Microfluidic chip device for optical force measurement and cell imaging using microfluidic chip configuration and dynamics

Abstract

To provide a microfluidic chip device for optical force measurement and cell imaging using a microfluidic chip configuration and dynamics.SOLUTION: An input and fluid flow up through the bottom of a chip 400 by using a manifold that avoids orthogonal re-orientation of fluid dynamics. The contents of a vial are located below the chip and pumped upwards and vertically directly into a first channel 410 of the chip. A long channel extends from the bottom of the chip to near the top of the chip. Then the channel takes a short horizontal turn 420 that nearly negates any influence of cell settling due to gravity and zero flow velocity at the walls. The fluid is pumped up to a horizontal analysis portion 440 that is close to the top of the chip that results in clearer imaging. A laser 480 may also suspend cells or particles in the channel during analysis, which prevents the cells or particles from settling.SELECTED DRAWING: Figure 4A

Description

Technical Field

[0001] The present invention generally relates to devices and methods for particle analysis and imaging of particles or cells in a fluid, and more particularly to devices and methods for particle imaging of a fluid using pressure, hydrodynamics, electrokinetics, and optical forces.

[0002] The present invention relates to a microfluidic chip in which injection is performed in a direction vertically upward, and a fluid vial is disposed under the chip in order to minimize sedimentation of particles before and at the analysis portion of the channel of the chip.

[0003] To implement the present invention, changes had to be made to existing microfluidic chip designs. For example, to keep the vial in-line perpendicular to the chip, a different interface with the chip had to be established compared to the prior art. Specifically, instead of an input tube interfacing with the chip via a port attached to the largest face of the chip (as typically done in a microfluidic lab-on-a-chip system), first, orthogonally, then across the chip, then pump the fluid, and then raise the chip. In one aspect of the present invention, a manifold is used to have an input and fluid that rise through the bottom of the chip, thereby avoiding horizontal reorientation of the fluid / hydrodynamics.

[0004] According to the present invention, the contents of the vial are placed under the chip and pumped directly, upwardly and vertically into the first channel of the chip. The long channel extends from the bottom of the chip to near the top of the chip. Then, the channel takes a short horizontal turn, but the new channel is very short and almost negates the effects of cell sedimentation due to gravity and zero flow velocity at the walls. Next, unlike the prior art, the fluid is pumped up to the analysis section. Thus, the horizontal analysis section is the highest channel / fluid point in the chip and is thus close to the top of the chip, resulting in less chip material (e.g., glass) between the microscope / camera and the sample than in the prior art, and thus sharper imaging is obtained. Also, a laser suspends the cells in this channel during analysis and prevents the cells from sedimenting.

Background Art

[0005] According to the prior art, a microfluidic chip vial containing cells or particles to be separated and / or analyzed is placed on the side and pumped horizontally into the channels in the microfluidic chip. First, the contents of the vial (e.g., particles or cells) are pumped in the upward vertical direction, then a u-turn is made to move downward, and then pumped horizontally into the chip (see, for example, U.S. Patent No. 9,594,071).

[0006] The connection to the chip is horizontal, which, combined with the dead volume in the connection (the empty space in a fluid connection which is to some extent inevitable), results in significant additional sedimentation due to gravity. Such a configuration also requires a relatively large diameter channel that is required by the connection, which, in addition to the dead volume, creates regions of relatively low velocity and further increases the problem of particle sedimentation. The current chip according to the invention eliminates the need for a relatively abrupt change from a large horizontal input channel and a large horizontal input channel to a relatively thin upflow in the first vertical chip channel. Such a configuration eliminates horizontal sedimentation and unnecessary directional changes that cause sedimentation. Orienting the cell at right angles to gravity such that the cell or particle cannot settle to the bottom of the horizontal channel and rather is always guided upwards by the flow solves the problem of sedimentation within the dead volume. This is not intuitive and requires a lot of experimentation to realize the problem before designing the current implemented solutions. Currently available microfluidic devices, in contrast to the present invention, incorporate custom or off-the-shelf connections on a polished surface and a larger area of glass, which generally forces any particles (e.g., cells) contained within the sample flow to immediately rotate and move horizontally as they enter the chip.

[0007] Also, in the 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 as compared to the vertical in-chip channels. The channels then flow upward and take a long horizontal turn. At this point, the cells tend to sediment to the bottom of the channel due to gravity. Also, they experience a lower velocity at the walls due to laminar flow conditions. Essentially, due to the parabolic velocity profile, the flow is highest in the middle of the channel and decreases to zero at or near the channel walls. After the first 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 pushed downward and eventually exit from the bottom of the chip.

[0008] Furthermore, due to prior art constraints, multiple horizontal runs are required to fix the cells at multiple locations within the channel. This causes a degradation in image quality as this then requires imaging through additional material at the edge of the chip. The prior art channels within the chip had to be pumped vertically upward, then horizontally, then in a zigzag nature for a proper cell or particle suspension, then under the chip, and then out of the chip. The zigzag channels are removed by the present invention.

[0009] There is also prior art regarding rendering 3D images of cells or particles in a fluid. For example, M. Habaza, M. Kirschbaum, C. Guernth-Marschner, G. Dardikman, I. Barnea, R. Korenstein, C. Duschl, 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.", February 27, 2014, 1:014002). However, the present invention claims to take multiple images of a cell or particle as the cell or particle moves through the fluid flow and passes through the focal plane of the imaging device, thereby eliminating 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 a cell or particle (see, for example, N. Lue et al., "Opt. Express", September 29, 2008, S16(20):16240-6), none of which use brightfield imaging as described herein and do not utilize fluid flow to provide cell positioning with respect to the image focal plane.

[0010] All prior art documents cited herein are incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0011] The present invention relates to a microfluidic chip in which injection is performed and a sample vial is placed under the chip in order to minimize particle sedimentation. Accordingly, the contents of the vial are located under the chip and are pumped directly upward and vertically into the channels of the chip. The long channels extend from the bottom of the chip to near the top of the chip. The channels then take a short horizontal turn, but the new channels are short enough to be negligible with respect to the effect of cell sedimentation due to zero flow velocity at the channel walls. And, contrary to the prior art, the sample is pumped up to the analysis section. Accordingly, the horizontal analysis portion is the highest channel / fluid point within the chip and is thus near the top of the chip, resulting in less glass between the microscope / camera and thus sharper imaging. The distance from the top of the chip to the analysis channel can be from 100 microns to 2 mm, but can also be on the order of 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, etc. In one embodiment, after the analysis portion of the chip, the sample, e.g., fluid, cells, and / or particles are pumped downward to the bottom of the chip and extruded outwards.

