WO2021127276A1

WO2021127276A1 - Cell sorting systems and methods for enhancing cell yield

Info

Publication number: WO2021127276A1
Application number: PCT/US2020/065750
Authority: WO
Inventors: Qiong PAN; Syed Husain; Lagnajeet PRADHAN; Efrat HAREL; Ivan Dimov; Colm Hunt; Khiem Do; Nick LEINDECKER
Original assignee: Orca Biosystems, Inc.
Priority date: 2019-12-18
Filing date: 2020-12-17
Publication date: 2021-06-24

Abstract

An apparatus and methods for using it to sort target particles are described. The apparatus comprises: (i) a substrate comprising a plurality of microchannels, where the plurality of microchannels is configured to hold a plurality of target particles immersed in a fluid; an extraction laser configured to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release a set of target particles from the respective microchannels, where the set of target particles is selected from the plurality of target particles; and a collection plate that is used for collecting the released set of target particles, where the collection plate is disposed relative to the substrate at a predetermined distance to enhance a yield of the released set of target particles.

Description

CELL SORTING SYSTEMS AND METHODS FOR ENHANCING CELL YIELD

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/949,922 filed December 18, 2019, which is incorporated by reference herein in its entirety for all purposes.

BACKGROUND

[0002] Techniques for sorting micron-sized particles have a variety of applications in chemical and biological analyses, clinical diagnostics, food and chemical processing, and environmental testing. Cell sorting techniques in particular have had a tremendous impact on biomedical research in a variety of fields by allowing researchers to study isolated, largely homogeneous sub-populations of cells. Existing cell sorting technologies include bulk sorting and single cell sorting approaches. Examples of existing bulk sorting techniques, in which all of the target cells are collected in a single step, include filtration, centrifugation, and magnetic cell sorting. Examples of existing single cell sorting technologies consist primarily of flow cytometry or fluorescence-activated cell sorting (FACS). While some cell sorting techniques can be very accurate, it is often difficult to determine if a sorted cell population is “pure”. Instead, the collected population is typically referred to as having been “enriched”, with single cell sorting techniques providing highly enriched cell populations that are typically more homogeneous than those obtained using bulk sorting methods. There remains an unmet need for improvements in both the accuracy and throughput of single cell sorting techniques to provide extremely homogeneous cell populations for use in genetically-engineered cell therapy applications and other biomedical research fields.

[0003] Methods and apparatus are described in the present disclosure that provide for high- throughput cell sorting with high accuracy and improved yields in terms of cell viability.

SUMMARY

[0004] Disclosed herein are apparatus for sorting target particles, the apparatus comprising: a substrate comprising a plurality of microchannels, wherein the plurality of microchannels is configured to hold a plurality of target particles immersed in a fluid; an extraction laser configured to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release a set of target particles from the respective microchannels, wherein the set of target particles is selected from the plurality of target particles; and a collection plate that is used for collecting the released set of target particles, wherein the collection plate is disposed relative to the substrate at a predetermined distance to enhance a yield of the released set of target particles.

[0005] In some embodiments, the predetermined distance is defined along a vertical axis extending between the collection plate and the substrate. In some embodiments, the predetermined distance is configured to influence a flight distance that the released set of target particles travels through air, after said target particles have been released from the respective microchannels. In some embodiments, the predetermined distance is configured to influence a time of flight that the released set of target particles travels through air, after said target particles have been released from the respective microchannels. In some embodiments, the predetermined distance is less than 2 mm. In some embodiments, the predetermined distance is at least 0.2 mm. In some embodiments, the predetermined distance is configured such that the flight distance is less than 2 mm. In some embodiments, the predetermined distance is configured such that the time of flight is less than 0.1 seconds. In some embodiments, each target particle of said set of target particles is coated with a layer of the fluid, upon immediate release from the respective microchannels. In some embodiments, the predetermined distance is configured to influence an extent of evaporation of the layer of the fluid as the released set of target particles travels through air, after said target particles have been released from the respective microchannels. In some embodiments, the predetermined distance is configured such that the layer of the fluid coated on each target particle does not evaporate completely before the released set of target particles reaches the collection plate. In some embodiments, the layer of the fluid coated on each target particle has a minimum thickness of at least 0.05 pm upon said target particle reaching the collection plate. In some embodiments, the coated layer of fluid serves to protect each target particle from an ambient environment as said target particle travels through the air. In some embodiments, the layer of the fluid coated on each target particle has (1) an initial pre evaporation thickness of at least 0.1 pm upon immediate release from the corresponding microchannel, and (2) a post-evaporation thickness of at least 0.05 pm upon said target particle reaching the collection plate. In some embodiments, the predetermined distance is determined based in part on a size of the plurality of target particles. In some embodiments, the predetermined distance is determined based in part on a size of the plurality of microchannels. In some embodiments, the predetermined distance is determined based in part on an evaporation rate of the layer of the fluid coated on the set of target particles. In some embodiments, the substrate and the collection plate are disposed substantially parallel to each other. In some embodiments, the collection plate is disposed below the substrate at the predetermined distance. In some embodiments, the yield of the released set of target particles is enhanced to at least 80% when the collection plate is disposed relative to the substrate at the predetermined distance. In some embodiments, the yield of the released set of target particles is higher in a first configuration in which the collection plate is disposed relative to the substrate at the predetermined distance, compared to a second configuration in which the collection plate is not disposed relative to the substrate at the predetermined distance. In some embodiments, the yield of the released set of target particles is higher by at least 10% in the first configuration compared to the second configuration.

[0006] The apparatus of claim 1, wherein the plurality of target particles comprises a plurality of live cells. In some embodiments, the released set of target particles comprises a released set of cells, and wherein the yield is measured by a state of the released set of cells when collected on the collection plate. In some embodiments, a useability of the released set of cells depends on the state of the released set of cells when collected on the collection plate. In some embodiments, an aqueous solution is flown over the collection plate, and wherein the aqueous solution is used for transporting the released set of target particles along the collection plate to a receiving port. In some embodiments, the receiving port is provided on or coupled to the collection plate. In some embodiments, the aqueous solution is flown over the collection plate at a speed ranging from about 1 mm/s to about 10 mm/s.

[0007] Also disclosed herein are methods for sorting target particles, the method comprising: providing (1) a substrate comprising a plurality of microchannels, (2) a collection plate, and (3) an extraction laser; providing a fluid containing a plurality of target particles to the substrate, wherein the plurality of microchannels is configured to hold said plurality of target particles immersed in said fluid; selecting a set of target particles from said plurality of target particles that are held in the plurality of microchannels; using the extraction laser to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release the set of target particles from the respective microchannels,; and using the collection plate to collect the released set of target particles, wherein the collection plate is disposed relative to the substrate at a predetermined distance to enhance a yield of the released set of target particles.

