JP7727702B2 - Microfluidic chip device for optical force measurement and cell imaging using microfluidic chip configuration and dynamics - Google Patents

Description

The present invention relates generally to devices and methods for particle analysis and imaging of particles or cells in fluids, and more particularly to devices and methods for particle imaging of fluids using pressure, hydrodynamics, electrokinetics, and optical forces.

The present invention relates to a microfluidic chip in which injections are made in a vertical upward direction and fluid vials are placed below the chip to minimize particle settling before and in the analytical portion of the chip's channels.

In order to implement the present invention, modifications had to be made to existing microfluidic chip designs. For example, to keep the vials vertically inline with the chip, a different interface with the chip had to be established compared to the prior art. Specifically, instead of input tubing interfacing with the chip through a port attached to the largest surface of the chip (as is typically done in microfluidic lab-on-a-chip systems), the fluid is pumped first perpendicularly, then across the chip, and then up the chip. In one aspect, the present invention uses a manifold to have the input and fluids rise through the bottom of the chip, thereby avoiding horizontal reorientation of the fluid/fluid dynamics.

According to the present invention, the contents of a vial are placed beneath the chip and pumped upward and vertically directly into the first channel of the chip. A long channel extends from the bottom of the chip to near the top of the chip. The channel then takes a short horizontal turn, but the new channel is very short, nearly negating the effects of cell settling due to gravity and zero flow velocity at the walls. Next, unlike prior art, the fluid is pumped up to the analysis section. Therefore, the horizontal analysis section is the highest channel/fluid point within the chip and is therefore closer to the top of the chip, resulting in less chip material (e.g., glass) between the microscope/camera and the sample than in prior art, and therefore clearer imaging. Additionally, a laser suspends the cells within this channel during analysis, preventing them from settling.

According to conventional techniques, microfluidic chip vials containing cells or particles to be separated and/or analyzed are positioned on their sides and pumped horizontally into channels within the microfluidic chip. The contents of the vial (e.g., particles or cells) are first pumped vertically upward, then make a U-turn to move downward, and then pumped horizontally into the chip (see, e.g., U.S. Patent No. 9,594,071).

The connections to the chip are horizontal, which, combined with dead volume at the connections (empty space in fluid connections that is unavoidable to some extent), results in significant additional settling due to gravity. Such a configuration also requires a relatively large diameter channel required by the connections, which, in addition to the dead volume, creates a region of relatively low velocity, further increasing the problem of particle settling. The current chip of the present invention eliminates the need for a large horizontal input channel and a rather abrupt transition from the large horizontal input channel to a relatively thin upward flow in the first vertical chip channel. This configuration eliminates unnecessary changes in direction that cause horizontal settling and sedimentation. Having cells enter the bottom edge of the chip also solves the problem of settling in the dead volume by orienting them perpendicular to gravity so that cells or particles cannot settle to the bottom of the horizontal channel but rather are always guided upward by the flow. This is not intuitive and required considerable experimentation to realize the problem before designing the current embodied solution. Currently available microfluidic devices, in contrast to the present invention, incorporate custom or commercially available connections on polished surfaces and larger areas of glass, which generally force any particles (e.g., cells) contained within the sample stream to immediately rotate and move horizontally upon entering the chip.

Also, in prior art, cells or particles have several horizontal runs on the microfluidic chip before reaching the analysis channel, which results in sedimentation. When the vial contents enter the chip and the channels within the chip, the contents are pumped horizontally compared to the vertical in-chip channels. The channels then flow upward, making a long horizontal turn. At this point, gravity tends to cause the cells to settle to the bottom of the channel. They also experience lower velocities at the walls due to laminar flow conditions. Essentially, due to a parabolic velocity profile, the flow is highest in the middle of the channel and decreases to zero at or near the channel wall. After the initial horizontal in-chip channel, the fluid takes a downward turn before the analysis channel, where the particles are imaged or separated. Due to this configuration, there is a relatively large distance between the microscope/camera and the analysis channel. In this typical prior art configuration, the particles are forced downward, eventually exiting the bottom of the chip.

Furthermore, due to limitations of the prior art, multiple horizontal runs were required, causing cells to settle in multiple locations within the channel. This, in turn, required imaging through additional material at the edge of the chip, resulting in reduced image quality. Prior art channels within the chip required pumping vertically upward, then horizontally, then in a zigzag manner for the appropriate cell or particle suspension to then be pumped down the chip and out of the chip. The zigzag channels are eliminated by the present invention.

There is also prior art for rendering 3D images of cells or particles in fluids. For example, M. Habaza, M. Kirschbaum, C. Guernth-Marschner, G. Dardikman, I. Barnea, R. Korenstein, C. Duschl, and N. T. Shaked, "Adv. Sci.", 2017, 4, 1600205, teaches capturing a cell, rotating it at high speed, and measuring the refractive index distribution within the cell using interferometry. Interferometry has also been used to analyze cells in microfluidic channels (see, for example, Y. Sung et al., "Phys. Rev. Appl.", 2014, 2, 27, 1:014002). However, the present invention claims to capture multiple images of a cell or particle as it moves through a fluid flow and passes the focal plane of an imaging device, thereby negating the need to capture the cell to render a 3D image. Other techniques have been taught, such as using a mechanical translation stage to move the cell or particle (e.g., N. Lue et al., Opt. Express, September 29, 2008, S16(20):16240-6), but none of them use bright-field imaging as described herein, nor do they utilize fluid flow to provide cell positioning relative to the image focal plane.

All prior art documents cited herein are incorporated by reference in their entirety.