[0012] The present invention further relates to a microfluidic chip in which the horizontal flow is minimized, particularly at the point where the fluid enters the chip channels. Prior art chips include a horizontal channel (non-analytical portion) of about 13 mm, and its injection port has a much larger diameter of about 2 mm, which exacerbates sedimentation due to the low velocity. The chips described herein preferably have a horizontal channel (non-analytical) of about 0.2 - 3.0 mm in a preferred embodiment, but the length of the horizontal channel can be in the range of 0.01 - 100.0 mm, such as 0.01 mm - 0.02 mm, 0.02 mm - 0.03 mm, 0.03 mm - 0.04 mm, etc. This is a result of the channel system of the present invention, which is of a different order of magnitude than the prior art and improves cell / particle flow and eliminates sedimentation.

[0013] Another aspect of the present invention relates to a microfluidic chip in which imaging is performed and the analysis is based on or near the corner of the chip, whereby the imaging from multiple viewpoints is improved because there is less glass and distance between the camera and the analysis channel. This also makes it possible to improve the detailed imaging by increasing the magnification using a higher numerical objective lens. Such design improvements reduce image distortion caused by glass (or other substances constituting the chip such as plastic or any transparent or translucent material) and distance (e.g., due to glass defects). The distance between the imaging device and the analysis channel may be from 100 microns to 2 mm, but may also be on the order of 100 mm, such as from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, etc.

[0014] Furthermore, the present invention relates to a microfluidic sorting chip that is separated downstream from an analysis channel and enables the sorting function to be actuated using both pressure and / or a laser (or other optical force) simultaneously or sequentially. In one aspect, the flow continues from the analysis channel for the sorting function. For example, particles are directed towards a vertical channel and then towards 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 the particles through the channel. Particles not directly acted upon by the optical force are diverted to an alternative channel, for example, by gravity, electrokinetic force, magnetic force, laminar flow lines, flow lines, a decrease in flow rate, orthogonal optical forces, or a vacuum applied to suck the particles into the alternative channel. In another aspect related to the analysis channel after sorting, the present invention enables directing the optical force from the back side of the chip (the laser or optical force is directed in the same flow direction), and in some aspects splitting this primary laser. In embodiments, the optical force and / or pressure may be applied in the opposite direction to the movement of the substance through the channel, for example, against the flow, or in the same direction as the movement of the substance within the channel, for example, with the flow. Sorting of cells or particles can be performed 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 enable single or multiple sorting regions.

[0015] In another aspect of the present invention, the manifold is connected to the vial such that the tube passes through the manifold and connects to the opposite vial or other container that contacts the substance (e.g., fluid) within the vial. The manifold enables the vial to be connected to the microfluidic chip while being stored under the chip, and / or enables the contents of the vial to be injected from the bottom of the chip, reducing some of the problems experienced by the prior art, such as the connection of the vial or the tube from the vial to the microfluidic chip, or the sedimentation of cells or particles communicating with the microfluidic chip.

[0016] In another aspect, the present invention is directed to a microfluidic chip holder, which has a structure guiding a light source including an integrated prism cavity, and when the prism is fitted, light is emitted at an angle with respect to the chip. This is a preferred method for illuminating a constrained geometric shape. In embodiments, the light source includes, but is not limited to, an optical fiber, a collimated light source, or a focused light source. This light source is particularly directed or oriented accurately towards the analysis channel.

[0017] In another aspect of the present invention, the device includes a first camera and a second imaging device oriented orthogonally to the channel view. The reasons for the second camera are various. In one aspect, the reason for the second imaging device is to assist in the visual alignment of a laser or optical force within the analysis channel. In another aspect, the data from the first camera can be recorded using the methods described herein. With the second camera, the data can be combined with the data from the first camera, and as a result, additional data can be obtained that can be used to more accurately extrapolate the position, size, shape, volume, etc. of the cell. This additional information about the same cell (or particle) increases the accuracy and scope 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 a group of cells or particles, imaged by an orthogonal camera and a camera placed towards the side of the chip or a camera within the flow. Using the algorithms described herein or elsewhere, including reversing or slowing down the flow and taking one or more images of a particular cell or particle, or a group of cells or particles, the present invention enables multiple images to be analyzed and processed. Thereby, it enables the determination of features / attributes / quantitative measurements such as cell volume, cell shape, nuclear position, nuclear volume, organelle or inclusion body position, etc. In another aspect of the present invention, the camera is oriented to image an image within an axis in the direction of the flow.

[0018] In one aspect, the present invention does not require a meandering or zigzag channel, as is preferred according to the prior art, to properly suspend the particles. For the verticality of the fluid and particles or cells injected into the chip, the zigzag channel is avoided by vertically integrated pumped particles that flow directly through the channel into a first horizontal channel (referred to herein as the second channel).

Brief Description of the Drawings

[0019] The accompanying drawings illustrate certain aspects of some embodiments of the present invention and should not be used to limit or define the present invention. The drawings, together with the description set forth herein, serve to explain the specific principles of the present invention.

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[0020] 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 practicing the present invention without departing from the scope or spirit of the present invention. Those skilled in the art will recognize that these features can be used alone or in any combination based on a given application or design requirements and specifications. Embodiments including various features may also be composed of or can be essentially composed of these various features. Other embodiments of the present invention will be apparent to those skilled in the art from the consideration of the present specification and the practice of the present invention. The description of the present invention provided is essentially exemplary or illustrative only, and thus, variations that do not depart from the essence of the present invention are intended to be within the scope of the present invention.

[0021] Before describing in detail at least one embodiment of the present invention, it is to be understood that the present invention is not limited to the details of the construction and arrangement of components described in the following description or shown in the drawings in its application. The present invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the terminology and phraseology used herein are for the purpose of description and should not be regarded as limiting.

[0022] Referring now to the drawings, FIG. 1A shows an overall view of the device taught herein. The sample vial 130 is disposed under the microfluidic chip 100 (also generally referred to herein as a substrate) and is not limited with respect to the number or size of the vials. The vial is configured to hold any type of sample that can be moved through the device, including but not limited to one or more substances such as fluids, liquids, gases, plasma, serum, blood, cells, platelets, particles, or combinations thereof. In the context of this specification, the terms "fluid" or "sample" can generally be used to refer to such one or more substances. The vial is connected either directly or indirectly using an air tube 110 that is operably in communication with the lower surface 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 onto one outer surface (e.g., the XZ plane in FIG. 1B) such as the edge of the microfluidic chip. The injection is shown in FIG. 1B along one of the planes shared by the minimum distance such as the XZ plane or the XY plane. In this particular embodiment, the length of side X160 is shorter than the lengths of side Y170 and side Z180. Such a configuration allows the sample to enter the channels on the chip with a minimum deviation (e.g., no right turn is required as in a typical case in the current state of the art).

[0023] In FIG. 1A, a manifold 120 is shown that connects 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 upwardly into the microfluidic chip. The manifold enables the injection of substances from the bottom of the chip, while electronics, flow sensors, and tubes (both liquid and air) are arranged to minimize cell sedimentation that optimizes throughput. The air tube 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 of the internal volume is atmospheric pressure. This enables pumping substances from or to an open container (a vacuum is required to pump from an open container). The manifold arranges electronics, flow sensors, and tubes (both liquid and air) to minimize the sedimentation of cells that optimize throughput.