[0008] Disclosed herein are apparatus for sorting target particles, the apparatus comprising: a substrate comprising a plurality of microchannels, wherein the plurality of microchannels is configured to hold a plurality of target particles immersed in a fluid; a collection plate that is used for collecting a released set of target particles, wherein the set of target particles is selected from the plurality of target particles; and an actuator stage coupled to the substrate and/or the collection plate, wherein the actuator stage is configured to move the substrate and/or the collection plate such that the collection plate is disposed relative to the substrate at a predetermined distance, and wherein the predetermined distance is configured to enhance a yield of the released set of target particles. [0009] In some embodiments, the apparatus further comprises an extraction laser configured to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release the set of target particles from the respective microchannels.

INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0012] FIG. 1 provides a schematic illustration (exploded view) of the components of a microchannel plate device for sorting cells or particles, as previously described in U.S. Patent Application Publication No. 2018/0353960 Al.

[0013] FIG. 2 provides a schematic illustration (assembled view) of the components of a microchannel plate device for sorting cells or particles, as previously described in U.S. Patent Application Publication No. 2018/0353960 Al.

[0014] FIG. 3 provides a photograph of a prototype microchannel plate device for sorting cells or particles.

[0015] FIG. 4 illustrates a single cell suspended in the fluid within a microchannel.

[0016] FIG. 5 provides a non-limiting example of a schematic for an optical apparatus for laser scanning and cell sorting utilizing two rotating polygon mirrors, as previously described in U.S. Patent Application Publication No. 2018/0353960 Al.

[0017] FIG. 6 provides a photograph of a prototype microchannel plate device for sorting cells or particles mounted in a laser scanning system configured to release selected cells or particles from microchannels in the device.

[0018] FIG. 7 provides a schematic illustration (cross-sectional view) of a microchannel plate device mounted at a predetermined distance above a cell collection plate device. [0019] FIG. 8 provides a plot of the calculated flight time for a cell that is released from the bottom of the microchannel plate device and travels to a cell collection plate device that is separated from the bottom of the microchannel plate device by a predetermined distance.

DETAILED DESCRIPTION

[0020] Disclosed herein are methods, devices, and systems for performing high-throughput sorting of cells or other target particles with high accuracy and improved yields. In some instances, the disclosed methods may comprise: (i) providing a substrate comprising a plurality of microchannels configured to capture cells or other target particles, a collection plate configured to receive cells or other target particles that are released from the microchannels of the substrate, and an extraction laser configured to scan a surface of the substrate and release cells or other target particles captured within the microchannels therein; (ii) contacting the substrate with a fluid containing a plurality of cells or target particles such that the cells or other target particles are captured and held within the plurality of microchannels; (iii) selecting a set of cells or target particles from said plurality of target particles that are held in the plurality of microchannels; (iv) using the extraction laser to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release the set of target particles from the respective microchannels; and (v) using the collection plate to collect the released set of cells or other target particles, where the collection plate is disposed relative to the substrate at a predetermined distance to enhance a yield, e.g ., a yield of viable cells in the collected cell population. In some instances, the yield of cells or other target particles is measured by a state of the released set of cells or target particles when collected on the collection plate. In some instances, a “useability” of the released set of cells or target particles depends on the state of the released set of cells or target particles when collected on the collection plate.

[0021] Also disclosed are devices and apparatus (or systems) for sorting cells or other target particles according to the disclosed methods. In some instances, the apparatus may comprise: (i) a substrate comprising a plurality of microchannels configured to capture and hold cells or target particles immersed in a fluid; (ii) an extraction laser configured to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release a selected set of cells or target particles from their respective microchannels; and a collection plate that is used for collecting the released set of cells or target particles, where the collection plate is disposed relative to the substrate at a predetermined distance to enhance a yield, e.g. , a yield of viable cells, of the released set of cells or other target particles. In some instances, the apparatus may further comprise an actuator stage coupled to the substrate and/or the collection plate, where the actuator stage is configured to move the substrate and/or the collection plate such that the collection plate is disposed relative to the substrate at a predetermined distance, and wherein the predetermined distance is configured to enhance a yield of the released set of cells or target particles.

[0022] Various aspects of the disclosed methods, devices, and systems described herein may be applied to any of the particular applications set forth below, or for any other type of cell sorting or particle-sorting application. It shall be understood that different aspects of the disclosed methods, devices, and systems can be appreciated individually, collectively, or in combination with each other.

[0023] Flight distance and flight time: A key aspect of the present disclosure is the optimization of the distance between the lower surface of a microchannel plate device from which cells or other particles are released and the upper surface of a collection plate configured to receive the release cells or other particles such that, e.g. , the yield of viable cells released from the microchannel plate device is enhanced. FIG. 7 provides a schematic illustration (cross- sectional view) of a microchannel plate device mounted at a predetermined distance above a cell collection plate device. The microchannel plate device comprising substrate 110 (comprising a plurality of microchannels) is adhered to a metal frame 130 by means of a seal 120. The microchannels are configured to trap single cells or particles that are suspended in a fluid which is placed in contact with the substrate. The microchannel plate device is positioned above a collection plate device configured to capture cells or other target particles that are released from the microchannels upon exposure to intense laser pulses, as described in more detail in U.S. Patent Application Publication No. 2018/0353960 Al. The upper surface of the collection plate substrate 750 may be positioned at a predetermined distance along a vertical axis from the lower surface of the microchannel plate substrate 110, as illustrated in FIG. 7, thereby defining a “flight distance” for cells or particles released from the microchannel plate substrate 110. In some instances, the predetermined distance is adjusted to optimize a yield for cells or particles released from the microchannel plate substrate 110 and captured in the thin layer of liquid 740 covering the collection plate substrate 750. In some instances, the predetermined distance is adjusted to optimize the yield of viable cells released from the microchannel plate substrate 110 and captured in the thin layer of liquid 740 covering the collection plate substrate 750, /. e. , by the minimizing the corresponding “flight time” of cells that are released from the microchannel plate substrate 110 and fall to the collection plate under the influence of gravitational forces, thereby also minimizing or preventing evaporation of a thin layer of fluid surrounding the cells that are extracted from the microchannel plate device. In some instances, the predetermined distance may be adjusted according to the type of cell or target particle to be sorted and collected. In some instances, the predetermined distance may be adjusted according to the size (e.g, diameter, longest cross-sectional dimension, and/or length) of the microchannels. In some instances, the predetermined distance may be adjusted according to the evaporation rate for a thin layer of fluid surrounding the released cells or particles that is exhibited under a given set of operating conditions.

