System and compositions for laser dissection and methods of using the same

Abstract

The disclosure relates to a system comprising a laser, a membrane, one or a plurality of cells positioned on or adherent to the membrane, and one or plurality of enclosure elements positioned around the one or plurality of cells and membrane, the enclosure elements defining a volume between the enclosure element and the membrane. The disclosure also relates to methods of using the system for microdissection of fixed and living cells on a single-cell level.

Description

SYSTEM AND COMPOSITIONS FOR LASER DISSECTION AND METHODS OF USING THE SAMECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 669,538, which was filed July 10, 2024, is titled “System and Compositions for Laser Dissection and Methods of Using the Same,” and is incorporated herein by reference in its entirety;FIELD

[0002] The disclosure relates to a system comprising a laser, a membrane, one or a plurality of cells positioned on or adherent to the membrane, and one or plurality of enclosure elements positioned around the one or plurality7of cells and membrane, the enclosure elements defining a volume between the enclosure element and the membrane. The disclosure also relates to methods of using the system for microdissection of fixed and living cells on a single-cell level.BACKGROUND

[0003] Current laser microdissection techniques have several flaws including a limited sample collection apparatus that routinely exposes samples to air. limited control of laser heat and position that damages biological samples, and an absence of configurations that allow microscopy of cells before and after isolation, particularly, those configurations that fail to enable high magnification and high-resolution imaging of biological samples.SUMMARY OF EMBODIMENTS

[0004] The disclosure relates to a device and system of using and making the device for the single-cell microdissection of cell culture biomaterial, including, but not limited to one or a plurality of cells or extracellular matrix. The disclosure also relates to a system comprising a laser source positioned proximate to a first side of a membrane opposite of a second side of the membrane upon which a cell or plurality of cells are positioned, and a controller operably connected to a camera and display; and a computer software product comprising instructions for a computer-implemented steps of: (i) creating a digitized image of the membrane through the camera and assigning one or a plurality7of positions on the screen corresponding to a position on the membrane; and (ii) directing the laser to cut the membrane at one or more predetermined positions around or adjacent to a cell or plurality of cells. The disclosure also relates to a system comprising a laser source positioned proximate to a first side of a membraneopposite of a second side of the membrane upon which a cell or plurality of cells are positioned, and a controller operably connected to a camera and display; and a computer software product comprising instructions for a computer-implemented steps of: (i) creating a digitized image of the membrane through the camera and assigning one or a plurality of positions on the screen corresponding to a position on the membrane; and (ii) directing a laser from the laser source to cut the membrane in one or more predetermined shapes around or adjacent to a cell or plurality of cells. The disclosure also relates to a system comprising a laser source positioned under a cell culture vessel (or, interchangeably, an incubation compartment or vessel), the cell culture vessel comprising a membrane upon which one or a plurality of cell are positioned or to which one or a plurality of cells are adherent. In some embodiments, the system further comprises one or a plurality of enclosure elements positioned above and around the membrane that define a volume around the one or plurality of cells in the cell culture vessel. In some embodiments, the one or plurality of enclosure elements comprise at least five metallic, plastic or semimetallic surfaces that are positioned in a three dimensional rectangular or substantially rectangular prism above and around the membrane and the cell or plurality of cells, wherein the edge of four of the five surfaces are in physical contact with the membrane forming a junction between the membrane and the enclosure elements or a solid support and the enclosure elements, wherein the solid support is positioned between the laser source and the membrane. In some embodiments, the system further comprises a solid support upon which the membrane rests with an aperture or transparent portion beneath the membrane that allows laser light from the laser to contact the one or a plurality of pre-determined positions on the membrane in a first operational mode. In some embodiments, the system further comprises a microscope operably aligned to capture light of a visual field of the membrane and / or the one or plurality of cells. In some embodiments, the microscope is operably equipped with a digital camera and magnification elements to visualize the visual field with a naked eye of a user.

[0005] The disclosure relates to a method of laser microdissection comprising: (a) assigning one or a plurality of predetermined positions on the membrane of the aforementioned systems that define a perimeter around a biomaterial, single cell or plurality of cells adhering to the membrane; (b) cutting the membrane around the perimeter of the membrane, resulting in a sample; and (c) collecting the sample. In some embodiments, the method further comprises analyzing the sample after the step of collecting. In some embodiments, the step of analyzing comprises performing flow cytometry, microscopy, digital imaging, mass spectrometry, photo spectrometry, nucleic acid sequencing, infrared scanning, immunohistochemistry, or combinations of two or more thereof after the step of collecting the sample. In someembodiments, the step of collecting the sample comprises positioning the sample in a region of the compartment or a region downstream of the compartment that is operably aligned with at least one interrogation device. In some embodiments, the interrogation device comprises one or a combination of two or more interrogation devices chosen from: a flow cytometer, a microscope, a digital imager such as a camera, a mass spectrometer, a photo spectrometer, a nucleic acid sequencer, an infrared or UV scanner with an infrared light or UV light, respectively.

[0006] The disclosure relates to a method of sorting one or a plurality of cells. The method comprises: (i) growing one or a plurality of cells in a tissue culture medium and within a system disclosed herein comprising an cell incubation vessel or compartment comprising one or a plurality of cells, wherein the one or plurality of cells are growing upon a biocompatible membrane; (ii) isolating a cell or plurality of cells using the composition or device disclosed herein to dissect the biocompatible membrane associated with the one or plurality of cells at a location chosen a user. In some embodiments, the method further comprises collecting a sample of cells after dissecting the biocompatible membrane with a laser disclosed herein; and, optionally, allowing the sample to flow in a fluid circuit to a collection chamber and exposing the cells to at least one probe specific for a biomarker on the surface of one or plurality of cells. In some embodiments, the method further comprises pooling the one or plurality7of cells in a collection vessel and subsequently interrogating the cell or plurality of cells by detection of the probe. In some embodiments, the step of interrogating the cell comprises exposing the cell or plurality of cells to a flow cytometer capable or sorting the cell or plurality of cells. In some embodiments, the step of interrogating the cell comprises exposing the cell or plurality of cells to a flow cytometer capable or sorting the cell or plurality of cells based upon the presence, absence and / or quantity of probe associated with a biomarker (e.g. such as a cell receptor) on the surface of the cell or plurality of cells. In some embodiments, the method comprises: (i) growing one or a plurality of cells in a tissue culture medium and within a system disclosed herein comprising an cell incubation vessel or compartment comprising one or a plurality of cells, wherein the one or plurality of cells are growing upon a biocompatible membrane; (ii) isolating a cell or plurality of cells using the composition or device disclosed herein to dissect the biocompatible membrane associated with the one or plurality of cells at a location chosen a user, such that the dissected biocompatible membrane is a sample; and (iii) exposing the sample to one or a combination of two or more interrogation devices. In some embodiments, the interrogation device comprises one or a combination of two or more interrogation devices chosen from: a flow cytometer, a microscope, a digital imager such as a camera, a massspectrometer, a photo spectrometer, a nucleic acid sequencer, an infrared or UV scanner with an infrared light or UV light, respectively. In some embodiments, the method of sorting further comprises exposing the sample to one or a combination of two or more of: one or plurality of probes, one or more wash solutions comprising at least about 30% ethanol, one or a plurality of dNTPs, one or a plurality of DNA or RNA polymerases.

[0007] The disclosure relates to methods of growing and analyzing one or a plurality of cells or one or more biomaterials, the cells and / or biomaterials positioned within an open or closed fluid circuit comprising a tissue culture compartment in fluid communication with a collection vessel, the tissue culture compartment and collection vessel in fluid communication with tubing, a tissue culture medium reservoir and a pump. In some embodiments, operating the pump in the system creates an adj ustable flow rate of tissue culture medium across or within the compartment and the collection vessel. In some embodiments, the tubing comprises at least two sets of tubing, a first set connecting the tissue culture reservoir to the incubation vessel or compartment, and a second set of tubing connecting the incubation vessel or compartment to a collection chamber operably aligned within at least one interrogation device. In some embodiments, the collection chamber comprises at least one reaction vessel operably aligned with at least one interrogation device. In some embodiments, the collection chamber comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more reaction vessels operably aligned to one or more interrogation devices, each reaction vessel in fluid communication with the closed fluid circuit.