[0024] By injecting the contents from the bottom of the chip, the present invention minimizes the horizontal movement of the inlet tube 140 and the outlet tube 145, which causes problems such as sedimentation of particles or cells in the channel 150. The outlet tube is offset from the inlet tube in the Z and X dimensions (FIG. 1B) in this example. In embodiments, the outlet tube is offset, straight, in-line, or angled with respect to the inlet tube rather than being offset. Such a configuration solves problems related to sedimentation that occur in the prior art, such as when a fluid combined with cells and / or particles is injected or pumped into the chip laterally in the horizontal direction where the fluid and hydrodynamics have to push upward in direction, eliminating the need for serpentine or zigzag vertical channels. Injection from the bottom of the manifold and the chip also enables additional elements, components, machines, or hardware to be placed under the chip (see FIG. 1A).

[0025] The manifold 120 functions by regulating the air pressure above the contents within the vial and providing the correct geometric shape for the flow sensor and electronics. Tubes such as fluid or air tubes pass through the manifold and connect to the vial on the opposite side. Pressurized air passes through one side of the manifold, creating a closed pressurized system within the manifold. By adjusting the pressure in the sealed area, the system allows for changes to parameters such as flow rate and hydrodynamics. The pressurized area is present in both the vial 130 and the manifold 120. In another aspect, since the vial is open to the ambient atmosphere, no air connection to the vial is required. This allows for sampling of a more diverse range of containers and sources. In such embodiments, a vacuum is applied to one or more other vials such that a pressure differential is created to drive fluid flow from the open container.

[0026] In one embodiment, pressure-based sample injection is used. The vial is filled with a sample in a fluid and sealed with either a lid or a tube connected to the chip. Before attaching the lid, the vial can be opened 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 gas and one for liquid. Optionally, the method embodiment further includes the step of providing a sample inlet line chip in communication with a sample inlet line in communication with the first channel.

[0027] In another embodiment, vacuum-based sample injection is used. The vial is filled with a sample in a fluid and sealed with either a lid or a tube connected to the chip. Before attaching the lid, the vial can be opened to air or sealed with a septum. In one aspect, the lid can include two connections, one for a fluid such as gas and one for liquid. Optionally, the fluid can be aspirated from the vial open to the atmosphere by applying a vacuum pressure to one or more other vials. Optionally, the method embodiment further includes the step of providing a sample inlet line chip in communication with a sample inlet line in communication with the first channel.

[0028] Figures 2A - B and 3A - B show chip holders 200, 300. The chip holders include a structure for guiding a light source 210 such as an optical fiber light source, a light - emitting diode, or a laser, and integrated prism - cavities 220, 320 to which prisms can be attached. The light source is guided or aligned to a desired position by an integrated structure or channel 240 within the chip holder. The built - in space for the optical fiber light source enables illumination such as illumination cone 250 even in a restricted geometric environment such as on the microfluidic chip 230, 330 or on a channel within the microfluidic chip. In a preferred embodiment, this light source is particularly accurately directed, or oriented, or focused towards the analysis channel 260. In a preferred embodiment, the chip holder includes a built - in space for the prisms 220, 320 and an optical fiber light source 210 that enables illumination with a restricted geometric shape. The chip further includes a hole or opening at the bottom of the holder 350 to accurately align a fluid tube as described herein. In one embodiment, adjustable screws are integrated into screw holes 360 on one or more surfaces for proper alignment.

[0029] FIG. 4A is a preferred embodiment of the microfluidic chip 400 described herein. As shown, the fluid first moves vertically upward through the first channel 410 and then communicates with the second horizontal channel 420. Another vertical channel 430 takes the fluid closer at the top of the chip where the fourth channel 440 is horizontal and includes an analysis channel as shown in FIG. 4. The channels are operably connected to each other to allow the sample to move from one channel to another through the system. In an embodiment, the sample can flow from the first channel to the second channel, to the third channel, to the fourth channel, or vice versa, or a combination thereof. A pump and / or vacuum device may be provided to provide positive and / or negative pressure to either or both of the openings and outlets of the channel to allow movement of substances through the channel. The analysis channel is close to one or more outer surfaces of the substrate, such as the face, edge, or side of the chip. For example, according to this configuration, the analysis channel is close to the top and side surfaces of the chip, thereby improving imaging and analysis through the substances of the microfluidic chip. In a preferred embodiment, the analysis channel is about 1 mm to about 2 mm from the top and side surfaces 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, 0.3 mm to 0.4 mm, etc. In other words, the analysis channel can be disposed within the upper 50%, 33%, 25%, 10%, or 5% of the substrate. The length of the 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 be 100 microns to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. 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.

[0030] FIG. 4B shows two imaging devices 450, such as machine vision cameras. In one embodiment, the camera may be disposed above the chip and oriented orthogonal to the analysis channel. In another embodiment, the camera may be disposed on the side of the chip and oriented orthogonal to the direction of flow, or at an angle to the direction of flow (e.g., above the channel, below the channel, or angled with respect to the side of the channel). In an embodiment, the camera can be arranged such that imaging is performed at any angle with respect to the flow of one or more substances, such as at right angles or 90 degrees with respect to the flow of one or more substances, or at 0 to 90 degrees, or 10 to 80 degrees, or 30 to 60 degrees. In another embodiment, two or more cameras can be used to image cells or particles within the analysis channel. For example, a camera may be above the chip and oriented orthogonal to the analysis channel. A second camera may be disposed on the side of the chip and oriented orthogonal to the direction of flow, or at an angle to the direction of flow (e.g., above the channel, below the channel, or angled with respect to the side of the channel). As shown in FIG. 4B, one or more light sources 460 can be used to illuminate the analysis channel 440, such light sources being disposed under the chip, illuminating the side of the chip, illuminating in the direction of flow or in the direction opposite to the direction of flow, illuminating above the chip, and illuminating the analysis channel obliquely.

[0031] Alternatively, using a dichroic mirror 840 or other suitable optical element, as shown in FIG. 8, light in a specific wavelength range can be selectively redirected and light in other wavelength ranges can be passed through. Thereby, the camera 810 can be arranged in a straight line with the analysis channel. As shown in FIGS. 8 and 9, several embodiments occur including arranging the optical force laser 830 and the camera 810 at the same end or opposite ends of the analysis region. Also, an illumination source 860 for the camera may 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.

[0032] 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 arranged along the flow of cells or opposite to the flow of cells. Also, the laser can be arranged and / or oriented orthogonal or oblique to the cell flow.