[0024] In some instances, a thin layer of fluid surrounding the cells or particles that are extracted from the microchannel plate device ( e.g ., a thin layer of the cell or particle suspension fluid) may range in thickness from about 0.01 pm to about 10 pm immediately upon release (i.e., a pre-evaporation thickness). In some instances, the layer of fluid surrounding the cells or particles that are extracted from the microchannel plate device may be at least at least 0.01 pm, at least 0.02 pm, at least 0.03 pm, at least 0.04 pm, at least 0.05 pm, at least 0.06 pm, at least 0.07 pm, at least 0.08 pm, at least 0.09 pm, at least 0.1 pm, at least 0.2 pm, at least 0.3 pm, at least 0.4 pm, at least 0.5 pm, at least 0.6 pm, at least 0.7 pm, at least 0.8 pm, at least 0.9 pm, at least 1.0 pm, at least 1.5 pm, at least 2.0 pm, at least 2.5 pm, at least 3.0 pm, at least 3.5 pm, at least 4.0 pm, at least 4.5 pm, at least 5.0 pm, at least 5.5 pm, at least 6.0 pm, at least 6.5 pm, at least 7.0 pm, at least 7.5 pm, at least 8.0 pm, at least 8.5 pm, at least 9.0 pm, at least 9.5 pm, or at least 10 pm upon release from the microchannel plate device (or pre-evaporation). In some instances, the layer of fluid surrounding the cells or particles that are extracted from the microchannel plate device may be at most 10.0 pm, at most 9.5 pm, at most 9.0 pm, at most 8.5 pm, at most 8.0 pm, at most 7.5 pm, at most 7.0 pm, at most 6.5 pm, at most 6.0 pm, at most 5.5 pm, at most 5.0 pm, at most 4.5 pm, at most 4.0 pm, at most 3.5 pm, at most 3.0 pm, at most 2.5 pm, at most 2.0 pm, at most 1.50 pm, at most 1.0 pm, at most 0.9 pm, at most 0.8 pm, at most 0.7 pm, at most 0.6 pm, at most 0.5 pm, at most 0.4 pm, at most 0.3 pm, at most 0.2 pm, at most 0.1 pm, at most 0.09 pm, at most 0.08 pm, at most 0.07 pm, at most 0.06 pm, at most 0.05 pm, at most 0.04 pm, at most 0.03 pm, at most 0.02 pm, or at most 0.01 pm upon release from the microchannel plate device (or pre-evaporation). Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, the thickness of the fluid layer surrounding each cell or target particle upon release may range from about 0.02 pm to about 1.0 pm. Those of skill in the art will recognize that the thickness of the fluid layer surrounding each cell or target particle upon release may have any value within this range, e.g., about 0.55 pm. In some instance, the thin layer of fluid surrounding the cells or particles that are extracted from the microchannel plate device serves to protect the cells or particles from the ambient environment as they travel from the microchannel plate device to the collection plate. [0025] In some instances, the layer of the fluid coated on or surrounding each cell or target particle has a minimum thickness of at least 0.01 pm upon said cell or target particle reaching the collection plate (i.e., a post-evaporation thickness). In some instances, the minimum thickness of the fluid layer surrounding each cell or target particle upon reaching the collection plate may be at least 0.2 nm, at least 0.4 nm, at least 0.6 nm, at least 0.8 nm, at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 0.01 mih, at least 0.02 mih, at least 0.03 mih, at least 0.04 mih, at least 0.05 mih, at least 0.06 mih, at least 0.07 mih, at least 0.08 mih, at least 0.09 mih, at least 0.1 mih, at least 0.2 mih, at least

0.3 mih, at least 0.4 mih, at least 0.5 mih, at least 0.6 mih, at least 0.7 mih, at least 0.8 mih, at least

0.9 mih, at least 1.0 mih, at least 1.5 mih, at least 2.0 mih, at least 4.0 mih, at least 6.0 mih, at least

8.0 mih, at least 10.0 mih, at least 20 mih, at least 30 mih, at least 40 mih, or at least 50 mih (i.e., the post-evaporation thickness). In some instances, the minimum thickness of the fluid layer surrounding each cell or target particle upon reaching the collection plate may be at most 50 pm, at most 40 pm, at most 30 pm, at most 20 pm, at most 10 pm, at most 8.0 pm, at most 6.0 pm, at most 4.0 pm, at most 2.0 pm, at most 1.5 pm, at most 1.0 pm, at most 0.9 pm, at most 0.8 pm, at most 0.7 pm, at most 0.6 pm, at most 0.5 pm, at most 0.4 pm, at most 0.3 pm, at most 0.2 pm, at most 0.1 pm, at most 0.09 pm, at most 0.08 pm, at most 0.07 pm, at most 0.06 pm, at most 0.05 pm, at most 0.04 pm, at most 0.03 pm, at most 0.02 pm, at most 0.01 pm, at most 9 nm, at most

8 nm, at most 7 nm, at most 6 nm, at most 5 nm, at most 4 nm, at most 3 nm, at most 2 nm, at most 1 nm, at most 0.8 nm, at most 0.6 nm, at most 0.4 nm, or at most 0.2 nm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, the minimum thickness of the fluid layer surrounding each cell or target particle upon reaching the collection plate (i.e., the post-evaporation thickness) may range from about 0.03 pm to about 1.5 pm. Those of skill in the art will recognize that the minimum thickness of the fluid layer surrounding each cell or target particle upon reaching the collection plate may have any value within this range, e.g ., about 0.74 pm.

[0026] In some instances, the predetermined distance between a lower surface of the microchannel plate device (e.g, the lower surface of substrate 110 in FIG. 7) and the collection plate device (e.g, the upper surface of collection plate substrate 750 in FIG. 7) may range from about 0.2 mm to about 5 mm. In some instances, the predetermined distance may be at least 0.2 mm, at least 0.4 mm, at least 0.6 mm, at least 0.8 mm, at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, or at least 5 mm. In some instances, the predetermined distance may be at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1 mm, at most 0.8 mm, at most

0.6 mm, at most 0.4 mm, at most 0.2 mm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the predetermined distance may range from about 0.4 mm to about 3 mm. In some instances, the predetermined distance may have any value within this range, e.g, about

1.75 mm. [0027] FIG. 8 provides an example of calculated values for the flight time versus separation distance (i.e., flight distance, or the predetermined distance between the microchannel plate device and the collection plate) for cells (or particles) released from the microchannel plate device and falling toward to the collection plate under the influence of gravity. The calculation assumes that the initial velocity of the released cells (or particles) is zero, and neglects air resistance. In some instances, the laser-induced extraction mechanism used to release cells or particles from the microchannels may impart a non-zero initial velocity to the cells or particles upon release.

[0028] In some instances, the flight time may range from about 0.25 msec to about 100 msec. In some instances, the flight time may be at least 0.25 msec, at least 0.5 msec, at least 1 msec, at least 5 msec, at least 10 msec, at least 20 msec, at least 30 msec, at least 40 msec, at least 50 msec, at least 60 msec, at least 70 msec, at least 80 msec, at least 90 msec, or at least 100 msec.

In some instances, the flight time may be at most 100 msec, at most 90 msec, at most 80 msec, at most 70 msec, at most 60 msec, at most 50 msec, at most 40 msec, at most 30 msec, at most 20 msec, at most 10 msec, at most 5 msec, at most 1 msec, at most 0.5 msec, or at most 0.25 msec. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the flight time may range from about 1 msec to about 20 msec. In some instances, the flight time may have any value within this range, e.g ., about 14.3 msec.

[0029] In some instances, the predetermined distance is adjusted to ensure that the thin layer of fluid does not evaporate completely as the cells or particles travel through the air (or other gaseous environment) separating the microchannel plate device and the collection plate device. [0030] In some instances, the predetermined distance may be adjusted to optimize a yield, e.g. , the yield of viable cells collected by the collection plate. In some instances, the yield of viable cells may be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%.

