiode), a photoresistor, a phototransistor, or any other photodetector known in the art. In some embodiments, the detector may include one or more cameras. For example, the camera may include a line scan camera, such as a TDI line scan camera. In some embodiments, the TDI line scan camera may include two or more vertically arranged rows of pixels, as shown with respect to Figures 8A-8D. The detector may be configured to rotate relative to the substrate to correct for tangential velocity blurring, as described herein. In some embodiments, the detector may be configured to rotate in response to, simultaneously with, or in anticipation of translation of the substrate relative to the optical system. For example, the detector may rotate such that the axis of the imaging field of view maintains a prescribed orientation relative to the axis of rotation of the substrate upon translation of the substrate relative to the optical axis in a direction neither directly toward nor away from the axis of rotation. The detector may be configured to rotate simultaneously with rotation of the line shaper element such that the imaging field of view maintains a prescribed orientation relative to the axis of the extended excitation light. The detector may be configured to rotate independently of the line shaper element.

The light path may include additional optical components not shown in FIG. 41. For example, the light path may include additional splitting, reflecting, focusing, magnifying, filtering, shaping, rotating, polarizing, or other optical elements.

One or more optical elements in the optical path may be disposed in a mount. The mount may be a rotational mount. The mount may be a kinematic mount. The mount may be a translational mount. The mount may be a fixed mount. In some embodiments, the mount may have one or more degrees of freedom. For example, the mount may have one or more of one-dimensional translation, two-dimensional translation, three-dimensional translation, one-dimensional rotation, two-dimensional rotation, or three-dimensional rotation.

The optical system of the present disclosure may further include one or more autofocus systems (not shown in FIG. 41). In some embodiments, each optical path in the optical system includes an autofocus system. The autofocus system may include an autofocus illumination source configured to direct an autofocus light through an objective lens to the surface. In some embodiments, the autofocus illumination source may include an infrared (IR) laser, such as a speckle-free IR laser. The autofocus light may pass through one or more optical elements in the optical path. In some embodiments, the optical path includes one or more optical elements for differentially reflecting or combining one or more of the excitation light, the emission light, or the autofocus light. The one or more optical elements may include one or more dichroic elements. The autofocus light may be reflected, refracted, or scattered from the surface toward the autofocus detector. The autofocus detector may be a position-sensitive detector. The autofocus light may coincide with the autofocus detector at discrete positions when the surface is focused on the emission detector (e.g., the camera shown in FIG. 41). The autofocus illumination source and autofocus detector may be configured such that a change in the position of the surface relative to the objective lens results in a change in the position of the autofocus illumination on the autofocus detector. For example, a change in the distance between the surface and the objective lens or a tilt of the surface relative to the objective lens may cause a displacement of the autofocus illumination position on the autofocus detector. The autofocus system may send a signal to the focusing system in response to the change in the position of the autofocus illumination on the autofocus detector. The focusing system may adjust the position of the surface relative to the objective lens such that the position of the autofocus illumination on the autofocus detector returns to a discrete position when the surface is focused on the emission detector.

The optical system of the present disclosure may be aligned such that the excitation light and the exit light pass through approximately the center of the optical element. In some embodiments, the excitation light may be aligned with respect to the line shaper element such that the position of the excitation light after passing through the line shaper does not change substantially upon rotation of the line shaper. The line shaper may be rotated during alignment, and the position of the excitation light source, the line shaper, or both may be adjusted to minimize movement of the position of the excitation light after passing through the line shaper upon rotation of the line shaper. In some embodiments, the position of the detector is aligned with respect to the rotating mount. For example, the detector is centered within the rotating mount by illuminating the center of the detector, rotating the rotating mount, and adjusting the position of the detector within the mount such that the position of the illumination does not move upon rotation of the rotating mount. In some embodiments, the position of the excitation light is aligned at two or more points, thereby defining both a position and an angle. In some embodiments, the position of the exit light is aligned at two or more points, thereby defining both a position and an angle.

One or more imaging heads of the present disclosure may be aligned with respect to the substrate. In some embodiments, the position of one or more imaging heads is adjusted in 0, 1, 2, or 3 translational dimensions (e.g., x, y, and z) and 0, 1, 2, or 3 rotational dimensions (e.g., α, β, and γ). In some embodiments, the position of one or more optical elements may be adjusted in any combination of translational or rotational dimensions. The optical system of the present disclosure may be coarsely aligned at low pump power. The alignment of the optical system of the present disclosure may be precisely aligned at higher pump power. In some embodiments, the alignment of the optical system may change upon increasing pump power. In some embodiments, the optical system may be aligned during one or more of a rotation of the substrate, a translation of the substrate, or a translation of one or more imaging heads. The optical system of the present disclosure may be aligned using any alignment method known in the art.

Computer Control Systems The present disclosure provides computer systems programmed to carry out the methods of the present disclosure. Figure 1 shows a computer system 101 programmed or otherwise configured to sequence a nucleic acid sample. The computer system 101 can coordinate various aspects of the methods and systems of the present disclosure.

The computer system 101 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 105, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 101 also includes a memory or storage location 110 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 115 (e.g., hard disk), a communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage, and/or electronic display adapters. The memory 110, storage unit 115, interface 120, and peripheral devices 125 are in communication with the CPU 105 via a communication bus (solid lines), such as a motherboard. The storage unit 115 may be a data storage unit (or data repository) for storing data. The computer system 101 may be operatively coupled to a computer network ("network") 130 with the aid of the communication interface 120. Network 130 may be the Internet, an Internet and/or an extranet, or an intranet and/or an extranet in communication with the Internet. Network 130 may be a telecommunications and/or data network in some cases. Network 130 may include one or more computer servers that may enable distributed computing, such as cloud computing. Network 130 may implement a peer-to-peer network in some cases with the help of computer system 101, allowing devices coupled to computer system 101 to operate as clients or servers.

The CPU 105 may execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 110. The instructions may be directed to the CPU 105, which may then program or otherwise configure the CPU 105 to perform the methods of the present disclosure. Examples of operations performed by the CPU 105 may include fetch, decode, execute, and writeback.

The CPU 105 may be part of a circuit, such as an integrated circuit. One or more other components of the system 101 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

Storage unit 115 can store files such as drivers, libraries, and saved programs. Storage unit 115 can store user data, such as user preferences and user programs. Computer system 101 can optionally include one or more additional data storage units external to computer system 101, such as located on a remote server in communication with computer system 101 via an intranet or the Internet.

Computer system 101 can communicate with one or more remote computer systems via network 130. For example, computer system 101 can communicate with a user's remote computer system. Examples of remote computer systems include personal computers (such as portable PCs), slate or tablet PCs (such as Apple® iPad, Samsung® Galaxy Tab, etc.), phones, smartphones (such as Apple® iPhone, Android-enabled devices, BlackBerry®, etc.), or personal digital assistants. A user can access computer system 101 via network 130.

The methods herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of computer system 101, such as memory 110 or electronic storage unit 115. Machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by processor 105. In some cases, the code may be retrieved from storage unit 115 and stored in memory 110 for immediate access by processor 105. In some circumstances, electronic storage unit 115 may be eliminated and machine executable instructions may be stored in memory 110.

The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or it may be compiled at run-time. The code may be provided in a programming language that may be selected to allow the code to be executed in a pre-compiled or compiled manner.

Aspects of the systems and methods provided herein, such as the computer system 101, may be embodied in programming. Various aspects of the technology may be considered "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data carried or embodied in some type of machine-readable medium. The machine-executable code may be stored in an electronic storage unit such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memory of a computer, processor, etc., or associated modules such as various semiconductor memories, tape drives, disk drives, etc., that may provide persistent storage at any time for software programming. All or a portion of the software may be communicated over the Internet or various other communication networks. Such communication may, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include optical, electrical, and electromagnetic waves, as used across physical interfaces between local devices, over wired and optical landline networks, and over various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media carrying the software. As used herein, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution, unless limited to persistent, tangible "storage" media.

Thus, a machine-readable medium such as a computer executable code can take many forms, including but not limited to a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any storage device of any computer (or computers), such as may be used to implement the databases shown in the drawings, for example. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire and optical fibers, including the wiring that constitutes a bus in a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, other magnetic media, CD-ROMs, DVDs or DVD-ROMs, other optical media, punch cards paper tapes, any other physical storage media with a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves transmitting data or instructions, cables or links transmitting such waves, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The computer system 101 may include or be in communication with, for example, an electronic display 135 that includes a user interface (UI) 140 for providing nucleic acid sequence information to a user. Examples of UIs include, but are not limited to, a graphical user interface (GUI) or a web-based user interface.

The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented via software when executed by the central processing unit 105.

Numbered Embodiments The following embodiments list non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is considered dependent or related to all preceding or succeeding numbered embodiments, regardless of their listed order. 1. A method for scanning a surface, comprising: (a) scanning a scan field including a portion of the surface using a scanning system, the scan field having a directionality relative to an axis of rotation of the surface; and (b) (i) rotating the surface about the axis of rotation of the surface, and (ii) rotating the scan field about an axis of rotation of the scan field such that the scan field substantially maintains its directionality relative to the axis of rotation of the surface before, during, or after translation of the surface relative to the scan field. 2. The method of embodiment 1, wherein the scan field has a substantially linear shape. 3. The method of embodiment 1, wherein the scan field has a major axis, and the directionality includes a line coinciding with the major axis of the scan field passing through the axis of rotation of the surface. 4. The method of embodiment 1, wherein the scan field follows a circular arc on the surface. 5. The method of embodiment 1, wherein scanning the surface comprises imaging the surface. 6. The method of embodiment 1, wherein the scan field includes an imaging field. 7. The method of embodiment 1, wherein the scan field follows a scan path on the surface, the scan path including an imaging path. 8. The method of embodiment 1, wherein the scanning system comprises an imaging system. 9. The method of embodiment 1, wherein the orientation includes a major axis of the scan field, the major axis being parallel to (i) an axis of rotation of the surface and (ii) a radial line passing through the axis of rotation of the scan field. 10. The method of embodiment 1, wherein the translation of the surface relative to the scan field includes translating in a direction that is not directly toward or away from the axis of rotation of the surface. 11. The method of embodiment 1, wherein the translation of the surface relative to the scan field includes translating along a translation path, and a line including a net movement along the translation path does not intersect both the scan field and the axis of rotation of the surface. 12. The method of embodiment 1, wherein the scan field rotates relative to the surface about the axis of rotation of the scan field. 13. The method of embodiment 12, wherein the axis of rotation of the scan field is approximately perpendicular to the surface. 14. The method of embodiment 12, wherein the axis of rotation of the scan field is approximately parallel to the axis of rotation of the surface. 15. The method of embodiment 12, wherein the axis of rotation of the scan field passes through the axis of symmetry of the scan field. 16. The method of embodiment 1, wherein the scan field is rotated by rotating an objective lens. 17. The method of embodiment 1, wherein the scan field is rotated by rotating a lens. 18. The method of embodiment 1, wherein the scan field is rotated by rotating a prism. 19. The method of embodiment 1, wherein the scan field is rotated by rotating a mirror. 20. The method of embodiment 1, wherein the scan field is rotated by rotating a camera. 21. The method of embodiment 1, wherein the scan field is rotated by rotating a diffractive optical element (DOE). 22. The method of embodiment 1, wherein the scan field is rotated using a motor. 23. The method of embodiment 1, wherein the surface is substantially circular and the scan field is translated along a chord of the surface. 24. The method of embodiment 12, wherein the surface is substantially circular and the axis of rotation of the scan field is translated along a chord of the surface. 25. The method of embodiment 24, wherein the chord does not pass through the axis of rotation of the surface. 26. The method of embodiment 1, wherein the scan field is translated by moving the surface. 27. The method of embodiment 1, wherein the scan field is translated by moving the scanning system. 28. The method of embodiment 1, wherein the scan field follows a circle on the surface. 29. The method of embodiment 1, wherein the scan field follows a spiral on the surface. 30. The method of embodiment 1, wherein the steps of rotating the surface and translating the surface are performed simultaneously. 31. The method of embodiment 1, wherein the translation of the surface is linear with respect to the axis of rotation of the surface. 32. The method of embodiment 1, wherein the translation of the surface is not generally circular with respect to the surface. 33. The method of embodiment 1, wherein the translation of the surface increases or decreases the distance between the axis of rotation of the scan field and the axis of rotation of the surface. 34. The method of embodiment 1, wherein the scanning system comprises an objective lens in optical communication with the surface. 35. The method of embodiment 1, wherein the scanning system comprises a camera. 36. The method of embodiment 1, wherein the scan field is in optical communication with the camera. 37. The method of embodiment 35, wherein the camera is a time delay integration (TDI) camera having a line rate. 38. The method of embodiment 35, wherein the camera is a multi-line TDI camera. 39. The method of embodiment 35, wherein the camera comprises an array of sensors, and the axis of rotation of the scan field passes through the center of the sensor array. 40. The method of embodiment 37, wherein the line rate is set such that the camera captures images as the scan field advances from a first position to a second position along the surface, the second position being adjacent to the first position. 41. The method of embodiment 37, in which the line rate is variable. 42. The method of embodiment 37, in which the line rate is higher when the objective lens is located further from the rotation axis of the surface. 43. The method of embodiment 1, in which the scanning system further comprises a tube lens. 44. The method of embodiment 34, in which the scanning system comprises two objective lenses in optical communication with the surface, namely the objective lens and a second objective lens. 45. The method of embodiment 44, in which the two objective lenses are on the same side of the surface with respect to a plane that is perpendicular to the surface and intersects the rotation axis of the surface. 46. The method of embodiment 44, in which the two objective lenses are on opposite sides of the surface with respect to a plane that is perpendicular to the surface and intersects the rotation axis of the surface. 47. The method of embodiment 44, in which the two objective lenses follow a circular path on the surface. 48. The method of embodiment 47, in which the circular paths are concentric. 49. The method of embodiment 48, in which the objective lens and the second objective lens follow alternating circular paths. 50. The method of embodiment 48, in which the objective lens follows a circular path closer to the axis of rotation and the second objective lens follows a circular path further from the axis of rotation of the surface. 51. The method of embodiment 44, in which the two objective lenses follow individual helical paths on the surface. 52. The method of embodiment 51, in which the helical paths are interleaved. 53. The method of embodiment 51, in which the helical paths are concentric, the objective lens follows a helical path closer to the axis of rotation of the surface and the second objective lens follows a helical path further from the axis of rotation of the surface. 54. The method of embodiment 44, wherein the objective lens follows a first path, the first path having a first width corresponding to the first width of the scanned field, and the second objective lens follows a second path, the second path having a second path width corresponding to the second width of the second scanned field. 55. The method of embodiment 54, wherein the first path width and the second path width overlap by 30% or less, 20% or less, 10% or less, 5% or less, 1% or less. 56. The method of embodiment 34, wherein the scanning system comprises four objective lenses in optical communication with the surface. 57. The method of embodiment 56, wherein the four objective lenses are positioned on the same side of the surface with respect to a plane perpendicular to the surface and intersecting the axis of rotation of the surface. 58. The method of embodiment 56, wherein a first two of the four objective lenses are located on a first side of the surface and a second two of the four objective lenses are located on a second side of the surface opposite the first side with respect to a plane perpendicular to the surface and intersecting the rotation axis of the surface. 59. The method of embodiment 34, wherein the scanning system comprises 5, 6, 7, 8, 9, 10, 11, 12, 13 or more objective lenses in optical communication with the surface. 60. The method of embodiment 1, wherein the surface is rotated at a constant angular velocity. 61. The method of embodiment 35, wherein the camera is configured to take images at a predetermined frequency and the surface is rotated relative to the objective lenses at a variable angular velocity. 62. The method of embodiment 35, wherein the angular velocity is changed such that at the predetermined frequency, the camera takes images when the scan field is at a first position and when the scan field is at a second position, the second position being adjacent to the first position. 63. The method of embodiment 1, further comprising illuminating a portion of the surface defined by the illumination field. 64. The method of embodiment 63, wherein the illumination field is illuminated using a laser. 65. The method of embodiment 63, wherein the illumination field is illuminated using a light emitting diode (LED) or a lamp. 66. The method of embodiment 64, wherein the output of the laser is adjusted to maintain a constant brightness on the surface and/or not to saturate the camera. 67. The method of embodiment 63, wherein the illumination field at least partially overlaps the scan field. 68. The method of embodiment 63, wherein the scan field encompasses the illumination field. 69. The method of embodiment 63, wherein the illumination field has a substantially similar shape to the scan field. 70. The method of embodiment 63, wherein the illumination field is substantially rectilinear in shape. 71. The method of embodiment 63, wherein the illumination field has a major axis. 72. The method of embodiment 63, wherein the scanning system further comprises a plurality of illumination fields. 73. The method of embodiment 72, wherein one or more of the plurality of illumination fields have a shape that is substantially rectilinear. 74. The method of embodiment 63, further comprising rotating the illumination field such that the illumination field maintains a predetermined orientation relative to the axis of rotation of the surface. 75. The method of embodiment 63, in which the illumination field maintains a predetermined orientation relative to the scan field. 76. The method of embodiment 74, in which the predetermined orientation comprises a line coinciding with a major axis of the illumination field passing through the axis of rotation of the surface. 77. The method of embodiment 74, in which the predetermined orientation comprises a major axis of the illumination field parallel to a radial line, the radial line passing through the axis of rotation of the surface and the axis of rotation of the illumination field. 78. The method of embodiment 63, in which the scan field and the illumination field are rotated together. 79. The method of embodiment 71, in which the major axis of the illumination field is parallel to the major axis of the scan field. 80. The method of embodiment 63, in which the illumination field rotates about the axis of rotation of the illumination field. 81. The method of embodiment 80, in which the axis of rotation of the illumination field is approximately perpendicular to the surface. 82. The method of embodiment 80, wherein the rotation axis of the illumination field is approximately parallel to the rotation axis of the surface. 83. The method of embodiment 80, wherein the rotation axis of the illumination field passes through the axis of symmetry of the illumination field. 84. The method of embodiment 80, wherein the rotation axis of the illumination field is the same as the rotation axis of the scan field. 85. The method of embodiment 63, wherein the illumination field is rotated by rotating a lens. 86. The method of embodiment 63, wherein the illumination field is rotated by rotating a diffractive optical element (DOE). 87. The method of embodiment 63, wherein the illumination field is rotated by rotating a prism. 88. The method of embodiment 63, wherein the illumination field is rotated by rotating a mirror. 89. The method of embodiment 63, wherein the illumination field is rotated by rotating a laser. 90. The method of embodiment 63, wherein the illumination field is rotated using a motor. 91. The method of embodiment 1, further comprising the step of scanning a second portion of the surface defined by the second scan field. 92. The method of embodiment 91, wherein the second scan field is scanned using a second scanning system. 93. The method of embodiment 91, wherein the second scanning system comprises a second objective lens in optical communication with the surface. 94. The method of embodiment 93, wherein the second objective lens is focused independently of the first objective lens. 95. The method of embodiment 93, wherein the second objective lens has a fixed position relative to the first objective lens. 96. The method of embodiment 91, wherein the second scan field is directional with respect to the axis of rotation of the surface. 97. The method of embodiment 84, wherein the second scan field is radially adjacent to the scan field. 98. The method of embodiment 91, wherein the scan field and the second scan field have the same orientation with respect to the axis of rotation of the surface. 99. The method of embodiment 91, wherein the second scan field is rotated independently of the scan field such that the second scan field maintains its orientation with respect to the axis of rotation of the surface. 100. The method of embodiment 91, wherein the second scan field is rotated in coordination with the scan field. 101. The method of embodiment 93, wherein the first objective lens and the second objective lens are part of a scanning module, and the scanning module is translated relative to the surface along a line extending radially from the axis of rotation of the surface. 102. The method of embodiment 93, wherein the surface is substantially circular, and at least one of the first objective lens or the second objective lens is not translated along a chord passing through the axis of rotation of the surface. 103. The method of embodiment 93, wherein the first objective lens and the second objective lens are on the same side of the surface with respect to a plane perpendicular to the surface and intersecting the axis of rotation of the surface, and both the first objective lens and the second objective lens are translated together toward or away from the axis of rotation of the surface. 104. The method of embodiment 93, wherein the first objective lens and the second objective lens are on opposite sides of the surface with respect to a plane perpendicular to the surface and intersecting the axis of rotation of the surface. 105. The method of embodiment 104, wherein (i) the first objective lens is translated towards the axis of rotation of the surface when the second objective lens is translated away from the axis of rotation of the surface, or (ii) the first objective lens is translated away from the axis of rotation of the surface when the second objective lens is translated towards the axis of rotation of the surface. 106. The method of embodiment 93, wherein the surface is substantially circular and the first objective lens and the second objective lens are translated along parallel chords on either side of a plane that is perpendicular to the surface and intersects the axis of rotation of the surface and is equidistant from the axis of rotation of the surface. 107. The method of embodiment 1, wherein the surface is mounted on a rotation module. 108. The method of embodiment 107, wherein the rotation module is translated relative to the scanning system. 109. The method of embodiment 107, wherein the rotation module is stationary and the scanning module is translatable. 110. The method of embodiment 107, wherein the scanning module is stationary and the rotation module is translatable. 111. The method of embodiment 107, wherein the rotating module is mounted to a track. 112. The method of embodiment 1, wherein the scanning module is mounted to the scanning module track. 113. The method of embodiment 112, wherein the scanning module track is linear. 114. The method of embodiment 107, wherein a plurality of surfaces are mounted to a plurality of rotating modules, and the plurality of rotating modules are mounted to a stage, and the stage is rotated to bring each of the rotating modules into optical communication with the scanning module. 115. The method of embodiment 107, wherein after scanning the surface, the rotating module is moved to the chemistry module. 116. The method of embodiment 107, further comprising translating the second rotating module so that a second surface is in optical communication with the scanning module. 117. The method of embodiment 1, wherein the surface comprises an array of nucleic acid colonies. 118. The method of embodiment 117, wherein the nucleic acid colonies are labeled with a fluorophore. 119. The method of embodiment 117, wherein the intensity of the fluorophores indicates the sequence of the nucleic acid colonies. 120. The method of embodiment 1, wherein a laser excites the fluorophores at a first wavelength and a camera detects emission from the fluorophores at a second wavelength. 121. The method of embodiment 1, wherein a laser illuminates the illumination field and a camera scans the scan field. 122. The method of embodiment 1, wherein two or more of the scanning, the rotation of the surface, the rotation of the scan field, and the translation are performed simultaneously. 123. The method of embodiment 1, wherein three or more of the scanning, the rotation of the surface, the rotation of the scan field, and the translation are performed simultaneously. 124. The method of embodiment 1, wherein the scanning, the rotation of the surface, the rotation of the scan field, and the translation are performed independently. 125. The method of embodiment 1, further comprising repeating steps (a) and (b). 126. 126. The method of embodiment 125, wherein steps (a) and (b) are repeated for each base in a nucleic acid polymerization reaction, thereby sequencing the nucleic acid.

