HK40037882B - Methods of processing tissue samples - Google Patents

Description

Method for processing tissue samples

The present application is a divisional application of an invention patent application having an application number of 201580056036.4, application date of 2015, 10-16, entitled "optical scanning system for in situ genetic analysis".

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an optical scanning system for in-situ genetic analysis.

Background

In conventional (i.e., wide-field) fluorescence microscopes, the entire sample is uniformly immersed in light from a light source. All parts of the sample in the light path are excited simultaneously and the resulting fluorescence is detected by a photodetector of the microscope or a camera comprising a large unfocused background part. In contrast, confocal microscopes use point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate the out-of-focus signal. Since only light generated by fluorescence very close to the focal plane can be detected, the optical resolution of the image, in particular in the depth direction of the sample, is much better than for a wide-field microscope. However, this increased resolution comes at the cost of reduced signal intensity since most of the light from the sample fluorescence is blocked at the pinhole — thus, typically requiring a long exposure time.

A disadvantage of some photoluminescence-based scanning instruments (or imaging systems) used in current fluorescence-based sequencing-by-synthesis (SBS) systems is that they have poor confocality (i.e. at most semi-confocality). These semi-confocal systems have a low signal-to-noise ratio (S/N ratio) and are therefore insufficient to eliminate out-of-focus features in the sample. In addition, current dithering focus tracking methods cannot maintain focus during imaging. Therefore, new methods are needed for imaging (or scanning) in photoluminescence-based SBS systems.

Disclosure of Invention

In various embodiments of the systems, methods, and apparatus within the scope of the appended claims, each of which has several aspects, no single one is responsible for the desirable attributes described herein. Without limiting the scope of the appended claims, some prominent features are described herein.

Systems and methods for performing fluorescence in situ sequencing are disclosed. That is, one embodiment provided herein is a confocal Time Delay Integration (TDI) line scan imaging system having a high S/N ratio and high confocality to produce a high resolution image of a sample. In one example, a confocal TDI line scan imaging system includes various pinhole and/or slit aperture mechanisms in front of the image sensor, where the various pinhole and/or slit aperture mechanisms are used to reject out-of-focus light. In another example, a confocal TDI line scan imaging system includes various pinhole and/or slit aperture mechanisms in intermediate image planes conjugate to the image sensor.

Also provided herein are structures that include focus tracking features that can be used to maintain focus during imaging. In one example, various configurations of focusing strips on a substrate in contact with a tissue sample to be imaged are provided. In another example, the strip is cut into a tissue sample, providing an exposed strip that can be used as a substrate for a focus tracking feature.

Also provided herein are flow cells and methods for processing tissue samples in the flow cells. That is, various configurations and methods are provided herein in which a tissue sample is placed within a reaction chamber of a flow cell during assembly of the flow cell, and then the tissue sample is chemically manipulated.

Also provided herein are flow cells for chemically manipulating tissue samples using open containers. In one example, a substantially "dry" imaging process may be used. In another example, a liquid immersion imaging process may be used.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

Drawings

Fig. 1 illustrates a side view of an example of a confocal imaging system according to one embodiment.

Fig. 2 illustrates another configuration of the confocal imaging system shown in fig. 1.

Fig. 3 illustrates a side view of an example of a sensor aperture mechanism of the confocal imaging system shown in fig. 1 and 2.

Fig. 4A and 4B illustrate side views of another example of a sensor aperture mechanism of the confocal imaging system shown in fig. 1 and 2.

Fig. 5A and 5B illustrate side views of yet another example of a sensor aperture mechanism of the confocal imaging system shown in fig. 1 and 2.

Fig. 6A and 6B illustrate a plan view and a cross-sectional view, respectively, of an example of a structure including a focusing stripe for improved focus tracking in an imaging process.

Fig. 7 illustrates a side view of the structure shown in fig. 6A and 6B when used in an imaging process.

Fig. 8 illustrates a side view of another example of a structure including focusing stripes for improved focus tracking in an imaging process.

FIG. 9 illustrates a side view of another technique for providing improved focus tracking during imaging.

Fig. 10A and 10B illustrate plan and cross-sectional views, respectively, of an example of a flow cell for holding and processing a tissue sample.

Fig. 11 illustrates a flow chart of an example of a method of processing a tissue sample using the flow cell shown in fig. 10A and 10B.

Fig. 12A and 12B illustrate a plan view and a cross-sectional view, respectively, of another example of a flow cell for holding and processing a tissue sample.

Fig. 13A and 13B illustrate additional side views of the flow cell shown in fig. 12A and 12B, and show tissue samples at different locations in a sequencing chamber.

Fig. 14A and 14B illustrate a plan view and a sectional view, respectively, of an example of a bonding portion of the flow cell shown in fig. 12A and 12B.

Fig. 15 illustrates a flow chart of an example of a method of processing a tissue sample using the flow cell shown in fig. 12A and 12B.

Fig. 16A and 16B illustrate side views of an example of a flow cell using an open container to hold a tissue sample and an example of a process of "dry" imaging a tissue sample therein.

Fig. 17A and 17B illustrate side views of the flow cell shown in fig. 16A and 16B and a liquid immersion process for imaging a tissue sample therein.

The various features shown in the drawings may not be drawn to scale. Accordingly, the dimensions of the various features may be arbitrarily expanded or reduced for clarity. Moreover, some of the figures may not depict all of the components of a given system, method, or apparatus.

Detailed Description

The detailed description set forth below in connection with the appended drawings is intended as a description of exemplary embodiments of the present invention and is not intended to represent the only embodiments in which the present invention may be practiced. The term "exemplary" used throughout this specification means "serving as an example, instance, or illustration," and should not be construed as preferred or advantageous over other exemplary embodiments. The detailed description includes specific details for the purpose of providing a thorough understanding of exemplary embodiments of the invention. In some cases, some devices are shown in block diagram form.