The present invention relates to a microfluidic chip in which injections are performed and sample vials are placed below the chip to minimize particle settling. Thus, the contents of the vial are located below the chip and pumped upward and vertically directly into the chip's channels. A long channel extends from the bottom of the chip to near the top of the chip. The channel then takes a short horizontal turn, but the new channel is short enough that the effects of cell settling due to zero flow velocity at the channel walls are negligible. Contrary to prior art, the sample is then pumped up to the analysis section. Thus, the horizontal analysis section is the highest channel/fluid point within the chip and is therefore closer to the top of the chip, resulting in less glass between the microscope/camera than in prior art and therefore clearer imaging. The distance of the analysis channel from the top of the chip can be between 100 microns and 2 mm, but can also be as large as 100 mm, such as between 100 microns and 200 microns, 200 microns and 300 microns, or 300 microns and 400 microns. In one embodiment, after the analysis portion of the chip, the sample, e.g., fluid, cells, and/or particles, is pumped down to the bottom of the chip and pushed out.

The present invention is further directed to microfluidic chips in which horizontal flow is minimized, particularly at the point where fluid enters the chip channels. Prior art chips include horizontal channels (non-analytical portions) of approximately 13 mm, with injection ports of much larger diameters of approximately 2 mm, exacerbating sedimentation due to low velocities. In preferred embodiments, the chips described herein have horizontal channels (non-analytical portions) of approximately 0.2-3.0 mm, although horizontal channel lengths can range from 0.01-100.0 mm, e.g., 0.01-0.02 mm, 0.02-0.03 mm, 0.03-0.04 mm, etc. This is a result of the channel system of the present invention, which is an order of magnitude different from the prior art, improving cell/particle flow and eliminating sedimentation.

Another aspect of the invention relates to microfluidic chips in which imaging and analysis are based at or near the corners of the chip, thereby improving imaging from multiple viewpoints because less glass and distance exists between the camera and the analysis channel. This also allows for the use of higher numerical objective lenses to improve detailed imaging by increasing magnification. Such design improvements reduce image distortion caused by glass (or other materials comprising the chip, such as plastic or any transparent or translucent material) and distance (e.g., due to glass imperfections). The distance between the imaging device and the analysis channel may be 100 microns to 2 mm, but may be as large as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, etc.

Additionally, the present invention relates to a microfluidic sorting chip, separated downstream from the analysis channel, that allows for the simultaneous or sequential use of both pressure and/or laser (or other optical force) to actuate the sorting function. In one aspect, flow continues from the analysis channel for the sorting function. For example, particles are directed into a vertical channel and then into a horizontal sorting channel. In one embodiment, optical force and/or pressure is applied in the direction of flow using the sorting channel to push particles through the channel. Particles not directly acted upon by optical force are diverted to an alternative channel, for example, by gravity, electrokinetic force, magnetic force, laminar flow lines, flow lines, a reduced flow rate, an orthogonal optical force, or a vacuum applied to suck the particles into the alternative channel. In another aspect related to the post-sorting analysis channel, the present invention allows for the directing of an optical force from the backside of the chip (where the laser or optical force is directed in the same flow direction) and, in some aspects, splitting this primary laser. In embodiments, optical force and/or pressure may be applied in the opposite direction of material movement through the channel, e.g., against the flow, or in the same direction as material movement in the channel, e.g., along with the flow. Cell or particle sorting can occur on a single device or on separate chips. For example, in FIG. 1B, the fifth channel before the outlet tube 145 includes one or more branches to allow for single or multiple sorting regions.

In another aspect of the invention, a manifold is connected to a vial such that tubing passes through the manifold and connects to a vial or other container on the opposite side that contacts the substance (e.g., fluid) in the vial. The manifold allows the vial to be connected to the microfluidic chip but stored below the chip, and/or the manifold allows the contents of the vial to be injected from the bottom of the chip, alleviating some of the problems experienced with prior art, such as settling of cells or particles when vials or tubing from the vial connect to or communicate with the microfluidic chip.

In another aspect, the present invention is directed to a microfluidic chip holder that includes a structure for directing a light source, including an integrated prism cavity, such that when the prism is engaged, the light exits at an angle relative to the chip. This is a preferred method for illuminating constrained geometries. In embodiments, the light source includes, but is not limited to, an optical fiber or a collimated or focused light source. The light source is specifically aimed or oriented precisely toward the analysis channel.

In another aspect of the invention, the device includes a second imaging device oriented orthogonally to the first camera and channel view. The reasons for the second camera vary. In one aspect, the reason for the second imaging device is to aid in visual alignment of laser or optical forces within the analysis channel. In another aspect, the methods described herein can be used to record data from the first camera. The second camera can combine the data with data from the first camera, resulting in additional data that can be used to more accurately extrapolate cell location, size, shape, volume, etc. This additional information about the same cell (or particle) increases the precision and range of measurement and analysis. In a further aspect, the second camera is combined with the first camera to enable 3D reconstruction of a cell or particle, or group of cells or particles, imaged by the orthogonal camera and a camera in the flow or positioned toward the side of the chip. Using algorithms described herein or elsewhere, including reversing or slowing the flow and taking one or more images of a particular cell or particle, or group of cells or particles, the present invention allows multiple images to be analyzed and processed, thereby allowing for the determination of characteristics/attributes/quantitative measurements such as cell volume, cell shape, nucleus location, nuclear volume, organelle or inclusion location, etc. In another aspect of the present invention, a camera is oriented to image an image in-axis in the direction of flow.

In one aspect, the present invention does not require serpentine or zigzag channels to keep particles properly suspended, as is preferred by the prior art. Due to the vertical nature of the fluids and particles or cells injected into the chip, zigzag channels are avoided by vertically integrated pumped particles flowing through the channel directly into a first horizontal channel (referred to herein as the second channel).