127. A scanning system comprising: a surface configured to rotate about an axis of rotation of the surface; a detector in optical communication with the surface, the detector having a scan field of view comprising a first portion of the surface; and an illumination source configured to illuminate an illumination area comprising a second portion of the surface, the illumination area and the scan field of view at least partially overlapping, the detector configured to maintain an orientation of the scan field of view relative to the axis of rotation of the surface during (i) rotation of the surface about the axis of rotation and (ii) translation of the surface relative to the scan field of view. 128. The scanning system of embodiment 127, wherein the scan field of view follows an arc on the surface. 129. The scanning system of embodiment 127, wherein scanning the surface comprises imaging the surface. 130. The scanning system of embodiment 127, wherein the scan field of view comprises an imaging field of view. 131. The scanning system of embodiment 127, wherein the scan field of view follows a scan path along the surface, the scan path comprising an imaging path. 132. The scanning system of embodiment 127, wherein the scanning system comprises an imaging system. 133. The scanning system of embodiment 127, wherein the detector comprises a line scan camera. 134. The scanning system of embodiment 133, wherein the line scan camera comprises a TDI line scan camera. 135. The scanning system of embodiment 134, wherein the TDI line scan camera images a first scan field on a first camera area. 136. The scanning system of embodiment 135, wherein the TDI line scan camera images a second scan field on a second camera area. 137. The scanning system of embodiment 134, wherein the TDI line scan camera images the first scan field on the first camera area and images the first scan field on the second camera area. 138. The scanning system of embodiment 137, wherein the first camera area and the second camera area detect different wavelengths. 139. The scanning system of embodiment 137, wherein the first camera area and the second camera area detect different dynamic ranges. 140. The scanning system of embodiment 127, wherein the surface is configured to translate along a translation axis relative to the scan field. 141. The scanning system of embodiment 140, wherein the translation axis intersects the rotation axis of the surface and the center point of the scan field. 142. The scanning system of embodiment 140, wherein the translation axis does not intersect the rotation axis of the surface and the center point of the scan field. 143. The scanning system of embodiment 142, wherein the orientation of the scan field changes from a first orientation to a second orientation relative to the rotation axis of the surface upon translation of the surface. 144. The scanning system of embodiment 143, wherein the scan field is configured to rotate about the rotation axis of the scan field relative to the rotation axis of the surface to correct the orientation of the scan field from the second orientation to the first orientation relative to the rotation axis of the surface. 145. The scanning system of embodiment 144, wherein the scan field is configured to rotate by rotating the objective lens. 146. The scanning system of embodiment 144, wherein the scan field is configured to rotate by rotating the lens. 147. The scanning system of embodiment 144, wherein the scan field is configured to rotate by rotating a prism. 148. The scanning system of embodiment 144, wherein the scan field is configured to rotate by rotating a mirror. 149. The scanning system of embodiment 144, wherein the scan field is configured to rotate by rotating a detector. 150. The scanning system of embodiment 144, wherein the scan field is configured to rotate by rotating a diffractive optical element (DOE). 151. The scanning system of embodiment 127, wherein the illumination source comprises a laser or a light emitting diode (LED). 152. The scanning system of embodiment 127, wherein the illumination source comprises a substantially circular illumination profile. 153. The scanning system of embodiment 127, wherein the substantially circular illumination profile is expanded along a single axis. 154. The scanning system of embodiment 153, wherein the substantially circular illumination profile is expanded along a single axis using a cylindrical lens. 155. The scanning system of embodiment 153, further comprising a plurality of illumination sources having a substantially circular illumination profile, the substantially circular illumination profile being magnified along a single axis.156. The scanning system of embodiment 127, wherein the illumination sources pass through a grating.157. The scanning system of embodiment 127, wherein the first portion of the surface is configured to move relative to the scan field.158. The scanning system of embodiment 157, wherein the first region of the first portion of the surface is configured to move at a first speed relative to the scan field and the second region of the first portion of the surface is configured to move at a second speed relative to the scan field.159. The scanning system of embodiment 158, wherein the first region is closer to the axis of rotation of the surface than the second region, and the first speed is slower than the second speed.160. The scanning system of embodiment 158, wherein an image of the first region is magnified by a first factor on the detector and an image of the second region is magnified by a second factor on the detector.161. The scanning system of embodiment 160, wherein the first magnification and the second magnification are different.162. The scanning system of embodiment 161, further comprising a lens whose lens axis is located in an optical path between the scan field and the detector, the lens axis not being perpendicular to the surface.163. The scanning system of embodiment 127, further comprising an objective lens located in an optical path between the scan field and the detector.164. The scanning system of embodiment 163, wherein the objective lens is in fluid contact with the surface.165. The scanning system of embodiment 163, wherein the objective lens and the surface are at different temperatures.166. The scanning system of embodiment 163, further comprising a temperature gradient across a fluid in contact with the surface and the objective lens.167. The scanning system of embodiment 166, wherein the objective lens comprises an insulating spacer in contact with the fluid.168. The scanning system of embodiment 167, wherein the insulating spacer comprises an air gap.169. The scanning system of embodiment 163, wherein the objective lens is heated to reduce the temperature gradient. 170. The scanning system of embodiment 163, wherein the objective lens is cooled to increase the temperature gradient. 171. The scanning system of embodiment 163, wherein the fluid is configured to exchange during rotation. 172. The method of embodiment 1, further comprising: (i) scanning a focal region of the surface using an autofocus system to generate a focal map of the focal region; and (ii) adjusting the focus of the surface relative to the scanning system based on the focal map while scanning the scan field. 173. The method of embodiment 172, wherein the surface rotates about the axis of rotation of the surface relative to the scan field while scanning a focal region of the surface using the autofocus system. 174. The method of embodiment 172, wherein the focal region comprises the scan field. 175. The method of embodiment 172, wherein the focal region comprises a field adjacent to the scan field. 176. The method of embodiment 175, wherein the focal region does not comprise the scan field. 177. The method of embodiment 172, wherein the focal region is scanned prior to scanning. 178. The method of embodiment 172, wherein the focal region is scanned during the scan.179. The scanning system of embodiment 164, wherein the objective lens is configured to maintain fluid contact with the surface while the surface is rotated about the axis of rotation of the surface relative to the objective lens.180. The scanning system of embodiment 164, wherein the objective lens is configured to move in a direction substantially perpendicular to the surface to move out of fluid contact with the surface and re-enter fluid contact.181. The scanning system of embodiment 180, wherein the objective lens is configured to retain a droplet of fluid attached to the objective lens when the objective lens moves out of fluid contact with the surface.182. The scanning system of embodiment 181, wherein the objective lens is configured to move an air bubble between the surface and the objective lens when the objective lens re-enters fluid contact with the surface.183. The scanning system of embodiment 182, further comprising an adapter attached to the objective lens and configured to facilitate movement of the air bubble.184. The scanning system of embodiment 163, further comprising a chamber surrounding the surface and an objective lens configured to maintain a higher humidity within the chamber compared to outside the chamber.185. The scanning system of embodiment 184, wherein the chamber comprises a reservoir below the surface configured to collect a fluid.186. The scanning system of embodiment 185, wherein the reservoir comprises a fluid level and is configured to maintain a substantially constant fluid level.187. The scanning system of embodiment 186, wherein the reservoir is configured to dispense an amount of fluid approximately equal to an amount of fluid collected by the reservoir.188. The scanning system of embodiment 185, wherein the top of the chamber is held at a first temperature, the objective lens is held at a second temperature, the surface is held at a third temperature, and the reservoir is held at a fourth temperature.189. The scanning system of embodiment 188, wherein the first temperature is higher than the second temperature.190. The scanning system of embodiment 188, wherein the third temperature is lower than the fourth temperature. The scanning system of embodiment 188, wherein the second temperature is higher than the third temperature and lower than the first temperature.