[0031] In some instances, the adjustment of the predetermined distance may result in an improvement in yield, e.g. , cell viability, of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,

80%, 90%, 100%, 125%, 150%, 175%, or 200% compared to that of a configuration in which the collection plate is not disposed relative to the microchannel substrate at the predetermined distance.

[0032] In some instances, the laser extraction apparatus may also comprise an environmental control chamber or other means for controlling the temperature, pressure, and/or humidity of the air space (or ambient environment) between microchannel plate device and the collection plate.

In some instances, environmental control may be used separately or in combination with optimization of flight distance to maximize the yield of viable cells in a collection of selected cells.

[0033] Examples of factors that may impact the viability of cells that are sorted and collected using the disclosed methods devices and systems include, but are not limited to, cell size, cell type, the flight distance and/or flight time between the microchannel plate device and the collection plate, the angle at which the cell strikes the collection plate (or layer of fluid flowing across the surface thereof), the flow rate of the fluid layer as it passes across the collection plate, and/or the ambient environment through which the cells travel between the microchannel plate device and the collection plate ( e.g ., the temperature, pressure, and/or humidity thereof).

[0034] Definitions: Unless otherwise defined, all of the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs.

[0035] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

[0036] As used herein, the term ‘about’ a number refers to that number plus or minus f0% of that number. The term ‘about’ when used in the context of a range refers to that range minus f0% of its lowest value and plus f0% of its greatest value.

[0037] Cells & other particles: As used herein, the term “cell” may refer to any of a variety of cells known to those of skill in the art. In some aspects, the term “cell” may refer to any adherent or non-adherent eukaryotic cell, mammalian cell, a primary or immortalized human cell or cell line, a primary or immortalized rodent cell or cell line, a cancer cell, a normal or diseased human cell derived from any of a variety of different organs or tissue types (e.g., a white blood cell, red blood cell, platelet, epithelial cell, endothelial cell, neuron, glial cell, astrocyte, fibroblast, skeletal muscle cell, smooth muscle cell, gamete, or cell from the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, small intestine), a distinct cell subset such as an immune cell, a CD8+ T cell, CD4+ T cell,

cancer stem cell,

Lgr5/6+ stem cell, undifferentiated human stem cell, a human stem cell that has been induced to differentiate, a rare cell (e.g, a circulating tumor cell (CTC), a circulating epithelial cell, a circulating endothelial cell, a circulating endometrial cell, a bone marrow cell, a progenitor cell, a foam cell, a mesenchymal cell, or a trophoblast), an animal cell (e.g, mouse, rat, pig, dog, cow, or horse), a plant cell, a yeast cell, a fungal cell, a bacterial cell, an algae cell, an adherent or non adherent prokaryotic cell, or in plural form, any combination thereof. In some aspects, the term “cell” may refer to an immune cell, e.g, a T cell, a cytotoxic (killer) T cell, a helper T cell, an alpha beta T cell, a gamma delta T cell, a T cell progenitor, a B cell, a B-cell progenitor, a lymphoid stem cell, a myeloid progenitor cell, a lymphocyte, a granulocyte, a Natural Killer cell, a plasma cell, a memory cell, a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a dendritic cell, and/or a macrophage, or in plural form, to any combination thereof.

[0038] In some instances, the disclosed methods, devices, and systems may be used for sorting particles other than cells. Examples include, but are not limited to, lipid vesicles, extracellular vesicles, microparticles, microbeads, chemical synthesis resin particles, glass microspheres, polymer microspheres, metal microspheres, ceramic microspheres, and the like, or any combination thereof.

[0039] In some instances, the average diameter or dimension of the cells or particles for which the disclosed methods, devices, and sorting systems may be used may range from about 0.5 pm to about 0.5 mm. In some instances, the average diameter or dimension of the cells or particles may be at least 0.5 pm, at least 1 pm, at least 2 pm, at least 3 pm, at least 4 pm, at least 5 pm, at least 6 pm, at least 7 pm, at least 8 pm, at least 9 pm, at least 10 pm, at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, or at least 0.5 mm. In some instances, the average diameter or dimension of the cells or particles may be at most 0.5 mm, at most 0.4 mm, at most 0.3 mm, at most 0.2 mm, at most 0.1 mm, at most 90 pm, at most 80 pm, at most 70 pm, at most 60 pm, at most 50 pm, at most 40 pm, at most 30 pm, at most 20 pm, at most 10 pm, at most 9 pm, at most 8 pm, at most 7 pm, at most 6 pm, at most 5 pm, at most 4 pm, at most 3 pm, at most 2 pm, at most 1 pm, or at most 0.5 pm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the average diameter or dimension of the cells or particles may range from about 5 pm to about 40 pm. In some instances, the average diameter or dimension of the cells or particles may have any value within this range, e.g ., about 12.4 pm.

[0040] MicroChannel plate device: FIG. 1 provides a schematic illustration (exploded view) of the components of a microchannel plate device for sorting cells or particles, as previously described in U.S. Patent Application Publication No. 2018/0353960 Al. In some instances, the device (shown in an assembled view in FIG. 2) comprises substrate 110, comprising a plurality of microchannels, which is adhered to a metal frame 130 by means of a seal 120. The microchannels are configured to trap single cells or particles that are suspended in a fluid which is placed in contact with the substrate. In some instances, the microchannel plate device illustrated in FIG. 1 and FIG. 2 may be part of a housing assembly that comprises additional components, as described in U.S. Patent Application Publication No. 2018/0353960 Al and co pending U.S. Provisional Application No. 62/898,357. [0041] The substrate may be fabricated from any of a variety of materials known to those of skill in the art. Examples include, but are not limited to, glass, fused silica, silicon, ceramic, a polymer, or any combination thereof.

[0042] In some instances, the substrate 110 may comprise any of a variety of shapes in two dimensions, e.g ., circular, square, rectangular, triangular, pentagonal, hexagonal, and so forth. In some instances, the diameter or longest dimension of the substrate may range from about 0.2 cm to about 150 cm. In some instances, the diameter or longest dimension of the substrate may be at least 0.2 cm, at least 0.5 cm, at least 1 cm, at least 5 cm, at least 10 cm, at least 20 cm, at least 30 cm, at least 40 cm, at least 50 cm, at least 60 cm, at least 70 cm, at least 80 cm, at least 90 cm, at least 100 cm, at least 110 cm, at least 120 cm, at least 130 cm, at least 140 cm, or at least 150 cm. In some instances, the diameter or longest dimension of the substrate may be at most 150 cm, at most 140 cm, at most 130 cm, at most 120 cm, at most 110 cm, at most 100 cm, at most 90 cm, at most 80 cm, at most 70 cm, at most 60 cm, at most 50 cm, at most 40 cm, at most 30 cm, at most 20 cm, at most 10 cm, at most 5 cm, at most 1 cm, at most 0.5 cm, or at most 0.2 cm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the diameter or longest dimension of the substrate may range from about 5 cm to about 120 cm. In some instances, the diameter or longest dimension of the substrate may have any value within this range, e.g. , about 24.5 cm. [0043] In some instances, the substrate may have a thickness ranging from about 0.1 mm to about 1 cm. In some instances, the thickness of the substrate may be at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm, at least 1.0 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, or at least 1 cm. In some instances, the thickness of the substrate may be at most 1 cm, at most 9 mm, at most 8 mm, at most 7 mm, at most 6 mm, at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1.0 mm, at most 0.9 mm, at most 0.8 mm, at most 0.7 mm, at most 0.6 mm, at most 0.5 mm, at most 0.4 mm, at most 0.3 mm, at most 0.2 mm, or at most 0.1 mm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the thickness of the substrate may range from about 0.2 mm to about 0.8 mm. In some instances, the thickness of the substrate may have any value within this range, e.g. , about 8.5 mm.