The accompanying drawings illustrate certain aspects of some embodiments of the invention and should not be used to limit or define the invention, and together with the written description, serve to explain certain principles of the invention.
FIG. 1A shows an overall view of the device configuration, including the chip, manifold, and vial. FIG. 1B shows a specific depiction of the orientation and position of the chip channels. 2A and 2B include diagrams illustrating a microfluidic chip holder according to the present invention. 2A and 2B include diagrams illustrating a microfluidic chip holder according to the present invention. 3A and 3B are diagrams showing angles and aspects of a tip holder according to the present invention. 3A and 3B are diagrams showing angles and aspects of a tip holder according to the present invention. 4A and 4B are diagrams illustrating an example of a cell path and imaging configuration for a chip. 4A and 4B are diagrams illustrating an example of a cell path and imaging configuration for a chip. FIG. 4C is a diagram illustrating an alternative cell path. FIG. 5 is a diagram illustrating on-chip multi-plane imaging and how it can be used to render 3D images and information. 6A and 6B are diagrams illustrating on-chip multi-planar imaging in fluid flow and how it can be used to render 3D images. 6A and 6B are diagrams illustrating on-chip multi-planar imaging in fluid flow and how it can be used to render 3D images. FIG. 7 shows how cells can be trapped and/or equilibrated within the analysis portion of the chip and imaged from multiple angles. FIG. 8 shows how the camera and illumination source are positioned along the direction of the laser and flow so that the particles are directed away from the camera. FIG. 9 shows how the camera and illumination source can be positioned along the direction of the laser and flow so that the particles move towards the camera.

The present invention has been described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Those skilled in the art will recognize that these features may be used alone or in any combination, based on the requirements and specifications of a given application or design. Embodiments incorporating various features may also consist of, or consist essentially of, these various features. Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. The description of the invention provided is merely exemplary or explanatory in nature and, thus, variations that do not depart from the essence of the invention are intended to be within the scope of the invention.

Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

Referring now to the drawings, FIG. 1A shows an overall view of the device taught herein. Sample vials 130 are positioned below the microfluidic chip 100 (also generally referred to herein as the substrate) and are not limited in number or size. The vials are configured to hold any type of sample that can be transferred through the device, such as one or more substances, including, but not limited to, fluids, liquids, gases, plasma, serum, blood, cells, platelets, particles, etc., or combinations thereof. In the context of this specification, the terms "fluid" or "sample" can be used generally to refer to such one or more substances. The vials are connected, directly or indirectly, using air tubing 110 that is in operative communication with the underside of the chip. They can also be connected by a manifold 120, as further shown in FIG. 1A. FIG. 1B shows a preferred embodiment of the microfluidic chip 100 in which substances, fluids, particles, and/or cells are injected into one exterior surface, such as the edge of the microfluidic chip (e.g., the XZ plane in FIG. 1B). Injection is shown in FIG. 1B along one of the planes shared by the smallest distance, such as the XZ or XY plane. In this particular embodiment, the length of side X 160 is shorter than the length of side Y 170 and the length of side Z 180. Such a configuration allows the sample to have minimal deviation as it enters the channels on the chip (e.g., no right turns are required, as is common in the current state of the art).

1A shows a manifold 120 connecting one or more vials 130 to a microfluidic chip 100 so that substances, fluids, particles, and/or cells can be injected or pumped vertically and upward into the microfluidic chip. The manifold allows for injection of substances from the bottom of the chip, while arranging the electronics, flow sensors, and tubing (both liquid and air) to minimize cell settling, optimizing throughput. The air tubing provides pressure or vacuum, which, when sealed, provides a closed system within the manifold device. In one embodiment, there is no seal between the vial and the manifold, and there is a distance indicating that the pressure in the internal volume is atmospheric. This allows for pumping of substances from or to a vessel open to the atmosphere (a vacuum is required to pump from an open vessel). The manifold arranges the electronics, flow sensors, and tubing (both liquid and air) to minimize cell settling, optimizing throughput.

By injecting contents from the bottom of the chip, the present invention minimizes horizontal movement of the inlet tube 140 and outlet tube 145, which can cause problems such as particle or cell settling within the channel 150. The outlet tube is offset from the inlet tube in the Z and X dimensions (Figure 1B) in this example. In embodiments, the outlet tube is offset, straight, in-line, or angled relative to the inlet tube. This configuration eliminates the need for serpentine or zigzag vertical channels, as the current configuration solves problems with settling that arise in prior art, such as when fluid combined with cells and/or particles is injected or pumped horizontally into the chip from the side, where the direction and fluid dynamics must be forced upward. Injection from the manifold and bottom of the chip also allows additional elements, components, machinery, or hardware to be placed below the chip (see Figure 1A).

The manifold 120 functions by regulating the air pressure above the contents in the vials and providing the correct geometry for the flow sensors and electronics. Tubes, such as fluid or air tubes, pass through the manifold and connect to the vials on the other side. Pressurized air passes through one side of the manifold, creating a closed, pressurized system within the manifold. By adjusting the pressure in the enclosed area, the system allows for changes to parameters such as flow rate and fluid dynamics. Pressurized areas are present in both the vials 130 and the manifold 120. In another aspect, no air connection is required to the vials, as they are open to the ambient atmosphere. This allows for sampling of a wider variety of containers and sources. In such an embodiment, a vacuum is applied to one or more other vials, creating a pressure differential to drive fluid flow from the open container.