[0008] The disclosure relates to the disclosed systems comprising a fluid circuit and methods of using the system in which a user introduces tissue culture medium into the device disclosed herein, wherein the device comprises a fluid circuit comprising: (a) a compartment with an inlet and an outlet in which cells are positioned for growth on a biocompatible membrane, and (b) a collection chamber comprising a plurality of reaction vessels, the plurality of reaction vessels are in fluid communication with the compartment inlet and the outlet and one or more reservoirs comprising tissue culture medium and / or reagent, such as a probe. In some embodiments, the device also comprises a first set of valves positioned in the fluid circuit between the inlet and the reaction vessels, wherein the first set of valves are adjustable in an open and closed position. In the open position, the first set of valves allows for fluid flow between the inlet and the incubation vessel or compartment. In some embodiments, the device also comprises a second set of valves positioned in the fluid circuit between the incubator compartment and the reaction vessel or outlet of the fluid circuit. In some embodiments, the second set of valves are adjustable in an open and closed position. In the open position, thesecond set of valves allow for fluid flow between the outlet and the tissue culture medium reservoir and pump. In some embodiments, the methods of the disclosure comprise introducing the tissue culture medium into the fluid circuit disclosed herein, operating a disclosed laser source to cut or dissect a portion of the biocompatible membrane from the membrane positioned in the incubation compartment to form a sample, positioning the sample into a collection vessel within the fluid circuit, and analyzing the cell or cells within the sample. In some embodiments, the cell or cells are optionally exposed to one or a plurality of reagents (such as probes) disclosed herein, optionally pooled in a second collection vessel or compartment and then counted or sorted based upon detection or presence of the probes. In some embodiments, at least a first and second probe form a pair corresponding to a pair of biomarkers on the cell, cells and / or biomaterials in the compartment or reaction vessels of the device. Upon detection of the pair of probes, cells or biomaterials exposed to the pair of probes exhibit a unique cell signature corresponding to the pair of probes and detectable by one or more interrogation devices. This unique signature can be used to identify, stratify or sort the cells prior to or subsequent to one or more interrogation steps. In some embodiments, the method of cell sorting comprises flow cytometry or mass cytometry.

[0009] In some embodiments, the disclosure relates to a system. In some embodiments, the system comprises (a) a biocompatible membrane comprising a top surface and a bottom surface; (b) one or a plurality of cells positioned on the top surface of the biocompatible membrane; (c) a laser source positioned beneath the membrane; and (d) one or a plurality of enclosure elements positioned above the top surface of the biocompatible membrane that define a compartment and a compartment volume around the biocompatible membrane and the one or plurality of cells. In some embodiments, the laser source is at a distance from about 1 millimeters to about 20,000 millimeters from the bottom surface of the biocompatible membrane. In some embodiments, the system comprises one or more mirrors in an optical path between the laser source and the solid support, the optical path created by a laser emitted from the laser source, and the mirrors adjustable in one or more planes such that the user may adjust the direction of the laser at one or a plurality of positions on the surface of the biocompatible membrane. In some embodiments, the laser source is positioned proximate to the surface of the biocompatible membrane opposite to the surface upon which the one or plurality of cells are adherent, and the user can adjust settings of the laser source to generate a laser at a predetermined point on the surface of the biocompatible membrane opposite to the cell, cells or biomaterials. In some embodiments, the system further comprises a microscope and camera in operable connection to a controller, memory, display and the laser source, the camera anmicroscope capable of creating a digital image of one or multiple fields one or both surfaces of the biocompatible membrane, such that a user may view, manipulate and adjust the laser in real-time when the system is in operation.

[0010] In some embodiments of the system, the system further comprises one or plurality of enclosure elements, the enclosure element or elements positioned over the biocompatible membrane thereby creating a compartment. In some embodiments, the compartment further comprises an inlet and an outlet on at least one side of the compartment which makes up one component of a fluid circuit. In some embodiments, the inlet and outlet are in fluid communication with a fluid circuit, such that fluid from the pump flows through the inlet and into the compartment and subsequently through the outlet into tubing and back to a tissue culture media and / or reagent reservoir, the reservoir and / or tubing operably connected to a pump capable of generating fluid flow through the fluid circuit that exposes the compartment and the biomaterials within the compartment to fluid. In some embodiments, the fluid circuit comprises a plurality of reservoirs, valves and the pump, such that a user may select a solution within a reservoir to pump across a surface of the biocompatible membrane. In some embodiments, the solution is tissue culture medium, a saline solution comprising one or more probes, or alcoholic solution that is sterile, isotonic and comprising a pH value that would not disturb the cell, plurality' of cells or the biomaterials prior to interrogation.

[0011] In some embodiments of the system, the membrane and the one or plurality of cells have a height of from about 2 microns to about 10 microns.

[0012] In some embodiments, the system further comprises a microscope operably connected to a controller, display and a camera. In some embodiments, the microscope is positioned at or proximate to the biocompatible membrane at a distance sufficient for magnification and visualization of the one or plurality of cells in a visual field.

[0013] In some embodiments of the system, the system comprises a microfluidic circuit comprising a pump, a cell culture media reservoir and heating element. In some embodiments, the pump is adjustable for generating a variable flow rate of cell culture medium through the microfluidic circuit.

[0014] In some embodiments, the system further comprises a collection vessel positioned distally from the compartment and in fluid communication with the compartment volume by a fluid conduit.

[0015] In some embodiments of the system, the vessel and fluid conduit connecting the incubation or incubator compartment, or vessel are free of air of greater than about 6% oxygen. In some embodiments, the vessel and / or incubator are free of a volume of air positioned overthe biocompatible membrane or sample, the volume of air free of greater than about 6% oxygen.

[0016] In some embodiments of the system, the system comprises a first operational mode in which light from the laser contacts the biocompatible membrane at an intersection point. In some embodiments, the electrostatic friction at the intersection point is less than about 2 nanoColumbs (nC).

[0017] In some embodiments of the system, the system is free of oil positioned at or proximate to the membrane surface or the sample surface.

[0018] In some embodiments of the system, the biocompatible membrane comprises polypropylene, PC (polycarbonate), PCL (poly(a-caprolactone)), PDMS (polydimethylsiloxane), PE (polyester), SiN (Silicon nitride). SiO2 (silicon dioxide), or a combination of a plurality thereof.

[0019] In some embodiments, the system further comprises a flow cytometer, a mass spectrometer, a digital scanner, or a combination of a plurality7thereof, operably connected to (and optionally in fluid connection with) the collection vessel or a collection compartment positioned downstream from the incubation compartment or incubation vessel.

[0020] In some embodiments of the system, the biocompatible membrane comprises one or a plurality7of UPCs, shapes or probes printed on the bottom surface at one or a plurality7of discrete positions beneath the one or plurality of cells. In some embodiments, methods of the disclosure comprise sorting biomaterials, a singe cell or a plurality of cells on the biocompatible membrane by a user associating the one or plurality' of UPCs, shapes or probes with one or a plurality' of samples; visualizing the one or plurality' of UPCs, shapes or probes and sorting the sample based upon the presence, absence or quantity of UPCs, shapes or probes. In some embodiments, the methods further comprise interrogating the biomaterials or cells on the biocompatible membrane and assigning a result to a sample based upon the presence, absence or quantity' of UPCs, shapes or probes. In some embodiments, the method further comprises positioning one or a plurality7of samples into one or more collection chambers or one or more reaction vessels within the collection chambers based upon the presence absence or quantity7of UPCs, shapes or probes prior to the step of analyzing or interrogating the samples. In some embodiments, the methods of the disclosure further comprise a step of creating a predetermined path for a laser to cut the biocompatible membrane around the one or plurality7of UPCs, shapes or probes, such that the user may associate a sample with the presence, absence or quantity7of UPCs, shapes or probes on the biocompatible membrane prior to cutting.

[0021] In some embodiments, the system further comprises a microscope operably connected to a controller, a display and a camera. The microscope is positioned at or proximate to the biocompatible membrane at a distance sufficient for magnification and visualization of the one or plurality of cells in a visual field. In some embodiments, the controller comprises a computer program product with instructions for at least one of: (a) pixelating an image of the one or plurality of cells and the biocompatible membrane; (b) determining one or a plurality’ of positions in the image around a cell or biomaterial that correspond to the pixels of the image and that define a point or series of points of a perimeter around a single cell, biomaterial or plurality of cells; (c) directing the laser to emit a pulse of light at or proximate to the point or series of points defining the perimeter, such that the biocompatible membrane is cut at the point or series of points with emission from the laser. In some embodiments, the system allows for cutting along the perimeter by a single, adjustable emission of a laser from the laser source by real-time visualization of the biocompatible membrane on the display. The system may further comprise a non-transient computer program product with instructions to capture, manipulate and direct interrogation of one or both of the real-time digitized images of the biocompatible membrane.