Sequencing

The systems and methods described herein can be used in conjunction with various nucleic acid sequencing techniques. These sequencing techniques include, but are not limited to, in situ sequencing techniques for reading sequence information from nucleic acids directly from cells or tissues ("Fluorescent in situ sequencing (FISESEQ) of RNA for gene expression in interaction cells and tissues" Nature protocols 10.3 (2015): 442-458; Lee, Je Hyuk, et al, "high ply nested RNA sequencing in situ" Science 343.6177 (2014): 1360-1363; and "Fluorescent in situ sequencing on polymers" Analytical biology 320.1 (2003): Mitra, Robi D, et al, the disclosures of which are incorporated herein by reference in their entirety). Particularly suitable techniques are those in which the nucleic acids are in fixed positions on the substrate (e.g. an array or a tissue sample) such that their relative positions do not change, and in which the substrate is repeatedly imaged. For example, the nucleic acid may be covalently or non-covalently attached to the substrate. Such an embodiment is particularly applicable where the images are obtained in different colour channels, for example coinciding with labels used to distinguish nucleotide base types from each other. In some embodiments, the method of determining the nucleotide sequence of a target nucleic acid can be an automated process. Preferred embodiments include sequencing by synthesis ("SBS") techniques.

SBS techniques typically involve enzymatic extension of nascent nucleic acid strands by repetitive addition of nucleotides to template strands. In the traditional approach of SBS, a single nucleotide monomer can be provided to a target nucleotide in the presence of a polymerase in each delivery. However, in the systems and methods described herein, multiple types of nucleotide monomers can be provided to a target nucleotide in the presence of a polymerase in each delivery.

SBS can utilize nucleotide monomers with a terminator moiety or nucleotide monomers lacking any terminator moiety. Methods of using nucleotide monomers lacking a terminator include, for example, pyrosequencing and sequencing using gamma-phosphate labeled nucleotides, as described in further detail below. In methods using nucleotide monomers lacking a terminator, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the mode of nucleotide delivery. For SBS techniques that utilize nucleotide monomers with a terminator moiety, the terminator may be effectively irreversible under the sequencing conditions used, as is the case with traditional Sanger sequencing using dideoxynucleotides, or the terminator may be reversible as is the case with the sequencing methods developed by Illumina, inc.

SBS techniques can utilize nucleotide monomers that have a tag moiety or nucleotide monomers that lack a tag moiety. Accordingly, incorporation (incorporation) events, such as fluorescence of the label, can be detected based on the identity of the label; the identity of the nucleotide monomer, such as molecular weight or charge; byproducts of incorporation of nucleotides, such as release of pyrophosphate; and so on. In embodiments where two or more different nucleotides are present in the sequencing reagent, the different nucleotides may be distinguishable from each other, or alternatively, two or more different labels may be indistinguishable under the detection technique used. For example, different nucleotides present in a sequencing reagent may have different labels, and they may be distinguished using suitable optics, as exemplified by the sequencing method developed by Illumina, inc.

Preferred embodiments include pyrosequencing techniques. Pyrophosphoric acid sequencing measures the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M., and Nyren, P. (1996) "Real-time DNA sequencing using detection of the phosphorylation reaction" Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing laser on DNA sequencing" Genome Res.11(1), 3-11; Ronaghi, M., Uhlen, M., and Nyren, P. (1998) "A sequencing method base on time" sequencing "ATP, and the release of ATP can be measured at the level of ATP, 35, 85, 320, 210, and optionally, 2, 3-11, features in an array) and the substrate can be imaged to capture a chemiluminescent signal resulting from the introduction of a nucleotide at the location of the nucleic acid on the substrate. After the substrate is treated with a particular nucleotide type (e.g., A, T, C or G), an image can be obtained. The images obtained after addition of each nucleotide type will differ in the features detected on the substrate. These differences in the images reflect the different sequence content of the features on the substrate. However, the relative position of each feature will remain unchanged in the image. The images may be stored, processed, and analyzed using the methods described herein. For example, for images obtained from different detection channels based on reversible terminator sequencing methods, images obtained after processing a substrate using each different nucleotide type can be processed in the same manner as exemplified herein.

In another exemplary type of SBS, cycle sequencing is achieved by stepwise addition of reversible terminator nucleotides that include, for example, a cleavable or degradable (e.g., photobleachable) dye label, as described, for example, in WO 04/018497 and U.S. patent No.7,057,026, the disclosures of which are incorporated herein by reference. Such a process is being commercialized by Illumina inc and is also described in WO 91/06678 and WO 07/123,744, each of which is incorporated herein by reference. Two of the terminators can be reversed, and the availability of fluorescently labeled terminators in which the fluorescent label is cleaved facilitates efficient Cycle Reversible Termination (CRT) sequencing. Polymerases can also be co-engineered (co-engineered) to efficiently incorporate and extend these modified nucleotides.

Preferably, in the reversible terminator-based sequencing embodiment, the tag does not substantially inhibit extension under SBS reaction conditions. However, the detection label may be removable, e.g., by cleavage or degradation. After incorporating the labels into the nucleic acid features on the array or other substrate, an image can be captured. In particular embodiments, each cycle involves the simultaneous delivery of four different nucleotide types to the substrate, and each nucleotide type has a spectrally distinct label. Four images can then be obtained, each using a detection channel selective to one of four different markers. Alternatively, different nucleotide types may be added sequentially, and images of the substrate may be obtained between each addition step. In such embodiments, each image will show the nucleic acid characteristics that have incorporated a particular type of nucleotide. Different images will or will not have different features due to the different sequence content of each feature. However, the relative positions of the features will remain unchanged in the image. Images obtained from such reversible terminator-SBS methods can be stored, processed, and analyzed as described herein. After the image capture step, the label can be removed and the reversible terminator moiety can be removed for subsequent cycles of nucleotide addition and detection. Removal of the markers (after they have been detected within a particular cycle, and before subsequent cycles) may provide the advantage of reducing cross-talk between background signals and cycles. Examples of useful marking and removal methods are described below.

In particular embodiments, some or all of the nucleotide monomers may comprise a reversible terminator. In such embodiments, the reversible terminator/cleavable phosphor may comprise a phosphor bonded to a ribose moiety via a 3' ester bond (Metzker, Genome Res.15: 1767-. Other methods have separated the terminator chemistry from the cleavage of the fluorescent label (Ruparal et al, Proc Natl Acad Sci USA 102: 5932-7(2005), the entire contents of which are incorporated herein by reference). Ruparael et al describe the development of a reversible terminator that uses small 3' allyl groups to block extension, but can be easily unblocked by short treatment with a palladium catalyst. The fluorophore is attached to the base via a photocleavable linker that can be easily cleaved by exposure to long-wave ultraviolet light for 30 seconds. Thus, disulfide bond reduction or photocleavage can be used as a cleavable linker. Another method of reversible termination is to use natural termination that occurs after placement of the bulk dye on the dntps. The presence of a charged bulk dye on a dNTP can act as an effective terminator by steric and/or electrostatic hindrance. The presence of one incorporation event prevents further incorporation unless the dye is removed. The cleavage of the dye removes the phosphor and effectively reverses the termination. Examples of modified nucleotides are also described in U.S. Pat. No.7,427,673 and U.S. Pat. No.7,057,026, the disclosures of which are incorporated herein by reference in their entirety.