[0044] The microchannels may be fabricated in the substrate using any of a variety of methods known to those of skill in the art, where in general the choice of fabrication technique is dependent on the choice of substrate material, and vice versa. Examples of suitable fabrication techniques include, but are not limited to, photolithography and wet chemical etching, deep reactive ion etching (DRIE), ion beam etching (or milling), micromolding, etc.

[0045] In some instances, the number of microchannels fabricated in the substrate may range from about 1 thousand to about 100 billion microchannels. In some instances, the number of microchannels may be at least 1 thousand, at least 10 thousand, at least 100 thousand, at least 1 million, at least 5 million, at least 10 million, at least 100 million, at least 200 million, at least

300 million, at least 400 million, at least 500 million, at least 600 million, at least 700 million, at least 800 million, at least 900 million, at least 1 billion, at least 10 billion, at least 20 billion, at least 30 billion, at least 40 billion, at least 50 billion, at least 60 billion, at least 70 billion, at least

80 billion, at least 90 billion, or at least 100 billion. In some instances, the number of microchannels may be at most 100 billion, at most 90 billion, at most 80 billion, at most 70 billion, at most 60 billion, at most 50 billion, at most 40 billion, at most 30 billion, at most 20 billion, at most 10 billion, at most 1 billion, at most 900 million, at most 800 million, at most 700 million, at most 600 million, at most 500 million, at most 400 million, at most 300 million, at most 200 million, at most 100 million, at most 90 million, at most 80 million, at most 70 million, at most 60 million, at most 50 million, at most 40 million, at most 30 million, at most 20 million, at most 10 million, at most 5 million, at most 1 million, at most 100 thousand, at most 10 thousand, or at most 1 thousand. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the number of microchannels contained within the substrate may range from about 5 million to about 100 million. In some instances, the number of microchannels may have any value within this range, e.g ., about 122 million.

[0046] In some instances, the distribution of microchannels across the surface of the substrate may comprise any random or non-random pattern. Examples of non-random patterns include, but are not limited to, a square grid, a rectangular grid, a triangular grid, a hexagonal grid, and the like. In some instances, the pattern or distribution of microchannels may vary across the surface of the substrate.

[0047] In some instances, the microchannels in the substrate may comprise any of a variety of cross-sectional shapes in two dimensions, e.g. , circular, square, rectangular, triangular, pentagonal, hexagonal, and so forth. In some instances, the diameter or longest cross-sectional dimension of the microchannels may range from about 5 pm to about 500 pm. In some instances, the diameter or longest cross-sectional dimension of the microchannels may be at least 5 pm, at least 10 pm, at least 15 pm, at least 20 pm, at least 25 pm, at least 30 pm, at least 35 pm, at least 40 pm, at least 45 pm, at least 50 pm, at least 75 pm, at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, or at least 500 pm. In some instances, the diameter or longest cross-sectional dimension of the microchannels may be at most 500 mih, at most 400 mih, at most

300 mih, at most 200 mih, at most 100 mih, at most 75 mih, at most 50 mih, at most 45 mih, at most

40 mih, at most 35 mih, at most 30 mm, at most 25 mih, at most 20 mih, at most 15 mih, at most 10 mih, or at most 5 mih. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the diameter or longest cross-sectional dimension of the microchannels may range from about 10 pm to about 200 pm. In some instances, the diameter or longest cross-sectional dimension of the microchannels may have any value within this range, e.g ., about 12.5 pm. In some instances, the diameter or longest cross-sectional dimension of the microchannels may vary across the surface of the substrate. In some instances, the substrate may comprise 1, 2, 3, 4, or 5, or more subsets of microchannels having different diameters or longest cross-sectional dimensions.

[0048] In some instances, the center-to-center spacing (or “pitch”) or edge-to-edge separation distance between adjacent microchannels may range from about 5 pm to about 500 pm. In some instances, the center-to-center spacing (or “pitch”) or edge-to-edge separation distance may be at least 5 pm, at least 10 pm, at least 15 pm, at least 20 pm, at least 25 pm, at least 30 pm, at least

35 pm, at least 40 pm, at least 45 pm, at least 50 pm, at least 75 pm, at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, or at least 500 pm. In some instances, the center-to-center spacing (or “pitch”) or edge-to-edge separation distance may be at most 500 pm, at most 400 pm, at most 300 pm, at most 200 pm, at most 100 pm, at most 75 pm, at most 50 pm, at most 45 pm, at most 40 pm, at most 35 pm, at most 30 pm, at most 25 pm, at most 20 pm, at most 15 pm, at most 10 pm, or at most 5 pm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the center-to-center spacing (or “pitch”) or edge-to-edge separation distance may range from about 20 pm to about 75 pm. In some instances, the center-to-center spacing (or “pitch”) or edge-to-edge separation distance may have any value within this range, e.g. , about 12.5 pm. In some instances, the center-to-center spacing (or “pitch”) or edge-to-edge separation distance between adjacent microchannels may vary across the surface of the substrate.

[0049] In general, the length (or height) of the microchannels will be equal to the thickness of the substrate. In some instances, the cross-sectional area of the microchannels may be constant over their entire length. In some instances, the cross-sectional area of the microchannels may vary with their length. For example, in some instances, the entrance and/or exit of the microchannel may be tapered such that the cross-sectional area of the microchannel at the top surface and/or bottom surface of the substrate is larger than that at the mid-point of the substrate.

[0050] Referring again to FIG. 1 and FIG. 2, the microchannel device may further comprise a seal 120 and a metal frame 130. In some instances, the seal 120 is placed between the substrate 110 and the metal frame 130. In some instances, the seal 120 may be an adhesive material. For example, in some instances the seal 120 may be an epoxy adhesive, including polyurethane, acrylic, and cyanoacrylate, which can be used as an adhesive for glass, metal, and plastics, etc.

The epoxy adhesive can be made flexible or rigid, transparent or opaque, fast-setting or slow setting. As depicted in FIG. 2, the substrate 110 and the metal frame 130 are in contact with the seal 120 in the assembled device. In one instance, the substrate 110, the seal 120, and the metal frame 130 may be assembled at a temperature of 15° C, 10° C, 5° C, or less. For example, the substrate 110, the seal 120, and the metal frame 130 may be assembled at a temperature of 5° C.