Further Numbered Embodiments
The following embodiments list non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is considered dependent or related to all preceding or succeeding numbered embodiments, regardless of their listed order. 1. A method for sequencing nucleic acid molecules, comprising: a. providing an array of nucleic acid molecules on an uncoated surface; b. dispersing a layer of solution over the uncoated surface at a rate of at least 1 nanoliter/second as measured at a temperature of 25 degrees Celsius, the solution comprising a reagent comprising at least one nucleotide that incorporates into a growing nucleic acid strand that is complementary to a nucleic acid molecule of the nucleic acid molecule array; and c. detecting one or more signals indicative of the nucleotide that is incorporated into the growing nucleic acid strand. 2. The method of embodiment 1, wherein the uncoated surface is exposed to the atmosphere. 3. The method of embodiment 1 or 2, wherein the layer comprises a first surface and a second surface, the first surface contacting the uncoated surface and the second surface contacting a gas. 4. The method of any one of embodiments 1 to 3, wherein the uncoated surface is not a flow cell. 5. The method of any one of embodiments 1 to 4, wherein the uncoated surface does not have a surface facing the uncoated surface. 6. The method of any one of embodiments 1 to 5, wherein the uncoated surface is substantially planar. 7. The method of any one of embodiments 1 to 6, wherein the layer has a thickness on the uncoated surface of less than about 100 micrometers (μm). 8. The method of any one of embodiments 1 to 7, wherein (b) comprises dispersing the solution on the uncoated surface throughout the non-solid gap. 9. The method of any one of embodiments 1 to 8, further comprising repeating (b) with a plurality of different solutions, each solution of the plurality of different solutions being dispersed over the uncoated surface using its own dedicated fluid. 10. The method of any one of embodiments 1 to 9, wherein the layer of solution is dispersed over the uncoated surface by rotating the uncoated surface. 11. The method of any one of embodiments 1 to 10, wherein the uncovered surface is rotated at a first angular velocity that directs the solution along a direction away from the central axis of rotation. 12. The method of any one of embodiments 1 to 11, wherein the solution comprises a fluid that is thixotropic. 13. The method of any one of embodiments 1 to 12, wherein the uncovered surface comprises a rim near the outer edge of the uncovered surface such that the amount of solution that flows beyond the outer edge in (b) is reduced. 14. The method of any one of embodiments 1 to 13, wherein the viscosity of the solution is selected such that less than about 50% of the solution dispensed in (b) flows beyond the outer edge of (b). 15. The method of any one of embodiments 1 to 14, wherein (c) is performed by rotating the uncovered surface at a second angular velocity while the uncovered surface is in close proximity to a camera. 16. The method of any one of embodiments 1 to 15, wherein the uncovered surface is bendable or flexible. 17. The method of any one of embodiments 1 to 16, wherein the uncovered surface is textured or patterned. 18. The method of any one of embodiments 1 to 17, wherein the layer of solution is distributed over the uncoated surface by passing the uncoated surface through a reservoir of solution and contacting the reservoir of solution. 19. The method of any one of embodiments 1 to 18, wherein (c) is performed by passing the uncoated surface under a camera. 20. The method of any one of embodiments 1 to 19, wherein the uncoated surface is moved through a series of solutions comprising the solution by moving against a plurality of rotating reels. 21. The method of embodiment 20, wherein the series of solutions includes a series of nucleotide solutions having sufficient reagents to incorporate one of the nucleotides (A, T/U, C, or G) into the growing nucleic acid strand. 22. The method of embodiment 21, wherein the uncoated surface is passed through and contacted with a wash solution after each of the nucleotide solutions. 23. The method of embodiment 22, wherein the uncoated surface is imaged after passing through each of the wash solutions. 24. The method of any one of embodiments 1 to 23, wherein the layer of solution is dispersed over the uncoated surface by spraying the solution over the surface. 25. The method of any one of embodiments 1 to 24, wherein the layer of solution is dispersed over the uncoated surface by subjecting the uncoated surface to vibration. 26. The method of any one of embodiments 1 to 25, wherein the layer of solution is dispersed over the uncoated surface by blowing a gas to move a predetermined amount of solution over the uncoated surface. 27. The method of any one of embodiments 1 to 26, wherein the layer of solution is dispersed over the uncoated surface by contacting the solution with a solid surface and moving the solid surface over the uncoated surface. 28. The method of any one of embodiments 1 to 27, wherein the uncoated surface is contained within a housing that surrounds an atmosphere, the atmosphere having a higher humidity than the surrounding atmosphere. 29. The method of any one of embodiments 1 to 28, wherein less than about 50% of the amount of the layer of solution dispersed over the uncoated surface evaporates prior to (c). 30. The method of any one of embodiments 1 to 29, wherein the solution comprises a reagent configured to reduce the evaporation rate of the solution. 31. The method of embodiment 30, wherein the solution comprises glycerol. 32. The method of any one of embodiments 1 to 31, wherein the uncoated surface is maintained at a temperature near the dew point. 33. The method of any one of embodiments 28 to 32, wherein the housing comprises a second surface separate from the uncoated surface, the second surface having a temperature that (i) promotes condensation on the second surface and/or (ii) inhibits condensation on or above the uncoated surface. 34. The method of embodiment 33, wherein the housing comprises walls that are shaped to direct condensation away from the uncoated surface. 35. The method of any one of embodiments 33 to 34, wherein a fluid flows within the housing to direct condensation away from the uncoated surface. 36. The method of any one of embodiments 1 to 35, wherein (c) is performed by a camera in fluid communication with the uncoated surface. 37. The method of embodiment 36, wherein the camera includes an adaptor configured to hold and/or replenish immersion fluid between the camera and the uncoated surface. 38. The method of embodiment 37, wherein the hydrophobicity or hydrophilicity of the adaptor is selected to hold a predetermined amount of fluid between the camera and the uncoated surface. 39. The method of any one of embodiments 36 to 38, further comprising removing one or more air bubbles trapped between the camera and the uncoated surface. 40. The method of any one of embodiments 36 to 39, wherein the camera has a numerical aperture of at least about 0.10. 41. The method of any one of embodiments 36 to 40, wherein the camera detects a single wavelength. 42. The method of any one of embodiments 36 to 41, wherein the camera has an intentional blur. 43. The method of any one of embodiments 1 to 42, further comprising repeating (b) and (c). 44. The method of any one of embodiments 1 to 43, wherein (b) and (c) are repeated for each of the four nucleotide solutions dispersed during (b). 45. 46. The method of any one of embodiments 1 to 45, wherein (b) is repeated at least twice within a period of less than about 30 seconds (s). 47. The method of any one of embodiments 1 to 46, wherein the solution comprises a plurality of nucleotides that are not reversibly terminating nucleotides. 48. The method of any one of embodiments 1 to 47, wherein the solution comprises a plurality of nucleotides that are labeled. 49. The method of embodiment 48, further comprising the step of cleaving the label from one of the plurality of labeled nucleotides after (c). 50. The method of any one of embodiments 1 to 49, wherein the solution comprises a plurality of nucleotides that are not labeled. 51. The method of any one of embodiments 1 to 50, further comprising the step of washing unincorporated nucleotides from the solution from the uncoated surface between (b) and (c). 52. 52. The method of any one of the preceding embodiments, further comprising the step of collecting at least a portion of the solution after (b). 53. The method of any one of the preceding embodiments, further comprising the step of recovering reagents from the solution after (b). 54. The method of any one of the preceding embodiments, wherein the solution comprises a plurality of nucleotides, and at least 50% of the nucleotides are natural nucleotides. 55. The method of any one of the preceding embodiments, wherein the one or more signals are fluorescent signals. 56. The method of any one of the preceding embodiments, wherein the solution comprises a polymerase, and the polymerase is natural. 57. The method of any one of the preceding embodiments, wherein the solution comprises a polymerase, and the polymerase is not replenished after each iteration of (b) and (c). 58. The method of any one of the preceding embodiments, wherein the solution comprises a polymerase, and the polymerase remains attached to the nucleic acid molecules after (c). 59. The method of any one of the preceding embodiments, wherein the array of nucleic acid molecules is attached to an uncoated surface. 60. The method of any one of the preceding claims, wherein the nucleic acids of the array of nucleic acid molecules are attached to beads disposed on an uncoated surface. 61. A method for processing a plurality of nucleic acid samples, comprising the steps of: (a) providing the plurality of nucleic acid samples, the plurality of nucleic acid samples comprising a first nucleic acid sample comprising a first set of nucleic acid molecules and a second nucleic acid sample comprising a second set of nucleic acid molecules, each sample of the plurality of nucleic acid samples having an identifiable sample source; (b) loading the first nucleic acid sample as a first array of the first set of nucleic acid molecules into a first region of a substrate and loading the second nucleic acid sample as a second array of the second set of nucleic acid molecules into a second region of the substrate, the first region being different from the second region; and (c) dispersing a solution across the substrate, the solution dispersing the solution across the first array or the second array. 62. The method of embodiment 61, wherein the nucleic acid sample comprises a nucleic acid molecule attached to a bead. 63. The method of embodiment 61, wherein the determining in (e) is performed without determining the barcode sequence of the nucleic acid molecule of the first array or the second array. 64. The method of embodiment 61, wherein the nucleic acid sample comprises a nucleic acid molecule attached to a bead. 65. The method of embodiment 61, wherein the determining in (e) is performed without determining the barcode sequence of the nucleic acid molecule of the first array or the second array. 66. The method of embodiment 61, wherein the determining in (e) is performed without determining the barcode sequence of the nucleic acid molecule of the first array or the second array. 67. The method of embodiment 61, wherein the nucleic acid sample comprises a nucleic acid molecule attached to a bead. 68. The method of embodiment 61, wherein the determining in (e) is performed without determining the barcode sequence of the nucleic acid molecule of the first array or the second array. 69. The method of embodiment 62, wherein the nucleic acid sample comprises a nucleic acid molecule attached to a bead. 60. The method of embodiment 62, wherein the nucleic acid sample comprises a nucleic acid molecule attached to a bead. 61. The method of embodiment 61, wherein the determining in (e) is performed without determining the barcode sequence of the nucleic acid molecule of the first array or the second array. 62. The method of embodiment 61, wherein the determining in (e) is performed without determining the barcode sequence of the nucleic acid molecule of the first array or the second array. 63. 65. The method of embodiment 61, wherein the first set of nucleic acid molecules and the second set of nucleic acid molecules do not have a barcode sequence indicative of an origin nucleic acid sample. 66. The method of embodiment 61, wherein the analyzing in (e) comprises sequencing the nucleic acid molecules of the first array or the second array. 67. The method of embodiment 66, wherein the solution comprises sufficient reagents to incorporate at least one nucleotide into a growing nucleic acid strand that is complementary to one of the nucleic acid molecules of the first array or the second array. 68. Repeating (c)-(e) with different nucleotides in the solution to provide sequencing information about the nucleic acid molecules.
69. The method of embodiment 61, wherein the plurality of nucleic acid samples comprises n number of nucleic acid samples, and (b) comprises the step of loading the n number of nucleic acid samples into n number of distinct regions of the substrate. 70. The method of embodiment 69, wherein n is at least 3. 71. The method of embodiment 69, wherein n is at least 5. 72. The method of embodiment 69, wherein n is at least 10. 73. The method of embodiment 61, wherein the first nucleic acid sample or the second nucleic acid sample comprises 1000 nucleic acid molecules. 74. The method of embodiment 73, wherein the first nucleic acid sample or the second nucleic acid sample comprises 10000 nucleic acid molecules. 75. The method of embodiment 74, wherein the first nucleic acid sample or the second nucleic acid sample comprises 100000 nucleic acid molecules. 76. 76. The method of embodiment 61, wherein (b) comprises depositing the first nucleic acid sample from a dispenser through an air gap onto the substrate. 77. The method of embodiment 61, wherein (b) comprises depositing the first nucleic acid sample onto the substrate through a closed flow cell. 78. The method of embodiment 61, wherein the first region and the second region have different sizes. 79. The method of embodiment 61, wherein the first region and the second region have the same size. 80. The method of embodiment 61, wherein the first region and the second region comprise different numbers of individually addressable locations on the substrate. 81. The method of embodiment 61, wherein the first region and the second region comprise the same number of individually addressable locations on the substrate. 82. The method of embodiment 61, wherein following (b), the first set of nucleic acid molecules are attached to a plurality of beads, and the plurality of beads are immobilized to the substrate. 83. 82. The method of embodiment 82, wherein one bead of the plurality of beads comprises a plurality of nucleic acid molecules attached thereto, the plurality of nucleic acid molecules comprising a colony of nucleic acid molecules. 84. The method of embodiment 83, wherein the colony of nucleic acid molecules is an amplification product derived from a nucleic acid molecule of the first set of nucleic acid molecules. 85. The method of embodiment 83, wherein the plurality of nucleic acid molecules is attached to the beads prior to (b), which comprises dispensing the plurality of beads onto the substrate. 86. The method of embodiment 82, wherein following (b), the second set of nucleic acid molecules is attached to a second plurality of beads, which second plurality of beads are immobilized onto the substrate. 87. The method of embodiment 61, wherein the substrate comprises a plurality of individually addressable locations. 88. The method of embodiment 87, wherein one individually addressable location of the plurality of individually addressable locations is configured to associate with a nucleic acid molecule of the nucleic acid molecules of the first array or the second array. 89. The method of embodiment 88, wherein the individually addressable locations are configured to associate with beads, the beads comprising the nucleic acid molecules attached thereto. 90. The method of embodiment 89, wherein the beads comprise a plurality of nucleic acid molecules comprising the nucleic acid molecules attached thereto. 91. The method of embodiment 90, wherein the plurality of nucleic acid molecules comprises a colony of nucleic acid molecules that is an amplification product derived from the nucleic acid molecules. 92. The method of embodiment 89, wherein the first set of nucleic acid molecules is attached to a first plurality of beads and the second set of nucleic acid molecules is attached to a second plurality of beads, and the first plurality of beads and the second plurality of beads are associated with the plurality of individually addressable locations. 93. The method of embodiment 92, wherein the first plurality of beads and the second plurality of beads are distinguishable. 94. The method of embodiment 93, wherein the first plurality of beads and the second plurality of beads emit signals of different wavelengths. 95. The method of embodiment 93, wherein the first plurality of beads and the second plurality of beads emit signals of different intensities. 96. The method of embodiment 92, further comprising, following (b), subjecting the individually addressable locations not associated with the first and second plurality of beads to conditions sufficient to preclude subsequent association of sample beads with the individually addressable locations not associated with the first and second plurality of beads. 97. The method of embodiment 96, further comprising, following (b), contacting the substrate with a plurality of blank beads such that the individually addressable locations not associated with the first and second plurality of beads are associated with blank beads. 98. The method of embodiment 97, wherein the plurality of blank beads has a higher affinity for the plurality of individually addressable locations than the first or second plurality of beads. 99. The method of embodiment 61, wherein the first and second nucleic acid samples are distinguishable by a fluorescent dye. 100. The method of embodiment 61, wherein the nucleic acid molecules each comprise a synthetic sequence of 6 bases or less in length. 101. The method of embodiment 100, wherein the synthetic sequence is 4 bases or less in length. 102. The method of embodiment 101, wherein the synthetic sequences are 2 bases or less in length. 103. The method of embodiment 102, wherein the synthetic sequences are 1 base or less in length. 104. The method of embodiment 100, wherein the total number of nucleic acid molecules is greater than the total number of unique synthetic sequences. 105. The method of embodiment 100, wherein a subset of nucleic acid molecules from the same nucleic acid sample of the plurality of nucleic acid samples each comprises a common synthetic sequence, which common synthetic sequence is different from the synthetic sequences of other subsets of nucleic acid molecules from different nucleic acid samples. 106. The method of embodiment 61, further comprising rotating the substrate relative to a reference axis of the substrate. 107. The method of embodiment 106, wherein the rotation is performed after the dispersing in (c). 108. The method of embodiment 106, wherein the rotation is performed during the dispersing in (c). 109. The method of embodiment 106, wherein the rotation is performed before the dispersing in (c). 110. The method of embodiment 106, wherein the dispersing in (c) comprises moving the solution from a first location on the substrate to a second location on the substrate due to centrifugal forces from the rotation, the first location and the second location having different radial distances from the reference axis. 111. The method of embodiment 106, wherein the first region and the second region are disposed on the substrate at a distance of at least 1 millimeter (mm) from the reference axis. 112. The method of embodiment 111, wherein the first region and the second region are disposed on the substrate at a distance of at least 1 centimeter (cm) from the reference axis. 113. The method of embodiment 61, wherein the first region and the second region are disposed radially around the substrate relative to a central axis of the substrate. 114. The method of embodiment 113, wherein the substrate comprises a plurality of radially alternating regions, including the first region and the second region, the plurality of radially alternating regions comprising a first set of regions of a first type and a second set of regions of a second type. 115. The method of embodiment 113, wherein the first set of regions is chemically distinct from the second set of regions. 116. The method of embodiment 113, wherein the first set of regions and the second set of regions are separated by a barrier. 117. The method of embodiment 113, wherein the first set of regions and the second type of regions are distinguishable only by a nucleic acid sample loaded into the first set of regions and the second set of regions. 118. The method of embodiment 61, wherein the first region and the second region are immediately adjacent. 119. The method of embodiment 61, wherein the first region and the second region are separated by another region on the substrate. 120. 121. The method of embodiment 61, wherein the first region and the second region overlap. 121. The method of embodiment 61, wherein in (e), the first subset and the second subset do not include a third subset of the nucleic acid molecules of the first array or the second array that is proximately located within 0.5 millimeters (mm) of a boundary between the first region and the second region. 122. The method of embodiment 61, wherein (b) is performed in a first station that is different from a second station in which (c) or (d) is performed. 123. The method of embodiment 61, wherein the substrate comprises a physical boundary, and the physical boundary is used as a reference for spatially indexing the substrate. 124. The method of embodiment 123, wherein the boundary comprises one or more of a depression, a notch, a physical feature, a dye, and an ink on the substrate. 125. The method of embodiment 123, wherein the boundary comprises a control nucleic acid sample. 126. The method of embodiment 61, wherein the first region and the second region are separated by a barrier on the substrate. 127. The method of embodiment 126, wherein the barrier remains fixed to the substrate during (c) or (d). 128. The method of embodiment 127, wherein the barrier remains fixed to the substrate during (c) and (d). 129. The method of embodiment 126, wherein the barrier is removable. 130. The method of embodiment 129, further comprising the step of removing the barrier after (b). 131. The method of embodiment 130, wherein the barrier dissolves. 132. The method of embodiment 130, wherein the barrier evaporates. 133. The method of embodiment 130, wherein the barrier sublimes. 134. The method of embodiment 130, wherein the barrier melts. 135. The method of embodiment 126, wherein the barrier comprises an injection molding guide. 136. 137. The method of embodiment 126, wherein the barrier comprises polyethylene glycol (PEG). 138. The method of embodiment 137, wherein the viscosity varies proportionally with temperature. 139. The method of embodiment 126, wherein the barrier comprises a fluid that is immiscible with a loading solution comprising the first and second nucleic acid samples. 140. The method of embodiment 126, wherein the barrier comprises hydrophobic regions, and the first and second regions comprise hydrophilic regions. 141. The method of embodiment 126, wherein the barrier comprises an air knife. 142. The method of embodiment 61, wherein prior to (b), the substrate is masked with one or more masks such that the substrate comprises a subset of one or more masked regions and a subset of one or more unmasked regions, the subset of one or more unmasked regions comprising the first and second regions. 143. 143. The method of embodiment 142, further comprising masking the substrate with the one or more masks prior to (b). 144. The method of embodiment 142, further comprising unmasking the substrate from the one or more masks following (b) and loading a third nucleic acid sample into a third region of the one or more masked regions. 145. The method of embodiment 61, wherein (b) comprises (i) masking the substrate with the one or more masks such that the substrate comprises a subset of one or more masked regions and a subset of one or more unmasked regions, the subset of one or more unmasked regions comprising the first region and the subset of one or more masked regions comprising the second region, (ii) loading the first nucleic acid sample, (iii) unmasking the substrate from the one or more masks, and (iv) loading the second nucleic acid sample. 146. 146. The method of embodiment 61, wherein (b) comprises contacting the substrate with a first loading fluid comprising the first nucleic acid sample and a second loading fluid comprising the second nucleic acid sample, the first loading fluid and the second loading fluid being immiscible. 147. The method of embodiment 61, wherein (b) comprises loading the first nucleic acid sample and the second nucleic acid sample simultaneously. 148. The method of embodiment 61, wherein (b) comprises loading the first nucleic acid sample and the second nucleic acid sample at separate times. 149. The method of embodiment 148, wherein the first nucleic acid sample is loaded prior to loading the second nucleic acid sample. 150. The substrate is dried between loading the first and second nucleic acid samples.
The method of embodiment 149. 151. The method of embodiment 61, wherein (b) comprises applying a magnetic field to direct the first nucleic acid sample to the substrate. 152. The method of embodiment 151, wherein the magnetic field is applied by one or more magnets. 153. The method of embodiment 151, wherein the first set of nucleic acid molecules is attached to a plurality of magnetic beads. 154. The method of embodiment 151, wherein the loading fluid comprising the first nucleic acid sample comprises a ferrofluid. 155. The method of embodiment 61, further comprising, prior to (b), activating the first region or the second region for loading using temperature. 156. The method of embodiment 61, further comprising, prior to (b), activating the first region or the second region for loading using electromagnetic radiation. 157. The method of embodiment 61, wherein the first region attracts the first nucleic acid sample. 158. 159. The method of embodiment 61, wherein the second region repels the first nucleic acid sample. 160. The method of embodiment 61, further comprising, following (b), washing nucleic acid molecules that are not associated with the first region or the second region from the substrate. 161. The method of embodiment 160, wherein the washing comprises aspiration. 162. 1. A method for processing a plurality of nucleic acid samples, comprising: (a) providing the plurality of nucleic acid samples, the plurality of nucleic acid samples comprising a first nucleic acid sample comprising a first set of nucleic acid molecules and a second nucleic acid sample comprising a second set of nucleic acid molecules; (b) loading the first nucleic acid sample onto a substrate to associate the first set of nucleic acid molecules with a first array of individually addressable locations; (c) imaging the substrate to identify the first array of individually addressable locations; (d) loading the second nucleic acid sample onto a substrate to associate the second set of nucleic acid molecules with a second array of individually addressable locations; (e) imaging the substrate to identify the second array of individually addressable locations; and (f) dispersing the solution across the substrate, 162. The method of embodiment 162, wherein the solution comprises a reagent sufficient to react with the nucleic acid molecules of the first array or the second array, (g) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecules of the first array or the second array, and (h) analyzing the first and second nucleic acid samples based at least in part on (i) the one or more signals and (ii) the location from the first array of individually addressable locations and the second array of individually addressable locations at which the one or more signals are detected, and (1) determining a first subset of the nucleic acid molecules of the first or second array as originating from the first nucleic acid sample, and (2) determining a second subset of the nucleic acid molecules of the first or second array as originating from the second nucleic acid sample. 163. The method of embodiment 162, wherein the analyzing in (h) comprises sequencing the nucleic acid molecules of the first or second array. The method of embodiment 163, wherein said solution comprises sufficient reagents to incorporate at least one nucleotide into a growing nucleic acid strand that is complementary to one of said nucleic acid molecules in said first array or said second array. 165. The method of embodiment 164, further comprising repeating (f)-(h) with different nucleotides in said solution to provide sequencing information about said nucleic acid molecule. 166. The method of embodiment 162, wherein said plurality of nucleic acid samples comprises n number of nucleic acid samples, and (b) comprises loading said n number of nucleic acid samples into n number of distinct regions of said substrate. 167. The method of embodiment 166, wherein n is at least 3. 168. The method of embodiment 166, wherein n is at least 5. 169. The method of embodiment 166, wherein n is at least 10. 170. The method of embodiment 162, wherein said first nucleic acid sample or said second nucleic acid sample comprises 1000 nucleic acid molecules. 171. The method of embodiment 170, wherein the first or second nucleic acid sample comprises 10,000 nucleic acid molecules. 172. The method of embodiment 171, wherein the first or second nucleic acid sample comprises 100,000 nucleic acid molecules. 173. The method of embodiment 162, wherein (b) comprises depositing the first nucleic acid sample onto the substrate through an air gap from a dispenser. 174. The method of embodiment 162, wherein (b) comprises depositing the first nucleic acid sample onto the substrate through a closed flow cell. 175. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations have different sizes. 176. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations have the same size. 177. 178. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations comprise a different number of individually addressable locations on the substrate. 179. The method of embodiment 162, wherein following (b), the first set of nucleic acid molecules are attached to a plurality of beads, and the plurality of beads are immobilized to the substrate. 180. The method of embodiment 179, wherein one bead of the plurality of beads comprises a plurality of nucleic acid molecules attached thereto, the plurality of nucleic acid molecules comprising a colony of nucleic acid molecules. 181. The method of embodiment 180, wherein the colony of nucleic acid molecules is an amplification product derived from a nucleic acid molecule of the first set of nucleic acid molecules. 182. The method of embodiment 180, wherein the plurality of nucleic acid molecules are attached to the beads prior to (b), and (b) comprises dispensing the plurality of beads to the substrate. 183. The method of embodiment 179, wherein following (b), the second set of nucleic acid molecules is attached to a second plurality of beads, and these second plurality of beads are immobilized to the substrate. 184. The method of embodiment 162, wherein the substrate comprises a plurality of individually addressable locations. 185. The method of embodiment 184, wherein one individually addressable location of the plurality of individually addressable locations is configured to associate with a nucleic acid molecule of the nucleic acid molecules of the first array or the second array. 186. The method of embodiment 185, wherein the individually addressable location is configured to associate with a bead, the bead comprising the nucleic acid molecule attached thereto. 187. The method of embodiment 186, wherein the bead comprises a plurality of nucleic acid molecules, the plurality of nucleic acid molecules comprising the nucleic acid molecule attached thereto. 188. The method of embodiment 187, wherein the plurality of nucleic acid molecules comprises a colony of nucleic acid molecules that is an amplification product derived from the nucleic acid molecules. 189. 190. The method of embodiment 189, wherein the first set of nucleic acid molecules is attached to a first plurality of beads, the second set of nucleic acid molecules is attached to a second plurality of beads, and the first and second plurality of beads are associated with the plurality of individually addressable locations. 191. The method of embodiment 190, wherein the first and second plurality of beads emit signals of different wavelengths. 192. The method of embodiment 190, wherein the first and second plurality of beads emit signals of different intensities. 193. The method of embodiment 189, further comprising the step of: subsequent to (b), subjecting the individually addressable locations not associated with the first and second plurality of beads to conditions sufficient to preclude subsequent association of sample beads with the individually addressable locations not associated with the first and second plurality of beads. 194. 195. The method of embodiment 190, further comprising contacting the substrate with a plurality of blank beads such that, following (b), the individually addressable locations not associated with the first and second plurality of beads are associated with blank beads. 196. The method of embodiment 162, wherein the first and second nucleic acid samples are distinguishable by fluorescent dyes. 197. The method of embodiment 162, wherein the nucleic acid molecules each comprise a synthetic sequence no greater than 6 bases in length. 198. The method of embodiment 197, wherein the synthetic sequence is no greater than 4 bases in length. 199. The method of embodiment 198, wherein the synthetic sequence is no greater than 2 bases in length. 200. The method of embodiment 199, wherein the synthetic sequence is no greater than 1 base in length. 201. The method of embodiment 197, wherein the total number of nucleic acid molecules is greater than the total number of unique synthetic sequences. 202. The method of embodiment 197, wherein a subset of nucleic acid molecules from the same nucleic acid sample of the plurality of nucleic acid samples each comprises a common synthetic sequence, which is different from the synthetic sequence of other subsets of nucleic acid molecules from different nucleic acid samples. 203. The method of embodiment 162, further comprising the step of rotating the substrate relative to a reference axis of the substrate. 204. The method of embodiment 203, wherein the rotation is performed after the dispersion in (f). 205. The method of embodiment 203, wherein the rotation is performed during the dispersion in (f). 206. The method of embodiment 203, wherein the rotation is performed before the dispersion in (f). 207. The method of embodiment 203, wherein the dispersion in (f) comprises moving the solution from a first location on the substrate to a second location on the substrate due to centrifugal force from the rotation, the first location and the second location having different radial distances from the reference axis. 208. The method of embodiment 203, wherein the first array of individually addressable locations and the second array of individually addressable locations are disposed on the substrate at a distance of at least 1 millimeter (mm) from the reference axis.209. The method of embodiment 208, wherein the first array of individually addressable locations and the second array of individually addressable locations are disposed on the substrate at a distance of at least 1 centimeter (cm) from the reference axis.210. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations are disposed radially around the substrate relative to a central axis of the substrate.211. The method of embodiment 210, wherein the substrate comprises a plurality of radially alternating arrays of individually addressable locations, including the first array of individually addressable locations and the second array of individually addressable locations, and the plurality of radially alternating arrays of individually addressable locations comprise a first set of regions of a first type and a second set of regions of a second type.212. 213. The method of embodiment 210, wherein the first set of regions is chemically distinct from the second set of regions. 214. The method of embodiment 210, wherein the first set of regions and the second set of regions are separated by a barrier. 215. The method of embodiment 210, wherein the first set of regions and the second type of regions are distinguishable only by a nucleic acid sample loaded into the first set of regions and the second set of regions. 216. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations are directly adjacent. 217. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations are individually addressable on the substrate.
217. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations overlap. 218. The method of embodiment 162, wherein in (h), the first subset and the second subset do not include a third subset of the nucleic acid molecules of the first array or the second array that is proximately located within 0.5 millimeters (mm) of a boundary of the first array of individually addressable locations and the second array of individually addressable locations. 219. The method of embodiment 162, wherein (b) is performed in a first station that is different from a second station in which (f) or (g) is performed. 220. The method of embodiment 162, wherein the substrate comprises a physical boundary, and the physical boundary is used as a reference for spatially indexing the substrate. 221. The method of embodiment 220, wherein the boundary comprises one or more of a depression, a notch, a physical feature, a dye, and an ink on the substrate. 222. The method of embodiment 220, wherein the boundary comprises a control nucleic acid sample. 223. The method of embodiment 162, wherein the first array of individually addressable locations and the second array of individually addressable locations are separated by a barrier on the substrate. 224. The method of embodiment 223, wherein the barrier remains fixed to the substrate during (f) or (g). 225. The method of embodiment 224, wherein the barrier remains fixed to the substrate during (f) and (g). 226. The method of embodiment 223, wherein the barrier is removable. 227. The method of embodiment 226, further comprising the step of removing the barrier after (b). 228. The method of embodiment 227, wherein the barrier dissolves. 229. The method of embodiment 227, wherein the barrier evaporates. 230. The method of embodiment 227, wherein the barrier sublimes. 231. The method of embodiment 227, wherein the barrier melts. 232. The method of embodiment 223, wherein the barrier comprises an injection molded guide. 233. The method of embodiment 223, wherein the barrier comprises polyethylene glycol (PEG). 234. The method of embodiment 223, wherein the barrier comprises a viscous solution. 235. The method of embodiment 234, wherein the viscosity varies proportionally with temperature. 236. The method of embodiment 223, wherein the barrier comprises a fluid that is immiscible with a loading solution comprising the first and second nucleic acid samples. 237. The method of embodiment 223, wherein the barrier comprises a hydrophobic region and the first and second regions comprise hydrophilic regions. 238. The method of embodiment 223, wherein the barrier comprises an air knife. 239. 240. The method of embodiment 162, wherein prior to (b), the substrate is masked with one or more masks such that the substrate comprises a subset of one or more masked regions and a subset of one or more unmasked regions, the subset of one or more unmasked regions comprising the first array of individually addressable locations and the second array of individually addressable locations. 240. The method of embodiment 239, further comprising the step of masking the substrate with the one or more masks prior to (b). 241. The method of embodiment 239, further comprising the steps of unmasking the substrate from the one or more masks and loading a third nucleic acid sample onto a third array of individually addressable locations of the one or more masked regions. 242. 243. The method of embodiment 162, wherein (b) comprises the steps of: (i) masking the substrate with one or more masks such that the substrate comprises one or more subsets of masked regions and one or more subsets of unmasked regions, the subset of one or more unmasked regions comprising the first array of individually addressable locations, and the subset of one or more masked regions comprising the second array of individually addressable locations; (ii) loading the first nucleic acid sample; (iii) unmasking the substrate from the one or more masks; and (iv) loading the second nucleic acid sample. 243. The method of embodiment 162, wherein (b) comprises contacting the substrate with a first loading fluid comprising the first nucleic acid sample and a second loading fluid comprising the second nucleic acid sample, the first loading fluid and the second loading fluid being immiscible. 244. 245. The method of embodiment 162, wherein (b) comprises loading the first and second nucleic acid samples simultaneously. 246. The method of embodiment 245, wherein (b) comprises loading the first and second nucleic acid samples at separate times. 247. The method of embodiment 246, wherein the first nucleic acid sample is loaded prior to loading the second nucleic acid sample. 248. The method of embodiment 162, wherein (b) comprises applying a magnetic field to direct the first nucleic acid sample to the substrate. 249. The method of embodiment 248, wherein the magnetic field is applied by one or more magnets. 250. The method of embodiment 248, wherein the first set of nucleic acid molecules is attached to a plurality of magnetic beads. 251. The method of embodiment 248, wherein the loading fluid comprising the first nucleic acid sample comprises a ferrofluid.252. The method of embodiment 162, further comprising the step of activating the first array of individually addressable locations or the second array of individually addressable locations for loading using temperature prior to (b).253. The method of embodiment 162, further comprising the step of activating the first array of individually addressable locations or the second array of individually addressable locations for loading using electromagnetic radiation prior to (b).254. The method of embodiment 162, wherein the first array of individually addressable locations attracts the first nucleic acid sample.255. The method of embodiment 162, wherein the array of individually addressable locations repels the first nucleic acid sample.256. The method of embodiment 162, wherein the substrate comprises a third array of individually addressable locations that repels the first nucleic acid sample.257. 257. The method of embodiment 162, further comprising the step of washing from said substrate nucleic acid molecules that are not associated with said first array of individually addressable locations or said second array of individually addressable locations following (b). 258. The method of embodiment 257, wherein said washing comprises aspiration. 1. A method for processing a plurality of diffusion samples, comprising the steps of: (a) providing the plurality of nucleic acid samples, each of the plurality of nucleic acid samples comprising a fluorescent dye; (b) separating the plurality of nucleic acid samples into a first set of one or more samples and a second set of one or more samples; (c) loading the first set of one or more samples onto a first set of regions on a substrate, with one sample per region in the first set of regions; (d) imaging the substrate to identify (i) locations within the first set of regions and (ii) locations within a second set of regions on the substrate, wherein the second set of regions is different from the first set of regions with which the first set of one or more samples are associated; and (e) imaging the second set of one or more samples onto the second set of regions on the substrate. 260. The method of embodiment 259, comprising: (a) loading a substrate onto a substrate with one sample per region in the second set of regions; (b) imaging the substrate to identify (i) locations within the first set of regions and (ii) locations within the second set of regions with which the second set of one or more samples are associated; (c) dispersing the solution across the substrate, the solution including sufficient reagents to react with nucleic acid molecules of the first set of one or more samples or the second set of one or more samples; (d) detecting one or more signals indicative of a reaction between the reagents and the nucleic acid molecules; and (e) analyzing each of the plurality of nucleic acid samples based at least in part on (i) the one or more signals and (ii) the location from the first set of regions and the second set of regions where the one or more signals are detected. 261. The method of embodiment 259, wherein the fluorescent dye is attached to a sequencing primer of the nucleic acid molecule of each of the plurality of nucleic acid samples. 262. The method of embodiment 259, further comprising the steps of: (j) loading a primer comprising a label onto the substrate; (ii) subjecting a nucleic acid molecule of one of the plurality of nucleic acid samples to conditions sufficient to interact with the primer; and (iii) detecting the presence of the nucleic acid molecule using the label. 262. The method of embodiment 259, wherein the analyzing in (i) comprises sequencing the nucleic acid molecules of the first set of regions or the second set of regions. 263. The method of embodiment 262, wherein the solution comprises reagents sufficient to incorporate at least one nucleotide into a growing nucleic acid strand that is complementary to one of the nucleic acid molecules of the first set of regions or the second set of regions. 264. The method of embodiment 263, further comprising the steps of repeating (g)-(i) with different nucleotides in the solution to provide sequencing information about the nucleic acid molecule. 265. 265. The method of embodiment 259, wherein the plurality of nucleic acid samples comprises n number of nucleic acid samples, and (c) comprises loading the n number of nucleic acid samples into n number of distinct regions of the substrate. 266. The method of embodiment 265, wherein n is at least 3. 267. The method of embodiment 265, wherein n is at least 5. 268. The method of embodiment 265, wherein n is at least 10. 269. The method of embodiment 259, wherein the nucleic acid sample comprises 1000 nucleic acid molecules. 270. The method of embodiment 269, wherein the nucleic acid sample comprises 10000 nucleic acid molecules. 271. The method of embodiment 270, wherein the nucleic acid comprises 100000 nucleic acid molecules. 272. The method of embodiment 259, wherein (c) comprises depositing the first set of one or more samples from a dispenser through a gap onto the substrate. 273. 274. The method of embodiment 259, wherein (c) comprises depositing the first set of one or more samples onto the substrate via a closed flow cell. 275. The method of embodiment 259, wherein the first set of regions and the second set of regions comprise a different number of individually addressable locations on the substrate. 276. The method of embodiment 259, wherein following (c), the first set of one or more samples are attached to a plurality of beads, and the plurality of beads are immobilized to the substrate. 277. The method of embodiment 276, wherein one bead of the plurality of beads comprises a plurality of nucleic acid molecules attached thereto, the plurality of nucleic acid molecules comprising a colony of nucleic acid molecules. 278. The method of embodiment 277, wherein the colony of nucleic acid molecules is an amplification product derived from a nucleic acid molecule of the first set of nucleic acid molecules. 279. 277. The method of embodiment 276, wherein the plurality of nucleic acid molecules are attached to the beads prior to (c), and (c) comprises dispensing the plurality of beads onto the substrate. 280. The method of embodiment 276, wherein following (c), the second set of nucleic acid molecules are attached to a second plurality of beads, and the second plurality of beads are immobilized on the substrate. 281. The method of embodiment 259, wherein the substrate comprises a plurality of individually addressable locations. 282. The method of embodiment 281, wherein one individually addressable location of the plurality of individually addressable locations is configured to associate with one of the nucleic acid molecules of the first array or the second array. 283.
284. The method of embodiment 283, wherein the individually addressable locations are configured to associate with beads, the beads comprising the nucleic acid molecules attached thereto. 285. The method of embodiment 284, wherein the beads comprise a plurality of nucleic acid molecules comprising the nucleic acid molecules attached thereto. 286. The method of embodiment 283, wherein the plurality of nucleic acid molecules comprises a colony of nucleic acid molecules that is an amplification product derived from the nucleic acid molecules. 287. The method of embodiment 283, wherein the first set of nucleic acid molecules is attached to a first plurality of beads and the second set of nucleic acid molecules is attached to a second plurality of beads, the first plurality of beads and the second plurality of beads being associated with the plurality of individually addressable locations. 288. The method of embodiment 287, wherein the first plurality of beads and the second plurality of beads are distinguishable. 289. The method of embodiment 287, wherein the first and second plurality of beads emit signals of different intensities.290. The method of embodiment 286, further comprising, following (b), subjecting the individually addressable locations not associated with the first and second plurality of beads to conditions sufficient to preclude association of subsequent sample beads to the individually addressable locations not associated with the first and second plurality of beads.291. The method of embodiment 286, further comprising, following (b), contacting the substrate with a plurality of blank beads such that the individually addressable locations not associated with the first and second plurality of beads are associated with blank beads.292. The method of embodiment 287, wherein the plurality of blank beads have a higher affinity for the plurality of individually addressable locations than the first or second plurality of beads.293. The method of embodiment 259, wherein the first and second nucleic acid samples are distinguishable by fluorescent dyes.294. 295. The method of embodiment 294, wherein the nucleic acid molecules each comprise a synthetic sequence that is 6 bases or less in length. 296. The method of embodiment 295, wherein the synthetic sequence is 4 bases or less in length. 297. The method of embodiment 296, wherein the synthetic sequence is 1 base or less in length. 298. The method of embodiment 294, wherein the total number of nucleic acid molecules is greater than the total number of unique synthetic sequences. 299. The method of embodiment 294, wherein a subset of nucleic acid molecules from a same nucleic acid sample of the plurality of nucleic acid samples each comprise a common synthetic sequence, which common synthetic sequence is different from the synthetic sequences of other subsets of nucleic acid molecules from different nucleic acid samples. 300. The method of embodiment 259, further comprising the step of rotating the substrate relative to a reference axis of the substrate. 301. The method of embodiment 300, wherein the rotation is performed after the dispersing in (g). 302. The method of embodiment 300, wherein the rotation is performed during the dispersing in (g). 303. The method of embodiment 300, wherein the rotation is performed prior to the dispersing in (g). 304. The method of embodiment 300, wherein the dispersing in (g) comprises moving the solution from a first location on the substrate to a second location on the substrate due to centrifugal forces from the rotation, the first location and the second location having different radial distances from the reference axis. 305. The method of embodiment 300, wherein the first set of regions and the second set of regions are disposed on the substrate at a distance of at least 1 millimeter (mm) from the reference axis. 306. The method of embodiment 305, wherein the first set of regions and the second set of regions are disposed on the substrate at a distance of at least 1 centimeter (cm) from the reference axis. 307. The method of embodiment 259, wherein the first set of regions and the second set of regions are disposed radially around the substrate relative to a central axis of the substrate. 308. The method of embodiment 307, wherein the substrate comprises a plurality of radially alternating arrays of individually addressable locations comprising the first set of regions and the second set of regions, the plurality of radially alternating arrays of individually addressable locations comprising a first set of regions of a first type and a second set of regions of a second type. 309. The method of embodiment 307, wherein the first set of regions is chemically distinct from the second set of regions. 310. The method of embodiment 307, wherein the first set of regions and the second set of regions are separated by a barrier. 311. The method of embodiment 307, wherein the first set of regions and the second type of regions are distinguishable only by a nucleic acid sample loaded into the first set of regions and the second set of regions. 312. The method of embodiment 259, wherein the first region and the second region are immediately adjacent. 313. The method of embodiment 259, wherein the first region and the second region are separated by another region on the substrate. 314. The method of embodiment 259, wherein the first region and the second region overlap. 315. The method of embodiment 259, wherein (e) is performed in a first station that is different from a second station in which (g) or (h) is performed. 316. The method of embodiment 259, wherein the substrate comprises a physical boundary, the physical boundary being used as a reference for spatially indexing the substrate. 317. The method of embodiment 316, wherein the boundary comprises one or more of a depression, a notch, a physical feature, a dye, and an ink on the substrate. 318. The method of embodiment 316, wherein the boundary comprises a control nucleic acid sample. 319. The method of embodiment 259, wherein the first region and the second region are separated by a barrier on the substrate. 320. The method of embodiment 319, wherein the barrier remains fixed to the substrate during (g) or (h). 321. The method of embodiment 320, wherein the barrier remains fixed to the substrate during (g) and (h). 322. The method of embodiment 319, wherein the barrier is removable. 323. The method of embodiment 320, further comprising the step of removing the barrier after (g). 324. The method of embodiment 321, wherein the barrier dissolves. 325. The method of embodiment 321, wherein the barrier evaporates. 326. The method of embodiment 321, wherein the barrier sublimes. 327. The method of embodiment 321, wherein the barrier melts. 328. The method of embodiment 319, wherein the barrier comprises an injection molding guide. 329. The method of embodiment 319, wherein the barrier comprises polyethylene glycol (PEG). 330. The method of embodiment 319, wherein the barrier comprises a viscous solution. 331. The method of embodiment 330, wherein the viscosity changes proportionally to temperature. 332. The method of embodiment 319, wherein the barrier comprises a fluid immiscible with a loading solution comprising the first set of one or more samples and the second nucleic acid sample. 333. The method of embodiment 319, wherein the barrier comprises a hydrophobic region and the first region and the second region comprise a hydrophilic region. 334. The method of embodiment 319, wherein the barrier comprises an air knife. 335. The method of embodiment 259, wherein prior to (g), the substrate is masked with one or more masks such that the substrate comprises one or more subsets of masked regions and one or more subsets of unmasked regions, the subsets of one or more unmasked regions comprising the first region and the second region. 336. The method of embodiment 335, further comprising the step of masking the substrate with the one or more masks prior to (g). 337. 338. The method of embodiment 334, further comprising, following (g), unmasking the substrate from the one or more masks and loading a third set of one or more samples onto a third one of the one or more masked regions. 338. The method of embodiment 259, wherein (g) comprises (i) masking the substrate with the one or more masks such that the substrate comprises a subset of one or more masked regions and a subset of one or more unmasked regions, the subset of one or more unmasked regions comprising the first region and the subset of one or more masked regions comprising the second region, (ii) loading the first set of one or more samples, (iii) unmasking the substrate from the one or more masks, and (iv) loading the second set of one or more samples. 340. The method of embodiment 259, wherein (g) comprises contacting the substrate with a first loading fluid comprising the first set of one or more samples and a second loading fluid comprising the second set of one or more samples, the first loading fluid and the second loading fluid being immiscible. 340. The method of embodiment 259, wherein (g) comprises loading the first set of one or more samples and the second set of one or more samples simultaneously. 341. The method of embodiment 259, wherein (g) comprises loading the first set of one or more samples and the second set of one or more samples at separate times. 342. The method of embodiment 341, wherein (g) the first set of one or more samples is loaded prior to loading the second set of one or more samples. 343. The method of embodiment 342, wherein the substrate is dried between loading the first set of one or more samples and loading the second set of one or more samples. 344. 345. The method of embodiment 344, wherein (g) comprises applying a magnetic field to direct said first set of one or more samples to said substrate. 346. The method of embodiment 344, wherein said first set of nucleic acid molecules are attached to a plurality of magnetic beads. 347. The method of embodiment 344, wherein the loading fluid comprising said first set of one or more samples comprises a ferrofluid. 348. The method of embodiment 259, further comprising the step of activating said first set of regions or said second set of regions for loading using temperature prior to (g). 349. The method of embodiment 259, further comprising the step of activating said first set of regions or said second set of regions for loading using electromagnetic radiation prior to (g). 350. The method of embodiment 259, wherein said first set of regions attracts said first set of one or more samples. 351. 352. The method of embodiment 259, wherein said set of regions repels said first set of one or more samples. 353. The method of embodiment 259, further comprising the step of washing from said substrate nucleic acid molecules not associated with said first set of regions or said second set of regions subsequent to (g). 354. The method of embodiment 353, wherein said washing comprises aspiration. 355. 356. A method for processing biological specimens, comprising: (a) moving a substrate through or along a reel, the surface of the substrate comprising an array having the biological specimens fixed thereto; (b) contacting the surface of the substrate with a reservoir comprising a solution, the solution comprising a plurality of probes; (c) subjecting the biological specimen to conditions sufficient to effect a reaction between one of the plurality of probes and the biological specimen, thereby binding the probe to the biological specimen; and (d) detecting one or more signals from the probes bound to the biological specimen, thereby analyzing the biological specimen, the substrate being moved through or along the reel in the same direction for at least two successive cycles of (b)-(d). 357. The method of embodiment 355, further comprising using a recirculation tank. 358. The dimensions of the substrate are determined by the size of the field of view of an imaging system used in (d).
358. The method of embodiment 355, wherein (a) is performed to contact the surface of the substrate with the reservoir. 359. The method of embodiment 355, further comprising the step of moving the substrate through or along a second reel. 360. The method of embodiment 355, further comprising the step of contacting the surface of the substrate with a second reservoir comprising a second solution. 361. The method of embodiment 360, wherein the second solution comprises a wash buffer. 362. The method of embodiment 360, wherein the second solution comprises a second probe, the method further comprising the step of subjecting the biological analyte to conditions sufficient to effect a reaction between the second probe and the biological analyte to bind the second probe to the biological analyte. 363. The method of embodiment 360, further comprising the step of contacting the surface of the substrate with n number of different reservoirs comprising n number of solutions. 364. The method of embodiment 355, further comprising repeating (b)-(d) during said transfer in (a) with additional reservoirs containing different solutions a sufficient number of times to complete analysis of said biological specimen. 365. The method of embodiment 364, wherein said biological specimen is a nucleic acid molecule, and said analysis comprises determining the sequence of said nucleic acid molecule. 366. The method of embodiment 355, wherein said probe comprises an oligonucleotide molecule. 367. The method of embodiment 366, wherein said oligonucleotide molecule comprises 1-10 bases in length. 368. The method of embodiment 366, wherein said oligonucleotide molecule comprises 10-20 bases in length. 369. The method of embodiment 366, wherein said probe comprises a dibase probe. 370. The method of embodiment 355, wherein said probe is labeled. 371. The method of embodiment 355, wherein said biological specimen comprises a nucleic acid molecule. 372. The method of embodiment 371, wherein said analysis comprises identifying the sequence of said nucleic acid molecule. 373. The method of embodiment 371, wherein the plurality of probes comprises a plurality of oligonucleotide molecules. 374. The method of embodiment 373, wherein (c) comprises performing a complementary binding reaction between the probe and the nucleic acid molecule to identify the presence of homology between the probe and the biological analyte. 375. The method of embodiment 371, wherein the plurality of probes comprises a plurality of nucleotides. 376. The method of embodiment 375, wherein (c) comprises subjecting the nucleic acid molecule to a primer extension reaction under conditions sufficient to incorporate at least one nucleotide from the plurality of nucleotides into a growing strand complementary to the nucleic acid molecule. 377. The method of embodiment 375, wherein the plurality of nucleotides comprises a nucleotide analog. 378. The method of embodiment 375, wherein the one or more signals indicate the incorporation of at least one nucleotide. 379. The method of embodiment 355, wherein the detection is performed using a sensor that continuously scans the array. 380. The method of embodiment 379, wherein the sensor linearly scans the array. 381. 382. The method of embodiment 355, further comprising using a tensioning mechanism to move the substrate through or along the reel. 383. The method of embodiment 355, wherein the substrate is textured or patterned. 384. The method of embodiment 355, wherein the array comprises a plurality of individually addressable locations, and the biological analyte is disposed at one individually addressable location of the plurality of individually addressable locations. 385. The method of embodiment 384, wherein the biological analyte is attached to a bead, and the bead is immobilized at the individually addressable location. 386. 386. A system for analyzing a biological analyte, comprising: a substrate containing a biological analyte, the substrate being maintained at or above a first temperature that is higher than an ambient temperature of an environment to which the substrate is exposed; and an optical imaging objective in optical communication with the substrate and exposed to the environment, the optical imaging objective being exposed to a temperature gradient between the first temperature of the substrate and the ambient temperature of the environment, the optical imaging objective comprising a first optical element and a second optical element adjacent to the first optical element, the second optical element being positioned further from the substrate than the first optical element, the first optical element being configured to be at least partially immersed in an immersion fluid in contact with the substrate, the second optical element being in optical communication with the substrate via the first optical element, and the first optical element being configured such that a second temperature of the second optical element is maintained at or below a predetermined threshold. 387. The system of embodiment 386, wherein the first optical element is a window configured to allow optical communication between the substrate and the second optical element. 388. The system of embodiment 387, wherein the window is substantially flat. 389. The system of embodiment 388, wherein the window is flat. 390. The system of embodiment 386, wherein the optical imaging objective comprises one or more spacers between optical elements and an outer layer surrounding the optical elements of the optical imaging objective, and a primary heat flux path from the substrate through the optical imaging objective to the environment includes conductive heat transfer from the substrate to the immersion fluid, to the first optical element, to the one or more spacers, to the outer layer, and convective heat transfer from the outer layer to the environment. 391. The system of embodiment 386, wherein the first temperature is at least 40 degrees Celsius. 392. The system of embodiment 386, wherein the first temperature is at least 50 degrees Celsius. 393. 394. The system of embodiment 386, wherein the first temperature is about 50 degrees Celsius. 395. The system of embodiment 386, wherein the predetermined threshold is an ambient temperature. 396. The system of embodiment 386, wherein the predetermined threshold is at most 30 degrees Celsius. 397. The system of embodiment 386, wherein the predetermined threshold is at most 25 degrees Celsius. 398. The system of embodiment 386, wherein at least 50% of the temperature gradient occurs within the first optical element and at least 70% of the temperature gradient occurs within the first optical element. 399. The system of embodiment 398, wherein at least 90% of the temperature gradient occurs within the first optical element. 400. The system of embodiment 386, wherein at least a portion of the first optical element is at a temperature of at least 40 degrees Celsius. 401. 402. The system of embodiment 386, wherein at least a portion of the first optical element is at a temperature of at least 50 degrees Celsius. 403. The system of embodiment 386, wherein at least a portion of the first optical element is at a temperature of about 50 degrees Celsius. 404. The system of embodiment 386, wherein the first optical element is at a temperature of at most 30 degrees Celsius. 405. The system of embodiment 386, wherein the first optical element is at a temperature of at most 25 degrees Celsius. 406. The system of embodiment 386, wherein the first optical element is at a temperature of about 20 degrees Celsius. 407. The system of embodiment 386, wherein the immersion fluid is maintained at a third temperature such that the substrate is maintained at or above the first temperature and the second temperature of the second optical element is maintained at or below the predetermined threshold. 408. The system of embodiment 407, further comprising a fluid flow unit configured to replenish the immersion fluid in contact with the substrate and the first optical element to maintain the third temperature of the volume of immersion fluid in contact with the substrate. 409. The system of embodiment 407, wherein the third temperature is at least 40 degrees Celsius. 410. The system of embodiment 407, wherein the third temperature is at least 50 degrees Celsius. 411. The system of embodiment 407, wherein the third temperature is about 50° C. 412. The system of embodiment 407, wherein the third temperature is within 5 degrees Celsius of the first temperature. 413. The system of embodiment 407, wherein the third temperature is an ambient temperature. 414. The system of embodiment 407, wherein the third temperature is at most 30 degrees Celsius. 415. The system of embodiment 407, wherein the third temperature is at most 25 degrees Celsius. 416. The system of embodiment 407, wherein the third temperature is at most 20 degrees Celsius. 417. The system of embodiment 386, wherein the optical imaging objective comprises an insulating spacer arranged between the first optical element and the second optical element, the insulating spacer configured to insulate heat transfer from the first optical element and the second optical element. 418. The system of embodiment 417, wherein the insulating spacer has a thermal resistance higher than a thermal resistance of the first optical element. 419. The system of embodiment 386, wherein the optical imaging objective comprises a cooling element configured to reduce a temperature of an outer layer of the optical imaging objective. 420. The system of embodiment 386, further comprising a fluid flow unit configured to dispense the immersion fluid onto the substrate. 421. The system of embodiment 420, wherein the fluid flow unit is configured to dispense the immersion fluid at a rate of less than about 1 milliliter per second. 422. 422. The system of embodiment 421, further comprising a container configured to at least partially surround the optical imaging objective lens with a cavity disposed between the optical imaging objective lens and a wall of the container, and a pressure unit configured to draw into the container a volume of the immersion fluid disposed outside the container after the optical imaging objective lens has come into contact with the immersion fluid. 423. The system of embodiment 422, wherein the dispensing unit is configured to replenish the immersion fluid in contact with the first optical element at a rate of at least 1 nanoliter per second. 424. The system of embodiment 420, wherein the dispensing unit is configured to dispense the immersion fluid onto the substrate before contacting the optical imaging objective lens with the immersion fluid. 425. 426. The system of embodiment 424, further comprising a container configured to at least partially surround the optical imaging objective lens with a cavity disposed between the optical imaging objective lens and a wall of the container, and a pressure unit configured to draw into the container a volume of the immersion fluid disposed outside the container after the optical imaging objective lens comes into contact with the immersion fluid. 426. The system of embodiment 386, further comprising a container configured to at least partially surround the optical imaging objective lens, a surface of the container interfacing with the immersion fluid, the surface being inclined with respect to a surface of the first optical element interfacing with the immersion fluid. 427. The system of embodiment 386, further comprising a casing at least partially surrounding the first optical element, the casing comprising a cavity adjacent to the first optical element, the cavity interfacing with the immersion fluid and configured to direct one or more gas bubbles in the immersion fluid away from the first optical element. 428. The system of embodiment 427, wherein the cavity is annular or surrounds the first optical element. 429. The system of embodiment 427, wherein the first optical element is substantially flat. 430. The system of embodiment 386, further comprising a movement unit operably coupled to the substrate or the optical imaging objective, the movement unit configured to move the substrate relative to the optical imaging objective. 431. The system of embodiment 430, wherein the movement is in a vector including a vertical component substantially perpendicular to a plane of the substrate. 432. The system of embodiment 430, wherein the movement is in a vector including a horizontal component substantially parallel to a plane of the substrate. 433. The system of embodiment 430, wherein the movement is linear. 434. The system of embodiment 430, wherein the movement is non-linear. 435. The system of embodiment 430, wherein the movement unit is configured to move the substrate during dispensing of the immersion fluid onto the substrate. 436. The optical imaging objective further comprises one or more computer processors operatively coupled to the optical imaging objective and the moving unit, the one or more computer processors being