Additional exemplary SBS systems and methods that may be used with the methods and systems described herein are described in U.S. patent application publication No.2007/0166705, U.S. patent application publication No.2006/0188901, U.S. patent No.7,057,026, U.S. patent application publication No.2006/0240439, U.S. patent application publication No.2006/0281109, PCT publication No. wo 05/065814, U.S. patent application publication No.2005/0100900, PCT publication No. wo 06/064199, PCT publication No. wo 07/010,251, U.S. patent application publication No.2012/0270305, and U.S. patent application publication No.2013/0260372, the disclosures of which are incorporated herein by reference in their entirety.

Some embodiments may utilize detection of four different nucleotides using less than four different labels. For example, SBS can be performed using the methods and systems described in the incorporated material of U.S. patent application publication No. 2013/0079232. As a first example, a pair of nucleotide types may be detected at the same wavelength, but distinguished based on differences in intensity of one member of the pair from the other members, or based on a change (e.g., via a chemical modification, photochemical modification, or physical modification) in one member of the pair, which results in the appearance or disappearance of a distinct signal compared to the detected signal of the other member of the pair. As a second example, three of four different nucleotide types can be detected under particular conditions, while the fourth nucleotide type lacks labels detectable under these conditions, or is minimally detected under these conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of a third nucleotide type into a nucleic acid can be determined based on the presence of its corresponding signal, and incorporation of a fourth nucleotide type into a nucleic acid can be determined based on the absence or minimal detection of any signal. As a third example, one nucleotide type may comprise a marker(s) detected in two different channels, while the other nucleotide type is detected in no more than one channel. The three exemplary configurations described above are not considered mutually exclusive and may be used in various combinations. An exemplary embodiment combining all three examples is a fluorescence-based SBS method that uses a first nucleotide type detected in a first channel (e.g., dATP with a label detected in the first channel when excited by a first excitation wavelength), a second nucleotide type detected in a second channel (e.g., dCTP with a label detected in the second channel when excited by a second excitation wavelength), a third nucleotide type detected in both the first and second channels (e.g., dTTP with at least one label detected in both channels when excited by the first excitation wavelength and/or the second excitation wavelength), and a fourth nucleotide type that lacks a label that is not or minimally detectable in either channel (e.g., no labeled dGTP).

In addition, sequencing data can be obtained using a single channel as described in the incorporated material of U.S. patent application publication No. 2013/0079232. In this so-called single channel sequencing method, a first nucleotide type is labeled, but the label is removed after the first image generation, and a second nucleotide type is labeled only after the first image generation. The third nucleotide type retains its label in the first and second images, and the fourth nucleotide type remains unlabeled in both images.

Some embodiments utilize sequencing by ligation (ligation) techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and recognize the incorporation of such oligonucleotides. Oligonucleotides typically have different labels that correlate with the identity of a particular nucleotide in the sequence to which the oligonucleotide hybridizes. As with other SBS methods, images can be obtained after processing of nucleic acid features on a substrate (e.g., an array or tissue) with labeled sequencing reagents. Each image will show nucleic acid features that have incorporated a specific type of label. Due to the different sequence content of each feature, different features will be present or absent in different images, but the relative positions of the features will remain unchanged in the images. Images obtained from ligation-based sequencing methods can be stored, processed, and analyzed as described herein. Exemplary SBS systems and methods that may be used with the methods and systems described herein are described in U.S. patent No.6,969,488, U.S. patent No.6,172,218, and U.S. patent No.6,306,597, the disclosures of which are incorporated herein by reference in their entirety.

Some embodiments may utilize nanopore sequencing (Deamer, D.W. & Akeson, M. "Nanopores and nucleic acids: protocols for amplified sequencing" Trends Biotechnol.18, 147-. In such embodiments, the target nucleic acid passes through the nanopore. The nanopore may be a synthetic pore or a biofilm protein, such as alpha-hemolysin. Each base pair can be identified by measuring the fluctuation in conductivity of the well as the target nucleic acid passes through the Nanopore (U.S. Pat. No.7,001,792; Soni, G.V. & Meller, "A.progressive beyond DNA sequencing using solid-state nanopores" Clin. chem.53, 1996-2001 (2007); health, K. "Nanopore-based single-molecule DNA analysis" Nanomed.2, 459-481 (2007); Cockroft, S.L., Chu, J., Amorin, M. & Ghadiri, M.R. "A single-molecule DNA detection DNA polymerase with single-molecule nucleic acid," J.Am. chem.818, 2008. 820, incorporated herein by reference in its entirety). Data obtained from nanopore sequencing can be stored, processed, and analyzed as described herein. In particular, the data may be processed as an image according to the exemplary processing of optical and other images described herein.

Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be detected by Fluorescence Resonance Energy Transfer (FRET) interaction between a fluorophore-containing polymerase and a gamma-phosphate labeled nucleotide, such as described in, for example, U.S. patent No.7,329,492 and U.S. patent No.7,211,414, both of which are incorporated herein by reference, or can be detected using a zero mode waveguide, such as described in, for example, U.S. patent No.7,315,019, both of which are incorporated herein by reference, and using a fluorescent nucleotide analog and an engineered polymerase, such as described in, for example, U.S. patent No.7,405,281 and U.S. patent application publication No.2008/0108082, both of which are incorporated herein by reference. Illumination may limit the volume of zeptolide order around the surface tethered polymerase so that incorporation of fluorescently labeled nucleotides can be observed in a low background (Leven, M.J. et al, "Zero-mode waveguides for single-molecule analysis at high concentrations" Science 299, 682-686 (2003); Lundquist, P.M. et al, "Parallel linkage detection of single molecules in real time" Optit.Lett.33, 1026-. Images obtained from these methods may be stored, processed, and analyzed as described herein.

Some SBS embodiments include detection of protons released when nucleotides are incorporated into the extension products. For example, sequencing based on detection of released protons may use electrical detectors and related Technologies commercially available from Ion Torrent (Guilford, CT, Life Technologies, inc.), or the sequencing methods and systems described in the following documents US 2009/0026082 a 1; US 2009/0127589 a 1; US 2010/0137143 a 1; or US 2010/0282617 a1, all incorporated herein by reference. The methods described herein for amplifying a target nucleic acid using kinetic exclusion can be readily applied to substrates for detecting protons. More specifically, the methods described herein can be used to generate clonal populations of amplicons for detecting protons.