[0033] One embodiment of the present invention includes a device for particle analysis (see, e.g., FIGS. 4-9). Embodiments of the present invention include 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 collimating 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. Embodiments of the present invention include a substrate having a first channel 410 extending vertically within the substrate such that a first plane traverses the first channel 410 substantially along its length, whereby a fluid sample is injected into the substrate / chip from the bottom of the chip and pushed upward by positive or negative pressure. Embodiments of the present invention include a second channel 420 disposed horizontally within the substrate that is orthogonal to the first channel and thus a second plane traverses the second channel 420 substantially along its length and the second plane is disposed orthogonal to the first plane. This second channel is in the horizontal direction of the chip. The second channel communicates directly or indirectly with the first channel. The second channel communicates directly or indirectly with a short upward 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 corners 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 an analysis channel. In one aspect, the camera 450 is oriented orthogonal to the flow direction within the fourth channel. Embodiments of the present invention include a focusing particle flow nozzle operably connected to the first channel. In another aspect of the present invention, the second channel is sized and passes through the nozzle before communicating with the third channel.

[0034] Figure 4C shows another embodiment of the sample path of the microfluidic chip. As shown here, the fluid moves vertically upward through the first channel 410 within the first chip, and then this channel communicates directly or indirectly, in this example at the top of the chip, with the second horizontal channel 420, which includes an analysis channel in this embodiment. According to this configuration, the analysis channel is close to the top of the chip, thereby improving imaging and analysis through the material of the microfluidic chip. In a preferred embodiment, the analysis channel is about 1 mm to about 2 mm from the upper and side surfaces 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, 0.3 mm to 0.4 mm, etc. In other words, the analysis channel can be arranged within the upper 50%, 33%, 25%, 10%, or 5% of the substrate. The length of the 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, 0.3 mm to 0.4 mm, etc. 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. In an embodiment, the substrate can include one or more analysis channels such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 analysis channels.

[0035] Also, as depicted in Figure 4C, a light source 480 such as a laser can be used to affect the flow of a substance such as a cell flow. The laser can be arranged along the cell flow or opposite to the cell flow. Also, the laser can be arranged and / or oriented orthogonal or oblique to the cell flow.

[0036] In FIGS. 1 and 4, a fluid stream containing cells or particles is directed vertically through a first channel. One or more substances (fluids, cells, and / or particles) enter through the opening of the first channel through the bottom of the chip and enter the substrate in the vertical direction. The first vertical channel has a length of 100 microns to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, etc. The first channel is followed by a second orthogonal / horizontal channel, which is shorter than the first channel in a preferred embodiment. The second channel may have a length of 250 microns to 100 mm, such as 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, 0.75 mm to 1.0 mm, etc. The third channel extends perpendicular and parallel to the first channel. The third channel may have a length of 50 microns to 100 mm, such as 0.05 mm to 0.1 mm, 0.1 mm to 0.15 mm, 0.15 mm to 0.2 mm, etc. The channels are arranged to be operably connected, either directly or indirectly, such that one or more substances can move through multiple channels. The typical direction of fluid flow is given by the flow arrows in FIGS. 4A and 4C, but it can also be reversed.

[0037] In a preferred embodiment, the fourth channel includes an analysis channel that is a channel with a length of 250 microns to 100 mm, such as 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, 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 also be as large as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns. In other words, the fourth channel can be arranged within the upper 50%, 33%, 25%, 10%, or 5% of the chip. An imaging device, such as a camera, is orthogonal to the fourth channel and can be sized from about 100 microns to 2 mm from the fourth channel, but may also be as large as 100 mm from the fourth channel, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns.

[0038] In one embodiment, a laser or other light source is present with a focusing lens element. FIG. 4A depicts the present invention where a laser 480 is activated to emit a laser beam and direct the beam through a focusing lens element into a fourth flow channel 440. The particles are aligned within the laser beam due to the gradient force that draws the particles towards the region of highest laser intensity. The laser scattering force propagates the particles in the direction of laser beam propagation (e.g., from left to right in FIG. 4A).

[0039] In another embodiment, a second camera or image capture device (see 450) is oriented orthogonal to the fourth channel in addition to the camera or image capture device oriented orthogonal to the fourth channel, here oriented on the side of the analysis (or fourth) channel and orthogonal or at any angle to the fourth channel, as shown for example in FIG. 4B. By acquiring one image of each cell in an orthogonal view, it becomes possible to calculate multiple cell characteristics, such as size and shape, in two dimensions and increase the amount of information that can be captured for each cell. This will also enable the calculation of volumetric characteristics of the cells, including shape, and provide insights into cells that are not symmetric with respect to 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 cells 550 can be constructed by combining the orthogonal images using existing 3D reconstruction algorithms such as the diffraction theory method or the illumination rotation method. The 3D model and analysis of the 3D model enable a more accurate analysis of the cells, such as cell size, shape, orientation, and other quantitative and qualitative measurements regarding particles or cells in the fourth channel of the microfluidic chip.

[0040] In FIG. 5, a portion of the analysis channel 540 is shown from two different planes, such as a plane that can be imaged from an imaging device (see, e.g., FIG. 4). In the first plane 520, a portion of the cell or particle 510 is imaged in a particular orientation. In the second plane 530, another portion of the same cell or particle is imaged from a different perspective than the other camera. This enables the calculation of multiple cell characteristics and generates a matrix of data per cell per camera and a 3D representation of the cell or particle 550.

[0041] 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 as the cell is moved, e.g., by a fluid flow 620. This is shown in FIG. 6A. In FIG. 6A, a portion of the cell or particle is imaged at multiple points in time and / or space from different directions and different perspectives. In this example, as the cell moves and rotates through the focal plane, it appears as different sizes in successive images (e.g., four example images per plane from two different cameras as shown in FIG. 6A). By using a 3D reconstruction algorithm, a more complex 3D rendering 650 of the particle or cell becomes possible. From such a rendering, specific attributes of the cell or particle (e.g., cell size, volume, nuclear position and size, as well as other organelles, and measurements or contours of cell topography) can be extrapolated. Furthermore, the rotation of the cell can be measured as a function of the torque based on optical forces, which is due to changes in biophysical or biochemical properties, including but not limited to refractive index, birefringence, or cell shape or morphology.

[0042] FIG. 6B depicts chip multi-plane imaging in which, for example, multiple images of the same cell are imaged over time as the cell moves through a focal plane. In such cases, the figure of the cell is referred to by those skilled in the art as a "slice" or "image slice", and the image slice is substantially the thickness of the optical plane being imaged. The thickness of the image plane or slice is determined, inter alia, by the optical magnification of the imaging system. At higher magnifications, the working distance of the objective lens decreases, resulting in the need for a 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, the cells or particles can be intentionally directed to or from the focal plane of the channel for imaging by any of moving the laser and / or camera, or adjusting the flow(s), or using hydrodynamic focusing. For example, the laser source can be moved a distance 660 using, for example, a piezoelectric actuator or a linear electro-optic stage. This can be done for each cell or for each 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 a gradient force that draws the particles towards the region of highest laser intensity. The laser scattering force propagates the particles in the direction of laser beam propagation. See FIG. 6B. By moving the laser, in this case along the X-axis, imaging of features in different parts of the cell, indicated by disk 680, becomes possible. This can represent, for example, the nucleus, organelles, inclusions, or other features of the cell or particle. The laser draws the cell towards its center as a result of the gradient force. Further, it is contemplated that two or more cameras can improve the detail and accuracy.