In one embodiment, pressure-based sample injection is used. A vial is filled with the sample in fluid and sealed with either a lid or tubing connected to the chip. Before attaching the lid, the vial can be open to air or sealed with a septum or other airtight device. In one aspect, the lid can include two connections, one for a fluid such as a gas and one for a liquid. Optionally, the method embodiment further includes providing a sample inlet line tip in communication with a sample inlet line in communication with the first channel.

In another embodiment, vacuum-based sample injection is used. A vial is filled with the sample in fluid and sealed with either a lid or tubing connected to the chip. Prior to attachment of the lid, the vial can be open to air or sealed with a septum. In one aspect, the lid can include two connections, one for a fluid such as a gas and one for a liquid. Optionally, fluid can be drawn from the open-to-air vial by applying vacuum pressure to one or more other vials. Optionally, the method embodiment further includes providing a sample inlet line tip in communication with a sample inlet line in communication with the first channel.

2A-B and 3A-B show chip holders 200, 300. The chip holders include a structure for guiding a light source 210, such as a fiber optic light source, a light emitting diode, or a laser, and an integrated prism cavity 220, 320 into which a prism can be mounted. The light source is guided or aligned to a desired location by an integrated structure or channel 240 within the chip holder. The built-in space for the fiber optic light source enables illumination, such as a cone of illumination 250, even in constrained geometric environments, such as on a microfluidic chip 230, 330 or a channel within a microfluidic chip. In a preferred embodiment, the light source is precisely aimed, directed, or focused specifically at the analysis channel 260. In a preferred embodiment, the chip holder includes built-in space for the prism 220, 320 and the fiber optic light source 210, enabling illumination with a constrained geometry. The chip further includes a hole or opening at the bottom of the holder 350 for precise alignment of fluidic tubes, as described herein. In one embodiment, adjustable screws are integrated into threaded holes 360 on one or more surfaces for proper alignment.

FIG. 4A illustrates a preferred embodiment of a microfluidic chip 400 described herein. As shown, fluid first travels vertically upward through a first channel 410 and then communicates with a second horizontal channel 420. Another vertical channel 430 takes fluid closer to the top of the chip, where a fourth channel 440 is horizontal, and includes the analysis channel, as shown in FIG. 4. The channels are in operative communication with one another to allow sample to move through the system from one channel to another. In embodiments, sample can flow from the first channel to the second channel, to the third channel, to the fourth channel, or vice versa, or combinations thereof. Pumps and/or vacuum devices can be provided to provide positive and/or negative pressure to either or both the openings and outlets of the channels to enable material movement through the channels. The analysis channel is proximate to one or more exterior surfaces of the substrate, such as the face, edge, or side of the chip. For example, with this configuration, the analysis channel is proximate to the top and sides of the chip, thereby improving imaging and analysis of materials throughout the microfluidic chip. In preferred embodiments, the analysis channel is about 1 mm to about 2 mm from the top and sides of the chip. However, the distance of the analysis channel from the top of the chip can be 0.1 mm to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, or 0.3 mm to 0.4 mm. Stated differently, the analysis channel can be located within the top 50%, 33%, 25%, 10%, or 5% of the substrate. The length of a horizontal analysis channel according to the present invention can be about 250 microns to about 10 mm. However, the length of the analysis channel can also be 100 microns to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, or 0.3 mm to 0.4 mm. In other words, the length of the analysis channel can be about 75% or less of the height, width, or length of the substrate/chip, for example, 50% or less, 33% or less, 25% or less, 10% or less, or 5% or less of the height, width, or length of the substrate/chip.

Figure 4B shows two imaging devices 450, such as machine vision cameras. In one embodiment, the cameras may be positioned above the chip and oriented perpendicular to the analysis channel. In another embodiment, the cameras may be positioned on the side of the chip and oriented perpendicular to the direction of flow or at an angle to the direction of flow (e.g., above the channel, below the channel, or at an angle to the side of the channel). In embodiments, the cameras can be positioned so that imaging is performed at any angle to the flow of one or more substances, such as perpendicular or at 90 degrees to the flow of one or more substances, or between 0 and 90 degrees, or between 10 and 80 degrees, or between 30 and 60 degrees, etc. In another embodiment, two or more cameras can be used to image cells or particles in the analysis channel. For example, a camera may be positioned above the chip and oriented perpendicular to the analysis channel. A second camera may be positioned on the side of the chip and oriented perpendicular to the direction of flow or at an angle to the flow (e.g., above the channel, below the channel, or at an angle to the side of the channel). As shown in Figure 4B, one or more light sources 460 can be used to illuminate the analysis channel 440; such light sources can be positioned below the chip, illuminate the side of the chip, illuminate in the direction of flow or opposite to the direction of flow, illuminate above the chip, or illuminate the analysis channel at an angle.

Alternatively, a dichroic mirror 840 or other suitable optical element can be used to selectively redirect light in certain wavelength ranges and pass light in other wavelength ranges, as shown in FIG. 8. This allows the camera 810 to be positioned in-line with the analysis channel. Several embodiments arise, including placing the optical power laser 830 and camera 810 at the same end or opposite ends of the analysis region, as shown in FIGS. 8 and 9. An illumination source 860 for the camera may also be required and can be directed in several ways, such as those shown in FIGS. 8 and 9. The light source can be a broad spectrum light source, such as one or more LEDs, or a narrow light source, such as a laser. The camera can be used as a single camera or as part of a multi-camera system, in combination with other viewpoints, as described herein.

Also, as depicted in FIG. 4A, a light source 480, such as a laser, can be used to affect the flow of cells. The laser can be positioned along the flow of cells, or opposite the flow of cells. The laser can also be positioned and/or oriented perpendicular or oblique to the flow of cells.