The above-described nucleic acid sequencing methods can advantageously be performed in a multiplex format, such that a plurality of different target nucleic acids are manipulated simultaneously. In particular embodiments, different target nucleic acids can be processed in a common reaction vessel or on the surface of a particular substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of incorporation events in a multiplexed manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an array format. In an array format, target nucleic acids can bind to a surface in a spatially distinguishable manner. The target nucleic acid can be bound by direct covalent attachment, attached to beads or other particles, or bound to a polymerase or other molecule attached to a surface. The array may contain a single copy (also referred to as a feature) of the target nucleic acid at each site, or there may be multiple copies of the same sequence at each site or feature. Multiple copies may be generated by amplification methods, such as bridge amplification or emulsion PCR, described in further detail below.

The methods described herein can use arrays having any one of a variety of densities of features, including, for example, at least about 10 features/cm²100 features/cm²500 features/cm²1,000 features/cm²5,000 features/cm²10,000 features/cm²50,000 features/cm²100,000 features/cm²1,000,000 features/cm²5,000,000 features/cm²Or higher. Other substrates may contain nucleic acid features at similar density ranges.

The methods described herein are advantageous in that they provide for the rapid and efficient detection of multiple target nucleic acids in a parallel fashion. Thus, the present disclosure provides integrated systems capable of preparing and detecting nucleic acids, such as those exemplified above, using techniques known in the art. Thus, the integrated system of the present disclosure may comprise fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, the system comprising components such as pumps, valves, reservoirs, fluid lines, and the like. The flow cell may be configured and/or used in an integrated system for detecting a target nucleic acid. Exemplary flow cells are described, for example, in US 2010/0111768A 1 and U.S. Pat. No.8,951,781, both of which are incorporated by referenceHerein. As exemplified by a flow cell, one or more fluidic components of the integrated system may be used for amplification methods and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more of the fluidic components of the integrated system may be used for the amplification methods described herein, and for the delivery of sequencing reagents in the sequencing methods exemplified above. Alternatively, the integrated system may comprise separate fluidic systems to perform the amplification method and to perform the detection method. Without limitation, examples of integrated sequencing systems capable of forming amplified nucleic acids and determining the sequence of the nucleic acids include, but are not limited to, MiSeq^TMThe platform (Illumina, inc., San Diego, CA), and the device described in U.S. patent No.8,951,781, which is incorporated herein by reference.

Confocal imaging system

Confocal TDI line scan imaging systems having high S/N ratios and high confocality to produce high resolution images are described below with reference to fig. 1, 2, 3, 4A, 4B, 5A and 5B.

In certain embodiments, a confocal TDI line scan imaging system includes a detector array that achieves confocality in the scan axis by limiting the scan axis dimension of the detector array. For example, confocality can be achieved in a single axis of the detector array, such that confocality occurs only in that dimension. Thus, in contrast to typical confocal systems where confocality is achieved in two dimensions, confocal TDI line scan imaging systems may be configured such that confocality is not achieved in more than one dimension.

Confocal TDI line scan imaging systems may also be configured to sequentially detect different portions of a sample by different subsets of elements of a detector array, where charge transfer between subsets of elements proceeds at a rate synchronized with and in the same direction as the apparent motion of the sample being imaged. For example, a confocal TDI line scan imaging system may scan a sample such that a frame transfer device produces successive video images of the sample through a stack of linear arrays aligned and synchronized with the apparent movement of the sample, whereby the stored charge moves with the image as it moves from one line to the next. The accumulation of charge can be integrated over the entire time required for the row of charge to move from one end of the detector to the serial register (or to the storage area of the device in the case of a frame transfer CCD). Exemplary confocal TDI line scan imaging systems are described, for example, in U.S. patent No.7329860, which is incorporated herein by reference. Fig. 1 illustrates a side view of an example of a confocal imaging system 100 according to some embodiments of the invention. Confocal imaging systems are, for example, TDI line scan imaging systems, which have high S/N ratios and high confocality to produce high resolution images.

The presently disclosed confocal imaging system 100 is suitable for use in a photoluminescence-based scanning instrument (or imaging system) for a fluorescence-based SBS system.

Confocal imaging system 100 includes a light source aperture 110, a beam splitter 112, a lens 114, a sensor aperture mechanism 130, and a TDI image sensor 146. In confocal imaging system 100, tissue sample 120 is disposed at a confocal plane 124 relative to lens 114. The tissue sample 120 is a sample tissue to be imaged (or scanned), for example, in an SBS process.

The sensor aperture mechanism 130 is positioned in an optically conjugate plane in front of the TDI image sensor 146 to substantially eliminate out-of-focus signals and provide high confocality. That is, various embodiments of the sensor aperture mechanism 130 include pinholes or slits to substantially eliminate out-of-focus signals. Substantially eliminating out-of-focus signals may be technically advantageous when used in an in-situ testing technique.

As introduced above, in situ sequencing techniques involve reading sequence information from nucleic acids directly from tissue without extracting the nucleic acids from the tissue. This may be in contrast to sequencing techniques that involve extracting nucleic acids from tissue in order to read sequence information from the extracted nucleic acids. Thus, in situ sequencing can provide a deeper understanding of the relationship between the genotype or gene expression of a cell and its morphology and local environment.

By calibrating the sensor aperture mechanism 130 to substantially eliminate out-of-focus signals, only light from the confocal plane that is just focused at the sensor aperture mechanism 130 is allowed to reach the image detector. Thus, the optical resolution of nucleic acids within a particular depth of tissue (from a confocal plane that is just focused at the slit) can be increased relative to a system that does not substantially eliminate out-of-focus signals. This type of optical sectioning simulates removing unwanted portions of tissue (without removing any tissue). Furthermore, the width of the slit (or the size of the pinhole) may be related to the resolution, with smaller slit widths (or smaller pinholes) providing higher resolution.