[0022] In some embodiments of the system, the cell or plurality of cells comprise one or more DNA barcodes on their surface. In some embodiments, methods of the disclosure comprise detecting the presence, absence or quantity of DNA barcodes are the cell surface in order to sort, characterize the cells. In some embodiments, the user may create a library’ of results or images of the cells after DNA barcode labeling of the cells, to create a library' of images and / or data (such as expression patterns of biomarkers) associated with the presence, absence or quantity of barcoding. In some embodiments, the user may correlate the presence, absence or quantity of barcoding with the presence, absence or quantity of expressed mRNA, DNA or protein in a sample. In some embodiments, the methods comprise sequencing one or a plurality of RNA or DNA molecules within a sample by' detecting or quantifying the use of a probe, such as the barcode. In some embodiments, single cell sequencing is performed in a sample with a single cell.

[0023] In some embodiments of the system, the fluid circuit is a closed system with one or more adjustable valves configured to increase or decrease the fluid flow through the microfluidic circuit and the one or plurality of disclosed compartments.

[0024] In some embodiments, the disclosure relates to a system. In some embodiments, the system comprises (a) a biocompatible membrane comprising a top surface and a bottom surface relative to a horizontal plane upon which a user operates the system; (b) a laser positionedbeneath the membrane; and (c) one or a plurality of enclosure elements positioned above the top surface of the biocompatible membrane that define a compartment and a compartment volume around the biocompatible membrane and the one or plurality of biomaterials, such as a cell. In some embodiments, the device further comprises (d) one or a plurality' of cells positioned on the top surface of the biocompatible membrane. In some embodiments, the lasers is at a distance of from about 10 millimeters to about 9,000 millimeters from the bottom surface of the biocompatible membrane.

[0025] In some embodiments, the disclosure relates to a device. In some embodiments, the device comprises (a) a biocompatible membrane comprising a first surface and a second surface; (b) a laser positioned proximate to the first surface; and (c) one or a plurality of enclosure elements positioned above the second surface of the biocompatible membrane that define a compartment and a compartment volume around the biocompatible membrane and biomaterials positioned on the second surface. In some embodiments, the device further comprises (d) one or a plurality' of cells positioned on the second surface of the biocompatible membrane. In some embodiments, the lasers is at a distance of from about 10 millimeters to about 9,000 millimeters from the first surface of the biocompatible membrane.

[0026] In some embodiments, the disclosure relates to a method of isolating a cell in a system or with a device disclosed herein. In some embodiments, the system comprises (a) a biocompatible membrane comprising a top surface and a bottom surface; (b) one or a plurality of cells positioned on the top surface of the biocompatible membrane; (c) a laser positioned beneath the bottom surface of the biocompatible membrane and at a distance of from about 10 millimeters to about 9,000 millimeters from the bottom surface of the biocompatible membrane; and (d) one or a plurality of enclosure elements positioned above the top surface of the biocompatible membrane that define an incubation compartment or vessel and a compartment volume around the biocompatible membrane and the one or plurality of cells. In some embodiments, the method comprises: (i) directing a laser from the laser source into the membrane and around a perimeter of biomaterial, such as one or a plurality of cells, such that the biocompatible membrane is cut at or around the perimeter.

[0027] In some embodiments, the method further comprises a step of (x) predetermining a set of positions on the membrane corresponding to a perimeter around a selected biomaterial and / or one or a plurality of cells. In some embodiments, step (x) is performed prior to step (i).

[0028] In some embodiments, the sy stem comprises a collection vessel positioned distally from the membrane and the compartment. In some embodiments, the collection vessel is in fluid communication with the compartment by at least one fluid conduit. The disclosure relatesto a method of analyzing a sample comprising (i) cutting a biocompatible membrane comprising a cell or plurality of cells to form a sample, (ii) allowing the sample to move to a collection vessel positioned via downstream fluid flow from the compartment in which the cutting occurs; and (iii) analyzing the biomaterial, cell or plurality of cells on the cut portion of membrane. In some embodiments, the methods comprise imaging the biomaterial, cell or the cells prior to step (i), and / or, optionally, imaging the biomaterial, cell or plurality of cells in the collection vessel during step (iii).

[0029] In some embodiments of the method, the one or plurality of enclosure elements comprise an inlet and an outlet on at least one side of the compartment, and the inlet and outlet are in fluid communication with a fluid circuit. In some embodiments, the method further comprises a step of allowing the fluid flow in the fluid circuit to carry the one or plurality of samples cut by step (i) through the outlet and into a collection vessel. In some embodiments of the method, the fluid flow rate is from about 10 microliters per second to about 50 microliters per second.

[0030] In some embodiments, the method further comprises a step of imaging the one or plurality of samples after step (i). In some embodiments of the method, the biocompatible membrane comprises one or a plurality of UPCs, barcodes, shapes, probes, or combinations of two or more thereof, printed on the bottom surface at one or a plurality7of discrete positions beneath the one or plurality of samples. In some embodiments, the method further comprises a step of detecting the one or plurality of UPCs, barcodes, shapes, probes, or combinations thereof. In some embodiments, the method further comprises a step of characterizing or isolating the biomaterial, cell, or plurality7of cells within a sample based upon the presence, absence or quantity of UPCs, barcodes, shapes, or probes, or a combination of a plurality thereof.

[0031] In some embodiments, the disclosure relates to a method of interrogating a cell or plurality of cells in a system or with a device disclosed herein. In some embodiments, the method comprises (i) directing a light beam from the laser into the membrane and around a perimeter of one or a plurality of cells.

[0032] In some embodiments, the method of interrogating a cell or plurality of cells further comprises a step (x) of predetermining a set of positions on the membrane corresponding to a perimeter around one or plurality of cells. In some embodiment, the step (x) is performed prior to step (i). In some embodiments of the method of interrogating a cell or plurality of cells, the system comprises a collection vessel positioned distally from the membrane and thecompartment, and the collection vessel is in fluid communication with the compartment by at least one fluid conduit.

[0033] In some embodiments, the method of interrogating a cell or plurality of cells further comprises a step of imaging the cells prior to step (i).

[0034] In some embodiments of the method of interrogating a cell or plurality of cells, the one or plurality of enclosure elements comprise an inlet and an outlet on at least one side of the compartment. In some embodiments, the inlet and outlet are in fluid communication with a microfluidic circuit. In some embodiments, the method of interrogating a cell or plurality of cells further comprises a step of allowing the fluid flow in the microfluidic circuit to carry the one or plurality of cells on the cut by step (i) through the outlet and into a collection vessel. In some embodiments, the fluid flow rate is from about 10 microliters per second to about 50 microliters per second, optionally in a volume of tissue culture medium.

[0035] In some embodiments, the method of interrogating a cell or plurality of cells further comprises exposing the cell to one or a plurality of antibodies or antibody fragments.

[0036] The disclosure also relates to methods of interrogating biomaterial, a single cell or plurality of cells. In some embodiments, the methods comprise growing a cell in the device disclosed herein on the biocompatible membrane disclosed herein. In some embodiments, comprise collecting one or a plurality of cells in culture and analyzing one or a plurality of UPCs, barcodes, shapes, probes, or combinations of a plurality thereof positioned on the biocompatible membrane to which the biomaterial, cell or cells are adherent. In some embodiments, the one or a plurality of UPCs, barcodes, shapes, probes, or combinations of a plurality thereof are printed on the bottom surface of the membrane at one or a plurality of discrete positions beneath the one or plurality of cells. In some embodiments, the method of interrogating a cell or plurality of cells further comprises a step of detecting the one or plurality of UPCs, barcodes, shapes, probes, or combinations thereof and optionally exposing the sample to one or a plurality' of probes in the fluid circuit before or after cutting the membrane. In some embodiments, the methods further comprise analyzing the biomaterial or position of the biomaterial relevant to a control by correlating the one or plurality of UPCs, barcodes, shapes, probes, or combinations thereof to a physical feature of the cells; and further charactenzing the biomaterial, cell or plurality of cells according to the presence, absence or quantity' of the one or plurality of UPCs, barcodes, shapes, probes, or combinations thereof. Methods may comprise identifying a cell type of identifying tissues by analyzing the presence, absence or quantity of a probes within the sample and determining a cell type by correlating the presence,absence or quantity of a probe in a sample with the the presence, absence or quantity of probe in a control.

[0037] The disclosure further relates to methods of detecting a probe corresponding to one or a plurality of physical features of collected cells. In some embodiments, the method of detecting a probe is conducted in a system or with a device herein. In some embodiments, the method comprises implementing the system as disclosed herein.

[0038] The disclosure relates to methods of labeling a cell. In some embodiments, the method of labeling a cell is conducted in a system or with a device herein. In some embodiments, the method comprises implementing the system as disclosed herein.