[0051] As represented in FIG. 2, the substrate 110, the seal 120, and the metal frame 130 are assembled when in operation. The temperature of the substrate 110, the seal 120, and the metal frame 130 may be increased during the operation of the device. At increased temperatures, the substrate 110, the seal 120, and the metal frame 130 may expand. In some instances, at increased temperatures, the expansion of the metal frame 130 exceeds the expansion of the substrate 110, thereby causing a tension to be applied across the surface of the substrate. Such tension across the surface of the substrate 110 may reduce sagging of the substrate 110 and thereby maintain planarity of the substrate. In some instances, the substrate 110 comprises of a first end, a second end, and a middle portion, and the first end, the second end, and the middle portion are substantially on the same plane. FIG. 3 provides a photograph of a prototype microchannel plate device for sorting cells or particles comprising the substrate mounted in a metal frame.

[0052] FIG. 4 illustrates a single cell 410 suspended in the fluid within a microchannel of substrate 110 by means of the meniscus 430 after contacting the substrate with a cell suspension.

Meniscus 430 is formed by surface tension and the wetting of the microchannel walls by fluid

420 of the cell suspension. In some instances, the geometry and/or dimensions of the microchannels may be adjusted according to the specific application in order to preferentially trap a single cell or a single target particle in each microchannel.

[0053] In some instances, as illustrated in FIG. 7, the microchannel plate device may further comprise a porous mesh 710 such as that described in co-pending U.S. Provisional Patent Application No. 62/898,357. Porous mesh 710 facilitates separation of cells or other target particles into a monolayer which may then be transferred to substrate 110 comprising the plurality of microchannels. In some instances, the porous mesh may be loaded by dispensing a cell suspension or particle suspension into fluid dispensing ports 730 integrated into a support frame 720, where the fluid dispensing ports are configured such that the dispensed cell suspension (or particle suspension) flows onto the top surface of the porous mesh 710. The device may be tilted at an inclination angle of, e.g ., between about 1 degree and about 10 degrees, to facilitate loading of single cells or particles into the pores of the porous mesh by means of capillary action. The loaded mesh may then be placed in contact with the top surface of the microchannel plate substrate 110, and suction is applied from below to draw single cells or particles out of mesh 710 and into the microchannels of substrate 110. As described above, substrate 110 may be mounted on a separate metal frame 130 by means of a seal 120.

[0054] Laser extraction systems: FIG. 5 provides a non-limiting schematic illustration of an optical apparatus for laser scanning and cell sorting utilizing two rotating polygon mirrors and a confocal detection technique, as described in more detail in U.S. Patent Application Publication No. 2018/0353960 Al. In some instances, the disclosed optical apparatus are configured to scan the microchannel plate device with light from a fluorescence excitation light source and detect fluorescence signals corresponding to those cells or particles trapped within the microchannels of the device that meet a predefined set of selection criteria, e.g ., expression of a fluorescently- labeled cell surface receptor or a GFP-labeled protein, in order to select a subset of the cells (or target particles) for extraction. In some instances, the disclosed optical apparatus are further configured to pulse a laser extraction beam in response to the detected fluorescence of a cell (or target particle) with an amount of energy sufficient to extract the cell (or target particle) from the microchannel and allow the cell to survive. In some instances, the amount of energy required to extract cells or other target particles may range from about 0.1 pj to about 1000 pj. In certain instances, the circuitry is configured to generate a plurality of pulses to extract a plurality of cells or target particles, where the amount of extraction energy delivered to each extracted target particle is within a range from about 1 pJ to about 50 pj, where a duration of the extraction energy delivered to the each extracted cell or target particle is within a range of about 0.1 ns to about 1000 ns, and where a peak extraction power delivered to the each of the plurality of extracted cells or target particles is within a range from about 0.1 W to about 10⁷ W.

[0055] The apparatus 1300 illustrated in FIG. 5 comprises a plurality of excitation light sources 1110 and 1112, a dichroic mirror 1120, a beam expander 1122, an excitation light mirror 1124, an extraction laser 1130, an extraction laser mirror 1132, a rotating polygon mirror 1140, an F-theta lens 1150, coupling optics 1162, a combination of beam-splitters, dichroic reflectors, and/or bandpass filters 1164, and a plurality of detectors 1170 and 1172.

[0056] Additionally, the apparatus 1300 may comprise a second rotating polygon mirror 1142 and a second F-theta lens 1152. The system may be configured such that the plurality of excitation light sources are directed to the microchannel plate device 100 by the first rotating polygon mirror 1140 and the first F-theta lens 1150, while the extraction laser is directed to the microchannel plate device 100 by the second rotating polygon mirror 1142 and the second F- theta lens 1152. The plurality of excitation light sources and the extraction beam may be located on the same side of the microchannel plate device 100. The plurality of excitation light sources and the extraction beam may be located on opposite sides of the microchannel plate device 100

[0057] The apparatus 1300 may further comprise a set of mirrors to direct light to the one or more detectors. The excitation light mirror 1124 may be a dichroic mirror. The confocal detection cavity may comprise mirrors 1166 and 1168. The mirrors may be flat mirrors. The mirrors may be concave mirrors. The mirrors may be spherical mirrors. The mirrors may be arranged in a confocal configuration.

[0058] In some instances, apparatus 1300 may further comprise an actuator stage coupled to the microchannel plate substrate 100 and/or a collection plate, where the actuator stage is configured to move the substrate and/or the collection plate such that the collection plate is disposed relative to the substrate at a predetermined distance, and wherein the predetermined distance is configured to enhance a yield of the released set of cells or target particles, as will be discussed in more detail below.

[0059] In some instances, the laser extraction apparatus may also further comprise an environmental control chamber or other means for controlling the temperature and/or humidity of the air space between microchannel plate substrate 100 and a collection plate configured to capture extracted cells (or particles) in order to minimize the evaporation rate of the thin layer of fluid surrounding cells released from the microchannel plate device, and thereby improve the yield of viable cells collected on the collection plate. In some instances, an environmental control chamber or other means for controlling the temperature and/or humidity may be used instead of, or in addition to, optimization of the predetermined distance (i.e., the flight distance / flight time) between the microchannel plate device and the collection plate, as will be discussed in more detail below.

[0060] In some instances, the laser extraction systems of the present disclosure may scan the substrate at a rate greater than 100,000 microchannels per second, greater than 250,000 microchannels per second, greater than 500,000 microchannels per second, greater than 1,000,000 microchannels per second, greater than 2,000,000 microchannels per second, greater than 3,000,000 microchannels per second, greater than 4,000,0000 microchannels per second, greater than 5,000,000 microchannels per second, greater than 10,000,000 microchannels per second, greater than 50,000,000 microchannels per second, greater than 100,000,000 microchannels per second, or greater than 150,000,000 microchannels per second. The laser extraction system may scan the substrate at a rate that is within a range defined by any two of the preceding values.