In operation, light source 150 passes through light source aperture 110, then through beam splitter 112, then through lens 114 and impinges on tissue sample 120 at confocal plane 124. The light source 150 is an excitation light source for illuminating the tissue sample 120 during an imaging (or scanning) procedure. In doing so, the tissue sample 120 emits some in-focus fluorescence 152, as well as some out-of-focus fluorescence 154, relative to the sensor aperture mechanism 130 and the TDI image sensor 146. The focused fluorescence 152 passes through the sensor aperture mechanism 130 and reaches the TDI image sensor 146, while the defocused fluorescence 154 is repelled by pinholes or slits in the sensor aperture mechanism 130. In one example, TDI image sensor 146 is a long linear sensor, such as a 3200 x 64 pixel sensor, to capture high resolution images of tissue sample 120.

Fig. 2 shows another configuration of the confocal imaging system 100 in which the sensor aperture mechanism 130 is positioned in an intermediate image plane 160 conjugate to the TDI image sensor 146. In this configuration of the confocal imaging system 100, an additional pair of lenses 162 is disposed between the sensor aperture mechanism 130 (which is at the intermediate image plane 160) and the TDI image sensor 146. More details of an example of a sensor aperture mechanism 130 for rejecting out-of-focus light are shown and described below with reference to fig. 3, 4A, 4B, 5A, and 5B.

Fig. 3 illustrates a side view of an example of the sensor aperture mechanism 130 of the confocal imaging system 100 shown in fig. 1 and 2. That is, fig. 3 shows an example of a TDI image sensor 146 that contains a 3200 × 64 array of pixels 148 (i.e., 3200 columns × 64 rows, with the first column being column # 1). In this example, the sensor aperture mechanism 130 includes two apertures that are switchable in position — one aperture for odd columns of the TDI image sensor 146 and another aperture for even columns of the TDI image sensor 146. That is, sensor aperture mechanism 130 includes a first aperture plate 132 including slits 134 and a second aperture plate 136 including slits 138. Aperture plate 132 and aperture plate 136 are formed of a material that is not optically transparent to the wavelengths present in confocal imaging system 100. For example, aperture plate 132 and aperture plate 136 may be formed from a glass substrate coated with a patterned opaque layer (e.g., chrome). In addition, the height and length of aperture plates 132 and 136 may depend on the overall size of TDI image sensor 146.

Both aperture plate 132 and aperture plate 136 may be positioned relative to a column of pixels 148 of TDI image sensor 146. The positions of aperture plate 132 and aperture plate 136 are mechanically switchable such that only one aperture plate is in front of TDI image sensor 146 at any given time. For example, aperture plate 132 and aperture plate 136 may be switchable in a rotational or displacement manner under the control of a controller (not shown). The aperture plate 132 is designed such that, when in front of the TDI image sensor 146, the position of the slit 134 substantially corresponds to the position of the odd pixel columns of the TDI image sensor 146. That is, the aperture plate 132 is open for odd pixel columns of the TDI image sensor 146 and blocks even columns. In contrast, aperture plate 136 is designed such that the position of slits 138 substantially corresponds to the position of the even pixel columns of TDI image sensor 146 when in front of TDI image sensor 146. That is, aperture plate 136 is open to even pixel columns of TDI image sensor 146 and blocks odd columns.

In aperture plate 132 and aperture plate 136, placing grooves corresponding to every other (i.e., every second) pixel column ensures sufficient off-focus light rejection. In addition, the sensor aperture mechanism 130 is not limited to two aperture plates. More than two aperture plates may be used if desired to further improve confocality, but with a compromise in reducing the scanning speed. For example, the sensor aperture mechanism 130 may include three aperture plates. The first aperture plate has slits at a first pixel column and then slits at every three pixel columns therebehind. The second aperture plate has slits at the second pixel column and then slits at every three pixel columns therebehind. The third aperture plate has a slit at a third pixel column and then a slit at every three pixel columns thereafter. Again, the positions of the three aperture plates are mechanically switchable such that at any given moment, only one aperture plate is in front of the TDI image sensor 146.

Slits 134 in aperture plate 132 and slits 138 in aperture plate 136 have a width w. The width w is determined by the size of the pixels 148 of the TDI image sensor 146. In confocal imaging system 100, the width w of slits 134 and 138 can be from about 1 μm to about 12 μm in one example, or about 9 μm in another example. The spacing between slits 134 in aperture plate 132 and slits 138 in aperture plate 136 may depend on the pitch p of pixels 148 of TDI image sensor 146. As a non-limiting example, the spacing between slits 134 in aperture plate 132 and slits 138 in aperture plate 136 may be substantially the same as the pitch p of pixels 148 of TDI image sensor 146. In addition, the length of slits 134 in aperture plate 132 and slits 138 in aperture plate 136 may depend on the overall size of TDI image sensor 146. As a non-limiting example, the length of slits 134 in aperture plate 132 and slits 138 in aperture plate 136 may be substantially the same as the width of TDI image sensor 146 along the same dimension of the length of slits 134 and slits 138.

The switching cycles of aperture plate 132 and aperture plate 136 are synchronized with the TDI line scan speed, particularly one switching cycle or an integer number of cycles in a TDI scan readout. In operation, in a first imaging or scanning half cycle, the aperture plate 132 is switched to a position in front of the TDI image sensor 146 whereby odd pixel columns of the TDI image sensor 146 are open and even pixel columns are blocked. In this half-cycle, image data of odd pixel columns of the TDI image sensor 146 is captured. Then, in the next imaging or scanning half cycle, aperture plate 132 is switched off and aperture plate 136 is switched to a position in front of TDI image sensor 146, whereby even pixel columns of TDI image sensor 146 are open and odd pixel columns are blocked. In this half-cycle, image data for even pixel columns of the TDI image sensor 146 is captured. The motion of aperture plate 132 and aperture plate 136 is synchronized with the high-speed TDI imaging process. In one example, aperture plate 132 and aperture plate 136 may be switched at a rate of about 5kHz to about 35 kHz.

Fig. 4A and 4B illustrate side views of another example of the sensor aperture mechanism 130 of the confocal imaging system 100 shown in fig. 1 and 2. In this example, only one aperture is used in front of the TDI image sensor 146, where one aperture can be laterally displaced to alternately allow or block odd and even pixel columns. In one example, the aperture plate 132 described with reference to fig. 3 is disposed in front of the TDI image sensor 146 and is mechanically displaced laterally during the imaging or scanning process. Fig. 4A shows aperture plate 132 and slits 134 in a first position relative to TDI image sensor 146, with odd pixel columns open and even pixel columns blocked. In contrast, fig. 4B shows aperture plate 132 and slits 134 in a second position relative to TDI image sensor 146, where even pixel columns are open and odd pixel columns are blocked.