[0039] The disclosure relates to a method of sorting one or a plurality of cells. In some embodiments, the method of sorting one or a plurality of cells is conducted in a system or with a device herein. In some embodiments, the method comprises implementing the system as disclosed herein. The disclosure also relates to methods of microdissecting one or a plurality of cells from tissue culture. In some embodiments, the method of microdissecting one or a plurality of cells from tissue culture is conducted in a system or a device herein, wherein a laser is programmed to emit a light beam at or around a point or series of points on the bottom of a membrane opposite to he periphery of a cell or plurality of cells targeted for interrogation and / or isolation. In some embodiments, the method comprises implementing the system disclosed herein by obtaining an image of the cell or plurality of cells.BRIEF DESCRIPTION OF DRAWINGS

[0040] FIGS. 1A through IE depict embodiments of a system incorporating a laser and fluid circuit.

[0041] FIGS 2 A through 2D depict additional embodiments of a system with two chips forming a

[0042] FIG. 2E depicts an embodiment of the disclosure comprising two, etched microchips that form a microfluidic circuit configured to cut (on the left side) a biocompatible membrane from the tissue culture compartment and collect (on the right side) the cut membrane for interrogation, characterization, collection into a separate body for transportation to a second device, spectrometry, sequencing, or cell sorting. In the second chip viewed on the right, the chip comprises two collection regions, the first collection region on the left to pool multiple samples cut from the incubation compartment on the left, and the second collection region comprising several branched, yet parallel collection vessels. The inset picture of FIG. 2E is a magnified view of a single collection vessel wherein multiple samples of the biocompatiblemembrane are collected. In this collection vessel, probes such as DNA barcodes, antibodies or other reagents can be exposed to the sample for labeling, sorting and / or further molecular characterization.

[0043] FIGS. 3 A through 3C depict full system embodiments wherein the incubator is in fluid communication with a cell culture reservoir, a pump, and tubing creating a closed tissue culture system.

[0044] FIG.4 depicts a series of software steps for image capture and interrogation of samples.

[0045] FIG. 5A through 5G depict various images of cells adherent to a biocompatible membrane within a system at indicated magnifications. The images depict how dissected membranes carry cells behave in different microfluidic environments during and after the microdissection process. FIG. 5A shows that under conditions outlined herein, shapes and perimeters can be overlayed on a digital image to guide a user and cut a biocompatible membrane.

[0046] FIG. 5B depicts a similar camera image of a biocompatible membrane comprising biomaterials, and how, under typical cutting conditions, conditions not aligned with this application, the cut sample is prevented from moving away from the membrane because of a lack of sufficient fluid flow and / or electrostatic friction. FIG.5C depicts another biocompatible membrane imaged under a microscope operably connected to a laser in a system disclosed herein. The outer edges of the membrane appear dry and rolled under typical condition not aligned with the parameters of this application. Under those conditions, while cutting shapes is accomplished, the membrane is damaged under the operating conditions by the heat generated by the power of the laser. FIG. 5D depicts a similar image to 5B where electrostatic friction prevents proper movement of the sample from the membrane after cutting.

[0047] FIG. 6A through 6E depict various embodiments of an enclosure element. FOG. 6 A depicts atop view of an enclosure element. FIG. 6B depicts an isometric view of an enclosure element. FIGS. 6C and 6D depict side view of an enclosure element. FIG. 6E depicts a bottom view of an enclosure element.

[0048] FIGS. 7A and 7B depict a close-up side view of a ramp within the enclosure element and accompanying channels.

[0049] FIGS 8A and 8B depict close-up side view of an outlet and outlet channel within an enclosure element.

[0050] FIGS. 9 A through 9E illustrate a chip.

[0051] FIG. 10A illustrates a contour, micro dissected membrane piece, at the arrowhead in liquid in a collection vessel.

[0052] FIGS. 10B is an image of a chip on membrane. The membrane includes induced holes (black arrows) and FIG. 10B illustrates that liquid stays in the chamber while inlet and outlet pump are synchronized.

[0053] FIG. 11 is an image of a micro dissected membrane piece abated into the fluid within the volume of a chip.

[0054] FIG. 12 A through 12D illustrate use of a standard no treated microscope glass slide to demonstrate the successful ablation and deposition into the collection chamber. FIG. 12A shows that areas of different sizes and shapes can be ablated from the glass slide. FIG. 12C shows that shapes can be repeatedly ablated from the glass slide. FIG. 12C shows ablated tissue is deposited in liquid of a collection chamber. FIG. 12D illustrates a schematic of the system used to obtain FIGS. 12A through 12C.DETAILED DESCRIPTION OF EMBODIMENTS

[0055] Before the present systems and methods are described, it is to be understood that the present disclosure is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purposes of describing the particular versions or embodiments only, and is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the methods, devices, and materials in some embodiments are now described. All publications disclosed herein are incorporated by reference in their entireties. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior invention.Definitions

[0056] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionaryor extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0057] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0058] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of.” or. when used in the claims, “consisting of.” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0059] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%. ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. In some embodiments herein, a value or range of values prefaced with “about” includes the value or ranges of values without the modifier “about.”

[0060] The term “at least’' prior to a number or series of numbers (e.g. “at least two”) is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context. When “at least” is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range. Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.

[0061] As used herein, the term “animal” includes, but is not limited to, humans and nonhuman vertebrates such as wild animals, rodents, such as rats, ferrets, and domesticated animals, and farm animals, such as dogs, cats, horses, pigs, cows, sheep, and goats. In some embodiments, the animal is a mammal. In some embodiments, the animal is a human. In some embodiments, the animal is a non-human mammal.

[0062] As used herein, the terms “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0063] Any probes may be used in concert with any of the devices, systems, kits, or methods disclosed herein. As used herein, the term “probe” refers to any molecule that may bind or associate, indirectly or directly, covalently or non-covalently, to any of the amino acids expressed by the immune cells disclosed herein and whose association or binding is detectable using the methods disclosed herein. In some embodiments, the probe is a fluorogenic, fluorescent, or chemiluminescent probe, an antibody, or an absorbance-based probe. In some embodiments, an absorbance-based probe, for example the chromophore pNA (para- nitroanaline), may be used as a probe for detection and / or quantification of a protein within the cells adherent to the membrane disclosed herein. In some embodiments, the probe comprises an amino acid sequence that is a natural or non-natural ligand of an protein of interest and / or an analog or salt thereof, including those analogs that comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%. at least about 96%, at least about 97%, at least about 98%. or at least about 99% sequence identity to amino acids disclosed herein. A probe may be immobilized, adsorbed, or otherwise non-covalently bound to a solid surface, such that upon exposure to an enzyme for a time period sufficient to associate with one or a plurality of amino acids expressed by immune cells disclosed herein. In some embodiments, association of the antigen to the amino acid sequence causes a biological change in the nature or chemical availability of oneor more probes such that the biological change enables detection of the association event. For instance, if the step of detecting comprises use of FRET, cleavage of the amino acid compositions disclosed herein cause one of the chromophore to emit a fluorescent light under exposure to a wavelength sufficient to activate such a fluorescent molecule. The intensity, length, or amplitude of a wavelength emitted from fluorescent marker can be measured and is, in some embodiments, proportional to the presence, absence or quantity of antigen present in the reaction vessel, thereby the levels of activation can be determined from detection of the intensity of or fluorescence at a known wavelength of light.

[0064] In some embodiments, methods of the disclosure relate to methods of interrogating a cell or plurality of cells with an activity-based probe after isolation of the membrane comprising the cell or cells through dissection and subsequent collection or capture. An “activity-based probe,” as used herein, refers to a certain embodiment of probe comprising a small molecule that binds to or has affinity for a molecule such as an activated amino acid that binds in the presence of cell or cells comprising the amino acid sequence, such that its bound or unbound state confers an activity readout observable by a user. In some embodiments, the activity-based probe covalently or non-covalently binds to a protein expressed on the surface of the cell or cells disclosed herein. In some embodiments, the binding of the activity-based probe modifies the physical or biological activity of a protein of interest. In some embodiments, the activity-based probe can be fluorescent or chemiluminescent. In some embodiments, the activity-based probe has a measurable activity of one value if the enzyme is inactive and another measurable activity if in an activated state.