[0061] In some instances, the laser extraction systems of the present disclosure may extract cells or target particles from the substrate at a rate greater than 50,000 cells or target particles per second, greater than 100,000 cells or target particles per second, greater than 250,000 cells or target particles per second, greater than 500,000 cells or target particles per second, greater than

600,000 cells or target particles per second, greater than 700,000 cells or target particles per second, greater than 800,000 cells or target particles per second, greater than 900,000 cells or target particles per second, greater than 1,000,000 cells or target particles per second, greater than

1,500,000 cells or target particles per second, greater than 2,000,000 cells or target particles per second, greater than 3,000,000 cells or target particles per second, greater than 4,000,000 cells or target particles per second, greater than 5,000,000 cells or target particles per second, greater than

10,000,000 cells or target particles per second, greater than 20,000,000 cells or target particles per second, greater than 40,000,000 cells or target particles per second, greater than 60,000,000 cells or target particles per second, greater than 80,000,000 cells or target particles per second, or greater than 100,000,000 cells or target particles per second. The laser extraction system may extract cells or target particles at a rate that is within a range defined by any two of the preceding values.

[0062] In some instances, the laser extraction systems of the present disclosure may extract the cells or target particles such that the extracted cells or target particles have a purity greater than 80%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, or greater than 99.5%. The laser extraction system may extract the cells or target particles such that the extracted cells or target particles have a purity that is within a range defined by any two of the preceding values.

[0063] Though shown as forming a single device in FIG. 5, system 1300 may be configured such that the fluorescence detection subsystem (comprising elements 1110, 1112, 1120, 1122, 1124, 1140, 1150, 1164, 1166, 1168, 1170, and 1172) are arranged as a fluorescence detection apparatus, and the extraction subsystem (comprising elements 1130, 1132, 1142, and 1152) are arranged as an extraction apparatus. In such an arrangement, the microchannel plate device 100 may be subjected to a fluorescence analysis as described herein using the fluorescence apparatus. Following the fluorescence analysis, the microchannel plate device 100 may be transferred to the extraction apparatus for extraction of locations of interest on the substrate identified during the fluorescence analysis. Proper alignment of the substrate in each of the fluorescence apparatus and the extraction apparatus may be achieved by referring to one or more fiducial markers on the substrate.

[0064] FIG. 6 provides a photograph of a prototype microchannel plate device for sorting cells or particles mounted in a laser scanning system configured to release selected cells or particles from microchannels in the device. [0065] Cell collection plate: As noted above, FIG. 7 provides a schematic illustration (cross- sectional view) of a microchannel plate device mounted at a predetermined distance above a cell collection plate device (which, in some instances, may be a component of the laser extraction apparatus), where the plane of the microchannel plate and the plane of the cell collection plate are positioned in a substantially parallel relative orientation.

[0066] Following scanning and extraction of a selected set of cells or particles 760 from the microchannel plate device, the cells or particles 760 released from one end of each selected microchannel fall under the influence of gravity and are collected by a collection plate comprising a substrate 750 covered with a thin layer of fluid 740. In some instances, the collection plate substrate 750 may be fabricated from any of a variety of materials known to those of skill in the art. Examples include, but are not limited to, glass, fused silica, silicon, ceramic, a polymer, or any combination thereof. In some instances, the thin layer of fluid 740 may comprise water, an aqueous solution, e.g ., a buffer solution, a cell growth medium, an organic solvent, or any combination thereof. In some instances, the thin layer of fluid 740 may be specifically formulated to minimize evaporation of the thin layer of fluid surrounding cells or other particles that are released from the microchannel plate device as they travel to the collection plate. In some instances, the thin layer of fluid 740 may be specifically formulated to maximize the viability of cells captured on the collection plate. In some instances, the collection plate substrate 750 may be in fluid communication with a receiving port such that following collection of a set of selected cells or particles in fluid 740, the fluid and selected cells or particles therein may be transported across the surface of the collection plate and drained from the device. In some instances, the receiving port is provided on or coupled to the collection plate. [0067] In order to harvest the collected cells or particles, the fluid in layer 740 may be flowing across the surface of collection plate substrate 750 at a rate ranging from about 0.1 mm/sec to about 20 mm/sec. In some instances, the flow rate may be at least 0.1 mm/sec, at least 0.2 mm/sec, at least 0.4 mm/sec, at least 0.6 mm/sec, at least 0.8 mm/sec, at least 1 mm/sec, at least 2 mm/sec, at least 3 mm/sec, at least 4 mm/sec, at least 5 mm/sec, at least 6 mm/sec, at least 7 mm/sec, at least 8 mm/sec, at least 9 mm/sec, at least 10 mm/sec, at least 15 mm/sec, or at least 20 mm/sec. In some instances, the flow rate may be at most 20 mm/sec, at most 15 mm/sec, at most 10 mm/sec, at most 9 mm/sec, at most 8 mm/sec, at most 7 mm/sec, at most 6 mm/sec, at most 5 mm/sec, at most 4 mm/sec, at most 3 mm/sec, at most 2 mm/sec, at most 1 mm/sec, at most 0.8 mm/sec, at most 0.6 mm/sec, at most 0.4 mm/sec, at most 0.2 mm/sec, or at most 0.1 mm/sec . Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the flow rate may range from about 2 mm/sec to about 15 mm/sec. Those of skill in the art will recognize that the flow rate may have any value within this range, e.g ., about 4.4 mm/sec.

[0068] As noted above, in some instances, the plane of microchannel plate device and the plane of the collection plate may be oriented in a substantially parallel fashion and offset relative to each other along a vertical axis. In some instances, the plane of the collection plate may be oriented at an angle relative to that of the microplate device, e.g. , such that cells or other particles released from the microchannel plate device strike the collection plate (or a fluid layer thereon) at an angle, or such that a fluid layer covering the top surface of the collection plate flows across the collection plate. In some instances, the plane of the microchannel plate device may be oriented relative to that of the collection plate device.

[0069] In some instances, the plane of the collection plate may be oriented relative to that of the microchannel plate device, or vice versa , such that the angle between them ranges from about 0 degrees (i.e., the two planes are parallel) to about 20 degrees. In some instances, the angle may be at least 0, at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 degrees. In some instances, the angle may be at most 20, at most 18, at most 16, at most 14, at most 12, at most 10, at most 8, at most 6, at most 4, at most 2, or at most 0 degrees. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the angle may range from about 0 degrees to about 12 degrees. Those of skill in the art will recognize that the angle may have any value within this range, e.g. , about 1.4 degrees.

[0070] In some instances, the plane of the collection plate may be oriented relative to that of the microchannel plate device, or vice versa , such that cells or other particles released from the microchannel plate device strike the collection plate (or a fluid layer thereon) at an angle ranging from about 0 degrees (i.e., perpendicularly) to about 20 degrees. In some instances, the angle may be at least 0, at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 degrees. In some instances, the angle may be at most 20, at most 18, at most 16, at most 14, at most 12, at most 10, at most 8, at most 6, at most 4, at most 2, or at most 0 degrees. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the angle may range from about 0 degrees to about 14 degrees. Those of skill in the art will recognize that the angle may have any value within this range, e.g. , about 2.5 degrees.