437. The system of embodiment 430, wherein the processor is individually or jointly programmed to (i) instruct the movement unit to move the substrate relative to the optical imaging objective lens during detection of the substrate by the optical imaging objective lens, and (ii) detect one or more signals from the biological analyte using the optical imaging objective lens. 437. The system of embodiment 430, wherein the processor is individually or jointly programmed to (i) instruct the movement unit to move the substrate relative to the optical imaging objective lens during detection of the substrate by the optical imaging objective lens, and (ii) detect one or more signals from the biological analyte using the optical imaging objective lens. 437. The system of embodiment 430, wherein the processor is individually or jointly programmed to (i) instruct the movement unit to move the substrate relative to the optical imaging objective lens during detection of the substrate by the optical imaging objective lens, and (ii) detect one or more signals from the biological analyte using the optical imaging objective lens. 438. The method of embodiment 437, comprising: (a) a first optical element disposed further from the substrate than a first optical element, the first optical element being at least partially immersed in an immersion fluid in contact with the substrate; (c) controlling or maintaining a second temperature of the first optical element to adjust a magnitude or position of the temperature gradient through the optical imaging objective such that a third temperature of the second optical element is maintained below a predetermined threshold; and (d) using the optical imaging objective to detect one or more signals from the biological analyte during movement of the substrate relative to the optical imaging objective. 439. The method of embodiment 438, wherein the first optical element is a window configured to allow optical communication between the substrate and the second optical element. 439. The method of embodiment 438, wherein the window is substantially flat. 440. The method of embodiment 439, wherein the window is flat. 441. 442. The method of embodiment 437, wherein the optical imaging objective comprises one or more spacers between optical elements and an outer layer surrounding the optical elements of the optical imaging objective, and a primary heat flux path from the substrate through the optical imaging objective to the environment includes conductive heat transfer from the substrate to the immersion fluid, to the first optical element, to the one or more spacers, to the outer layer, and convective heat transfer from the outer layer to the environment. 443. The method of embodiment 437, wherein the first temperature is at least 40 degrees Celsius. 444. The method of embodiment 437, wherein the first temperature is at least 50 degrees Celsius. 445. The method of embodiment 437, wherein the predetermined threshold is an ambient temperature. 446. The method of embodiment 437, wherein the predetermined threshold is at most 30 degrees Celsius. 447. The method of embodiment 437, wherein the predetermined threshold is at most 25 degrees Celsius. 448. The method of embodiment 437, wherein the predetermined threshold is about 20 degrees Celsius. 449. The method of embodiment 437, wherein at least 50% of the temperature gradient occurs within the first optical element and at least 70% of the temperature gradient occurs within the first optical element. 450. The method of embodiment 449, wherein at least 90% of the temperature gradient occurs within the first optical element. 451. The method of embodiment 437, wherein at least a portion of the first optical element is at a temperature of at least 40 degrees Celsius. 452. The method of embodiment 437, wherein at least a portion of the first optical element is at a temperature of at least 50 degrees Celsius. 453. The method of embodiment 437, wherein at least a portion of the first optical element is at a temperature of about 50 degrees Celsius. 454. The method of embodiment 437, wherein at least a portion of the first optical element is at ambient temperature. 455. The method of embodiment 437, wherein the first optical element is at a temperature of at most 30 degrees Celsius. 456. The method of embodiment 437, wherein the first optical element is at a temperature of at most 25 degrees Celsius. 457. The method of embodiment 437, wherein the first optical element is at a temperature of about 20 degrees Celsius. 458. The method of embodiment 437, wherein the immersion fluid is maintained at a third temperature such that the substrate is maintained at or above the first temperature and the second temperature of the second optical element is maintained below the predetermined threshold. 459. The method of embodiment 458, further comprising the step of maintaining the third temperature of the volume of the immersion fluid in contact with the substrate using a fluid flow unit configured to replenish the immersion fluid in contact with the substrate and the first optical element. 460. The method of embodiment 458, wherein the third temperature is at least 40 degrees Celsius. 461. The method of embodiment 458, wherein the third temperature is at least 50 degrees Celsius. 462. The method of embodiment 458, wherein the third temperature is about 50 degrees Celsius. 463. The method of embodiment 458, wherein the third temperature is within 5 degrees Celsius of the first temperature. 464. The method of embodiment 458, wherein the third temperature is an ambient temperature. 465. The method of embodiment 458, wherein the third temperature is at most 30 degrees Celsius. 466. The method of embodiment 458, wherein the third temperature is at most 25 degrees Celsius. 467. The method of embodiment 458, wherein the third temperature is at most 20 degrees Celsius. 468. The method of embodiment 437, wherein the optical imaging objective lens comprises an insulating spacer disposed between the first optical element and the second optical element, the insulating spacer being configured to insulate heat transfer from the first optical element and the second optical element. 469. The method of embodiment 468, wherein the insulating spacer has a thermal resistance higher than a thermal resistance of the first optical element. 470. The method of embodiment 437, wherein the optical imaging objective comprises a cooling element configured to reduce a temperature of an outer layer of the optical imaging objective. 471. The method of embodiment 437, further comprising dispensing the immersion fluid onto the substrate using a fluid flow unit. 472. The method of embodiment 471, wherein the fluid flow unit is configured to dispense the immersion fluid at a rate of less than about 1 milliliter per second. 473. The method of embodiment 472, further comprising at least partially surrounding the optical imaging objective with a container comprising a cavity disposed between the optical imaging objective and a wall of the container, and drawing into the container a predetermined amount of the immersion fluid disposed outside the container using a pressure unit after the optical imaging objective has come into contact with the immersion fluid. 474. The method of embodiment 473, wherein the dispensing unit is configured to replenish the immersion fluid in contact with the first optical element at a rate of at least 1 nanoliter per second. 475. The method of embodiment 471, wherein the dispensing unit is configured to dispense the immersion fluid onto the substrate before contacting the optical imaging objective with the immersion fluid. 476. The method of embodiment 475, further comprising at least partially surrounding the optical imaging objective with a container, the container comprising a cavity disposed between the optical imaging objective and a wall of the container, and drawing into the container a predetermined amount of the immersion fluid disposed outside the container using a pressure unit after the optical imaging objective has come into contact with the immersion fluid. 477. The method of embodiment 437, further comprising at least partially surrounding the optical imaging objective with a container, a surface of the container interfacing with the immersion fluid, the surface being inclined with respect to a surface of the first optical element interfacing with the immersion fluid. 478. The method of embodiment 437, further comprising at least partially surrounding the first optical element with a casing, the casing comprising a cavity adjacent to the first optical element, the cavity interfacing with the immersion fluid and configured to direct one or more gas bubbles in the immersion fluid away from the first optical element. 479. The method of embodiment 478, wherein the cavity is annular or surrounds the first optical element. 480. The method of embodiment 478, wherein the first optical element is substantially flat. 481. The method of embodiment 437, wherein a movement unit is operably coupled to the substrate or the optical imaging objective, the movement unit being configured to move the substrate relative to the optical imaging objective. 482. The method of embodiment 481, wherein the movement is in a vector including a vertical component substantially perpendicular to a plane of the substrate. 483. The method of embodiment 481, wherein the movement is in a vector including a horizontal component substantially parallel to a plane of the substrate. 484. The method of embodiment 481, wherein the movement is linear. 485. The method of embodiment 481, wherein the movement is non-linear. 486. The method of embodiment 481, wherein the movement unit is configured to move the substrate during dispensing of the immersion fluid onto the substrate. 487. The method of embodiment 481, further comprising one or more computer processors operably coupled to the optical imaging objective and the movement unit, the one or more computer processors being individually or jointly programmed to (i) instruct the movement unit to move the substrate relative to the optical imaging objective during detection of the substrate by the optical imaging objective, and (ii) detect one or more signals from the biological analyte using the optical imaging objective. 488. 489. A method for storing a substrate with a surface coated with nucleic acid molecules, comprising: (a) providing a substrate having a surface comprising a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules from one or more nucleic acid samples; (b) contacting the substrate comprising the surface comprising the first set of nucleic acid molecules with a second set of nucleic acid molecules under conditions sufficient to provide a treated surface in which at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized to nucleic acid molecules of the second set of nucleic acid molecules, wherein the second set of nucleic acid molecules are not the sample nucleic acid molecules; and (c) storing the substrate having the treated surface for a period of at least one hour. 489. The method of embodiment 488, further comprising the step of removing the nucleic acid molecules of the second set of nucleic acid molecules from the treated surface subsequent to (c). 490. The method of embodiment 489, further comprising the step of using the first set of nucleic acid molecules immobilized on the surface for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of nucleic acid molecules or derivatives thereof, or a combination thereof, following said removal. 491. The method of embodiment 489 or 490, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by enzymatic degradation. 492. The method of embodiment 489 or 490, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by denaturation with a chemical or thermal stimulus. 493. The method of embodiment 492, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface using a chemical stimulus. 494. The method of embodiment 493, wherein the chemical stimulus comprises sodium hydroxide. 495. 496. The method of any one of embodiments 488-495, wherein during storage of the engineered surface, each nucleic acid molecule of the first set of nucleic acid molecules that is hybridized to a nucleic acid molecule of the second set of nucleic acid molecules is not hybridized to other nucleic acid molecules. 497. The method of any one of embodiments 488-496, wherein the engineered surface is stored at a temperature of about 18° C. to about 30° C. 498. The method of any one of embodiments 488-497, wherein the engineered surface is stored for at least 6 hours. 499. The method of embodiment 498, wherein the engineered surface is interpolated for at least 24 hours. 500. The method of embodiment 499, wherein the engineered surface is stored for at least 2 days. 501. The second set of nucleic acid molecules is hybridized to a nucleic acid molecule of the second set of nucleic acid molecules.
502. The method of any one of embodiments 488-501, wherein each nucleic acid molecule of the second set of nucleic acid molecules comprises a sequence substantially complementary to a sequence of the first set of nucleic acid molecules. 503. The method of embodiment 502, wherein the sequence of the first set of nucleic acid molecules comprises at least 6 bases. 504. The method of any one of embodiments 488-503, wherein the nucleic acid molecules of the first set of nucleic acid molecules are immobilized on the surface at independently addressable locations. 505. The method of embodiment 504, wherein the independently addressable locations are substantially planar. 506. The method of embodiment 504 or 505, wherein the independently addressable locations comprise one or more wells. 507. The method of any one of embodiments 488-506, wherein the nucleic acid molecules of the first set of nucleic acid molecules are immobilized on the surface of the substrate according to a predetermined pattern. 508. 509. The method of any one of embodiments 488-508, wherein each nucleic acid molecule of the first set of nucleic acid molecules comprises the same nucleic acid sequence. 510. The method of any one of embodiments 488-509, wherein the first set of nucleic acid molecules comprises one or more different nucleic acid sequences. 511. The method of embodiment 510, wherein the first set of nucleic acid molecules comprises a first subset of nucleic acid molecules comprising a first nucleic acid sequence and a second subset of nucleic acid molecules comprising a second nucleic acid sequence, wherein the first and second nucleic acid sequences are different. 512. The method of embodiment 511, wherein the first subset of nucleic acid molecules and the second subset of nucleic acid molecules both comprise a third nucleic acid sequence. 513. The method of embodiment 512, wherein the third nucleic acid sequence comprises a poly(T) sequence. 514. 515. The method of any one of embodiments 488-513, wherein the second set of nucleic acid molecules comprises DNA nucleotides. 516. The method of any one of embodiments 488-513, wherein the second set of nucleic acid molecules comprises RNA nucleotides. 517. The method of any one of embodiments 488-516, wherein each nucleic acid molecule of the second set of nucleic acid molecules comprises at least 6 bases. 518. The method of any one of embodiments 488-517, wherein the surface of the substrate is substantially planar. 519. The method of any one of embodiments 488-518, wherein the substrate comprises one or more particles immobilized thereon. 520. 521. A method for nucleic acid processing, comprising the steps of: (a) providing a substrate having a processing surface on which a first set of nucleic acid molecules are immobilized, wherein at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized to nucleic acid molecules of a second set of nucleic acid molecules, the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules from one or more nucleic acid samples, the second set of nucleic acid molecules are not the sample nucleic acid molecules, and the substrate with the processing substrate is stored for a period of at least one hour; and (b) removing the nucleic acid molecules of the second set of nucleic acid molecules from the processing surface. 522. The method of embodiment 520, further comprising the step of using the first set of nucleic acid molecules immobilized on the surface for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of nucleic acid molecules or derivatives thereof, or a combination thereof, after (b). 523. 523. The method of any one of embodiments 520-522, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by enzymatic degradation. 524. The method of embodiment 523, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by denaturation by chemical or thermal stimulation. 525. The method of embodiment 524, wherein a chemical stimulation is used to remove the nucleic acid molecules of the second set of nucleic acid molecules from the engineered surface. 526. The method of any one of embodiments 520-525, wherein during storage of the engineered surface, each nucleic acid molecule of the first set of nucleic acid molecules that is hybridized to a nucleic acid molecule of the second set of nucleic acid molecules is not hybridized to other nucleic acid molecules. 527. 528. The method of any one of embodiments 520-526, wherein at least 95% of the nucleic acid molecules of said first set of nucleic acid molecules are hybridized to a nucleic acid molecule of said second set of nucleic acid molecules. 528. The method of any one of embodiments 520-527, wherein said treated surface is stored at a temperature of about 18° C. to about 30° C. 529. The method of any one of embodiments 520-528, wherein said treated surface is stored for a period of at least 6 hours. 530. The method of embodiment 529, wherein said treated surface is stored for a period of at least 24 hours. 531. The method of embodiment 530, wherein said treated surface is stored for a period of at least 2 days. 532. The method of any one of embodiments 520-531, wherein each nucleic acid molecule of said second set of nucleic acid molecules comprises a sequence substantially complementary to a sequence of said first set of nucleic acid molecules. 533. The method of embodiment 532, wherein the sequence of said first set of nucleic acid molecules comprises at least 6 bases. 534. 535. The method of any one of embodiments 520 to 533, wherein the nucleic acid molecules of the first set of nucleic acid molecules are immobilized on the surface at independently addressable locations. 535. The method of embodiment 534, wherein the independently addressable locations are substantially planar. 536. The method of embodiment 534 or 535, wherein the independently addressable locations comprise one or more wells. 537. The method of any one of embodiments 520 to 536, wherein the nucleic acid molecules of the first set of nucleic acid molecules are immobilized on the surface of the substrate according to a predetermined pattern. 538. The method of any one of embodiments 520 to 537, wherein the density of the first set of nucleic acid molecules on the surface is at least 1 million molecules/mm2. 539. The method of any one of embodiments 520 to 538, wherein each nucleic acid molecule of the first set of nucleic acid molecules comprises the same nucleic acid sequence. 540. The method of any one of embodiments 520 to 539, wherein the first set of nucleic acid molecules comprises one or more different nucleic acid sequences. 541. 542. The method of embodiment 541, wherein the first set of nucleic acid molecules comprises a first subset of nucleic acid molecules comprising a first nucleic acid sequence and a second subset of nucleic acid molecules comprising a second nucleic acid sequence, where the first and second nucleic acid sequences are different. 543. The method of embodiment 542, wherein the first subset of nucleic acid molecules and the second subset of nucleic acid molecules both comprise a third nucleic acid sequence. 544. The method of any one of embodiments 520-543, wherein the second set of nucleic acid molecules comprises DNA nucleotides. 545. The method of any one of embodiments 520-543, wherein the second set of nucleic acid molecules comprises RNA nucleotides. 546. The method of any one of embodiments 520-543, wherein the second set of nucleic acid molecules comprises a mixture of RNA and DNA nucleotides. 547. 548. The method of any one of embodiments 520-546, wherein each nucleic acid molecule of the second set of nucleic acid molecules comprises at least 6 bases. 549. The method of any one of embodiments 520-548, wherein the surface of the substrate is substantially planar. 550. The method of any one of embodiments 520-548, wherein the substrate comprises one or more particles immobilized thereon. 551. A kit comprising a substrate comprising an engineered surface, the engineered surface comprising a plurality of pairs of bound nucleic acid molecules, each pair of the plurality of pairs comprising a first nucleic acid molecule of a first set of nucleic acid molecules that is at least partially hybridized to a second nucleic acid molecule of a second set of nucleic acid molecules, the first set of nucleic acid molecules being immobilized on the surface, at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules being paired with a nucleic acid molecule of the second set of nucleic acid molecules, and the nucleic acid molecules of the first set of nucleic acid molecules being configured to capture sample nucleic acid molecules from one or more nucleic acid samples when a nucleic acid molecule of the first set of nucleic acid molecules is not paired with a nucleic acid molecule of the second set of nucleic acid molecules. 551. The kit of embodiment 550, wherein the treated surface is stored for at least 24 hours. 552. The kit of embodiment 551, wherein the treated surface is stored for at least 2 days. 553. The kit of any one of embodiments 550-552, wherein during storage of the treated surface, each nucleic acid molecule of the first set of nucleic acid molecules in each of the plurality of pairs does not hybridize to other nucleic acid molecules. 554. The kit of any one of embodiments 550-553, further comprising a chemical stimulus configured to remove a second nucleic acid molecule from the treated surface. 555. The kit of embodiment 554, wherein the chemical stimulus comprises sodium hydroxide. 556. The kit of any one of embodiments 550-555, wherein at least 95% of the nucleic acid molecules of the first set of nucleic acid molecules are at least partially hybridized to a nucleic acid molecule of the second set of nucleic acid molecules. 557. 558. The kit of any one of embodiments 550 to 556, wherein the engineered surface is stored at a temperature of about 18° C. to about 30° C. 559. The kit of embodiment 558, wherein the second nucleic acid molecule comprises a sequence substantially complementary to a sequence of the first nucleic acid molecule. 559. The kit of embodiment 558, wherein the sequence of the first nucleic acid molecule comprises at least 6 bases. 560. The kit of embodiment 558 or 559, wherein the sequence of the second nucleic acid molecule comprises at least 6 bases. 561. The kit of any one of embodiments 550 to 560, wherein the first nucleic acid molecule and the second nucleic acid molecule comprise the same number of nucleotides. 562. The kit of any one of embodiments 550 to 561, wherein the first nucleic acid molecule and the second nucleic acid molecule comprise a different number of nucleotides. 563. The kit of any one of embodiments 550 to 562, wherein the nucleic acid molecules of the first set of nucleic acid molecules are immobilized on the surface at independently addressable locations. 564. The kit of embodiment 563, wherein the independently addressable locations are substantially planar. 565. The kit of embodiment 563 or 564, wherein the independently addressable locations comprise one or more wells. 566. The kit of any one of embodiments 550 to 565, wherein the density of the first set of nucleic acid molecules on the surface is at least 1 million molecules/mm2. 567. The kit of any one of embodiments 550 to 566, wherein each nucleic acid molecule in the first set of nucleic acid molecules comprises the same nucleic acid sequence. 568. The kit of any one of embodiments 550 to 567, wherein the first set of nucleic acid molecules comprises one or more different nucleic acid sequences. 569. The kit of embodiment 568, wherein the first set of nucleic acid molecules comprises a first subset of nucleic acid molecules comprising a first nucleic acid sequence and a second subset of nucleic acid molecules comprising a second nucleic acid sequence, wherein the first and second nucleic acid sequences are different. 570. 570. The kit of embodiment 571, wherein the third nucleic acid sequence comprises a poly(T) sequence. 572. The kit of any one of embodiments 550-571, wherein the second set of nucleic acid molecules comprises DNA nucleotides. 573. The kit of any one of embodiments 550-571, wherein the second set of nucleic acid molecules comprises RNA nucleotides. 574. The kit of any one of embodiments 550-571, wherein the second set of nucleic acid molecules comprises a mixture of RNA and DNA nucleotides. 575. The kit of any one of embodiments 550-571, wherein each nucleic acid molecule in the second set of nucleic acid molecules comprises at least one nucleotide.
575. The kit of any one of embodiments 550 to 575, wherein the surface of the substrate is substantially planar. 576. The kit of any one of embodiments 550 to 575, wherein the surface of the substrate comprises a plurality of wells. 577. The kit of any one of embodiments 550 to 576, wherein the surface of the substrate comprises one or more particles immobilized thereon. 578. The kit of any one of embodiments 550 to 577, wherein the substrate comprises one or more particles immobilized thereon. 579. 580. A kit comprising a substrate comprising a surface having a first set of nucleic acid molecules immobilized thereon, the first set of nucleic acid molecules being one or more first nucleic acid molecules configured to capture sample nucleic acid molecules from one or more nucleic acid samples, and a solution comprising a second set of nucleic acid molecules, the second set of nucleic acid molecules comprising one or more second nucleic acid molecules that are not the sample nucleic acid molecules, the second set of nucleic acid molecules being selected such that upon contacting the solution with the surface, at least 70% of the one or more first nucleic acid molecules bind to a second nucleic acid molecule of the second set of nucleic acid molecules to generate one or more pairs of bound nucleic acid molecules, each pair of the one or more pairs comprising (i) a first nucleic acid molecule of the first set of nucleic acid molecules and a second nucleic acid molecule of the second set of nucleic acid molecules, and (ii) a section of substantially complementary sequence. 581. The kit of embodiment 579, further comprising a chemical stimulus configured to remove second nucleic acid molecules from the surface. 582. The kit of any one of embodiments 579-581, wherein upon contacting the solution with the surface, at least 90% of the one or more first nucleic acid molecules of the first set of nucleic acid molecules bind to a second nucleic acid molecule of the second set of nucleic acid molecules. 583. The kit of any one of embodiments 579-582, wherein each nucleic acid molecule of the first set of nucleic acid molecules in each pair of the one or more pairs does not hybridize to other nucleic acid molecules. 584. The kit of any one of embodiments 579-583, wherein the section of substantially complementary sequences of each pair of the one or more pairs comprises a first sequence of a first nucleic acid molecule of the one or more first nucleic acid molecules and a second sequence of a second nucleic acid molecule of the one or more second nucleic acid molecules, the first sequence being substantially complementary to the second sequence. 585. The kit of embodiment 584, wherein the first sequence and the second sequence each comprise about 6-20 bases. 586. The kit of any one of embodiments 579-585, wherein a first nucleic acid molecule of the one or more first nucleic acid molecules and a second nucleic acid molecule of the one or more second nucleic acid molecules have the same number of nucleotides. 587. The kit of any one of embodiments 579-586, wherein a first nucleic acid molecule of the one or more first nucleic acid molecules and a second nucleic acid molecule of the one or more second nucleic acid molecules have a different number of nucleotides. 588. The kit of any one of embodiments 579-587, wherein the first set of nucleic acid molecules are immobilized on the surface at independently addressable locations. 589. The kit of embodiment 588, wherein the independently addressable locations are substantially planar. 590. The kit of embodiment 588 or 589, wherein the independently addressable locations comprise one or more wells. 591. 592. The kit of any one of embodiments 579 to 590, wherein the first set of nucleic acid molecules are immobilized on the surface according to a predetermined pattern. 593. The kit of any one of embodiments 579 to 592, wherein the density of the first set of nucleic acid molecules on the surface is at least 1 million molecules/mm2. 594. The kit of embodiment 593, wherein the first set of nucleic acid molecules comprises a first subset of nucleic acid molecules comprising a first nucleic acid sequence and a second subset of nucleic acid molecules comprising a second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence being different. 595. The kit of embodiment 594, wherein the first subset of nucleic acid molecules and the second subset of nucleic acid molecules both comprise a third nucleic acid sequence. 596. The kit of embodiment 595, wherein the third nucleic acid sequence comprises a poly(T) sequence. 597. The kit of any one of embodiments 579 to 596, wherein the second set of nucleic acid molecules comprises DNA nucleotides. 598. The kit of any one of embodiments 579 to 596, wherein the second set of nucleic acid molecules comprises RNA nucleotides. 599. The kit of any one of embodiments 579 to 596, wherein the second set of nucleic acid molecules comprises a mixture of RNA and DNA nucleotides. 600. The kit of any one of embodiments 579 to 599, wherein each nucleic acid molecule in the second set of nucleic acid molecules comprises at least 6 bases. 601. The kit of any one of embodiments 579 to 600, wherein the surface of the substrate is substantially planar. 602. The kit of any one of embodiments 579 to 601, wherein the surface of the substrate comprises a plurality of wells. 603. The kit of any one of embodiments 579 to 602, wherein the substrate comprises one or more particles immobilized thereon. 604. 604. A method for storing a substrate with a surface coated with nucleic acid molecules, comprising: (a) providing a substrate having a surface comprising a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules from one or more nucleic acid samples, each nucleic acid molecule of the first set of nucleic acid molecules comprising a first nucleic acid sequence and a second nucleic acid sequence, the second nucleic acid sequence being substantially complementary to the first nucleic acid sequence; (b) generating a treated surface by subjecting the surface to conditions sufficient to bind the first nucleic acid sequence of a nucleic acid molecule of the first set of nucleic acid molecules to the second nucleic acid sequence of the nucleic acid molecule to provide a fixed hairpin molecule; and (c) storing the substrate with the treated surface for a period of at least one hour. 605. The method of embodiment 604, further comprising the step of separating the second sequence from the first sequence of the fixed hairpin molecule subsequent to (c). The method of embodiment 605, wherein the separation comprises enzymatic degradation or denaturation using a chemical or thermal stimulus.607. The method of embodiment 606, wherein the chemical stimulus comprises sodium hydroxide.608. The method of any one of embodiments 605-607, further comprising a system that uses the first set of nucleic acid molecules immobilized on the surface following the separation for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of nucleic acid molecules or derivatives thereof, or combinations thereof.609. The method of any one of embodiments 605-608, wherein each nucleic acid molecule of the first set of nucleic acid molecules comprises a cleavable base, and the cleavable base is located between the first sequence and the second sequence of the nucleic acid molecule.610. 609. The method of embodiment 609, further comprising the step of cleaving the nucleic acid molecule at the cleavable base after separating the second sequence from the first sequence of the fixed hairpin molecule, thereby removing the second sequence of the nucleic acid molecule from the surface. 611. The method of any one of embodiments 604-610, wherein during storage of the treated surface, each nucleic acid molecule of the first set of nucleic acid molecules does not hybridize to other nucleic acid molecules. 612. The method of any one of embodiments 604-611, wherein during storage of the treated surface, at least 70% of the nucleic acid molecules of the first set of nucleic acid molecules are present as fixed hairpin molecules. 613. The method of any one of embodiments 604-612, wherein the treated surface is stored at a temperature of about 18° C. to about 30° C. 614. The method of any one of embodiments 604-613, wherein the treated surface is stored for at least 6 hours. 615. The method of embodiment 614, wherein the treated surface is stored for at least 24 hours. 616. 617. The method of any one of embodiments 604-615, wherein the first sequence and the second sequence each comprise at least 6 bases. 617. The method of any one of embodiments 604-616, wherein the nucleic acid molecules of the first set of nucleic acid molecules are immobilized on the surface at independently addressable locations. 618. The method of embodiment 617, wherein the independently addressable locations are substantially planar. 619. The method of embodiment 617 or 618, wherein the independently addressable locations comprise one or more wells. 620. The method of any one of embodiments 604-619, wherein the density of the first set of nucleic acid molecules on the surface is at least 1 million molecules/mm2. 621. The method of any one of embodiments 604-620, wherein the first set of nucleic acid molecules comprises one or more different nucleic acid sequences. 622. 623. The method of embodiment 622, wherein the first set of nucleic acid molecules comprises a first subset of nucleic acid molecules comprising the first and second nucleic acid sequences, and a second subset of nucleic acid molecules comprising a third and fourth nucleic acid sequence, wherein the third nucleic acid sequence is substantially complementary to the fourth nucleic acid sequence, and the first nucleic acid sequence is different from the third and fourth nucleic acid sequences. 623. The method of embodiment 622, wherein the first subset of nucleic acid molecules and the second subset of nucleic acid molecules both comprise a fifth nucleic acid sequence. 624. The method of embodiment 623, wherein the fifth nucleic acid sequence comprises a poly(T) sequence. 625. The method of any one of embodiments 604-624, wherein the surface of the substrate is substantially planar. 626. The method of any one of embodiments 604-625, wherein the surface of the substrate comprises a plurality of wells. 627. The method of any one of embodiments 604-626, wherein the substrate comprises one or more particles immobilized thereon. 628. A method for storing a substrate having a surface coated with nucleic acid molecules, comprising the steps of: (a) providing a substrate having a surface having a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules from one or more nucleic acid samples, each nucleic acid molecule of the nucleic acid molecules of the first set of nucleic acid molecules comprising a first nucleic acid sequence; (b) providing a second set of nucleic acid molecules, wherein each nucleic acid molecule of the second set of nucleic acid molecules comprises a second nucleic acid sequence that is substantially complementary to the first nucleic acid sequence, and wherein the second set of nucleic acid molecules are not the sample nucleic acid molecules; and (c) providing a substrate having a surface having a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules from one or more nucleic acid samples, each nucleic acid molecule of the second set of nucleic acid molecules comprising a first nucleic acid sequence that is substantially complementary to the first nucleic acid sequence, and wherein the second set of nucleic acid molecules are not the sample nucleic acid molecules. 629. The method of embodiment 628, wherein the first nucleic acid sequence hybridized to the second nucleic acid sequence is at least partially denatured between about 50° C. and 60° C. 630. The method of embodiment 628 or 629, further comprising the step of: (a) contacting the surface comprising the first set of nucleic acids with the second set of nucleic acid molecules to produce an engineered surface on which at least 70% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized to a nucleic acid molecule of the second set of nucleic acid molecules; and (d) storing the engineered surface for at least 1 hour, wherein for each nucleic acid molecule of the first set of nucleic acid molecules hybridized to a nucleic acid molecule of the second set of nucleic acid molecules, the first nucleic acid sequence hybridizes to the second nucleic acid sequence, and the first nucleic acid sequence hybridized to the second nucleic acid sequence is at least partially denatured between about 40° C. and 60° C. 631. The method of embodiment 628 or 629, further comprising the step of: (a) removing the nucleic acid molecules of the second set of nucleic acid molecules from the engineered surface. 631. Following the removal, the first set of nucleic acid molecules immobilized on the surface is used for hybridization capture, single nucleotide polymorphism (SNP) genotyping, sequencing library capture, synthesis of nucleic acid molecules, on-surface amplification, downstream processing or analysis of nucleic acid molecules or derivatives thereof, or a combination thereof.
632. The method of embodiment 630 or 631, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by enzymatic degradation. 633. The method of embodiment 630 or 631, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by denaturation with a chemical or thermal stimulus. 634. The method of embodiment 633, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by denaturing the first nucleic acid sequence hybridized to the second nucleic acid sequence. 635. The method of embodiment 633 or 634, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the engineered surface by heating the engineered surface to about 40°C to 60°C. 636. The method of any one of embodiments 633-635, wherein the nucleic acid molecules of the second set of nucleic acid molecules are removed from the treated surface by heating a solution in contact with the treated surface to about 40° C. to 60° C. 637. The method of any one of embodiments 633-636, wherein a chemical stimulus is used to remove the nucleic acid molecules of the second set of nucleic acid molecules from the treated surface. 638. The method of embodiment 637, wherein the chemical stimulus comprises sodium hydroxide. 639. The method of any one of embodiments 628-638, wherein during storage of the treated surface, each nucleic acid molecule of the first set of nucleic acid molecules that is hybridized to a nucleic acid molecule of the second set of nucleic acid molecules is not hybridized to other nucleic acid molecules. 640. The method of any one of embodiments 628-639, wherein at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized to a nucleic acid molecule of the second set of nucleic acid molecules. 641. 642. The method of any one of embodiments 628-641, wherein the treated surface is stored at a temperature of about 18° C. to about 30° C. 643. The method of embodiment 642, wherein the treated surface is stored for at least 6 hours 644. The method of embodiment 643, wherein the treated surface is stored for at least 24 hours 645. The method of embodiment 628-644, wherein the second set of nucleic acid molecules is provided to the surface in solution 646. The method of embodiment 628-645, wherein the first nucleic acid sequence and the second nucleic acid sequence each comprise at least 6 bases 647. The method of embodiment 628-646, wherein a given nucleic acid molecule of the first set of nucleic acid molecules and a given nucleic acid molecule of the second set of nucleic acid molecules comprise the same number of nucleotides 648. 649. The method of any one of embodiments 628-648, wherein a given nucleic acid molecule of said first set of nucleic acid molecules and a given nucleic acid molecule of said second set of nucleic acid molecules comprise a different number of nucleotides. 650. The method of embodiment 649, wherein said independently addressable locations are substantially planar. 651. The method of embodiment 649 or 650, wherein said independently addressable locations comprise one or more wells. 652. The method of any one of embodiments 628-651, wherein said first set of nucleic acid molecules are immobilized on said surface according to a predetermined pattern. 653. The method of any one of embodiments 628-652, wherein the density of said first set of nucleic acid molecules on said surface is at least 1 million molecules/mm2. 654. The method of any one of embodiments 628-653, wherein said first set of nucleic acid molecules comprises one or more different nucleic acid sequences. 655. 655. The method of embodiment 655, wherein the first set of nucleic acid molecules comprises a first subset of nucleic acid molecules comprising the first nucleic acid sequence and a second subset of nucleic acid molecules comprising a third nucleic acid sequence, the first and third nucleic acid sequences being different. 656. The method of embodiment 655, wherein the first subset of nucleic acid molecules and the second subset of nucleic acid molecules both comprise a fourth nucleic acid sequence. 657. The method of embodiment 656, wherein the fourth nucleic acid sequence comprises a poly(T) sequence. 658. The method of any one of embodiments 628-657, wherein the second set of nucleic acid molecules comprises DNA nucleotides. 659. The method of any one of embodiments 628-657, wherein the second set of nucleic acid molecules comprises RNA nucleotides. 660. The method of any one of embodiments 628-657, wherein the second set of nucleic acid molecules comprises a mixture of RNA and DNA nucleotides. 661. 662. The method of any one of embodiments 628-660, wherein each nucleic acid molecule of said second set of nucleic acid molecules comprises at least 6 bases. 662. The method of any one of embodiments 628-661, wherein said surface of said substrate is substantially planar. 663. The method of any one of embodiments 628-662, wherein said surface of said substrate comprises a plurality of wells. 664. The method of any one of embodiments 628-663, wherein said substrate comprises one or more particles immobilized thereon. 665. 665. A method for detecting or analyzing an analyte, comprising: (a) providing an open substrate having a central axis, the open substrate comprising an array of analytes fixed adjacent to the open substrate, at least one analyte of the array of analytes being bound to a probe; and (b) performing a nonlinear scan of the open substrate using a detector system to detect at least one signal or signal change from the bound probe, the detector system comprising a line scan camera and an illumination source, the illumination source configured to generate an illuminated region on the open substrate, the open substrate comprising a first region and a second region, the first region and the second region (i) comprising different subsets of the array of analytes, (ii) being at different radial positions of the open substrate relative to the central axis, and (iii) being spatially resolved by the detector system, the bound probe being disposed in the first region of the open substrate, and the nonlinear scan being performed during a relative nonlinear movement between the open substrate and one or both of (i) the line scan camera and (ii) the illuminated region. The method of embodiment 665, wherein the illuminated area has a maximum dimension of at most about 2 millimeters.667. The method of embodiment 665 or 666, wherein the illuminated area has a maximum width of at most about 0.5 millimeters.668. The method of any one of embodiments 665 to 667, wherein the line scan camera is a time-delay integrated line scan camera.669. The method of any one of embodiments 665 to 668, wherein the illumination source comprises a laser.670. The method of embodiment 669, wherein the laser is a continuous wave laser.671. The method of embodiment 669 or 670, wherein the detector system comprises an optical element configured to change the shape of the light beam emitted by the laser.672. The method of embodiment 671, wherein the optical element comprises a cylindrical lens.673. The method of any one of embodiments 665 to 668, wherein the illumination source comprises a light emitting diode.674. The method of any one of embodiments 665 to 673, wherein the open substrate is rotating during (b). 675. The method of embodiment 674, wherein the line scan camera of the detector system is stationary during (b). 676. The method of embodiment 674, wherein the line scan camera of the detector system is rotating during (b). 677. The method of embodiment 675 or 676, wherein the illuminated area is rotating during (b). 678. The method of embodiment 677, wherein the illuminated area is rotating at the same speed as the line scan camera during (b). 679. The method of embodiment 674, wherein the line scan camera of the detector system is radially translated across the open substrate during (b). 680. The method of embodiment 674 or 679, wherein the illuminated area is radially translated across the open substrate during (b). 681. The method of any one of embodiments 665 to 673, wherein the open substrate is stationary during (b). 682. The method of embodiment 681, wherein during (b), the line scan camera of the detector system rotates.683. The method of embodiment 681 or 682, wherein during (b), the illuminated area rotates.684. The method of embodiment 683, wherein during (b), the illuminated area rotates at the same speed as the line scan camera.685. The method of embodiment 681, wherein during (b), the line scan camera is stationary.686. The method of embodiment 685, wherein during (b), the illuminated area of the detector system rotates.687. The method of any one of embodiments 665 to 686, wherein the detector system further comprises a prism, and wherein the prism rotates during (b).688. The method of any one of embodiments 665 to 687, wherein the detector system is configured to detect a signal from the illuminated area using the line scan camera.689. 690. The method of any one of embodiments 665-689, wherein the array of analytes comprises a second analyte bound to a further probe, the further probe being disposed in the second region of the open substrate, and during (b) at least one signal or signal change is detected from the further probe simultaneously with the at least one signal or signal change detected from the bound probe. 691. The method of any one of embodiments 665-690, wherein the detector system compensates for velocity differences at different radial positions of the array relative to the central axis within a scan region. 692. The method of any one of embodiments 665-690, wherein the detector system comprises an optical imaging system having an anamorphic magnification gradient substantially transverse to a scan direction along the open substrate, the anamorphic magnification gradient at least partially compensating for tangential velocity differences substantially perpendicular to the scan direction. 693. The method of any one of embodiments 665-692, wherein (b) comprises reading two or more regions on the open substrate at two or more different scan velocities, respectively, to at least partially compensate for tangential velocity differences in the two or more regions.693. The method of any one of embodiments 665-692, wherein (b) further comprises using an immersion objective lens in optical communication with the detector system and the open substrate to detect the at least one signal or signal change, the immersion objective lens being in contact with a fluid in contact with the open substrate.694. The method of embodiment 693, wherein the fluid is in a container, and an electric field is used to adjust the hydrophobicity of one or more surfaces of the container to retain at least a portion of the fluid in contact with the immersion objective lens and the open substrate.695. 695. The method of any one of embodiments 665-694, wherein the array of analytes comprises nucleic acid molecules, the plurality of probes comprises fluorescently labeled nucleotides, and at least one of the fluorescently labeled nucleotides is bound to at least one of the nucleic acid molecules via a nucleotide complementary bond. 696. The method of any one of embodiments 665-695, wherein the open substrate is substantially planar. 697. The method of any one of embodiments 665-696, wherein an analyte of the array of analytes is immobilized adjacent to the open substrate via one or more binders. 698. The method of any one of embodiments 665-697, wherein the open substrate comprises at least 100,000 binders, and one binder of the at least 100,000 binders immobilizes one analyte of the array of analytes immobilized adjacent to the open substrate. 699. The method of any one of embodiments 665-698, wherein one analyte of the array of analytes is bound to a bead, and the bead is immobilized to the open substrate. 700. 701. The method of any one of embodiments 665-699, wherein one analyte of said array of analytes comprises a nucleic acid molecule. 701. The method of any one of embodiments 665-700, wherein said plurality of probes comprises a plurality of oligonucleotide molecules. 702. The method of any one of embodiments 665-700, wherein said plurality of probes comprises a plurality of nucleotides or analogs thereof.
The method of any one of embodiments 665-700. 703. 703. An apparatus for analyte detection or analysis, comprising: a housing configured to receive an open substrate having an array of analytes fixed adjacent thereto, at least one analyte of the array of analytes being bound to a probe; and a detector system comprising a line scan camera and an illumination source, the illumination source configured to generate an illumination area on the open substrate, the open substrate comprising a first area and a second area, the first area and the second area (i) comprising a subset of the array of fixed analytes, (ii) being at different radial positions of the open substrate relative to the central axis, and (iii) being spatially resolved by the detector system, the bound probe being disposed in the first area of the open substrate, the detector system programmed to perform a non-linear scanning of the open substrate and to detect at least one signal or signal change from the bound probe in the first area of the open substrate, the non-linear scanning being performed during a relative non-linear movement between the open substrate and one or both of (i) the line scan camera and (ii) the illumination area. The apparatus of embodiment 703, wherein the illumination area has a maximum dimension of at most about 2 millimeters.705. The apparatus of embodiment 703 or 704, wherein the illumination area has a maximum width of at most about 0.5 millimeters.706. The apparatus of any one of embodiments 703 to 705, further comprising a processor programmed to instruct the detector system to compensate for velocity differences at different radial positions of the array relative to the central axis within a scan area.707. The apparatus of embodiment 706, wherein the processor is programmed to instruct the detector system to scan two or more regions on the open substrate at two or more different scan velocities, respectively, to at least partially compensate for tangential velocity differences in the two or more regions.708. The apparatus of any one of embodiments 703 to 707, further comprising one or more optical elements configured to generate an anamorphic magnification gradient substantially transverse to a scan direction along the open substrate, the anamorphic magnification gradient at least partially compensating for tangential velocity differences substantially perpendicular to the scan direction.709. 708. The apparatus of embodiment 708, further comprising a processor programmed to adjust the anamorphic magnification gradient to compensate for different imaged radial positions relative to the central axis. 710. The apparatus of any one of embodiments 703 to 709, wherein the line scan camera is a time-delay-integrated line scan camera. 711. The apparatus of any one of embodiments 703 to 710, wherein the illumination source comprises a laser. 712. The apparatus of embodiment 711, wherein the laser is a continuous wave laser. 713. The apparatus of embodiment 711 or 712, wherein the detector system comprises an optical element configured to change a shape of a light beam emitted by the laser. 714. The apparatus of embodiment 713, wherein the optical element comprises a cylindrical lens. 715. The apparatus of any one of embodiments 703 to 710, wherein the illumination source comprises a light emitting diode. 716. The apparatus of any one of embodiments 703 to 715, wherein the detector system and the rotation unit are located in different regions of the apparatus. 717. The apparatus of any one of embodiments 703 to 716, further comprising a rotation unit configured to rotate the detector system or an element thereof, the detector system being programmed to detect the at least one signal from the coupling probe while the line scan camera of the detector system is rotating.718. The apparatus of embodiment 717, wherein the detector system is programmed to detect the at least one signal from the coupling probe while the illumination area of the detector system is rotating.719. The apparatus of embodiment 718, wherein the detector system is programmed to detect the at least one signal from the coupling probe while the line scan camera and the illumination area are rotating at the same speed.720. The apparatus of any one of embodiments 703 to 719, wherein the detector system is programmed to detect the at least one signal from the coupling probe while the open substrate is stationary.721. The apparatus of any one of embodiments 703 to 719, wherein the detector system is programmed to detect the at least one signal from the coupling probe while the open substrate is rotating.722. 723. The apparatus of any one of embodiments 703-716, wherein the detector system is programmed to detect the at least one signal from the coupling probe while the line scan camera translates radially across the open substrate. 724. The apparatus of any one of embodiments 703-716, wherein the detector system is programmed to detect the at least one signal from the coupling probe while the illumination area translates radially across the open substrate. 725. The apparatus of any one of embodiments 703-724, further comprising an immersion objective in optical communication with the detector system and the open substrate, the immersion objective configured to contact a fluid in contact with the open substrate. 726. 726. The apparatus of embodiment 725, further comprising a container configured to hold the fluid and an electric field application unit configured to adjust the hydrophobicity of one or more surfaces of the container to hold at least a portion of the fluid in contact with the immersion objective lens and the open substrate. 727. The apparatus of embodiment 725 or 726, wherein the immersion objective lens separates a first environment from a second environment, the first environment and the second environment having different operating conditions. 728. The apparatus of embodiment 727, wherein the immersion objective lens forms a seal between the first environment and the second environment. 729. The apparatus of any one of embodiments 703 to 728, wherein the open substrate is substantially planar. 730. The apparatus of any one of embodiments 703 to 729, wherein one analyte of the array of analytes is fixed adjacent to the open substrate via one or more binders. 731. 731. The device of any one of embodiments 703-730, wherein the open substrate comprises at least 100,000 binders, and one binder of the at least 100,000 binders immobilizes an analyte of the array of analytes immobilized adjacent to the open substrate. 732. The device of any one of embodiments 703-731, wherein an analyte of the array of analytes is bound to a bead, and the bead is immobilized to the open substrate. 733. The device of any one of embodiments 703-732, wherein an analyte of the array of analytes comprises a nucleic acid molecule. 734. The device of any one of embodiments 703-733, wherein the plurality of probes comprises a plurality of oligonucleotide molecules. 735. The device of any one of embodiments 703-733, wherein the plurality of probes comprises a plurality of nucleotides or analogs thereof. 736. 1. A computer readable medium having stored thereon non-transitory instructions that, when executed, cause one or more computer processors to perform a method for detecting or analyzing analytes, the method including the steps of: providing an open substrate about a central axis, the open substrate comprising an array of analytes fixed adjacent to the open substrate, at least one analyte of the array of analytes being bound to a probe; and performing a non-linear scan of the open substrate using a detector system to detect at least one signal or signal change from the bound probe, the detector system comprising a line scan camera and an illumination source. 737. The computer-readable medium of embodiment 736, wherein the illumination source is configured to generate an illuminated region on the open substrate, the open substrate comprising a first region and a second region, the first region and the second region (i) comprising different subsets of the array of analytes, (ii) being at different radial positions of the open substrate relative to the central axis, and (iii) being spatially resolved by the detector system, the coupling probes being disposed in the first region of the open substrate, and the nonlinear scanning being performed during a relative nonlinear motion between the open substrate and one or both of (i) the line scan camera and (ii) the illuminated region. 738. The computer-readable medium of embodiment 736 or 737, wherein the illumination source comprises a laser. 739. The computer-readable medium of embodiment 738, wherein the laser is a continuous wave laser. 740. The computer readable medium of embodiment 738 or 739, wherein the detector system comprises an optical element configured to change the shape of the light beam emitted by the laser.741. The computer readable medium of embodiment 740, wherein the optical element comprises a cylindrical lens.742. The computer readable medium of embodiment 736 or 737, wherein the illumination source comprises a light emitting diode.743. The computer readable medium of any one of embodiments 736 to 742, wherein the open substrate is stationary during the detection.744. The computer readable medium of embodiment 743, wherein the line scan camera of the detector system is rotating during the detection.745. The computer readable medium of embodiment 744, wherein the illuminated area is rotating during the detection.746. The computer readable medium of embodiment 745, wherein the illuminated area is rotating at the same speed as the line scan camera during the detection.747. The computer readable medium of embodiment 743, wherein the line scan camera is radially translated across the open substrate during the detection.748. The computer readable medium of embodiment 747, wherein the illuminated area is translated radially across the open substrate during the detection.749. The computer readable medium of any one of embodiments 736 to 742, wherein the open substrate is rotated during the detection.750. The computer readable medium of embodiment 749, wherein the line scan camera of the detector system is stationary during the detection.751. The computer readable medium of embodiment 750, wherein the illuminated area of the detector system is rotating during the detection.752. The computer readable medium of embodiment 749, wherein the line scan camera of the detector system is rotating during the detection.753. The computer readable medium of embodiment 752, wherein the illuminated area is rotating during the detection.754. The computer readable medium of embodiment 753, wherein the illuminated area is rotating at the same speed as the line scan camera during the detection.755. The computer readable medium of embodiment 749, wherein the line scan camera is translated radially across the open substrate during the detection. 756. The computer readable medium of embodiment 755, wherein the illuminated area is translated radially across the open substrate during the detection. 757. The computer readable medium of any one of embodiments 736-756, wherein the detector system further comprises a prism, and wherein the prism is rotated during the detection. 758. The computer readable medium of any one of embodiments 736-757, wherein the detector system is configured to detect signals from the illuminated area using the line scan camera. 759. The computer readable medium of any one of embodiments 736-758, wherein the detector system compensates for velocity differences at different radial positions of the array relative to the central axis within a scan region. 760. Any one of embodiments 736-759, wherein the detector system comprises an optical imaging system having an anamorphic magnification gradient substantially transverse to a scan direction along the open substrate, the anamorphic magnification gradient at least partially compensating for tangential velocity differences substantially perpendicular to the scan direction.
A computer readable medium. 761. The computer readable medium of any one of embodiments 736 to 760, wherein the detecting comprises reading two or more regions on the open substrate at two or more different scan speeds, respectively, to at least partially compensate for tangential velocity differences in the two or more regions. 762. The computer readable medium of any one of embodiments 736 to 761, wherein the detecting further comprises using an immersion objective lens in optical communication with the detector system and the open substrate to detect the at least one signal or signal change, the immersion objective lens being in contact with a fluid that is in contact with the open substrate. 763. The computer readable medium of embodiment 762, wherein the fluid is in a container, and an electric field is used to adjust the hydrophobicity of one or more surfaces of the container to retain at least a portion of the fluid in contact with the immersion objective lens and the open substrate. 764. 765. The computer readable medium of any one of embodiments 736-764, wherein the array of analytes comprises nucleic acid molecules, and the plurality of probes comprises fluorescently labeled nucleotides, at least one fluorescently labeled nucleotide of the fluorescently labeled nucleotides binding to at least one nucleic acid molecule of the nucleic acid molecules via a nucleotide complementary bond. 765. The computer readable medium of any one of embodiments 736-764, wherein the open substrate is substantially planar. 766. The computer readable medium of any one of embodiments 736-765, wherein an analyte of the array of analytes is immobilized adjacent to the open substrate via one or more binders. 767. The computer readable medium of any one of embodiments 736-766, wherein the open substrate comprises at least 100,000 binders, and one binder of the at least 100,000 binders immobilizes an analyte of the array of analytes immobilized adjacent to the open substrate. 768. 768. The computer readable medium of any one of embodiments 736-768, wherein one analyte of said array of analytes is bound to a bead, and said bead is immobilized on said open substrate. 769. The computer readable medium of any one of embodiments 736-768, wherein one analyte of said array of analytes comprises a nucleic acid molecule. 770. The computer readable medium of any one of embodiments 736-769, wherein said plurality of probes comprises a plurality of oligonucleotide molecules. 771. The computer readable medium of any one of embodiments 736-769, wherein said plurality of probes comprises a plurality of nucleotides or analogs thereof.