One embodiment of the present invention includes a device for particle analysis (see, e.g., Figures 4-9). The embodiment of the present invention includes at least one camera 450 for capturing images of particles or cells within a microfluidic channel (e.g., 440). In one embodiment, a laser or other optical force 480, such as a collimated light source, operable to generate at least one collimated light source beam is included. The at least one collimated light source beam includes at least one beam cross section. The embodiment of the present invention includes a substrate having a first channel 410 extending vertically within the substrate, such that a first plane intersects the first channel 410 substantially along its length, thereby allowing a fluid sample to be injected into the substrate/chip from the bottom and forced upward by positive or negative pressure. The embodiment of the present invention includes a second channel 420 disposed horizontally within the substrate, such that a second plane intersects the second channel 420 substantially along its length, the second plane being disposed orthogonal to the first plane. This second channel is horizontal to the chip. The second channel communicates directly or indirectly with the first channel. The second channel communicates directly or indirectly with a short, upwardly directed, vertical third channel 430 that brings the channel network closer to the top of the chip. The third channel communicates directly or indirectly with a fourth horizontal channel 440 located near the top and/or corner of the chip. In a preferred embodiment, the fourth channel is the channel closest to the top of the chip. In one embodiment, the fourth channel is the analysis channel. In one aspect, a camera 450 is oriented perpendicular to the flow direction in the fourth channel. An embodiment of the invention includes a focused particle flow nozzle operably connected to the first channel. In another aspect of the invention, the second channel undergoes resizing and passes through the nozzle before communicating with the third channel.

Figure 4C shows another embodiment of a sample path for a microfluidic chip. As shown here, fluid travels vertically upward through a first chip through a first channel 410, which then communicates directly or indirectly with a second horizontal channel 420, in this example at the top of the chip, which in this embodiment includes an analysis channel. This configuration places the analysis channel close to the top of the chip, thereby improving imaging and analysis through the materials of the microfluidic chip. In a preferred embodiment, the analysis channel is about 1 mm to about 2 mm from the top and sides of the chip. However, the distance of the analysis channel from the top of the chip can be 0.1 mm to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, or 0.3 mm to 0.4 mm. Stated differently, the analysis channel can be positioned within the top 50%, 33%, 25%, 10%, or 5% of the substrate. The length of a horizontal analysis channel according to the present invention can be about 250 microns to about 10 mm. However, the length of the analysis channel may be between 100 microns and 100 mm, such as between 0.1 mm and 0.2 mm, between 0.2 mm and 0.3 mm, or between 0.3 mm and 0.4 mm. In other words, the length of the analysis channel may be about 75% or less of the height, width, or length of the substrate/chip, for example, 50% or less, 33% or less, 25% or less, 10% or less, or 5% or less of the height, width, or length of the substrate/chip. In embodiments, a substrate may include one or more analysis channels, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 analysis channels.

Also, as depicted in FIG. 4C, a light source 480, such as a laser, can be used to affect a material stream, such as a cell stream. The laser may be positioned along the cell stream, or opposite the cell stream. The laser may also be positioned and/or oriented perpendicular or oblique to the cell stream.

1 and 4, a fluid stream containing cells or particles is directed vertically through a first channel. One or more substances (fluid, cells, and/or particles) enter through the bottom of the chip at the opening of the first channel and vertically enter the substrate. The first vertical channel is 100 microns to 100 mm in length, such as 0.1 mm to 0.2 mm, or 0.2 mm to 0.3 mm. The first channel is followed by a second orthogonal/horizontal channel, which in preferred embodiments is shorter than the first channel. The second channel may be 250 microns to 100 mm in length, such as 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, or 0.75 mm to 1.0 mm. The third channel runs perpendicular and parallel to the first channel. The third channel may be 50 microns to 100 mm in length, such as 0.05 mm to 0.1 mm, 0.1 mm to 0.15 mm, or 0.15 mm to 0.2 mm. The channels are arranged in direct or indirect operable communication to allow one or more substances to travel through the channels. Typical directions of fluid flow are given by the flow arrows in Figures 4A and 4C, but can be reversed.

In a preferred embodiment, the fourth channel comprises an analysis channel that is a channel 250 microns to 100 mm in length, such as 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, or 0.75 mm to 1.0 mm. In this embodiment, the fourth channel is the channel closest to the top of the chip. The distance of the fourth channel from the top of the chip, measured vertically, may be 100 microns to 2 mm, but may be as great as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, or 300 microns to 400 microns. In other words, the fourth channel may be located within the top 50%, 33%, 25%, 10%, or 5% of the chip. The imaging device, such as a camera, is perpendicular to the fourth channel and is approximately 100 microns to 2 mm from the fourth channel, but can be as large as 100 mm from the fourth channel, such as 100 microns to 200 microns, 200 microns to 300 microns, or 300 microns to 400 microns.

In one embodiment, a laser or other light source is present in conjunction with a focusing lens element. Figure 4A depicts the present invention in which a laser 480 is activated, emitting a laser beam and directing the beam through a focusing lens element into a fourth flow channel 440. Particles are aligned within the laser beam due to gradient forces that attract particles toward areas of highest laser intensity. Laser scattering forces cause particles to propagate in the direction of laser beam propagation (e.g., from left to right in Figure 4A).