[0065] As used herein, the terms “fluorogenic” and “fluorescent” probe refer to any molecule (dye, quantum dot, peptide, or fluorescent marker) that emits a known and / or detectable wavelength of light upon exposure to a known wavelength of light. In some embodiments, the probes are covalently or non-covalently attached to a fluorogenic probe. In some embodiments, the attachment of the fluorogenic probe to the immune cells create a chimeric molecule or two associated molecules capable of a fluorescent emission or emissions upon exposure of the amino acid sequence a known wavelength of light, such that exposure to the wavelength of light creates a reaction product which is quantifiable in the presence of a fluorimeter. In some embodiments, light from the fluorogenic probe is fully quenched upon exposure to the known wavelength of light before association of the disclosed amino acid sequences to the fluorogenic probe emits a known wavelength of light, the intensity of which is quantifiable by absorbance readings or intensity levels in the presence of a fluorimeter. In some embodiments, the fluorogenic probe is a coumarin-based dye or rhodamine-based dyewith fluorescent emission spectra measurable or quantifiable in the presence of or exposure to a predetermined wavelength of light. In some embodiments, the fluorogenic probe comprises rhodamine. In some embodiments, the fluorogenic probe comprises rhodamine-100. Coumarin-based fluorogenic probes are known in the art, for example in US Pat Nos. 7,625,758 and 7,863,048, which are herein incorporated by reference in their entireties. In some embodiments, the fluorogenic probes are a component to, covalently bound to, non-covalently bound to, intercalated with one or a plurality of amino acid sequences disclosed herein or a protein of interest. In some embodiments, the fluorogenic probes are chosen from ACC or AMC. In some embodiments, the fluorogenic probe is a fluorescein molecule. In some embodiments, the fluorogenic probe is capable of emitting a resonance wave detectable and / or quantifiable by a fluorimeter after exposure to one or a plurality of immune cells disclosed herein. “Fluorescence microscopy,” which uses the fluorescence to generate an image, may be used to detect the presence, absence, or quantity of a fluorescent probe. In some embodiments, fluorescence microscopy comprises measuring fluorescence resonance energy' transfer (FRET) within a FRET-based assay.

[0066] A "chemiluminescent probe” refers to any molecule (dye, peptide, or chemiluminescent marker) that emits a known and / or detectable wavelength of light as the result of a chemical reaction. Chemiluminescence differs from fluorescence or phosphorescence in that the electronic excited state is the product of a chemical reaction rather than of the absorption of a photon. Non-limiting examples of chemiluminescent probes are luciferin and aequorin molecules. In some embodiments, a chemiluminescent molecule is covalently or non-covalently attached to an immune biomarker disclosed herein, such that the excited electronic state can be quantified to determine directly to the amino acid sequences disclosed. In some embodiments, an immune biomarker is CD8, CD4. CD3. CD43, CD44 or variants thereof.

[0067] The term “score” as used herein is a numerical value that may be assigned or generated after normalization of the value based upon the presence, absence, or quantity' of deposition of a probe associated with a cell in the sample of a subject in respect to the amount of . In some embodiments, the score is normalized in respect to a control data value.

[0068] As used herein, the term “stratifying” refers to sorting cells into different classes or groups based upon one or more features identified by microscopy or other observations of microdissected membrane comprising cells. For example, stratifying a population of cells with a certain pathology involves assigning, masking or otherwise characterizing the cells in animage based upon the presence, absence or quantity of physical features associated with the pathology and on the basis of the severity of the disease (e.g.. mild, moderate, advanced, etc.).

[0069] As used herein, the term ‘'subject,” “individual” or “patient,” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mammal. Cells of the disclosure can be mammalian cells, insect cells, prokaryotic cells, plant cells or combinations thereof. In some embodiments, the cells are derived from a subject prior to seeding and propagation in the systems disclosed herein.

[0070] As used herein, the term “threshold” refers to a defined value by which a normalized score can be categorized. By comparing to a preset threshold, a subject, with corresponding qualitative and / or quantitative data corresponding to a normalized score, can be classified based upon whether it is above or below the preset threshold.

[0071] As used herein, “cell culture” means growth, maintenance, transfection, or propagation of cells, tissues, or their products. As used herein, “culture medium” refers to any solution capable of sustaining the growth of the cell or cells, or any solution with which cells or exogenous nucleic acids are mixed before being applied to cells in vitro. In some embodiments, culture medium means solution capable of sustaining the grow th of adherent cells in vitro.

[0072] Any probes may be used in concert with any of the devices, systems, kits, or methods disclosed herein. As used herein, the term “probe” refers to any molecule that may bind or associate, indirectly or directly, covalently or non-covalently, to any of the biomaterial present in a sample disclosed herein and whose association or binding is detectable using the methods disclosed herein. In some embodiments, the probe is a fluorogenic, fluorescent, or chemiluminescent probe, an antibody, or an absorbance-based probe. In some embodiments, an absorbance-based probe, for example the chromophore pNA (para-nitroanaline), may be used as a probe for detection and / or quantification of a protein or molecule of interest. In some embodiments, the probe comprises an amino acid sequence that is a natural or non-natural ligand of a cell or plurality of cells disclosed herein and / or an analog or salt thereof, including those analogs that comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to amino acids indicated in the Table 1 below. A probe may be immobilized, adsorbed, or otherwise non- covalently bound to a solid surface, such that upon exposure to a biomaterial disclosed hereinfor a time period sufficient to associate with one or a plurality of nucleic acid sequence or amino acid sequence expressed by the cell or plurality of cells disclosed herein. In some embodiments, association of the a probe to the biomaterial causes a biological change in the nature or chemical availability of one or more probes such that the biological change enables detection of the association event. For instance, if the step of detecting comprises use of FRET, cleavage of the amino acid compositions disclosed herein cause one of the chromophore to emit a fluorescent light under exposure to a wavelength sufficient to activate such a fluorescent molecule. The intensity, length, or amplitude of a wavelength emitted from fluorescent marker can be measured and is, in some embodiments, proportional to the presence, absence or quantity of antigen present in the reaction vessel, thereby the levels of activation can be determined from detection of the intensity of or fluorescence at a known wavelength of light.

[0073] An ’‘activity-based probe,” as used herein, refers to a certain embodiment of probe comprising a small molecule that binds to or has affinity for biomaterial disclosed herein such as an amino acid that binds a cell or plurality of cells, or biomaterial disclosed herein in the presence of such probe, such that its bound or unbound state confers an activity’ readout observable by a user. In some embodiments, the activity-based probe covalently or non- covalently binds to a toxin disclosed herein or derivative herein. In some embodiments, the binding of the activity' -based probe modifies the physical or biological activity' of the toxin. In some embodiments, the activity-based probe can be fluorescent or chemiluminescent. In some embodiments, the activity-based probe has a measurable activity of one value if the enzyme is inactive and another measurable activity7if in an activated state.

[0074] As used herein, the terms “fluorogenic” and “fluorescent” probe refer to any molecule (dye, quantum dot, peptide, or fluorescent marker) that emits a known and / or detectable wavelength of light upon exposure to a known wavelength of light. In some embodiments, the probes are covalently or non-covalently attached to a fluorogenic probe. In some embodiments, the attachment of the fluorogenic probe to the one or plurality of cells disclosed herein create a chimeric molecule or two associated molecules capable of a fluorescent emission or emissions upon exposure of the amino acid sequence a known wavelength of light, such that exposure to the wavelength of light creates a reaction product which is quantifiable in the presence of a fluorimeter. In some embodiments, light from the fluorogenic probe is fully quenched upon exposure to the know n wavelength of light before association of the disclosed amino acid sequences to the fluorogenic probe emits a known wavelength of light, the intensity of which is quantifiable by absorbance readings or intensity levels in the presence of a fluorimeter. In some embodiments, the fluorogenic probe is acoumarin-based dye or rhodamine-based dye with fluorescent emission spectra measurable or quantifiable in the presence of or exposure to a predetermined wavelength of light. In some embodiments, the fluorogenic probe comprises rhodamine. In some embodiments, the fluorogenic probe comprises rhodamine- 100. Coumarin-based fluorogenic probes are known in the art, for example in US Pat Nos. 7,625,758 and 7,863,048, which are herein incorporated by reference in their entireties. In some embodiments, the fluorogenic probes are a component to, covalently bound to, non-covalently bound to, intercalated with one or a plurality of amino acid sequences or toxins disclosed herein. In some embodiments, the fluorogenic probes are chosen from ACC or AMC. In some embodiments, the fluorogenic probe is a fluorescein molecule. In some embodiments, the fluorogenic probe is capable of emitting a resonance wave detectable and / or quantifiable by a fluorimeter after exposure to one or a plurality’ of immune cells disclosed herein. “Fluorescence microscopy,” which uses the fluorescence to generate an image, may be used to detect the presence, absence, or quantity’ of a fluorescent probe. In some embodiments, fluorescence microscopy comprises measuring fluorescence resonance energy transfer (FRET) within a FRET-based assay.