[0043] The embodiment of the present invention shown in FIG. 7 is a static mode in which the particles or cells 710 are stopped at a specific differential holding position by balancing the optical force 730 and the fluid force 735. The optical force may be applied, for example, by a laser or a collimated light source. Images can be taken by a plurality of airplanes as shown in FIGS. 5 and 6A - B. The flow sensor is used to measure the flow rate at which each particle stops in the flow for a given laser power. Since the optical force and the fluid force are in equilibrium, the fluid drag force (i.e., from the flow velocity and the channel dimensions) is equal to the optical force. In this way, the characteristics of each cell can be measured sequentially. Although not a high - throughput measurement system, this embodiment of the present invention enables detailed observation and imaging of the captured cells, as well as dynamic changes in the optical force resulting from biochemical or biological changes in the cells. A reagent flow containing chemicals, biochemicals, cells, or other standard biological substances can be introduced into the flow channel to interact with the captured cells. These dynamic processes can be quantitatively monitored by measuring changes in the optical force during experiments on single or multiple cells.

[0044] In one embodiment, a camera or other imaging device is oriented and / or focused toward and / or away from the flow direction of the analysis channel so as to be in series and parallel with the flow of the analysis channel (see, e.g., FIGS. 8 and 9). FIG. 8 shows a camera 810 along an 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 the light 865 generated by the irradiation source 860 to pass through to enable imaging. The camera is oriented parallel to the fluid flow 870 such that the cells or particles 880 move away from the camera in one embodiment. The irradiation source is oriented in a direction orthogonal to the channel and the laser. A second dichroic 845 that 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 the irradiation source such that the laser is orthogonal to the channel and the irradiation source is parallel to the channel. The second dichroic still guides both the laser light and the visible light through the channel.

[0045] An alternative implementation is shown in FIG. 9. In this case, a camera or imaging device 910 is oriented such that the cells or particles 980 move toward the camera in the fluid flow 970. Thus, the camera and the laser 930 are on the same side of the channel, while the irradiation source 960 is at the end on the opposite side of the channel. Two dichroics 940 and 945 are used to direct the laser light 935 and the illumination light 965 toward the channel, direct the illumination light toward the camera, and deflect the laser light away from the illumination source. An alternative embodiment of this configuration switches the positions of the laser and the camera such that the laser is orthogonal to the channel and the irradiation source is parallel to the channel. The second dichroic 945 then guides the laser light through the channel and irradiates the camera with the light.

[0046] Optionally, embodiments of the present invention further include at least one optical element operable to generate a standard TEM00 mode beam, a standard TEMOO 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 between the optical force source and the fourth channel. 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 source of the optical force may include a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam.

[0047] Optionally, the device comprises a microfluidic channel, a laser light source focused by an optical system, and an electric field source operably connected to the microfluidic channel via an electrode, and causes particles in a liquid to flow through the microfluidic channel and manipulates the laser light and the electric field to act jointly 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 comprises a microfluidic channel configured to supply a dielectrophoresis (DEP) field inside the channel via (1) an electrode system or (2) an insulator DEP system, and a laser light source focused inside the microfluidic channel by an optical system, and flows a plurality of particles in a liquid into the microfluidic channel and operates the laser light and the field together on the particles in the microfluidic channel to capture the particles or modify their velocities, and the DEP field is linear or non-linear. Another possible embodiment of the device comprises a microfluidic channel including an inlet and a plurality of outlets, and a laser light source optically focused across the microfluidic channel at a critical angle adapted to the velocity of the flow in the microfluidic channel so as to generate an optical force on the particles while maximizing the residence time of the selected particles in the laser light, and thus separating the particles to the plurality of outlets, and the laser light is operable to apply a force to the particles flowing through the microfluidic channel, thereby separating the particles to the plurality of outlets.

[0048] Optionally, embodiments of the present invention further include at least one particle interrogation unit in communication with one or more of the channel(s), particularly the fourth channel, etc. The particle interrogation unit includes a standard illuminator, a standard optical system, and a 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 spectrofluorometry 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 spectroscopy detector.

[0049] At least one particle interrogation unit that communicates with a fourth channel comprises a laser force-based device or devices that facilitate the identification, selection, and sorting of cellular diseases. In one aspect, the unit utilizes the unique differences in optical pressure resulting from changes in particle size, shape, refractive index, or morphology as a means of separating and characterizing particles. In one embodiment, a near-infrared laser beam exerts a physical force on the cell, which is then measured. When the optical force via radiation pressure is balanced against the hydrodynamic drag force on the particle, changes in particle velocity can be brought about based on the unique differences, for use in discriminating between different particles or for changes in a population of particles. The balance between the hydrodynamic and optical forces can also be used to vary the relative positions of the particles with respect to each other based on the unique properties of the particles, thereby effecting physical separation. Another embodiment of the interrogation unit includes devices 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 includes at least one beam cross-section.

[0050] One embodiment of the present invention includes combining some of the above-described design elements in a single device. Embodiments also include methods of using such devices. An example of such an integrated device is shown in FIG. 1. The illustrated embodiment of the present invention is a five-layer structure, and all five layers are bonded to each other to result in a solid microfluidic chip, although the chip may be a single structure as opposed to the bonded layers. The chip can be constructed using a number of standard materials including fused silica, crown glass, borosilicate glass, soda lime glass, sapphire glass, cyclic olefin polymer (COP), poly(dimethyl)siloxanes (PDMS), OSTE, polystyrene, poly(methyl)methacrylate, polycarbonate, other plastics or polymers. However, it is not limited thereto. This chip allows for sample input, hydrodynamic focusing, optical interrogation, imaging, analysis, sample output, and clear optical access for laser light to enter and exit the region. The chip in the embodiment can also be 3D printed, molded, or otherwise shaped.

[0051] Optionally, at least one particle type includes a plurality of particle types. Each particle type of the plurality of particle types includes respective intrinsic properties and respective induced properties. Optionally, the intrinsic properties include size, shape, refractive index, morphology, intrinsic fluorescence, and / or aspect ratio. Optionally, the induced properties include deformation, angular orientation, rotation, rotation speed, antibody-labeled fluorescence, aptamer-labeled fluorescence, DNA-labeled fluorescence, stain-labeled fluorescence, differential retention measurement, and / or gradient force measurement. Embodiments of this method further include identifying and separating a plurality of particles according to each particle type based on at least one of the intrinsic properties and the induced properties. Optionally, embodiments of this method further include interrogating or manipulating the sample flow. Optionally, interrogating the sample flow includes determining at least one of the intrinsic properties such as those and the induced properties of the particle type, and measuring the particle velocity of the plurality of particles. Measurement of at least one of the endogenous properties can be used for a series of applications including, but not limited to, virus quantification, process development and monitoring, sample release assay, adventitious agent testing, clinical diagnosis, biomarker discovery, determination of antibody or protein productivity for process development and monitoring, determination of the efficacy, quality, or activation state of cells produced as CAR T and other oncology applications and cell-based therapies including stem cells, determination of the effect of chemicals, bacteria, viruses, antibacterial or antiviral agents on a particular cell population, and determination of the disease state or potentiality of research or clinical cell samples. Optionally, the optical force source includes at least one beam axis, and the sample flow includes a sample flow axis. The step of determining at least one of the intrinsic properties and the induced properties of the particle type and the step of measuring the particle velocity of the plurality of particles together include the step of offsetting the beam axis from the sample flow axis. Optionally, the step of determining at least one of the intrinsic properties and the induced properties of the particle type and the step of measuring the particle velocity of the plurality of particles together include the step of calculating the inclination and trajectory of a plurality of particles deviating from the sample flow axis towards at least one beam axis.