In another embodiment, a second camera or image capture device (see 450) is oriented to the side of the analysis (or fourth) channel, in addition to the camera or image capture device oriented orthogonal to the fourth channel, here oriented orthogonally or at any angle relative to the fourth channel, as shown, for example, in FIG. 4B. Acquiring one image of each cell in an orthogonal view allows for the calculation of multiple cell properties, such as size and shape, in two dimensions, increasing the amount of information that can be captured about each cell. This also allows for the calculation of volumetric properties of cells, including total volume and shape, and may provide insight into cells that are not symmetric about an axis parallel to the direction of flow (the Z-axis as depicted in FIG. 5). Using two or more cameras, a basic 3D model of the cell 550 can be constructed by combining the orthogonal images using existing 3D reconstruction algorithms, such as diffraction theory or illumination rotation methods. The 3D model and analysis of the 3D model enable more accurate analysis of cells, such as cell size, shape, orientation, and other quantitative and qualitative measurements related to particles or cells in the fourth channel of a microfluidic chip.

In Figure 5, a portion of the analysis channel 540 is shown from two different planes, such as the planes that may be imaged from an imaging device (see, e.g., Figure 4). In the first plane 520, a portion of a cell or particle 510 is imaged at a particular orientation. In the second plane 530, another portion of the same cell or particle is imaged from a different perspective with another camera. This allows for the calculation of multiple cell properties, producing a matrix of data per cell per camera and a 3D representation of the cell or particle 550.

As different portions of the cell pass through the focal planes of the imaging device (e.g., 630 (XZ focal plane) and 640 (YZ focal plane)), different slices of the cell can be imaged, for example, as the cell is moved by a fluid flow 620. This is shown in FIG. 6A. In FIG. 6A, portions of the cell or particle are imaged from different directions and different viewpoints at multiple points in time and/or space. In this example, as the cell moves and rotates through the focal plane, it appears as different sizes in successive images (such as four example images per plane from two different cameras, as shown in FIG. 6A). Using 3D reconstruction algorithms, more complex 3D renderings 650 of the particle or cell are possible. From such renderings, specific attributes of the cell or particle can be extrapolated (e.g., cell size, volume, location and size of the nucleus and other organelles, and measurements or contours of cell topography). Furthermore, cell rotation can be measured as a function of optical force-based torque due to changes in biophysical or biochemical properties, including, but not limited to, refractive index, birefringence, or cell shape or morphology.

Figure 6B, for example, depicts chip multiplane imaging, in which multiple images of the same cell are imaged over time as the cell moves through the focal plane. In such cases, the view of the cell is referred to in the art as a "slice" or "image slice," and the image slice is essentially the thickness of the optical plane being imaged. The thickness of the image plane or slice is determined, among other things, by the optical magnification of the imaging system. At higher magnifications, the working distance of the objective lens decreases, resulting in the need to have the lens closer to the cell or particle being imaged. In one embodiment, a laser or other optical force 670 can be used to affect the flow of cells within the analysis channel. In a preferred embodiment, cells or particles can be intentionally guided into or out of the focal plane of the channel for imaging by either moving the laser and/or camera, adjusting the flow(s), or adjusting their position using hydrodynamic focusing. For example, the laser source can be moved a distance 660 using, for example, a piezoelectric actuator or a linear electro-optomechanical stage. This can be done cell-by-cell or population-by-population. The hydrodynamic focusing of the cells can be altered to affect the initial position and trajectory of the cells. For example, particles can be aligned or oriented within the focal plane of the laser beam due to gradient forces that draw the particles toward the areas of highest laser intensity. Laser scattering forces cause the particles to propagate in the direction of laser beam propagation. See Figure 6B. Moving the laser, in this case in the X-axis, allows for imaging of features in different parts of the cell, represented by disk 680. This may represent, for example, the nucleus, organelles, inclusions, or other features of the cell or particle. The laser draws the cell toward its center as a result of the gradient forces. Additionally, it is contemplated that two or more cameras may provide increased detail and precision.

The embodiment of the present invention shown in Figure 7 is a static mode in which a particle or cell 710 is arrested at a specific differential retention position by balancing an optical force 730 and a fluidic force 735. The optical force may be applied, for example, by a laser or collimated light source. Images can be taken in multiple planes, as in Figures 5 and 6A-B. A flow sensor is used to measure the flow rate at which each particle is arrested in the flow for a given laser power. Because the optical and fluidic forces are balanced, the fluidic drag force (i.e., from the flow rate and channel dimensions) is equal to the optical force. In this way, properties of each cell can be measured sequentially. While not a high-throughput measurement system, this embodiment of the present invention allows for close observation and imaging of trapped cells and dynamic changes in optical forces resulting from biochemical or biological changes in the cells. Reagent streams containing chemicals, biochemicals, cells, or other standard biological materials can be introduced into the flow channel and allowed to interact with the trapped cells. These dynamic processes can be quantitatively monitored by measuring changes in optical forces during experiments with single cells or multiple cells.

In one embodiment, a camera or other imaging device is aimed and/or focused in the direction of and/or opposite the flow of the analysis channel so that it is in-line with and parallel to the flow of the analysis channel (see, e.g., Figures 8 and 9). Figure 8 shows a camera 810 in line with the analysis channel 820 and a laser or collimated light source 830. A dichroic mirror or similar device 840 reflects the laser light 835 away from the camera to prevent damage, but allows light 865 generated by the illumination source 860 to pass through and enable imaging. The camera is oriented parallel to the fluid flow 870, such that cells or particles 880, in one embodiment, move away from the camera. The illumination source is oriented perpendicular to the channel and laser. A second dichroic 845, which passes the laser light and reflects the illumination light, is used to pass both the illumination light and the laser light through the channel. An alternative embodiment of this configuration switches the positions of the laser and illumination source so that the laser is perpendicular to the channel and the illumination source is parallel to the channel. The second dichroic still directs both the laser light and the visible light through the channel.