[0075] A "chemiluminescent probe” refers to any molecule (dye, peptide, or chemiluminescent marker) that emits a known and / or detectable wavelength of light as the result of a chemical reaction. Chemiluminescence differs from fluorescence or phosphorescence in that the electronic excited state is the product of a chemical reaction rather than of the absorption of a photon. Non-limiting examples of chemiluminescent probes are luciferin and aequorin molecules. In some embodiments, a chemiluminescent molecule is covalently or non-covalently attached to an immune biomarker disclosed herein, such that the excited electronic state can be quantified to determine directly to the amino acid sequences disclosed.

[0076] As used herein, “conservative” amino acid substitutions may be defined as set out in Tables A, B, or C below; Protein or biomaterial interrogated in this disclosure may comprise amino acid sequences. Amino acid sequences disclosed herein may include those amino acid sequences that include or a plurality of conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.Table A - Conservative Substitutions ISide Chain Characteristics Amino Acid AliphaticNon-polar GA P I LV FPolar - uncharged CSTMNQPolar - charged D E K RAromatic H F WYOther NQDE

[0077] Alternatively, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table B.Table B — Conservative Substitutions IISide Chain Characteristic Amino AcidNon-polar (hydrophobic)Aliphatic: AL I V P.Aromatic: F W YSulfur-containing: MBorderline: GYUncharged-polarHydroxyl: S TYAmides: N QSulfhydryl: CBorderline: GYPositively Charged (Basic): K R HNegatively Charged (Acidic): D E

[0078] Alternately, exemplary conservative substitutions are set out in Table C.Table C - Conservative Substitutions IIIOriginal Residue Exemplary SubstitutionAla (A) Vai Leu He MetArg (R) Lys HisAsn (N) GinAsp (D) GluCys (C) Ser Thr

[0079] As used herein, the term ‘'sample” refers generally to a limited quantity of something selected which is intended to be similar to and represent a larger amount of that selection. In the present disclosure, a sample is a collection of cells and, in some embodiments, adherent to the biocompatible membrane. In some embodiments, the cells are derived from subject fluid, whole blood, plasma, swab, particular isolated cell type (such as peripheral blood mononuclear cells (PBMCs)) brushing, scraping, biopsy, fixed cells, adherent cells, a single adherent cells, removed tissue, surgical resection, or formalin fixed, and paraffin embedded (FFPE), or archival biomaterials that is to be tested. In some embodiments, the sample is a cell or plurality of cells adherent to a biocompatible membrane wherein the cell or plurality of cells exhibit known or control physical features and / or phenotype. As used herein, '‘control sample” or '‘reference sample” refer to samples with a known presence, absence, or quantity of biomaterial being measured, that is used for comparison against an experimental sample. In some embodiments, the sample may be a tissue sample from a mammal, a human, or a preexisting cell hne. In some embodiments, the subject is exhibiting a symptom of a particular disorder or suspected of having a disorder with a dysfunction of cellular phenotype.

[0080] In some embodiments, as will be clear from context, the term “sample” refers to a preparation of biomaterial, including cells and / or cellular matrix, that is obtained by dissecting (e.g.. by removing one or more components of and / or by adding one or more agents to) a cell culture growing adherently on a biocompatible membrane. In some embodiments, the sample is a homogenous population of cells in tissue culture growing on the system. In some embodiments, the sample is a proteinaceous deposit on a biocompatible membrane. In some embodiments, the sample is processed prior to, simultaneously with, or subsequent to dissection or cutting of the membrane by a laser disclosed herein. In some embodiments, the sample comprises a heterogenous mixture of multiple cell types. In some embodiments, the sample is a portion of a biocompatible membrane comprising cells adherent to one side of the membrane. In some embodiments, the sample comprises a portion of the biocompatible membrane and a single cell or plurality of cells in solution. In such embodiments, the sample may be a “processed sample,” which comprises fixed cells to the biocompatible membrane or non-adherent cells and the biocompatible membrane. For example, methods of the disclosure comprise dissecting or cutting the biocompatible membrane but, before analysis of the sample is performed, the sample the cell and / or proteinaceous biomaterial is chemically treated to become removed or fixed to the membrane for preparation of further interrogation steps. Insome embodiments, the methods disclosed herein do not comprise a processed sample. In some embodiments, the sample is a single cell or plurality of cells bound to a biocompatible membrane in a predetermined shape. In some embodiments, the predetermined shape is predetermined by a user operating the laser to cut a certain shape around the cells, and then assigning a physical feature of the cell to the shape, such that collection and / or analysis of the sample is facilitated and organized by the shape. In some embodiments, the sample comprises a square, semi- circular or circular shape, polygonal, elliptical or semi-elliptical in shape. In some embodiments, systems and device comprise a first set of samples comprising a first shape of membrane upon which one or a plurality of cells comprising a first physical feature are adhere, a second set of samples comprising a second shape of membrane upon which one or a plurality of cells comprising a second physical feature adhere.

[0081] '‘Solid support” refers to any solid (flexible or rigid) substrate onto which it is desired to immobilize one or more biocompatible membranes or onto which it is desired to isolate or interrogate a sample cut form the membrane. The substrate may be biological, non- biological, organic, inorganic or a combination thereof, and may be in the form of particles, strands, precipitates, gels, sheets, tubings, spheres, containers, capillaries, prisms, pads, slices, films, plates, slides, inserts, etc, having any convenient shape, including disc, sphere, circle, etc. In some embodiments, the solid support is a slide, such as a silicon or glass slide that is configured to be mounted on a microscope stand. In some embodiments, the solid support comprises a first and second component, wherein the first component is a slide and the second component is a chip, insert or cartridge, whereby the first and second components are configured for attachment to each other and form a compartment having a volume. In some embodiments, the compartment forms a compartment into which a first set of tubing is attached to form a closed component of a disclosed fluid circuit.Device or System

[0082] The disclosure relates to a device and system, the device or system comprising at least a first tissue culture incubation chamber operably linked and in fluid communication with a fluid circuit comprising a cell culture medium reservoir, a pump, and a collection chamber; wherein the tissue culture incubation chamber (or interchangeably incubation compartment) is in fluid communication with the collection chamber through one or a plurality of apertures, conduits and / or openings (such as an outlet) positioned within the fluid circuit. The disclosure relates to a system comprising: (i) at least a first tissue culture incubation chamber operably linked and in fluid communication with a fluid circuit comprising a cell culture mediumreservoir, a pump, and a collection chamber; (ii) a microscope comprising a solid support (such as a stand) upon which the first tissue culture incubation chamber is positioned, wherein the microscope is operably linked to a camera configured to image one or a plurality of cells growing in the first tissue culture incubation chamber on a membrane; and (iii) a laser source operably linked to a power source, controller and processor. In some embodiments the laser source comprises an aperture capable of emitting a laser beam positioned at or proximate to the bottom of the solid support, such that, in a first operable mode, laser light emitted from the aperture passes through the solid support and onto a side of the membrane opposite to the side of the membrane comprising biomaterials, such as cells. In some embodiments, the laser aperture is positioned within about 2 mm to about 12,000 mm from the membrane in the first tissue culture incubation chamber. In some embodiments, the first tissue culture incubation chamber is defined by the solid support acting as a bottom side of a compartment (parallel to the solid support) and an enclosure element comprising the sidewalls and top side of the compartment. The compartment acting as the first tissue culture incubation chamber comprises a membrane upon which cells grow. In a first operative mode, laser light from the aperture is directed at or around a point or series of points around one or a plurality of cells growing on the membrane resulting in a microdissected membrane comprising biomaterial or the one or plurality of cells. In a second operative mode, fluid flows across, under and / or above the surface of the membrane transfers the microdissected membrane to a collection chamber adjacent to and in fluid communication with the compartment. In a third operative mode, a user can interrogate the one or plurality of cells on the microdissected membrane in the collection chamber. Interrogation can include one or a combination of: imaging the biomaterial or one or plurality of cells, labeling the biomaterial or one or plurality of cells, classifying / stratifying the biomaterial or one or plurality of cells, and / or exposing the biomaterial or cells to mass spectrometry, and / or exposing the biomaterial or cells to cell sorting.