[Example]
[Example 1]
Imaging of nucleic acid molecule sequencing FIG. 42 shows an example of an image generated by imaging a substrate with immobilized analytes. A biological analyte, e.g., a nucleic acid molecule, is immobilized on a substrate 310 comprising a substantially planar array. The substantially planar array comprises a plurality of individually addressable locations 320, each of which comprises a biological analyte, e.g., one or more nucleic acid molecules. The individually addressable locations 320 may be arranged randomly or in an ordered pattern. The biological analyte may be attached to beads immobilized on the array. A single bead may comprise a plurality of analytes, such as at least 10, 20, 30, 40, 50, 100, 150 or more analytes. The beads may be associated with the individually addressable locations. A plurality of fluorescent probes (e.g., a plurality of fluorescently labeled A, T, C, or G) are dispensed on the substrate 310. In some embodiments, the substrate is configured to rotate about a central axis, and the fluid flow unit comprises fluid channels configured to dispense a solution containing a plurality of probes to the array, and during rotation of the substrate, the solution is centrifugally directed along a direction away from the central axis to contact the biological analyte. In other embodiments, the substrate is not rotated. The substrate 310 is then subjected to conditions sufficient to allow a reaction between at least one of the plurality of probes and the biological analyte and to bind the at least one probe to the biological analyte. Unbound probes are washed away. Binding of the at least one probe to the biological analyte is detected using photometry, which includes imaging at least a portion of the substrate 310 (e.g., via scanning or fixed field imaging) and measuring a signal at each individually addressable location 320. Nucleic acid molecules containing nucleotides complementary to the fluorescent probes are fluorescent at the individually addressable locations 320. The operation may then be repeated, matching signals from the image with signals from previous images of the same substrate to generate a trace of signals in time for each biological analyte at each individually addressable location 320. The sequence of the multiple fluorescent probes is known for each iteration of the process, producing a known sequence of analyte at each of the individually addressable locations 320 .