In operation, in a first imaging or scanning half cycle, the aperture plate 132 is positioned in front of the TDI image sensor 146 such that odd pixel columns are open and even pixel columns are blocked, as shown in fig. 4A. In this half-cycle, image data of odd pixel columns of the TDI image sensor 146 is captured. Then, in the next imaging or scanning half cycle, the position of the aperture plate 132 is mechanically shifted in front of the TDI image sensor 146 so that even pixel columns are opened and odd pixel columns are blocked, as shown in fig. 4B. In this half-cycle, image data for even pixel columns of the TDI image sensor 146 is captured. Again, the motion of the aperture plate 132 may be synchronized with the high-speed TDI imaging process, where the switching rate may be from about 5kHz to about 35 kHz.

Fig. 5A and 5B illustrate side views of yet another example of the sensor aperture mechanism 130 of the confocal imaging system 100 shown in fig. 1 and 2. In this example, the sensor aperture mechanism 130 is a stationary, electronically controlled spatial light modulator 140. Spatial light modulator 140 may be, for example, a Liquid Crystal Display (LCD) based device or a micro-electro-mechanical system (MEMS) mirror device. The window or slit 142 may be electronically disposed in the spatial light modulator 140. The size, number and location of the windows or slits 142 in the spatial light modulator 140 are electronically controlled.

In the confocal imaging system 100, the spatial light modulator 140 can be used in two states. For example, FIG. 5A shows a first state of spatial light modulator 140 in which windows or slits 142 are electronically open, which are substantially aligned with odd columns of pixels 148 of TDI image sensor 146. In contrast, FIG. 5B shows a second state of spatial light modulator 140 in which windows or slits 142 are electronically open, which are substantially aligned with even columns of pixels 148 of TDI image sensor 146. The switching frequency of spatial light modulator 140 is synchronized with the high-speed TDI imaging process. In one example, the switching frequency of spatial light modulator 140 is about 5kHz to about 35 kHz. In the confocal imaging system 100, the spatial light modulator 140 is not limited to two states, and may be two or more states.

Focusing tracking mechanism in imaging process

Certain embodiments of the present invention provide structures that include focus tracking features that can be used to maintain focus during imaging, as described below with reference to fig. 6A, 6B, 7, 8, and 9. For example, the presently disclosed focus tracking mechanism is suitable for assisting laser-based focusing techniques.

Fig. 6A and 6B illustrate a plan view and a cross-sectional view, respectively, of an example of a structure 600 including focusing stripes for improved focus tracking in an imaging process. In this example, structure 600 includes a bottom substrate 610 and a top substrate 612 with a gap 614 disposed therebetween. The tissue sample 120 may be placed on either or both of the bottom substrate 610 or the top substrate 612. The bottom substrate 610 and the top substrate 612 may be, for example, glass, plastic, or silicon substrates. A set of focusing strips 616 is disposed on the side of top substrate 612 facing gap 614. The focusing strips 616 may be formed of, for example, chromium, gold, or other semiconductor-friendly, highly reflective material. The focusing strips 616 may be formed on the top substrate 612 using standard photolithographic processes. Each focusing stripe 616 has a thickness t and a width w. In one example, the focusing strips 616 have a thickness t of about 50nm and a width w of about 50 μm. The focusing strips 616 are disposed on a pitch p. In one example, the pitch p of the focusing strips 616 is about 1100 μm.

In fig. 6 and elsewhere herein, the strip shape is exemplified as a reference or light guide. However, it should be understood that other shapes and designs may be used in addition to or instead of straps. Fig. 7 illustrates a side view of the structure 600 shown in fig. 6A and 6B when used in an imaging process. Figure 7 shows an application that allows imaging through a substrate. Fig. 7 illustrates a structure 600 associated with a lens 618 and a lens focused beam 620, where the lens 618 and the lens focused beam 620 may be a laser-based focusing mechanism. In this example, imaging is performed through the top substrate 612, and wherein the focusing strips 616 are arranged along the scan direction (see fig. 6A). Focusing strips 616 are used to aid focus tracking, where each focusing strip 616 has a physical relationship with tissue sample 120. That is, focusing strips 616 provide physical features in substantially the same plane of tissue sample 120 on which lens focused beam 620 can be focused.

Fig. 8 illustrates a side view of another example of a structure 600 including focusing strips 616 for improved focus tracking in an imaging process. Figure 8 shows an application that does not allow imaging through the substrate. In this example, the top substrate 612 is omitted and the tissue sample 120 is placed on the upper surface of the bottom substrate 610. Focusing strips 616 are disposed on the upper surface of bottom substrate 610, which abuts tissue sample 120. The lens 114 and light source 150 used in the fluorescence imaging process are disposed on the exposed side of the tissue sample 120. In contrast, lens 618 and laser-based lens focused beam 620 (as depicted in fig. 7) are disposed on the bottom substrate 610 side of tissue sample 120.

In this configuration, a lens 618, which is a laser-based focusing mechanism, and a lens focusing beam 620 use focusing strips 616 on the bottom substrate 610. A feedback loop is provided from the laser-based focusing mechanism to the fluorescence imaging mechanism. That is, lens 618, lens focusing beam 620, and focusing strips 616 are used to generate a focus error signal 630 to the fluorescence imaging mechanism (i.e., lens 114 and light source 150). The focus error signal 630 is used to maintain focus during the imaging (or scanning) process.

FIG. 9 illustrates a side view of another technique for providing improved focus tracking during imaging. In this example, tissue sample 120 is placed on top of bottom substrate 610, and tape 122 is cut into tissue sample 120 to expose the tape of bottom substrate 610. The exposed strips of substrate may be used as focusing features by, for example, a laser-based focusing mechanism (e.g., lens 618 and lens focused beam 620).

Flow cell for processing tissue samples

Currently, cell culture processes in flow cell chambers are not optimal. Certain embodiments of the present invention provide flow cells and methods for processing tissue samples, as described below with reference to fig. 10A-17B.

In particular embodiments, advantageous features of a flow cell for processing tissue include, but are not necessarily limited to, one or more of (1) at least temporary access to a surface of the flow cell on which a tissue sample is allowed to be placed, (2) facilitating assembly of flow cell components to at least partially enclose the tissue sample in a flow chamber that allows fluid to contact the tissue sample and allows a detection area for viewing the tissue sample to be formed, and (c) facilitating disassembly to allow the tissue sample to be removed for subsequent analysis (e.g., intact tissue or intact portions thereof) or for reuse of the flow cell. In certain embodiments, the integrity of the flow cell will be substantially the same after disassembly and reassembly. In some embodiments, no tools are required for assembly or disassembly. However, in some cases, a hand tool may be provided for ease of use, without the need for a power tool.