[0083] The disclosure also relates to a system comprising a device for microdissection of tissue or cells within a tissue culture environment and / or a fixed tissue environment, wherein the device comprises a first tissue culture incubation chamber comprising a tissue culture growth region and an interrogation or collection region. The disclosure also relates to a system comprising a cell culture region comprising one or a plurality of cell reactor surfaces housed within one or a plurality of compartments (or, interchangeably, a first tissue culture incubation chamber). In some embodiments, the cell culture region comprises a first compartment comprising at least one cell reactor surface, the at least one cell reactor surface in fluid connection with a first and second media line, the first media line in fluid communication witha first media inlet, the second media line in fluid communication to a first media outlet. In some embodiments, the cell culture unit comprises a single cell culture chamber comprising multiple partitions, each partition independently removable and independently in fluid connection with the first and the second media line and each partition or set of partitions defining a distinct compartment. In some embodiments, the cell culture region comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more compartments, each compartment separated by and / or defined by one or more partitions. In some embodiments, the compartments are configured in a grid or linear patern. In some embodiments, the compartments are configured to grow a cell or cells on a flat or substantially horizontal surface. In some embodiments, each partition separating one compartment from another compartment may be removed such that the cell reactor surface of a first compartment is or becomes contiguous with a cell reactor surface of a second compartment. The removal of one or more partitions allows for an increased surface area onto which cells from one compartment (such as the first compartment) may proliferate and / or grow into another compartment (such as the second compartment) during a method of culturing. In some embodiments, the cell culture region comprises a set of side walls defining a single surface area divided among 2, 3. 4, 5. 6, 7. 8, 9, 10, 11, 12 or more compartments each compartment with at least one or a plurality of cell reactor surfaces, each surface comprising one or a plurality of biocompatible membranes onto which a cell or cells are positioned. In some embodiments, each compartment comprises at least a first cell reactor surface onto which a membrane is positioned. In some embodiments one or a plurality of cells adheres to the membrane.

[0084] In some embodiments, the system comprises a device comprising a first compartment comprising one or a plurality of cell reactor surfaces, each cell reactor surface comprising a membrane upon which adherent cells grow. In some embodiments, the system further comprises a laser positioned proximate to or near the first reactor surface such that the path of light emited from the laser is orthogonal to or substantially orthogonal to the membrane. In a first operable mode, the laser source emits laser light at a wavelength and a frequency from a distance to the membrane sufficient to cut a region of the membrane. In some embodiments, in a first operable mode, the laser source emits laser light at a wavelength and a frequency from a distance to the membrane sufficient to cut a region of the membrane submerged in solution at an intersection point or points free of static electricity of no more than about 2, 3, 4, or 5 nC. In some embodiments, the region of the membrane cut is predetermined by a user operating a non-transient computer program product operably connected to a controller and processor within the system and electrically connected to the laser source orwithin the laser source, the computer program product comprising instructions for: determining settings for the intensity, frequency, wavelength, position and duration of light beam emission. In some embodiments, the system further comprises a camera operably linked to a microscope for capture of digital images of one or a plurality of cells adherent to the membrane. In some embodiments, the computer program product comprises instructions for pixelating a captured image of the membrane, defining a mask around the image, generating a real-time predetermined path for a laser and operating the laser source in order to cut the membrane at or proximate to the predetermined path. In some embodiments, the laser source is positioned from about 5 to about 1500 mm from the cell reactor surface or the first tissue culture compartment. In some embodiments, a cartridge, chip or slide is positioned on the opposite side of the cell reactor surface or membrane of the system relative to the position of the laser source, the cartridge, chip or slide forming a closed fluid circuit on one face of the membrane or glass surface comprising the cell reactor surface, when positioned adjacent to or proximate to the membrane and / or solid support,

[0085] The disclosure also relates to a system configured to image cells in culture or fixed on a slide, coverslip, or biocompatible membrane; and operate a laser source within the system to produce a laser and cut a region of the membrane from intact membrane or ablate in culture from the slide or coverslip; in either case, within a compartment positioned on a cell reactor surface upon which a cell or plurality of cells grow or are positioned in a fixed state. In some embodiments, the system comprises a display, controller, camera, microscope and laser source which is configured to select at least about two or more of a plurality of functionalities of a laser source and microscope based upon user preferences for microdissection, collection and capture of one or more cells and / or biomaterials positioned on the region of the biocompatible membrane. The two or more functionalities are shown in relation to a visual representation of the two or more input modalities on the display. In some embodiments, the system is configured to provide a display signal to a display device operably connected to the microscope with the system. The display signal comprises a visual overlay of a field of view controlled by the user. In some embodiments, the display signal comprises a signal sufficient to create a digital image of a field of view comprising a portion of the cell reactor surface comprising a cell culture and / or biomaterials encompassing a target area on the membrane or slide. In some embodiments, the display signal further comprises a digital graphics option that makes up a visual overlay for the digital image of the field of view corresponding to a location or series of locations assigned to segmented image and capable of being aligned to the actual physical features on or within the target area.

[0086] Various embodiments of the present disclosure further provide a system comprising, a tissue culture chamber within the compartment, a microscope, and a laser source operably linked by a controller, processor and a memory. In some embodiments, the system further comprises one or more input devices for the microscope e.g. a foot pedal and / or handles, a display device, and a microscope comprising a solid support or stand upon which the tissue culture chamber is positioned. The system may comprise one or more additional, optional features, such as a base unit (that comprises the system), a microphone (for a voice recognitionbased control mechanism), a first adjustable arm upon which the microscope is attached, and a second adjustable arm operably linked to a controller an computer program product with executable instructions for repositioning one or a plurality of collected samples from a collection vessel to a nearby location for analytical processing. In some embodiments, the system further comprises one or more display devices. For example, the microscope may comprise ocular eyepieces through which a user may view or adjust the field of view and / or the magnification of the field of view, the field of view enabling proper alignment of the laser from the laser source to a spot or perimeter around a sample on the slide, coverslip or biocompatible membrane.

[0087] Embodiments of the present disclosure relate to a system, a method and a computer program product that are suitable for a laser dissection of a membrane comprising one or a plurality of cells. In some embodiments, the microscope comprises a lighting system and / or an auxiliary display. In some embodiments, the microscope comprises one or more optical magnification components that are used to magnify a view of a sample. In modem microscopes, the optical magnification is often provided for a camera or an imaging sensor of the microscope.

[0088] There are a variety of different types of microscopes. One non-limiting example is disclosed in https: / / www.leica-microsystems.eom / products / light-microscopes / p / leica-lmd7, the contents of which are incorporated by reference in its entirety.

[0089] In some embodiments, the microscope comprises a base to stabilize the microscope in place, a stage to position a biocompatible membrane, slide or coverslip in place, and one or more partitions that define an interior space around a cell reactor surface and an exterior space distal from the position of the sample and stage. One or more of the partitions comprise an interior portion, a frame portion and an exterior portion. The interior portion of the partition is positioned in the closed portion of the system; the frame portion spans a wall of the culture system separating the interior of the culture system to the exterior of the system; and the exterior portion is positioned outside of the system. In some embodiments, a seal operably fits around the frame portion of one or more of the partitions such that removal of the partitiondoes not introduce pathogens to and / or does not expose the environment outside of the tissue culture system to the interior of the tissue culture system. In some embodiments, an underside of a cell reaction surface is sealed by positioning of a cartridge disclosed herein or, interchangeably, a “chip” that is adjacently positioned on the underside of the cell reaction surface, the mating of the chip and the sample surface provide a volume through which fluid communication can occur in a fluid circuit. The setup allows for imaging and directional cutting or ablating of biomaterials on one side of a slide, covershp or biocompatible membrane positioned on the stage or platform, and it allows for collection of the ablated biomaterial or cut membrane in the fluid circuit on the opposite side of the slide, coverslip or biocompatible membrane. In some embodiments, fixed tissue, such as from a subject, is provided on a glass slide or coverslip. In still other embodiments, biomaterials (including cells or molecules absorbed to a membrane) are not fixed but submerged in a solution such as a cell culture media on a biocompatible membrane clipped to the slide of a stage and / or partitions facing the side of the system available to the microscope (imaging device) and the laser source. In some embodiments, the biocompatible membrane is positioned within a compartment positioned between the cartridge or chip and the laser source. The region of the compartment upon which biomaterial is positioned is named a cell reactor surface. In some embodiments, living cells at this position can be exposed to fixing agents to fix the biomatierals to the membrane or slide. In other embodiments, the biomaterials may be exposed to cell culture fluid to maintain its biologically active state. In some embodiments, pre-fixed slides may be positioned at the cell reactor surface.