[Example 2]
Diagnostic Procedures for Nucleic Acid Incorporation A diagnostic procedure is performed to determine whether the probe has bound to a biological analyte (e.g., a nucleic acid molecule). FIG. 43 shows exemplary data from such a diagnostic procedure performed at about 29 gigabase pairs (Gbp) from about 183 million beads. A substrate, for example similar to that shown at 310 in FIGS. 15-23, includes an array configured to immobilize a biological analyte. The biological analyte may be attached to beads immobilized in the array. A single bead may include multiple analytes, such as at least 10, 20, 30, 40, 50, 100, 150 or more analytes. The biological analyte is optionally genomic DNA from E. coli. In some cases, human DNA may be used as the biological analyte. In some cases, the biological analyte is a shotgun library of DNA from a clonal population. In some cases, the substrate is configured to rotate about a central axis. In other embodiments, the substrate is not configured to rotate and may be stationary. In other embodiments, the substrate is not configured to rotate and may be movable laterally or vertically as described elsewhere herein. In some cases, a fluid flow unit including a fluid channel is used to dispense a solution including a plurality of probes (e.g., fluorescently labeled nucleotides) onto the array, and during rotation of the substrate, the solution is centrifugally directed along a direction away from the central axis to contact the biological analyte under conditions sufficient to bind at least one probe (e.g., a nucleotide) of the plurality of probes to the biological analyte. In other cases, the probe may be dispensed onto the substrate via a mist, spray, pressurized gas (e.g., bubbling gas) system, etc., as described elsewhere herein. The substrate 310 is then subjected to conditions sufficient to perform a reaction between at least one probe of the plurality of probes and the biological analyte and to bind the at least one probe to the biological analyte. Unbound probes are washed away. Binding of the at least one probe to the biological analyte is detected using a photometric method including imaging at least a portion of the substrate. Nucleic acid molecules including nucleotides complementary to the fluorescent probes are fluorescent at the individually addressable locations. One or more of the processes may be repeated or repeated in cycles.