Fig. 10A and 10B illustrate a plan view and a cross-sectional view, respectively, of an example of a flow cell 1000 for holding a tissue sample and performing any of various types of reaction chemistry (e.g., SBS chemistry). In this example, flow cell 1000 includes a bottom substrate 1010 and a top substrate 1012 coupled together using O-rings 1014. The O-ring 1014 may be formed of viton, silicone, or any other material having process compatibility. That is, the bottom base 1010 has a groove 1016 for receiving the O-ring 1014 and the top base 1012 has a groove 1018 for receiving the O-ring 1014. When assembled, the O-rings 1014 fit into the grooves 1016, 1018 of the bottom base 1010 and the top base 1012 and are sandwiched between the bottom base 1010 and the top base 1012. The O-ring 1014 is sized such that when the bottom base 1010, top base 1012, and O-ring 1014 are assembled together, there is a space or gap between the bottom base 1010 and the top base 1012. In this space or gap, O-ring 1014 defines a reaction chamber 1020 in flow cell 1000. In addition, the top substrate 1012 has an inlet 1022 and an outlet 1024 for flowing a liquid (e.g., a reagent) into and/or through the reaction chamber 1020 of the flow cell 1000. Further, in one example, bottom base 1010, top base 1012, and O-ring 1014 may be held together using screws 1026. Fig. 10B also shows tissue sample 120 within reaction chamber 1020 of flow cell 1000.

Fig. 11 illustrates a flow chart of an example of a method 1100 of processing a tissue sample using the flow cell 1000 shown in fig. 10A and 10B. Method 1100 may include, but is not limited to, the following steps.

At step 1110, a first substrate of a flow cell is provided. For example, a bottom substrate 1010 of the flow cell 1000 is provided.

At step 1115, a sample tissue is placed on a first substrate. For example, tissue sample 120 is placed on bottom substrate 1010 of flow cell 1000.

At step 1120, a second substrate is provided and assembled to the first substrate, wherein a reaction chamber is formed around the sample tissue. For example, top base 1012 is provided and assembled to bottom base 1010 using O-ring 1014 and screws 1026. In doing so, O-ring 1014 defines a reaction chamber 1020 around tissue sample 120.

At step 1125, the sample tissue is chemically manipulated. For example, the tissue sample 120 is chemically manipulated (e.g., SBS chemical manipulation) using the inlet 1022 and outlet 1024 to flow fluid into and/or through the reaction chamber 1020 of the flow cell 1000. In this example, the imaging or scanning process of the tissue sample 120 may occur through the bottom substrate 1010 and/or the top substrate 1012.

The methods may comprise imaging steps with sequencing or other nucleic acid detection techniques (such as those set forth elsewhere herein). Alternatively, the method may comprise the step of obtaining a picture, image or other representation of the physical form or structure of the tissue sample. The representation may be obtained via light field, fluorescence or other microscopy techniques, and may optionally be aided by the use of dyes or labels. The comparison of the representation to the spatially resolved nucleic acid detection results can be used to locate genetic information having identifiable characteristics of tissue. Exemplary methods for spatial detection of nucleic acids that can be modified for use in the devices and methods described herein are described in U.S. patent application publication No.2014/0066318 a1 and PCT application publication No. wo 2014/060483 a1, both of which are incorporated herein by reference.

Fig. 12A and 12B illustrate a plan view and a cross-sectional view, respectively, of another example of a flow cell 1200 for holding a tissue sample and performing any of various types of reaction chemistry, such as SBS chemistry. In this example, flow cell 1200 includes a bottom substrate 1210 and a top substrate 1212. Bottom substrate 1210 and top substrate 1212 are bonded together with an adhesive layer 1214 sandwiched therebetween. Openings are provided in the adhesive layer 1214 to form reaction chambers 1216 in the flow cell 1200, more details of which are shown in fig. 14A and 14B. Additionally, an inlet 1218 and an outlet 1220 are disposed in the top substrate 1212. Inlet 1218 and outlet 1220 are used to flow a liquid (e.g., a reagent) into and/or through reaction chamber 1216 in flow cell 1200.

Adhesive layer 1214 is used to couple bottom substrate 1210 and top substrate 1212 together. In one example, the adhesive layer 1214 is a layer of double-sided tape, such as Ultraviolet (UV) curable double-sided tape.

Within reaction chamber 1216 of flow cell 1200, a tissue sample may be placed on the top substrate, the bottom substrate, or both substrates. For example, fig. 12B shows the tissue sample 120 within the reaction chamber 1216 and on the bottom substrate 1210. In another example, and referring now to fig. 13A, tissue sample 120 within reaction chamber 1216 is on top substrate 1212. In yet another example, and referring now to fig. 13B, within reaction chamber 1216, a first tissue sample 120 is on bottom substrate 1210 and a second tissue sample 120 is on top substrate 1212.

Reference is now made to fig. 14A and 14B, which are a plan view and a cross-sectional view, respectively, of an example of an adhesive layer 1214 as an adhesive portion of the flow cell 1200 shown in fig. 12A and 12B. That is, fig. 14A and 14B show an opening 1230 in the adhesive layer 1214 that is used to form the reaction chamber 1216 of the flow cell 1200. In one example, the thickness of adhesive layer 1214 is about 100 μm.

Fig. 15 illustrates a flow chart of an example of a method 1500 of processing a tissue sample using the flow cell 1200 shown in fig. 12A and 12B. Method 1500 may include, but is not limited to, the following steps.

At step 1510, a first substrate of a flow cell is provided. For example, a bottom substrate 1210 of the flow cell 1200 is provided.

At step 1515, a sample tissue is placed on the first substrate. For example, the tissue sample 120 is placed on the bottom substrate 1210 of the flow cell 1200.

At step 1520, a second substrate is provided and then coupled to the first substrate using an adhesive layer, wherein the adhesive layer defines a reaction chamber around the sample tissue. For example, a top substrate 1212 is provided and then coupled to a bottom substrate 1210 using an adhesive layer 1214 (e.g., uv-cured double-sided tape), wherein an opening 1230 in the adhesive layer 1214 forms a reaction chamber 1216 around the tissue sample 120. In the case of uv-cured double-sided tape, the uv-curing operation may occur during this step of forming a bond between the adhesive layer 1214 and the bottom and top substrates 1210 and 1212.