[0071] Systems: In some instances, the systems of the present disclosure may comprise one or more of the disclosed microchannel plate devices, one or more imaging and/or laser scanning/extraction modules (or sub-systems), one or more collection plates, or any combination thereof. [0072] In some instances, the systems of the present disclosure may further comprise an actuator stage (or translation stage) configured to control the orientation of the collection plate and/or microchannel plate device with three or more degrees of freedom ( e.g ., x position, y position, z (or vertical) position, or a rotation about the x-, y-, or z-axis. In some instances, the actuator stage may be configured to allow manual or automated adjustment or control of the distance between the microchannel plate (e.g., the lower surface thereof) and the collection plate

(e.g, the upper surface thereof), the cell or particle flight distance, the relative orientation of the collection plate and the microchannel plate (or vice versa), or any combination thereof.

[0073] In some instances, the systems of the present disclosure may further comprise one or more processors, computer memory devices, computer data storage devices, user input devices

(e.g, keyboards, joysticks, etc.), display devices (e.g, LCD or flat screen monitors), or any combination thereof. In some instances, the computer memory devices may comprise one or more software programs or sets of encoded instructions which, when executed by one or more processors, cause the system to execute one or more of the disclosed methods for cell (or particle) sorting and separation, including, but not limited to, execution of image processing tasks, system operation and control tasks, data processing and display task, data storage tasks, and the like. In some instances, the disclosed systems may be configured to access data stored in a local computer, a local server-based computer system, or a cloud-based database. In some instances, the disclosed systems may be configured to upload data to a local computer, a local server-based computer system, or a cloud-based database. In some instances, the disclosed systems may be configured to perform processing tasks (i.e., tasks encoded for by one or more software programs) on a local processor or computer, on a local server-based computer system, and/or in the cloud.

[0074] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in any combination in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An apparatus for sorting target particles, the apparatus comprising: a substrate comprising a plurality of microchannels, wherein the plurality of microchannels is configured to hold a plurality of target particles immersed in a fluid; an extraction laser configured to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release a set of target particles from the respective microchannels, wherein the set of target particles is selected from the plurality of target particles; and a collection plate that is used for collecting the released set of target particles, wherein the collection plate is disposed relative to the substrate at a predetermined distance to enhance a yield of the released set of target particles.

2. The apparatus of claim 1, wherein the predetermined distance is defined along a vertical axis extending between the collection plate and the substrate.

3. The apparatus of claim 1, wherein the predetermined distance is configured to influence a flight distance that the released set of target particles travels through air, after said target particles have been released from the respective microchannels.

4. The apparatus of claim 1, wherein the predetermined distance is configured to influence a time of flight that the released set of target particles travels through air, after said target particles have been released from the respective microchannels.

5. The apparatus of claim 1, wherein the predetermined distance is less than 2 mm.

6. The apparatus of claim 1, wherein the predetermined distance is at least 0.2 mm.

7. The apparatus of claim 3, wherein the predetermined distance is configured such that the flight distance is less than 2 mm.

8. The apparatus of claim 4, wherein the predetermined distance is configured such that the time of flight is less than 0.1 seconds.

9. The apparatus of claim 1, wherein each target particle of said set of target particles is coated with a layer of the fluid, upon immediate release from the respective microchannels.

10. The apparatus of claim 9, wherein the predetermined distance is configured to influence an extent of evaporation of the layer of the fluid as the released set of target particles travels through air, after said target particles have been released from the respective microchannels.

11. The apparatus of claim 10, wherein the predetermined distance is configured such that the layer of the fluid coated on each target particle does not evaporate completely before the released set of target particles reaches the collection plate.

12. The apparatus of claim 11, wherein the layer of the fluid coated on each target particle has a minimum thickness of at least 0.05 pm upon said target particle reaching the collection plate.

13. The apparatus of any one of claims 9 through 12, wherein the coated layer of fluid serves to protect each target particle from an ambient environment as said target particle travels through the air.

14. The apparatus of any one of claims 9 through 11, wherein the layer of the fluid coated on each target particle has (1) an initial pre-evaporation thickness of at least 0.1 pm upon immediate release from the corresponding microchannel, and (2) a post-evaporation thickness of at least 0.05 pm upon said target particle reaching the collection plate.

15. The apparatus of claim 1, wherein the predetermined distance is determined based in part on a size of the plurality of target particles.

16. The apparatus of claim 1, wherein the predetermined distance is determined based in part on a size of the plurality of microchannels.

17. The apparatus of claim 10, wherein the predetermined distance is determined based in part on an evaporation rate of the layer of the fluid coated on the set of target particles.

18. The apparatus of claim 1, wherein the substrate and the collection plate are disposed substantially parallel to each other.

19. The apparatus of claim 1, wherein the collection plate is disposed below the substrate at the predetermined distance.

20. The apparatus of claim 1, wherein the yield of the released set of target particles is enhanced to at least 80% when the collection plate is disposed relative to the substrate at the predetermined distance.

21. The apparatus of claim 1, wherein the yield of the released set of target particles is higher in a first configuration in which the collection plate is disposed relative to the substrate at the predetermined distance, compared to a second configuration in which the collection plate is not disposed relative to the substrate at the predetermined distance.

22. The apparatus of claim 21, wherein the yield of the released set of target particles is higher by at least 10% in the first configuration compared to the second configuration.

23. The apparatus of claim 1, wherein the plurality of target particles comprises a plurality of live cells.

24. The apparatus of claim 23, wherein the released set of target particles comprises a released set of cells, and wherein the yield is measured by a state of the released set of cells when collected on the collection plate.

25. The apparatus of claim 24, wherein a useability of the released set of cells depends on the state of the released set of cells when collected on the collection plate.

26. The apparatus of claim 1, wherein an aqueous solution is flown over the collection plate, and wherein the aqueous solution is used for transporting the released set of target particles along the collection plate to a receiving port.

27. The apparatus of claim 26, wherein the receiving port is provided on or coupled to the collection plate.

28. The apparatus of claim 26, wherein the aqueous solution is flown over the collection plate at a speed ranging from about 1 mm/s to about 10 mm/s.

29. A method for sorting target particles, the method comprising: providing (1) a substrate comprising a plurality of microchannels, (2) a collection plate, and (3) an extraction laser; providing a fluid containing a plurality of target particles to the substrate, wherein the plurality of microchannels is configured to hold said plurality of target particles immersed in said fluid; selecting a set of target particles from said plurality of target particles that are held in the plurality of microchannels; using the extraction laser to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release the set of target particles from the respective microchannels,; and using the collection plate to collect the released set of target particles, wherein the collection plate is disposed relative to the substrate at a predetermined distance to enhance a yield of the released set of target particles.

30. An apparatus for sorting target particles, the apparatus comprising: a substrate comprising a plurality of microchannels, wherein the plurality of microchannels is configured to hold a plurality of target particles immersed in a fluid; a collection plate that is used for collecting a released set of target particles, wherein the set of target particles is selected from the plurality of target particles; and an actuator stage coupled to the substrate and/or the collection plate, wherein the actuator stage is configured to move the substrate and/or the collection plate such that the collection plate is disposed relative to the substrate at a predetermined distance, and wherein the predetermined distance is configured to enhance a yield of the released set of target particles.

31. The apparatus of claim 30, further comprising: an extraction laser configured to emit and scan an extraction beam over distal portions of the plurality of microchannels in order to release the set of target particles from the respective microchannels.