[0052] Those skilled in the art will recognize that the disclosed features can be used alone, in any combination, or omitted, based on the requirements and specifications of a given application or design. When an embodiment refers to "comprising" a particular feature, 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 the consideration of the specification and the practice of the invention.

[0053] In particular, it should be noted that when 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. Variations that do not depart from the essence of the invention are intended to be included within the scope of the invention, and the specification and examples are intended to be exemplary or illustrative in nature and not restrictive. Further, all references cited in this disclosure are hereby individually incorporated herein by reference in their entirety, and thus are intended to provide an efficient way to supplement the possible disclosure of the invention and to provide a background that details the level of those skilled in the art.

Claims

1. 1. A device comprising: a substrate comprising a plurality of channels configured to transport one or more substances; and a collimated light source capable of interacting with said one or more substances, The plurality of channels include: a first channel oriented vertically upward relative to gravity along a Y axis within the substrate; a second channel in operative communication with the first channel and disposed horizontally within the substrate along an X-axis; a third channel in communication with the second channel and oriented vertically with respect to gravity in an upward direction along a Y axis within the substrate; a fourth channel in communication with the third channel and disposed horizontally along an X-axis 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 substances to travel through the substrate from the first channel to the second channel, the third channel, and the fourth channel; the second channel and the fourth channel are shorter in length than the first channel, and the collimated light source is oriented to interact with the one or more substances in the fourth channel; the first channel is oriented upward along a Y-axis; configured to receive the one or more substances by ejection via pressure or vacuum driven flow upward against gravity through a bottom horizontal planar surface having an opening into the first channel in a vertical direction; device.

2. the one or more substances are injected into the first channel disposed vertically within the substrate through a bottom horizontal planar surface having an opening into the first channel; said bottom horizontal planar surface having a smaller or equal surface area in a vertical direction compared to a surface area on a vertical plane of the chip to maintain directional and volumetric continuity with said first channel; The device of claim 1 .

3. the first channel is disposed on an outer surface of the substrate, the one or more materials extending vertically into the substrate; The device of claim 1 comprising an opening arranged in a manner to provide a path for vertical movement within the first channel.

4. the one or more substances are injected vertically into the first channel through a bottom horizontal planar surface having an opening into the first channel, the first channel being disposed vertically within the first channel to maintain directional and volumetric continuity with the first channel; The device of claim 1 .

5. the one or more substances are injected vertically into the first channel through a bottom horizontal planar surface having an opening into the first channel, the first channel being disposed vertically within the first channel to maintain directional and volumetric continuity with the first channel; the first channel and the opening are shaped, sized and oriented in a manner to maintain directional and volumetric continuity; The device of claim 1 .

6. 10. The device of claim 1, wherein the collimated light source is oriented to propagate in a direction opposite to, perpendicular to, or oblique to a direction of movement of one or more substances in the fourth channel.

7. The device of claim 1 , wherein the fourth channel enables imaging and analysis of particles or cells in multiple focal planes.

8. The device of claim 1 , wherein the fourth channel enables imaging and analysis of particles or cells in multiple focal planes during movement of the one or more substances.

9. The device of claim 1 , wherein the fourth channel allows imaging and analysis of particles or cells during transfer of the one or more substances.

10. The device of claim 1 , wherein the fourth channel enables imaging and analysis of particles or cells from multiple angles and / or orientations during movement of the one or more substances.

11. 10. The device of claim 1, wherein the fourth channel enables imaging and analysis of particles or cells from multiple focal planes, angles, and / or orientations by one or more imaging devices during movement of the one or more substances.

12. The device of claim 1 , further comprising one or more electrical, optical, and / or fluidic forces for moving cells or particles within the one or more channels.

13. 10. The device of claim 1, further comprising one or more electrokinetic, electrophoretic, and / or dielectrophoretic (DEP) forces for moving cells or particles in one or more channels.

14. Further comprising one or more imaging devices; at least one of the imaging devices is capable of being moved to change the focal plane imaged in the fourth channel; The device of claim 1 .

15. a cell or particle in the fourth channel can be moved by a change in optical and / or fluidic force; the area of ​​the cell imaged in the fourth channel changes as the particle moves. The device of claim 1 .

16. the fourth channel is disposed at a distance from two or more outer surfaces of the substrate; allowing imaging and analysis of multiple image slices of a cell or particle as the focal plane moves relative to the cell or particle; The device of claim 1 .

17. the fourth channel is spaced apart from two or more outer surfaces of the substrate; allowing imaging and analysis of multiple image slices of a migrating cell or particle as it moves through the focal plane; The device of claim 1 .

18. the fourth channel is spaced apart from two or more outer surfaces of the substrate; The device of claim 1 , which enables imaging and analysis of multiple image slices of floating or stationary cells or particles.

19. The device of claim 1 , wherein the one or more substances can be moved by pressure, vacuum, peristalsis, electrokinetic forces, electrophoretic forces, magnetic forces, optical forces, or any combination thereof.

20. 10. The device of claim 1, further comprising a dichroic mirror for directing light to or away from an imaging device that images and / or analyzes cells or particles in the fourth channel.

21. 10. The device of claim 1, further comprising a dichroic mirror for directing a collimated or focused light source to interact with cells or particles in the fourth channel.

22. The device of claim 1 , further comprising a dichroic mirror for directing a collimated or focused light source away from an imaging device, another light source, or another portion of the device.

23. The device of claim 1 , wherein the plurality of channels includes a fifth channel disposed horizontally, vertically, or diagonally within the substrate.

24. The device of claim 1 , wherein the plurality of channels includes a fifth channel that divides into two or more channels or wells for sorting cells or particles.

25. The device of claim 1 , wherein the fourth channel is disposed closer to a top of the substrate than any of the first channel, the second channel, or the third channel.

26. The device of claim 1 , wherein the fourth channel is located between 100 microns and 100 mm from a top of the substrate.

27. The device of claim 1 , wherein the first channel has a length in the range of 0.1 mm to 100.0 mm.

28. The device of claim 1 , wherein the second channel has a length in the range of 0.1 mm to 100.0 mm.

29. The device of claim 1 , wherein the third channel has a length in the range of 0.05 mm to 100.0 mm.

30. The device of claim 1 , wherein the fourth channel has a length in the range of 0.1 mm to 100.0 mm.

31. The device of claim 1 , wherein the first channel has a longer length than the second channel, the third channel, or the fourth channel.