From the image, a signal 2320 is measured for each individually addressable location or multiple individually addressable locations. An average signal 2330 of the multiple individually addressable locations can also be obtained for each cycle. Because the probes applied to the substrate are known for each cycle, the average signal 2330 can be plotted as a function of the known nucleotide sequence 2310. Additionally, the standard deviation of the signal 2340 can also be plotted for each cycle. The plot 2300 can then provide information regarding the nucleic acid sequence of the biological specimen. One or more of these operations can be performed in real time.

[Example 3]
Scanned Image Patterns of Biological Specimens FIG. 44 shows exemplary data of a diagnostic procedure that informs quality control metrics of scanned imaging. The substrate can undergo rotation, similar to that shown in 310. The substrate can be rotatable about a central axis in some cases. In other embodiments, the substrate may not be rotatable or rotated. The substrate includes biological specimens, such as human and E. coli shotgun libraries. In one example, the substrate includes a shotgun library and about 15% synthetic monoplates spiked into the sample. In such an example, the shotgun library and synthetic monoplates may be labeled (e.g., fluorescently). In other examples, the shotgun library and synthetic monoplates are associated with beads that may be associated with the substrate (e.g., via a linker). In some cases, the beads may be associated with the substrate in a pattern. In some cases, a subset of beads on the substrate may be detected in a pattern, such as a spiral pattern (e.g., following a scan path). The library and synthetic monoplates can be directly detected using optical measurements. In another example, a plurality of probes is applied to a substrate, and the substrate is subjected to conditions sufficient to effect a reaction between at least one of the plurality of probes and the biological analyte to bind the at least one probe to the biological analyte, and one or more signals are detected from the at least one probe bound to the biological analyte.

Diagnostic metrics of the imaging segments can be calculated. Figures 44A-44F show plots showing image or process metrics at different individually addressable locations (e.g., varying R and Θ on a circular substrate). Each scanned field is shown as a small circle on each plot (panels A-F). Images can then be analyzed for the number of reads per image (panel A), the percentage of reads passing the filter (panel B), the average first incorporation signal of nucleotides (panel C), droop (signal loss per cycle, panel D), lag phasing, which may indicate false negatives, e.g., the proportion of clonal populations that do not progress per cycle (panel E), and read phasing, which may indicate false positives, e.g., the proportion of clonal populations that misprogress per cycle (panel F). Uniform signal levels and read/lag phasing across R and Θ, in this case, indicate consistent fluid and biochemical reactions over the course of many incorporation cycles and predict high quality sequence reads.

[Example 4]
Linearity and Accuracy of Homopolymers In single nucleotide flow-based sequencing by synthesis chemistry, it is necessary to determine the length of the synthesized homopolymer in order to determine the sequence. The homopolymers can be of various lengths and can contain the same sequence of nucleotides (e.g., one nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, and ten nucleotides, where all the nucleotides are the same, i.e., all A, all T, all C, all G, etc.). Figure 45A shows exemplary data of flow-based sequencing by synthesis. Many homopolymers of different lengths were bound to a substrate. Complementary probes were added to the substrate, the substrate was washed and imaged, and the process was repeated. The signal was measured from each bead position. As can be visualized in the plot, the signal from the image is very linear with homopolymer length, up to the 9 nucleotides tested here. Thus, the signal from the obtained images (e.g., individually addressable positions) can be used to determine homopolymer lengths of up to 5 bases with sufficiently high accuracy and low noise (>99% accuracy).

[Example 5]
Nucleic acid molecule sequencing and signal processing Nucleic acid molecules from biological specimens, e.g., E. coli, are immobilized on a substrate containing a substantially planar array. Sequencing by synthesis was performed using flow-based chemistry. Imaging was performed as described elsewhere herein. FIG. 45B shows the signal distribution of a set of hundreds of colonies, each colony being a replica of a single synthesized monotonic plate. The x-axis is labeled with the length of sequencing after each cycle (e.g., each chemical flow step). In FIG. 45C, the same data is processed with a parametric model, which accounts for the different template counts (amplitudes) and background levels for each colony. The signal is deconvoluted with a model of lead and lag phasing, which also accounts for the global signal loss per cycle. In the example shown here, the nominal phasing was 0.54% lag, 0.41% read, and 0.45% signal loss. Residual systematic variation may be due to signal variations with sequence context and can be further corrected using other algorithms (not shown).

[Example 6]
Sequencing of a shotgun library from E. coli A substrate containing a substantially planar array is immobilized with nucleic acid molecules from a biological specimen, for example E. coli. Sequencing by synthesis was performed using flow-based chemistry. Imaging was performed as described elsewhere herein. Images were then processed. Figure 46A shows individual aligned reads to a segment of the E. coli reference genome. Figure 46B shows a plot obtained from image processing of aligned read depth for each position in the E. coli genome for a set of shotgun reads. The x-axis shows the coverage level at each E. coli reference key position, and the y-axis shows the frequency.

[Example 7]
Calculation of Reel-to-Reel Dimensions Flexible substrates containing biological specimens can be designed to improve throughput for processing nucleic acid molecules. In one example, biological specimens are nanoimprinted onto a flexible substrate, such as a film that is pulled through a first reel to contact the flexible substrate with a reservoir containing a solution containing multiple probes. The dimensions of the film can be modulated to match the detector (e.g., optical sensor). In some cases, the length of the film can be wound onto a reel. The film can be about 85 meters long and 7 millimeters (mm) wide, resulting in an area of about 6000 square centimeters ( ^cm2 ). Compared to a flat circular substrate with a diameter of 5.9 centimeters (cm), the usable area of the film can be more than 60 times larger than the usable area of the flat circular substrate. Given an optical sensor speed of 10 centimeters per second (cm/s), the entire film can be imaged within about 14 minutes. Alternatively, the dimensions of the film (e.g., length and width) can be modulated to improve detection rate, imprint rate, contact area, etc.

[Example 8]
Preparation of a Substrate for Sequencing Nucleic acid molecules may be sequenced using the methods and systems provided herein. The substrate used in the sequencing process may be substrate 310. The substrate may include a substantially planar array that may include a plurality of individually addressable locations 320. The plurality of individually addressable locations may be arranged randomly or in an ordered pattern. At least a subset of the plurality of individually addressable locations may be bound to a plurality of nucleic acid molecules in preparation for the sequencing process. The individually addressable locations of the subset of the plurality of individually addressable locations may be arranged randomly or in an ordered pattern. The substrate may be configured to rotate about a central axis. A fluid flow unit comprising a fluid channel may be coupled to the substrate and configured to dispense a solution to the array. When the solution is dispensed during the rotation of the substrate, the solution may be centrifugally directed along a direction away from the central axis and contacted with one or more biological analytes (e.g., nucleic acid molecules) bound to the substrate. However, the substrate may not be rotated during the preparation or the sequencing process. In some cases, the substrate may be subjected to continuous rotation during the preparation and execution of the sequencing process. In other cases, the substrate may be stationary during at least a portion of such a process.

The preparation of a substrate for sequencing a nucleic acid molecule may include dispensing one or more nucleic acid molecules onto the substrate. Dispensing of the nucleic acid molecules onto the substrate may be performed in an ordered or random manner. The nucleic acid molecules bound to the substrate may be directly or indirectly immobilized to the substrate. For example, the nucleic acid molecules may be coupled to a plurality of particles, which may be directly immobilized to the substrate (e.g., via one or more oligonucleotide molecules herein or another mechanism). The plurality of particles may include a plurality of beads. A given particle of the plurality of particles may include one or more nucleic acid molecules bound thereto. For example, a given particle of the plurality of particles may include a clonal population of nucleic acid molecules bound thereto. In one example, the plurality of particles includes a plurality of primer molecules bound thereto, the plurality of primer molecules configured to hybridize to a sequence of the nucleic acid molecules of the library of nucleic acid molecules. The nucleic acid molecules of the library of nucleic acid molecules may be bound to the plurality of particles via hybridization of the plurality of primer molecules to a sequence of the nucleic acid molecules. An amplification process can be performed to amplify the nucleic acid molecules bound to the plurality of particles, which can provide one or more clonal populations of the nucleic acid molecules bound to the plurality of particles. The amplification process can include emulsion PCR. Following the amplification process, the plurality of particles can be dispensed (e.g., in an ordered or random manner) onto a substrate (e.g., as described herein). Alternatively, the plurality of particles can be dispensed onto a substrate prior to interaction with the nucleic acid molecules of the library of nucleic acid molecules, and the amplification process can be performed with the plurality of particles immobilized on the substrate.