An alternative implementation is shown in Figure 9. In this case, a camera or imaging device 910 is oriented so that cells or particles 980 move in a fluid stream 970 toward the camera. Thus, the camera and laser 930 are on the same side of the channel, while the illumination source 960 is at the opposite end of the channel. Two dichroics 940 and 945 are used to direct the laser light 935 and illumination light 965 into the channel, directing the illumination light toward the camera and diverting the laser light away from the illumination source. An alternative embodiment of this configuration switches the positions of the laser and camera so that the laser is perpendicular to the channel and the illumination source is parallel to the channel. A second dichroic 945 then directs the laser light through the channel, illuminating the light onto the camera.

Optionally, embodiments of the present invention further include at least one optical element between the optical force source and the fourth channel operable to generate a standard TEM00 mode beam, a standard TEM00 mode beam, a standard TEM10 mode beam, a standard Hermite-Gaussian beam mode, a standard Laguerre-Gaussian beam mode, a Bessel beam, or a standard multimode beam. Optionally, the at least one optical element includes a standard cylindrical lens, a standard axicon, a standard concave mirror, a standard toroidal mirror, a standard spatial light modulator, a standard acousto-optic modulator, a standard piezoelectric mirror array, a diffractive optical element, a standard quarter-wave plate, and/or a standard half-wave plate. Optionally, the optical force source may include a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam.

Optionally, the device includes a microfluidic channel, a laser light source focused by an optical system, and an electric field source operably connected to the microfluidic channel via electrodes, where particles in a liquid are caused to flow through the microfluidic channel, and the laser light and electric field are manipulated to jointly act on the particles in the microfluidic channel, thereby separating the particles based on size, shape, refractive index, charge, charge distribution, charge mobility, dielectric constant, and/or deformability. In yet another embodiment, the device includes a microfluidic channel configured to provide a dielectrophoretic (DEP) field inside the channel via (1) an electrode system or (2) an insulator DEP system, and a laser light source focused within the microfluidic channel by an optical system, where a plurality of particles in a liquid are caused to flow through the microfluidic channel, and the laser light and field are jointly operated on the particles in the microfluidic channel to trap the particles or modify their velocity, where the DEP field is linear or nonlinear. Another possible embodiment of the device includes a microfluidic channel including an inlet and multiple outlets, and a laser light source optically focused to traverse the microfluidic channel at a critical angle matched to the velocity of flow within the microfluidic channel, such that an optical force is exerted on the particles while maximizing the residence time of selected particles within the laser light, thereby separating the particles into the multiple outlets, the laser light being operable to apply a force to particles flowing through the microfluidic channel, thereby separating the particles into the multiple outlets.

Optionally, embodiments of the present invention further include at least one particle interrogation unit in communication with one or more of the analysis channel(s), particularly the fourth channel. The particle interrogation unit includes a standard illuminator, standard optics, and standard sensor. Optionally, the at least one particle interrogation unit includes a standard bright-field imaging device, a standard light scattering detector, a standard single-wavelength fluorescence detector, a standard spectroscopic fluorescence detector, a standard CCD camera, a standard CMOS camera, a standard photodiode, a standard photomultiplier tube, a standard photodiode array, a standard chemiluminescence detector, a standard bioluminescence detector, and/or a standard Raman spectroscopic detector.

At least one particle interrogation unit in communication with the fourth channel comprises a laser-force-based device or devices that facilitate identification, selection, and sorting of cellular diseases. In one aspect, the unit utilizes inherent differences in optical pressure resulting from changes in particle size, shape, refractive index, or morphology as a means of particle separation and characterization. In one aspect, a near-infrared laser beam exerts a physical force on cells, which is then measured. When optical force via radiation pressure is balanced against fluid drag on the particles, it results in a change in particle velocity that can be used to distinguish different particles or to alter particle populations based on the inherent difference. The balance between fluid and optical forces can also be used to change the relative positions of particles relative to one another based on their intrinsic properties, thereby resulting in physical separation. Another embodiment of the interrogation unit includes a device for particle analysis and/or separation, such as at least one collimated light source operable to generate at least one collimated light source beam. The at least one collimated light source beam comprises at least one beam cross-section.

One embodiment of the present invention includes combining several of the design elements described above into a single device. Embodiments also include methods of using such devices. An example of such an integrated device is shown in FIG. 1. While the illustrated embodiment of the invention is a five-layer structure, with all five layers bonded together to result in a solid microfluidic chip, the chip may be a single structure as opposed to bonded layers. The chip can be constructed using a number of standard materials, including, but not limited to, fused silica, crown glass, borosilicate glass, soda-lime glass, sapphire glass, cyclic olefin polymer (COP), poly(dimethyl)siloxane (PDMS), OSTE, polystyrene, poly(methyl)methacrylate, polycarbonate, and other plastics or polymers. The chip allows for sample input, hydrodynamic focusing, optical interrogation, imaging, analysis, sample exit, and clear optical access for laser light to enter and exit the area. The chip in embodiments can also be 3D printed, molded, or otherwise shaped.