32. The device of claim 1 further comprising a cell or particle interrogation unit.

33. The device of claim 1 further comprising a cell or particle collection channel.

34. 10. The device of claim 1, further comprising an imaging device selected from at least one of a bright field imager, a light scattering detector, a single wavelength fluorescence detector, a spectroscopic fluorescence 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), and / or coherent Stokes Raman spectroscopy (CSRS).

35. a tube end for injecting the one or more substances into an opening disposed on an exterior surface of the substrate to provide a path for the one or more substances to enter the substrate and travel within the first channel. The device of claim 1 .

36. 1. A device comprising a substrate having a plurality of internal fluidic channels and a collimated light source capable of interacting with migrating cells and / or particles, the plurality of internal fluid channels being a first channel of a plurality of internal channels oriented vertically upwardly with respect to gravity along a Y-axis with respect to the substrate, the cells or particles flowing upwardly through the first channel along a length of the first channel; an opening disposed on an outer surface of the substrate and in operative communication with the first channel such that one or more substances can enter and move vertically, upwardly against gravity, within the first channel; a second channel of the plurality of internal channels disposed within the substrate oriented horizontally along an X-axis relative to the substrate and in operable communication with the first channel, the cells or particles flow horizontally through the second channel along a length of the second channel, the second channel having a shorter length than the first channel, the collimated light source oriented to interact with the one or more substances within the second channel, the first channel oriented upwards relative to gravity along a Y-axis and configured to receive the migrating cells and / or particles vertically upwards relative to gravity through an opening via injection via pressure or vacuum driven flow; comprising a substrate; A device for moving cells and / or particles in a fluid.

37. 37. The device of claim 36, wherein the one or more substances are injected vertically into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel to maintain directional and volumetric continuity with the first channel.

38. the one or more substances are injected into the first channel disposed vertically within the substrate through a bottom horizontal planar surface having an opening into the first channel; the bottom horizontal planar surface has a smaller surface area in a vertical direction compared to a surface area on the vertical plane of the chip to maintain directional and volumetric continuity with the first channel; 37. The device of claim 36.

39. 37. The device of claim 36, wherein the first channel is disposed on an outer surface of the substrate and comprises an opening positioned to provide a path for the one or more substances to vertically enter the substrate and travel vertically within the first channel.

40. the one or more substances are injected into the first channel vertically disposed within the first channel through a bottom horizontal planar surface having an opening into the first channel in a vertical direction to maintain directional and volumetric continuity with the first channel; 37. The device of claim 36.

41. the one or more substances are injected into the first channel vertically disposed within the first channel through a bottom horizontal planar surface having an opening into the first channel in a vertical direction to maintain directional and volumetric continuity with the first channel; the first channel and the opening are shaped, sized and oriented in a manner to maintain directional and volumetric continuity; 37. The device of claim 36.

42. 37. The device of claim 36, wherein the collimated or focused light source is oriented to propagate in the same direction as, opposite to, perpendicular to, or oblique to the direction of movement of one or more substances in the second channel.

43. 37. The device of claim 36, wherein the second channel allows imaging and analysis of particles or cells in multiple focal planes.

44. 37. The device of claim 36, wherein the second channel enables imaging and analysis of particles or cells in multiple focal planes during movement of the one or more substances.

45. 37. The device of claim 36, wherein the second channel allows imaging and analysis of particles or cells during transfer of the one or more substances.

46. 37. The device of claim 36, wherein the second channel enables imaging and analysis of particles or cells from multiple angles and / or orientations during movement of the one or more substances.

47. 37. The device of claim 36, wherein the second channel enables imaging and analysis of particles or cells from multiple focal planes, angles, and / or orientations by one or more imaging devices during movement of the one or more substances.

48. 37. The device of claim 36, further comprising one or more electrical, optical, and / or fluidic forces for moving cells or particles within the one or more channels.

49. 37. The device of claim 36, further comprising one or more electrokinetic, electrophoretic, and / or dielectrophoretic (DEP) forces for moving cells or particles within the one or more channels.

50. further comprising one or more imaging devices; at least one of the imaging devices is capable of being moved to change the focal plane imaged in the second channel; 37. The device of claim 36.

51. Cells or particles in the second channel can be moved by changes in optical and / or fluidic forces; the area of ​​the cell imaged in the second channel changes as the particle moves.

37. The device of claim 36.

52. 37. The device of claim 36, wherein the second channel is positioned at a distance from two or more outer surfaces of the substrate that enables imaging and analysis of multiple image slices of a cell or particle as a focal plane moves relative to the cell or particle.

53. the second channel is spaced apart from two or more outer surfaces of the substrate; allowing imaging and analysis of multiple image slices of a migrating cell or particle as it moves through the focal plane; 37. The device of claim 36.

54. the second channel is spaced apart from two or more outer surfaces of the substrate; Allows imaging and analysis of multiple image slices of suspended or stationary cells or particles, 37. The device of claim 36.

55. 37. The device of claim 36, wherein the one or more substances can be moved by pressure, vacuum, peristalsis, electrokinetic forces, electrophoretic forces, magnetic forces, optical forces, or any combination thereof.

56. 37. The device of claim 36, further comprising a dichroic mirror for directing light to or away from an imaging device that images and / or analyzes cells or particles in the second channel.

57. 37. The device of claim 36, further comprising a dichroic mirror for directing the collimated light source to interact with cells or particles in the second channel.

58. 37. The device of claim 36, further comprising a dichroic mirror for directing the collimated light source away from an imaging device, another light source, or another portion of the device.

59. 37. The device of claim 36, wherein the plurality of internal fluid channels includes a third channel disposed horizontally, vertically, or diagonally within the substrate.

60. 37. The device of claim 36, wherein the plurality of internal fluidic channels includes a third channel that divides into two or more channels or wells for sorting cells or particles.

61. 37. The device of claim 36, wherein the second channel is located closer to a top of the substrate than the first channel.

62. 37. The device of claim 36, wherein the second channel is located between 100 microns and 100 mm from a top of the substrate.

63. 37. The device of claim 36, wherein the first channel has a length in the range of 0.1 mm to 100.0 mm.

64. 37. The device of claim 36, wherein the second channel has a length in the range of 0.1 mm to 100.0 mm.

65. 37. The device of claim 36, wherein the first channel has a longer length than the second channel.

66. 37. The device of claim 36, further comprising a cell or particle interrogation unit.

67. 37. The device of claim 36, further comprising a cell or particle collection channel.

68. 37. The device of claim 36, further comprising an imaging device selected from at least one of a bright field imager, a light scattering detector, a single wavelength fluorescence detector, a spectroscopic fluorescence 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), and / or coherent Stokes Raman spectroscopy (CSRS).

69. the one or more substances enter the substrate; and a tube end configured to inject the one or more substances into an opening disposed in an exterior surface of the substrate to provide a path for travel within the first channel.