At step 1525, the sample tissue is chemically manipulated. For example, using the inlet 1218 and outlet 1220, a liquid is flowed into and/or through the reaction chamber 1216 of the flow cell 1200 and the tissue sample 120 is subjected to a chemical operation (e.g., SBS chemical operation). In this example, the imaging or scanning process of tissue sample 120 may occur through bottom substrate 1210 and/or top substrate 1212. Again, imaging may be performed as part of the nucleic acid detection technique, and/or to determine the shape or form of the tissue sample.

Fig. 16A and 16B illustrate side views of an example of a flow cell 1600 that uses an open container to hold a tissue sample and an example of a process of "dry" imaging a tissue sample therein. In this example, flow cell 1600 includes an open vessel 1610. Two or more tubes are disposed relative to open vessel 1610 as its inlet(s) and/or outlet(s). For example, tube 1612 and tube 1614 are disposed relative to open vessel 1610 with one end of tube 1612 and one end of tube 1614 within open vessel 1610. That is, tube 1612 and tube 1614 are used to flow liquid 1620 (e.g., reagents) into and/or through open vessel 1610. In addition, fig. 16A and 16B illustrate tissue sample 120 within open vessel 1610.

In imaging the tissue sample 120 in the open vessel 1610, fig. 16A shows the open vessel 1610 filled with liquid 1620 and the chemical operations taking place on the tissue sample 120. Referring now to fig. 16B, upon completion of a chemical operation using tube 1612 and tube 1614, open vessel 1610 substantially drains liquid 1620, and then an imaging or scanning process of tissue sample 120 occurs through the air gap without liquid 1620. That is, fig. 16B shows a substantially "dry" imaging process. Some minimum amount of moisture content may be maintained in open vessel 1610 such that tissue sample 120 may not be completely dry.

Referring now to fig. 17A and 17B, a liquid immersion imaging process may be used. For example, fig. 17A shows open vessel 1610 filled with liquid 1620 and the chemical operations taking place on tissue sample 120. An imaging lens (e.g., lens 114) is positioned outside of open vessel 1610 and is not submerged in liquid 1620. Upon completion of the chemical operation, fig. 17B shows open vessel 1610 still filled with liquid 1620, and the imaging lens (e.g., lens 114) is lowered into open vessel 1610 to be exposed and submerged in liquid 1620. In this example, the imaging or scanning process of the tissue sample 120 occurs without an air gap. Without an air gap, resolution and S/N ratio can be improved, and focusing is easier.

In the foregoing detailed description with reference to fig. 1-17B, the terms "top", "bottom", "above", "below" and "upper" are used throughout the description with reference to the relative positions of the components of the structure and/or flow cell (e.g., the relative positions of the top and bottom substrates of the flow cell) with the understanding that the structure and/or flow cell are functional regardless of their orientation in space.

The foregoing detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the disclosure. Other embodiments having different structures and operations do not depart from the scope of the present disclosure. The terms "invention" and the like are used with reference to certain specific examples of many alternative aspects or embodiments of applicants 'invention set forth in the specification, and neither its use nor its absence is intended to limit the scope of applicants' invention or the claims. This specification is divided into two parts for the convenience of the reader only. The title content should not be construed as limiting the scope of the invention. The definitions are intended to be part of the description of the invention. It will be understood that various details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

In this application, conditional language such as "may," "can," "might," or "may" is generally intended to mean that some embodiments include certain features, elements and/or steps, while other embodiments do not, unless expressly stated otherwise or understood otherwise in the context of usage. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for determining whether such features, elements and/or steps are included or are to be performed in any particular embodiment, with or without user input or prompting. Throughout this application, various publications, patents and/or patent applications have been cited. These publications are incorporated by reference into this application in their entirety.

The term "comprising" is intended to be open-ended, and includes not only the recited elements, but also any additional elements.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims.

The various operations of the methods described above may be performed by any suitable means capable of performing the operations, such as various hardware and/or software components, circuits, and/or modules. Generally, any of the operations shown in the figures can be performed by corresponding functional means capable of performing the operations.

Information and signals may be represented using any of a variety of different technologies and techniques. For example, data, instructions, commands, information, signals, bits, symbols, and chips that may be referenced throughout the above description may be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields or particles, or any combination thereof.

The various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. The described functionality may be implemented in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the embodiments of the invention.

The various illustrative blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with the following: a general purpose processor, a Digital Signal Processor (DSP), an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

The steps of a method or algorithm described in connection with the embodiments disclosed herein, as well as the functions, may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. If implemented as software, the functions may be stored on and transmitted over as one or more instructions or code on a tangible, non-transitory computer-readable medium. The software modules may reside in: random Access Memory (RAM), flash memory, Read Only Memory (ROM), electrically programmable ROM (eprom), electrically erasable programmable ROM (eeprom), registers, hard disk, a removable disk, a CD ROM, or any other form of storage medium known in the art. A storage medium is coupled to the processor such that the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. Disk and disc, as used herein, includes Compact Disc (CD), laser disc, optical disc, Digital Versatile Disc (DVD), floppy disk and blu-ray disc where disks usually reproduce data magnetically, while discs reproduce data optically with lasers. Combinations of the above should also be included within the scope of computer-readable media. The processor and the storage medium may reside in an ASIC. The SIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

For purposes of summarizing the disclosure, certain aspects, advantages and novel features of the invention have been described herein. It is to be understood that not necessarily all such advantages may be achieved in accordance with any particular embodiment of the invention. Thus, the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

Various modifications to the above-described embodiments will be readily apparent, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (6)

1. A method of processing a tissue sample, the method comprising:

providing a first substrate of a flow cell;

placing a sample tissue on the first substrate;

providing a second substrate and assembling the second substrate to the first substrate, wherein a reaction chamber is formed around the sample tissue; and

imaging the sample tissue in the reaction chamber.

2. The method of claim 1, wherein the reaction chamber comprises a spacer disposed between the first substrate and the second substrate.

3. The method of claim 2, wherein the spacer comprises an O-ring.

4. The method of claim 2, wherein the spacer comprises an adhesive layer.

5. The method of claim 1, wherein the flow cell comprises a liquid and an imaging lens is immersed in the liquid.

6. The method of claim 5, wherein the flow cell substantially drains liquid and the imaging lens is no longer immersed in the liquid.