The substrate may include one or more types of adaptors or primers suitable for binding and amplifying nucleic acid molecules. These adaptors may be attached to the substrate in a pattern or without a pattern. The pattern may include areas that are attractive to the adaptors as well as areas that are repulsive to the adaptors. Examples of patterns that may be applied to the substrate include spiral patterns, single or concentric rings, and checkerboard patterns. In one example, the substrate is divided into two parts (e.g., a disk-shaped substrate is bisected to provide two parts), one of which includes a first region that is attracted to a first adaptor type and the other of which includes a second region that is attracted to a second adaptor type, where the first and second adaptor types are not the same. The adaptors may be dispensed onto the substrate via a dispensing head that may provide a specific local concentration of nucleic acid molecules to the substrate in a predefined pattern. In the case of continuous process amplification, the concentration may be such that the binding rate of the nucleic acid molecules to a local volume (e.g., spot) is substantially less than the amplification doubling rate (at least about 4-fold). This may ensure that most seeds are fully amplified before the second seed in the spot. Loading may be repeated with moderate seeding efficiency, such as about 10% seeding efficiency. Patterns such as repeated rings and spirals may be generated. Continuous seeding may be used to ensure that most spots are seeded and nearly monoclonal amplified (e.g., at least about 90% of spots and at least about 90% monoclonal). Alternatively, templates may be seeded on an unpatterned surface at a concentration such that the seeding density is at least about 50k/ ^mm2 , 100k/ ^mm2 , 500k/ ^mm2 , 1M, 2M, 4M or more. Solid-phase amplification may be performed during or after seeding.

Amplification of nucleic acid molecules (e.g., bound to a plurality of particles, such as a plurality of particles bound to a substrate) may include PCR, bridge amplification, recombinase polymerase amplification, Wildfire amplification, template walking amplification, strand displacement amplification, rolling circle amplification, or any other useful method. The amplification method may include kinetic exclusion amplification. For example, amplification reagents may undergo a reaction from a given nucleic acid molecule to a product amplification site, each having a clonal population of amplicons, and the reaction may include simultaneously transporting nucleic acid molecules comprising the given nucleic acid molecule to the site at an average transport rate and amplifying the nucleic acid molecules transported to the site at an average amplification rate, where the average amplification rate exceeds the average transport rate. Nanoball sequencing methods may also be used in combination with the methods and systems provided herein. For example, the nucleic acid molecule may include a template fragment, and an adapter sequence may be ligated to the fragment to result in circularization of the fragment. The circular fragment may then be amplified using rolling circle amplification, thereby providing ligated amplified fragments that may be compacted into a nucleic acid nanoball.

[Example 9]
Sequencing Nucleic Acid Molecules Using Blocked or Terminal Nucleotides Nucleic acid molecules can be sequenced using the methods and systems provided herein. The nucleic acid molecules can be immobilized on a substrate (e.g., directly or via a support such as a bead, which can include a plurality of nucleic acid molecules, e.g., a clonal population of nucleic acid molecules, attached thereto). The substrate (e.g., substrate 310 as described herein) can include a substantially planar array, which can include a plurality of individually addressable locations (e.g., individually addressable locations 320 as described herein). The plurality of individually addressable locations can be randomly arranged or arranged in an ordered pattern. The nucleic acid molecules can be associated with the individually addressable locations of the array. For example, beads to which the nucleic acid molecules are attached can be associated with the individually addressable locations of the array. The nucleic acid molecules can be attached to the array via oligonucleotides, such as adaptors or primer molecules (e.g., via a support attached to the substrate). The substrate may be configured to rotate about a central axis, and a fluid flow unit including fluid channels configured to dispense solutions may be coupled to the substrate such that, during rotation of the substrate, the solutions are centrifugally directed along a direction away from the central axis into contact with the biological analytes (e.g., nucleic acid molecules). Alternatively, the substrate may not be rotated.

The nucleic acid molecule may include a double-stranded region, which may include a sequence complementary to a first strand adapter sequence and a second strand adapter sequence. The nucleic acid molecule may include a target sequence (e.g., a library insert sequence), which may be adjacent to one or more adapter sequences and one or more other sequences, such as one or more barcode or identifier sequences, primer sequences, or other sequences. The nucleic acid molecule may be derived from a sample, such as a sample containing a biological fluid (e.g., blood or saliva). The nucleic acid molecule may include deoxyribonucleic acid or ribonucleic acid. For example, the nucleic acid molecule may include genomic DNA.

Sequencing of a nucleic acid molecule may proceed by providing a first nucleotide complementary to an available position of the nucleic acid molecule. The first nucleotide may include a blocking or terminating group, such as a reversible terminator. The blocking or terminating group (e.g., a reversible terminator) may be coupled to the first nucleotide via the sugar moiety, e.g., to the 3' position of the sugar moiety. The blocking or terminating group may include an azido moiety. For example, the blocking or terminating group may be a 3'-O-azidomethyl blocking group. Alternatively, the blocking or terminating group may be another group, e.g., a small stable group, that does not significantly affect the incorporation of the subsequent nucleotide into the template. The first nucleotide may be labeled (e.g., may be attached to a fluorescent label). Alternatively, the first nucleotide may be unlabeled (e.g., may not be attached to a fluorescent label). The first nucleotide may be provided in a first solution (e.g., a reaction mixture), which may include one or more additional nucleotides. The first solution may be supplied to the substrate through a fluid channel of a fluid flow unit coupled to the substrate (e.g., during rotation of the substrate or while the substrate is stationary). The first solution may include a plurality of identical nucleotides, including the first nucleotide. Alternatively, the first solution may include a first plurality of identical nucleotides, including the first nucleotide and a second plurality of identical nucleotides, where the first nucleotide and the second nucleotide of the second plurality of identical nucleotides may have different chemical structures. For example, the first nucleotide and the second nucleotide may include different bases (e.g., canonical bases, e.g., A, G, C, and U/T), labels (e.g., fluorescent labels), linkers (e.g., linkers that link a label to the base, sugar, or phosphate moiety of the nucleotide), sugar moieties (e.g., sugar moieties with or without blocking or terminating groups), or combinations thereof. In one example, the first solution includes a first plurality of identical nucleotides including a first nucleotide, a second plurality of identical nucleotides, a third plurality of identical nucleotides, and a fourth plurality of identical nucleotides, each of the plurality of identical nucleotides including a base of a different canonical type (e.g., A, G, C, and U/T). Each nucleotide of each of the plurality of identical nucleotides may include a blocking or terminating portion, which may be the same or different for the different types of nucleotides. Each nucleotide of the plurality of identical nucleotides may be unlabeled. Alternatively, all or a portion of each nucleotide of a given plurality of identical nucleotides may be labeled (e.g., with a fluorescent label). For example, all or a portion of each nucleotide of each of the plurality of identical nucleotides may be labeled. The first solution may include other reagents for carrying out the reaction, such as a buffer, a cation, an enzyme (e.g., a polymerase enzyme), or other reagents.

The nucleic acid molecule coupled to the substrate and the substantially planar array of first nucleotides can be subjected to conditions sufficient to incorporate the first nucleotide into an available position of the nucleic acid molecule (e.g., into a growing strand coupled to a nucleic acid strand containing a target sequence). A blocking or terminating group of the first nucleotide can prevent the incorporation of additional nucleotides (e.g., of the same type, such as in the case of a homopolymeric sequence, or of a different type).

Incorporation of the first nucleotide into the nucleic acid molecule can be detected via imaging, for example, by imaging a label or a label of a reporter moiety attached to the first nucleotide. The array can be interrogated with a detector, such as an optical detector. Imaging can be performed while the substrate is rotating or while the substrate is stationary. Imaging can include scanning or fixed field imaging. For example, the optical detector can be translated and/or rotated relative to the substrate during imaging. Imaging can detect a signal (e.g., a fluorescent emission) of the label (e.g., the first nucleotide or a reporter moiety attached thereto). The signal can be indicative of the type of nucleotide incorporated into the nucleic acid molecule. Alternatively, the signal can be indicative of the type of reporter moiety attached to the nucleic acid molecule and thus the type of nucleotide incorporated into the nucleic acid molecule.

Further details of such methods are described in the Examples below. After detection of incorporation of the first nucleotide into the nucleic acid molecule, the process can be repeated using a second solution containing a second nucleotide, and so on, to determine the sequence of the nucleic acid molecule.

[Example 10]
Sequencing Nucleic Acid Molecules Using Non-Terminal Nucleotides Nucleic acid molecules can be sequenced using the methods and systems provided herein. The nucleic acid molecules can be immobilized on a substrate (e.g., directly or via a support such as a bead, which can include a plurality of nucleic acid molecules, e.g., a clonal population of nucleic acid molecules, attached thereto). The substrate (e.g., substrate 310 as described herein) can include a substantially planar array, which can include a plurality of individually addressable locations (e.g., individually addressable locations 320 as described herein). The plurality of individually addressable locations can be randomly arranged or arranged in an ordered pattern. The nucleic acid molecules can be associated with the individually addressable locations of the array. For example, beads to which the nucleic acid molecules are attached can be associated with the individually addressable locations of the array. The nucleic acid molecules can be attached to the array via oligonucleotides, such as adaptors or primer molecules (e.g., via a support attached to the substrate). The substrate may be configured to rotate about a central axis, and a fluid flow unit including fluid channels configured to dispense solutions may be coupled to the substrate such that, during rotation of the substrate, the solutions are centrifugally directed along a direction away from the central axis into contact with the biological analytes (e.g., nucleic acid molecules). Alternatively, the substrate may not be rotated.

The nucleic acid molecule may include a double-stranded region, which may include a sequence complementary to a first strand adapter sequence and a second strand adapter sequence. The nucleic acid molecule may include a target sequence (e.g., a library insert sequence), which may be adjacent to one or more adapter sequences and one or more other sequences, such as one or more barcode or identifier sequences, primer sequences, or other sequences. The nucleic acid molecule may be derived from a sample, such as a sample containing a biological fluid (e.g., blood or saliva). The nucleic acid molecule may include deoxyribonucleic acid or ribonucleic acid. For example, the nucleic acid molecule may include genomic DNA.

Sequencing of a nucleic acid molecule may proceed by providing a first nucleotide complementary to an available position of the nucleic acid molecule. The first nucleotide may be a non-terminal nucleotide (e.g., may not include a blocking or terminating group). The first nucleotide may be labeled (e.g., may be attached to a fluorescent label). Alternatively, the first nucleotide may be unlabeled (e.g., may not be attached to a fluorescent label). The first nucleotide may be provided in a first solution (e.g., a reaction mixture), which may include one or more additional nucleotides. The first solution may be supplied to the substrate through a fluid channel of a fluid flow unit coupled to the substrate (e.g., during rotation of the substrate or while the substrate is stationary). The first solution may include a plurality of identical nucleotides, including the first nucleotide. Alternatively, the first solution may include a first plurality of identical nucleotides, including the first nucleotide and a second plurality of identical nucleotides, where the first nucleotide and the second nucleotide of the second plurality of identical nucleotides may have different chemical structures. For example, the first nucleotide and the second nucleotide may include different bases (e.g., canonical bases, e.g., A, G, C, and U/T), labels (e.g., fluorescent labels), linkers (e.g., linkers that link a label to a base, sugar, or phosphate moiety of the nucleotide), sugar moieties, or combinations thereof. In one example, the first solution includes a first plurality of identical nucleotides comprising the first nucleotide, a second plurality of identical nucleotides, a third plurality of identical nucleotides, and a fourth plurality of identical nucleotides, each of the plurality of identical nucleotides including a base of a different canonical type (e.g., A, G, C, and U/T). Each nucleotide of the plurality of identical nucleotides may be unlabeled. Alternatively, all or a portion of each nucleotide of a given plurality of identical nucleotides may be labeled (e.g., with a fluorescent label). For example, all or a portion of each nucleotide of each of the plurality of identical nucleotides may be labeled. In one example, the first solution may include a plurality of nucleotides comprising the first nucleotide, each nucleotide including the same canonical base. The plurality of nucleotides may include a plurality of labeled nucleotides and a plurality of unlabeled nucleotides. For example, at least 20% of the plurality of nucleotides in the first solution can be labeled nucleotides. Any percentage of the plurality of nucleotides can be labeled nucleotides. The first solution can include other reagents for carrying out the reaction, such as a buffer, a cation, an enzyme (e.g., a polymerase enzyme), or other reagents.

The nucleic acid molecule coupled to the substrate and the substantially planar array of first nucleotides can be subjected to conditions sufficient to incorporate the first nucleotide into an available position of the nucleic acid molecule (e.g., into a growing strand coupled to a nucleic acid strand containing a target sequence). The absence of a blocking or terminating group can facilitate the incorporation of additional nucleotides (e.g., of the same type, such as in the case of a homopolymeric sequence, or of a different type) into positions adjacent to the position at which the first nucleotide is incorporated.

If the first solution contains nucleotides containing the same base (e.g., canonical bases, e.g., A, G, C and U/T), the detection of the incorporation of the first nucleotide and optionally (e.g., if the target sequence contains a homopolymeric sequence) one or more further nucleotides can be detected by imaging a label coupled to the first nucleotide and/or one or more further nucleotides or by detecting a label of a reporter moiety provided on the nucleic acid molecule (e.g., a reporter moiety configured to specifically bind to a nucleotide of a given type). The label coupled to the incorporated nucleotide can be removed after detection, e.g., before contacting the nucleic acid molecule with a second solution containing the second nucleotide (e.g., by contacting the incorporated nucleotide with a cleavage reagent). The label coupled to the reporter moiety can be removed as well. Alternatively, the sequencing process can proceed without cleaving the label associated with the nucleotide incorporated in the nucleic acid molecule.

When the first solution includes nucleotides containing different bases, detection of incorporation of the first nucleotide and possibly (e.g., when the target sequence includes a homopolymer sequence) one or more further nucleotides may be detected by imaging a label attached to the first nucleotide and/or one or more further nucleotides, which label may be different from other labels attached to nucleotides containing different types of bases. For example, the first nucleotide may include a first type of label and the second nucleotide included in the first solution may include a second type of label. The different labels may provide different signals, such as different fluorescent signatures, such that detection of the fluorescent signature of the label coupled to the first nucleotide indicates incorporation of the first nucleotide and not the second nucleotide. Alternatively, a labeled reporter moiety may be used to detect incorporation of a given type of nucleotide.

The array can be interrogated with a detector, such as an optical detector. Imaging can be performed while the substrate is rotating or while the substrate is stationary. Imaging can include scanning or fixed field imaging. For example, the optical detector can be translated and/or rotated relative to the substrate during imaging. Imaging can detect a signal (e.g., fluorescence emission) of a label (e.g., the first nucleotide or a reporter moiety attached thereto). The signal can be indicative of the type of nucleotide incorporated into the nucleic acid molecule. Alternatively, the signal can be indicative of the type of reporter moiety attached to the nucleic acid molecule and thus the type of nucleotide incorporated into the nucleic acid molecule.

Further details of such methods are described in the Examples below. After detection of incorporation of a first nucleotide into a nucleic acid molecule, the process can be repeated using a second solution containing additional nucleotides, etc., to determine the sequence of the nucleic acid molecule.

[Example 11]
Detection of nucleotide incorporation using a reporter moiety As described in the previous examples, detection of nucleotide incorporation into a nucleic acid molecule can include detection of a label coupled to the nucleotide. Alternatively, detection of nucleotide incorporation can include detection of a label coupled to a reporter moiety.

The labeled (e.g., fluorescently labeled) reporter moieties may be provided to nucleic acid molecules coupled to a substantially planar array of a substrate (e.g., via a particle). The first nucleotide may be incorporated into the nucleic acid molecule (e.g., as described in the previous examples). The first nucleotide may include a blocking or terminating moiety. Alternatively, the first nucleotide may be a non-terminal nucleotide. The first reporter moiety may be provided in a first solution (e.g., a first solution that provides the first nucleotide to the nucleic acid molecule for incorporation therein) or a second solution that is provided to the nucleic acid molecule (e.g., after removal of the first solution by centrifugation and application of an optional wash solution). The second solution may be provided during rotation of the substrate or while the substrate is stationary. The first reporter moiety may include an antibody. The first reporter moiety may include a fluorescent label. The first reporter moiety may be configured to bind to a nucleotide incorporated into the nucleic acid molecule. For example, the first reporter moiety may be base specific. The first reporter moiety may be configured to bind to a nucleotide that includes a blocking group or a terminating group. For example, the first reporter moiety can be a base-specific 3'-block-dependent first reporter moiety, such as a base-specific 3'-block-dependent fluorescently labeled antibody. The first reporter moiety can be configured to bind to the first nucleotide. The first reporter moiety can be configured not to bind to any type of nucleotide other than the first nucleotide. The solution (e.g., the second solution) containing the first reporter moiety can include a plurality of identical first reporter moieties that include the first reporter moiety. Also, the solution containing the first reporter moiety can include a plurality of identical second reporter moieties specific to a second nucleotide type (e.g., a second plurality of identical nucleotides), a plurality of identical third reporter moieties specific to a third nucleotide type (e.g., a third plurality of identical nucleotides), and a plurality of identical fourth reporter moieties specific to a fourth nucleotide type (e.g., a fourth plurality of identical nucleotides). Each of the plurality of identical reporter moieties can include a different type of label. The first reporter moiety and the nucleic acid molecule may be subjected to conditions sufficient to bind to the first reporter moiety and the first nucleotide incorporated into the nucleic acid molecule. Unbound reporter moieties may be removed (e.g., via removal of the second solution by centrifugation and optional application of a wash solution). The array may be examined with a detector, such as an optical detector. Imaging may be performed while the substrate is rotating or while the substrate is stationary. Imaging may include scanning or fixed field imaging. For example, the optical detector may be translated and/or rotated relative to the substrate during imaging. Imaging may detect a signal (e.g., fluorescence emission) of the label of the first reporter moiety. The signal may indicate the type of reporter moiety bound to the nucleic acid molecule and thus the type of nucleotide incorporated into the nucleic acid molecule.

Following imaging, the nucleic acid molecules coupled to the array may be subjected to conditions sufficient to remove the first reporter moiety coupled to the first nucleotide. For example, a wash solution may be provided that may include a reagent configured to cleave the blocking or terminating group from the first nucleotide and remove the first reporter moiety. Following the cleavage/wash process, the first nucleotide may no longer include the blocking or terminating group such that the incorporation and detection process may be repeated one or more times. In this manner, the sequence of the nucleic acid molecules bound to the array may be determined. This process may be used to identify the sequence of a plurality of nucleic acid molecules, such as one or more clonal populations of nucleic acid molecules coupled to the array. For example, this process may be used to identify the sequence of a plurality of different clonal populations of nucleic acid molecules bound to a plurality of beads bound to a plurality of individually addressable locations of a substantially planar array of a substrate.

While preferred embodiments of the present invention have been illustrated and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art at this point without departing from the present invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. It is therefore contemplated that the present invention shall cover such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims (20)

1. A method for dispensing a sample onto a surface of a substrate, comprising:
dispensing a sample solution including a plurality of beads along a path onto the surface of the substrate via a sample dispenser during relative movement between the surface of the substrate and the sample dispenser;
a surface of the substrate comprising a plurality of individually addressable locations for receiving and securing the plurality of beads;
a surface of the substrate comprising a first location and a second location;
the first location has a higher affinity for one bead of the plurality of beads than the second location;
the first location comprises an individually addressable location of the plurality of individually addressable locations;
Following said dispensing step, said one bead is immobilized at said first location. The method of claim 1, wherein the surface of the substrate is substantially flat. The method of claim 1, wherein the surface of the substrate is patterned using surface chemistry. The method of claim 1, wherein the path is formed in a substantially spiral pattern. The method of claim 4, wherein the sample solution is dispensed onto the surface of the substrate in the substantially spiral pattern in a radially outward direction relative to a central axis of the surface of the substrate. The method of claim 4, wherein the sample solution is dispensed onto the surface of the substrate in the substantially spiral pattern in a radially inward direction relative to a central axis of the surface of the substrate. a path width of the path is greater than a path pitch of the path;
The method of claim 4 , wherein the path pitch is the distance between a center of the path at a first position and a center of the path at a second position after one revolution of the surface of the substrate. a path width of the path is smaller than a path pitch of the path;
The method of claim 4 , wherein the path pitch is the distance between a center of the path at a first position and a center of the path at a second position after one revolution of the surface of the substrate. a path width of the path is substantially the same as a path pitch of the path;
The method of claim 4 , wherein the path pitch is the distance between a center of the path at a first position and a center of the path at a second position after one revolution of the surface of the substrate. The method of claim 1, wherein at least one of the surface of the substrate and the sample dispenser is moving during the dispensing step. During the dispensing step, (i) the surface of the substrate rotates about an axis of rotation, and (ii) the sample dispenser moves relative to the axis of rotation of the surface of the substrate, or the movement of the surface of the substrate causes the axis of rotation to move relative to the sample dispenser.
The method of claim 10. The method of claim 11, wherein the surface of the substrate rotates at a rotational frequency of 5 revolutions per minute (rpm) or less. the plurality of beads comprises a first subset of beads and a second subset of beads;
each bead of the first subset of beads comprises a template nucleic acid sequence bound to the bead, and each bead of the second subset of beads does not comprise a template nucleic acid sequence bound to the bead;
The method of claim 1. 14. The method of claim 13, wherein following the dispensing step, the first subset of beads are immobilized at individually addressable locations among the plurality of individually addressable locations and the second subset of beads are not immobilized at the plurality of individually addressable locations. The method of claim 1, wherein the single bead contains multiple nucleic acid molecules having sequence identities. The method of claim 1, wherein the plurality of individually accessible locations comprises at least 50,000,000,000 individually addressable locations. a surface of the substrate comprising a set of first locations and a set of second locations different from the set of first locations;
The method of claim 1 , wherein the set of first locations includes the first location and the set of second locations includes the second location. 18. The method of claim 17, wherein the first location does not contact another first location of the set of first locations. the set of first locations carries a positive charge with an affinity toward a negative charge;
20. The method of claim 18, wherein said one bead comprises a nucleic acid molecule bound to said one bead, said nucleic acid molecule carrying said negative charge. the first set of locations comprises (3-aminopropyl)triethoxysilane (APTMS);
20. The method of claim 19, wherein the set of second locations comprises hexamethyldisilazane (HMDS).

Images