37. The device of claim 36.

70. 37. The device of claim 36, wherein the one or more substances can be moved by pressure, vacuum, peristalsis, electrokinetic forces, electrophoretic forces, magnetic forces, optical forces, or any combination thereof to sort cells or particles into one or more distinct regions or wells.

71. 37. The device of claim 36, wherein the one or more substances are capable of being moved by pressure, vacuum, peristalsis, electrokinetic forces, electrophoretic forces, magnetic forces, optical forces, or any combination thereof to sort cells or particles into one or more distinct regions or wells.

72. 1. A method for evaluating a biological particle for use in cellular immunotherapy, comprising: a) injecting one or more biological particles into a first channel of a substrate comprising a plurality of channels configured to transport one or more biological particles, the plurality of channels comprising: i) a first channel oriented vertically upward relative to gravity along a Y axis within the substrate; ii) a second channel disposed horizontally within the substrate along an X-axis and in operative communication with the first channel; iii) a third channel in communication with the second channel and oriented vertically upward relative to gravity along a Y axis with respect to the substrate; iv) a fourth channel disposed horizontally along an X-axis within the substrate and in communication with the third channel; b) evaluating the biological particle via an optical force-based measurement in the fourth channel using a collimated light source capable of interacting with the biological particle, The horizontal channel has a length shorter than the first channel; the first channel, the second channel, the third channel, and the fourth channel are arranged in a manner to provide a path for movement of the one or more biological particles through the substrate from the first channel to the second channel to the third channel to the fourth channel; injecting the one or more biological particles via pressure or vacuum driven flow into a first channel oriented vertically upward along a Y axis in the substrate relative to gravity through a bottom horizontal plane having an opening into the first channel; The biological particle comprises a T cell, an engineered T cell, including a CAR-T cell; The optical force-based measurements are used to obtain information regarding morphology, motility, binding affinity, binding profile, effects on other biological particles, effects on target cells, susceptibility to external forces such as biological, biochemical, chemical, physical, and temperature effects; Evaluating the biological particles includes characterizing T cell receptors, regulatory T cells, allogeneic T cells, or bionic T cells. The method includes the steps of:

73. The evaluation includes a quantitative measurement of the amount of change in the T cells; Providing predictive or prescriptive analytics; 73. The method of claim 72.

74. 73. The method of claim 72, wherein a lower horizontal 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 planar surface of the substrate to maintain directional and volumetric continuity with the first channel.

75. the one or more biological particles are injected into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening into the first channel; The bottom horizontal planar surface is vertically smaller than the vertical planar surface area of ​​the chip. having a smaller or equal surface area to maintain directional and volumetric continuity with the first channel; 73. The method of claim 72.

76. the first channel is disposed on an outer surface of the substrate; the one or more biological particles having openings arranged in a manner to provide a path for vertically entering the substrate and moving vertically within the first channel; 73. The method of claim 72.

77. 73. The method of claim 72, further comprising a collimated or focused light source oriented to interact with biological particles or cells in the fourth channel.

78. 73. The method of claim 72, wherein the collimated or focused light source is oriented to propagate in the direction of, opposite to, perpendicular to, or oblique to the direction of movement of one or more biological particles in the fourth channel.

79. 73. The method of claim 72, wherein the fourth channel enables imaging and analysis of the one or more biological particles or cells during movement and from multiple focal planes, angles, and / or orientations by one or more imaging devices.

80. 73. The method of claim 72, further comprising one or more electrical, optical, and / or fluidic forces for moving biological particles or cells within the one or more channels.

81. the plurality of channels includes a fifth channel; The fifth channel is divided into two or more channels or wells for sorting cells or particles; 73. The method of claim 72.

82. 73. The method of claim 72, further comprising an imaging device selected from at least one of a bright field imager, a light scattering detector, a single wavelength fluorescence detector, a spectroscopic fluorescence 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), and / or coherent Stokes Raman spectroscopy (CSRS).

83. 73. The method of claim 72, wherein LFC (laser power cytology) is used to evaluate the biological particles or cells.

84. 84. The method of claim 83, wherein the use of LFC includes a combination of microfluidics and light-induced pressure to perform measurements including optical force, pressure, size, and velocity on a cell-by-cell basis.

85. The use of LFC has been used to characterize biological particles; The properties include morphology, motility, interaction of the biological particle with other physiological components, deformability (cytoskeleton changes), or response of the biological particle to environmental changes; 84. The method of claim 83.

86. 74. The method of claim 73, wherein the evaluation of the biological particle comprises measuring complexes of one or more cell therapy product cells interacting with one or more target cells or particles.

87. 1. A method for evaluating a biological particle for use in cellular immunotherapy, comprising: a) injecting one or more biological particles into a first channel of a substrate comprising a plurality of channels configured to transport one or more biological particles, The plurality of channels include: i) a first channel oriented vertically upward along a Y axis in said substrate; ii) a second channel disposed horizontally within the substrate along an X-axis and in operative communication with the first channel; iii) a third channel oriented vertically upward along a Y axis in the substrate relative to gravity and in communication with the second channel; (iv) a fourth channel disposed horizontally along the X-axis within the substrate and in communication with the third channel; and b) evaluating the biological particles for use in cellular immunotherapy via optical force-based measurements using a collimated light source capable of interacting with the biological particles in the fourth channel, The horizontal channel has a length shorter than the first channel; the first channel, the second channel, the third channel, and the fourth channel are arranged to provide a path for movement of the one or more biological particles through the substrate from the first channel, to the second channel, to the third channel, to the fourth channel; injecting the one or more biological particles via pressure or vacuum driven flow into a first channel oriented vertically upwards along a Y-axis within the substrate through a bottom horizontal planar surface having an opening into the first channel; the biological particles include T cells, engineered T cells, and include CAR T cells; and evaluating the biological particles via LFC (laser power cytology) measurements; The LFC measurements are used to obtain information regarding morphology, motility, binding affinity, binding profile, effects on other biological particles, effects on target cells, susceptibility to external forces such as biological, biochemical, physical, and temperature effects; Evaluating the biological particles includes characterizing T cell receptors, regulatory T cells, allogeneic T cells, or bionic T cells. The method includes the steps of:

88. 88. The method of claim 87, wherein the use of LFC comprises a combination of microfluidics and light-induced pressure to perform measurements including optical force, pressure, size, and velocity on a cell-by-cell basis.

89. The use of said LFC is used to characterize biological particles; The properties include morphology, motility, interaction of the biological particle with other physiological components, deformability (cytoskeleton changes), or the response of the biological particle to environmental changes.

88. The method of claim 87.

90. 88. The method of claim 87, wherein said evaluation of biological particles comprises measuring complexes of one or more cell therapy product cells interacting with one or more target cells or particles.

91. The evaluation of biological particles may include characterization of cells for efficacy assays; 88. The method of claim 87, comprising characterization of cells for potency assays, CAR-T cell affinity assays, CAR-T cell binding assays, detection of T cell biomarkers, or label-free detection of T cell activation.

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