Optionally, the at least one particle type comprises a plurality of particle types. Each particle type of the plurality of particle types comprises a respective intrinsic property and a respective induced property. Optionally, the intrinsic property comprises size, shape, refractive index, morphology, intrinsic fluorescence, and/or aspect ratio. Optionally, the induced property comprises deformation, angular orientation, rotation, rotation rate, antibody label fluorescence, aptamer label fluorescence, DNA label fluorescence, dye label fluorescence, differential retention metric, and/or gradient force metric. This method embodiment further comprises identifying and separating the plurality of particles according to their respective particle types based on at least one of the intrinsic property and the induced property. Optionally, this method embodiment further comprises interrogating or manipulating the sample flow. Optionally, interrogating the sample flow comprises determining at least one of the intrinsic property and the induced property of the particle type, and measuring particle velocities of the plurality of particles. The measurement of at least one intrinsic property may be used for a range of applications, including, but not limited to, virus quantification, process development and monitoring, sample release assays, adventitious agent testing, clinical diagnostics, biomarker discovery, determining antibody or protein productivity for process development and monitoring, determining the potency, quality, or activation state of cells produced as cell-based therapies, including CAR T and other oncology applications and stem cells, determining the effect of chemicals, bacteria, viruses, antibacterial or antiviral agents on specific cell populations, and determining the disease state or likelihood of a research or clinical cell sample. Optionally, the optical force source includes at least one beam axis, and the sample flow includes a sample flow axis. Determining at least one of the intrinsic property and the induced property of the particle type and measuring the particle velocities of the plurality of particles together includes offsetting the beam axis from the sample flow axis. Optionally, determining at least one of the intrinsic property and the induced property of the particle type and measuring the particle velocities of the plurality of particles together includes calculating the slope and trajectory of the plurality of particles deviating from the sample flow axis toward the at least one beam axis.

Those skilled in the art will recognize that the disclosed features may be used singly, in any combination, or omitted, depending on the requirements and specifications of a given application or design. When an embodiment is referred to as "comprising" certain features, it should be understood that the embodiment can alternatively "consist of" or "consist essentially of" any one or more of the features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of this specification and practice of the invention.

It should be noted in particular that where a range of values is provided herein, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is intended that variations that do not depart from the essence of the invention be within its scope, and that the specification and examples be considered exemplary or illustrative in nature. Furthermore, all references cited in this disclosure are individually incorporated herein by reference in their entirety, and are thus intended to provide an efficient manner of supplementing the enabling disclosure of the present invention as well as to provide a background that will inform those skilled in the art.

Claims (12)

1. A method for analyzing one or more cells or particles, comprising:
Injecting the one or more cells or particles into a substrate comprising a plurality of channels configured to transport the one or more cells or particles;
The plurality of channels includes a first channel disposed in the substrate perpendicular to gravity;
a second channel disposed horizontally within the substrate and in operative communication with the first channel;
a third channel communicating with the second channel and disposed vertically in the substrate in an upward direction relative to gravity;
a fourth channel communicating with the third channel and disposed horizontally within the substrate;
the first channel, the second channel, the third channel, and the fourth channel are arranged to provide a path for the one or more cells or particles to move through the substrate from the first channel to the second channel, to the third channel, and to the fourth channel;
injecting one or more cells or particles into a first channel disposed vertically within the substrate through a bottom horizontal planar surface having an opening to the first channel to maintain directional and volumetric continuity with said first channel;
The analyzing step includes:
a) during the movement of biological particles, and
b) from multiple focal planes,
c) from multiple angles,
d) from multiple directions,
e) using a plurality of imaging devices,
or f) a combination thereof;
imaging the one or more cells or particles;
method. 2. The method of claim 1, wherein the bottom horizontal planar surface has a smaller or the same area as the surface area on the vertical plane of the chip to maintain directional and volumetric continuity with the first channel in the vertical direction. The method of claim 1 , wherein the analysis comprises a quantitative measurement of the amount of change in the cells or particles to provide a predictive or normative analysis. 2. The method of claim 1, wherein the bottom horizontal planar surface has at least one shorter length from one edge to another edge compared to at least one length from one edge to another edge on a vertical plane of the front substrate to maintain directional and volumetric continuity with the first channel. the one or more cells or particles are injected into the first channel vertically disposed within the substrate through a bottom horizontal plane having an opening to a first flow path;
2. The method of claim 1, wherein the bottom horizontal planar surface has a smaller or the same area in the vertical direction as compared to the surface area on the vertical plane of the chip to maintain directional and volumetric continuity with the first channel. The method of claim 1 , further comprising one or more electrical, optical, and/or fluidic forces to move one or more cells or particles in one or more channels. 10. The method of claim 1, wherein the imaging comprises the use of a bright field imager, a light scattering detector, a single wavelength fluorescence detector, a spectrofluorescence detector, a CCD camera, a CMOS camera, a photodiode, a photodiode array (PDA), a spectrometer, a photomultiplier tube or tube array, a photodiode array, a chemiluminescence detector, a bioluminescence detector, a standard Raman spectroscopic detection system, surface-enhanced Raman spectroscopy (SERS), coherent anti-Stokes Raman spectroscopy (CARS), coherent Stokes Raman spectroscopy ( CSRS ), or a combination thereof. The analysis
determining the efficacy, quality, and activation state of the cells produced for cell-based therapy;
determining the quality of the cells for efficacy assays; and/or
determining the effect of a chemical, bacterial, viral, antibacterial, or antiviral agent on a particular cell population;
The method of claim 1 . 9. The method of claim 8 , wherein the cells produced for cell-based therapy are CAR-T cells or stem cells. 10. The method of claim 1, wherein the analysis includes determining the effect of one or more chemical, biological, biochemical, physical forces, temperature, bacteria, viruses, antibacterial, or antiviral agents on the cells or particles . The method of claim 1 , further comprising determining a disease state of the one or more cells or particles. 10. The method of claim 1, wherein the analysis comprises one or more of determining morphology, cell size, cell shape, refractive index, birefringence, intrinsic fluorescence, aspect ratio, deformation, angular orientation, rotation, rotation rate, antibody label fluorescence, aptamer label fluorescence, DNA label fluorescence, dye label fluorescence, differential retention metric, and / or gradient force metric.