[0090] In some embodiments, the biomaterials comprise one or a plurality of cells, and, in some embodiments, cell density of each compartment within the cell reactor region is from about 0.1 to about 10 million cells per mL of fluid (e.g. cell culture media, if the cells are unfixed). In some embodiments, the cell density of each compartment is from about 0.1 to about 10 million cells per mL of fluid. In some embodiments, the cell density' of each compartment is from about 0.5 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 1.0 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 2 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 3 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 4 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 5 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 6 to about 10 millioncells per mL of fluid. In some embodiments, the cell density of each compartment is from about 7 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 8 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 9 to about 10 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 0.1 to about 20 million cells per mL of fluid. In some embodiments, the cell density of each compartment is from about 0. 1 to about 50 million cells per mL of fluid.

[0091] In some embodiments, the systems disclosed herein comprise a cell density of from about 0.01 million to about 10 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.03 million to about 5 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.07 million to about 5 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.03 million to about 5 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.001 million to about 5 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.002 million to about 4 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.003 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.004 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density7of from about 0.005 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.006 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.007 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.001 million to about 4 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.001 million to about 3 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.003 million to about 3 million cells per square centimeter of surface area of cell reactor surface.

[0092] The disclosure relates to a system comprising a cell reactor comprising a cell reactor surface positioned within a fluid circuit, the fluid circuit comprising a pump, cell culture medium reservoir and a first length of tubing in fluid communication with the cell reactor surface carrying cell culture medium from the cell culture reservoir to the cell reactor surface. In some embodiments, the system comprises a second length of tubing in fluid connection with the cell reactor surface carrying cell culture medium from the cell reactor surface to the cell culture medium reservoir. In some embodiments, the fluid flow is achieved by the pump which generates fluid pressure within the fluid circuit and a flow rate of tissue culture medium through the fluid circuit. In some embodiments, the pump is operably electrically connected to a controller and a display that allows a user to set the pump speed and thus set a predetermined fluid flow per second across the cell reactor surface. In some embodiments, the display displays the approximate value of the fluid flow rate across the cell reactor surface. In some embodiments, the reservoir comprises multiple volumes of solution each independently linked in fluid communication to the cell reactor by a series of valves and tubing, such that each volume may comprises a different solution comprising tissue culture medium or other reagents desirable to culture biomaterials within the cell reactor, label biomaterials within the cell reactor and / or stain or fix biomaterials within the cell reactor to the biocompatible membrane prior to cutting.

[0093] One embodiment of the system is depicted in FIG. 1 A. FIG. 1 A depicts a plurality of cells (labeled as “tissue7’) growing on a side of a biocompatible membrane proximate to an enclosure element (e g. a chip) that defines a compartment, the compartment comprising a volume within which the plurality7of cells grows in culture. The left panel of FIG. 1 A illustrates a first set of tubing depicted on the left side of the chip extends from a culture medium source and pump (not depicted) into the compartment, and a second set of tubing extends from the compartment and into a fluid circuit (the whole fluid circuit not depicted).The first and second sets of tubing carry tissue culture medium from one side of the compartment to the other, generating a flow rate of the culture medium across the side of the biocompatible membrane upon which the plurality7of cells grow. The flow of cell culture medium not only provides nutrients and growth factors to the plurality cells, but also creates a flow rate of fluid on one surface of the biocompatible membrane, such that any7biocompatible membrane cut in a shape by the laser can be carried by the fluid flow out of the compartment. The first set and second set of tubing would be present in the view of the right panel of FIG. 1A but are not illustrated for sake of clarity. In this embodiment, a solid support, depicted as a silicon slide, provides support for the biocompatible membrane to rest. The solid support also provides support forthe biocompatible membrane to form a sixth side of the compartment to define the interior volume. A laser source (not depicted) generates a laser beam directed through the solid support and at a point on the biocompatible membrane opposite to the side of the biocompatible membrane upon which the plurality of cells grows. The Figure depicts dotted lines in an irregular shape, the shape representing the perimeter around a target area on the membrane within which a user is interested to magnify, interrogate and / or examine further. In some embodiments, the user performs disclosed methods to label or to visualize protein expression, quantify RNA or protein expression patterns, count cells or subcellular organelles, or examine extracellular contents with the target area. The laser is directed to the perimeter of the irregular shape and is mounted on a movable element (not depicted) in order to trace a path around the perimeter. The right panel of FIG. 1 A depicts the piece of cut membrane comprising the target area falling into the cell culture media within the compartment. Any resultant cut biocompatible membrane is capable of falling into the fluid flow within the fluid circuit positioned underneath the biocompatible membrane and within the cartridge or chip to carry the cut membrane to a point within the fluid circuit distal to the compartment. In the alternative, after a cutting step, the irregular shape of biocompatible membrane may fall into the compartment, flow rate can be decreased to a flow rate to trap the shape in the compartment and manually obtained by opening the fluid circuit and removing the membrane from the compartment. Examination or interrogation of the cut membrane target area can be completed by transferring the cut membrane to another slide or another location within the fluid circuit. In some embodiments, the cut membrane can be transported via fluid flow in the fluid circuit to a collection region of the system comprising a volume adjacent to a syringe plunger, then deposited by depression of a syringe (either manually or mechanically through a pump) into a test tube, plate or other vessel enabling interrogation by one or more analytical processes. In some embodiments, fluid volume in the fluid circuit can be directionally shunted to one or more secondary or auxiliary collection vessels, such as a well within a 96-well plate. The method of cutting a membrane or ablating biomaterials from a biocompatible membrane can be repeated serially such that multiple samples from the same biocompatible membrane can be collected and placed in adjacent vessels of the 96 well plate, enabling independent interrogation of multiple samples of biomaterials from biocompatible membrane. Additionally, because a user of the system may select certain biomaterials from certain regions within the biocompatible membrane, interrogation results can be correlated to spatial positioning of the biomaterials within an original sample. This enables microdissection of multiple areas and rich data acquisition of multiple regions of fixed or live tissue. In one non-limiting example, massspectrometry can be performed on one or a plurality of cells and that mass spectrometry data can be correlated to spatial positioning of the cells within regions or subregions of the biocompatible membrane or slide. Because cell morphologies are known, known cell types or even single cells, can be isolated collected and interrogated such that datasets for individual cells or cell populations can be catalogued and, if desired, correlated to spatial position within a sample.

[0094] FIG. IB depicts a similar embodiment of the system depicted in FIG. 1A. A solid support glass slide 15 is positioned laterally between a laser source and a biocompatible membrane 14. The glass slide may be held in place by a stand. A portion of the biocompatible membrane 14 is depicted as transparent to conveniently demonstrate growth of a plurality of cells 12 positioned on the bottom side of the membrane. A boxed-shape microchip 13 with a hollow interior rests on the bottom side of the membrane 14, such that the cell-side of the membrane on the slide 15, and the sides of the microchip define interior sidewalls of a compartment. The interior of the compartment defines a volume into w hich cell culture medium is pumped from one side of the compartment to the other side of the compartment. FIG. IB depicts that the compartment is part of a closed fluid circuit comprising a first set of tubing 16 carrying cell culture medium into the compartment and a second set of tubing 17 carrying cell culture medium from the compartment and into the fluid circuit. In the depicted embodiment, both the first and second set of tubing is positioned on the side of the solid support A laser beam 10 emitted from a laser source is mounted on a movable element and allows the laser source to move in a clockwise or counterclockwise direction and / or in any position within a predetermined plane, such that the beam can create a series of contact points that cut the biocompatible membrane in any predetermined path. In FIG. IB, the path of the laser is predetermined by software instruction to create a perimeter (delineated by a dashed lime overlayed) defining a target area on the biocompatible membrane.

[0095] FIG. 1C depicts another embodiment of the disclosed system. A solid support glass slide 105 is positioned laterally betw een a laser source and a biocompatible membrane 104. A portion of the biocompatible membrane 104 is depicted as transparent to conveniently demonstrate growth of a plurality of cells 102 positioned on the bottom side of the membrane. A hollow boxed-shape chip without one side proximate to the cells 103 with a hollow interior rests on the bottom side of the membrane 104, such that the cell-side of the membrane on the slide 105, and the sides of the microchip define interior sidewalls of a compartment. The interior of the compartment defines a volume into which cell culture medium is pumped from one side of the compartment to the other side of the compartment. FIG. 1C depicts a fluid flowproceeding from the left to right of the system, but the embodiment depicts a first set of tubing 106 carrying cell culture medium into the compartment and a second set of tubing 107 carrying cell culture medium from the compartment and into the fluid circuit. In the depicted embodiment, both the first and second set of tubing is positioned on the side of the solid support closest to the laser source. The system may comprise one or more gaskets and / or seals wrapped around the first and / or second set of tubing, such that the tubing is a component in the closed fluid circuit providing an inlet at the interior of the compartment in respect to the first set of tubing and an outlet at the interior on the opposite side of the compartment and marking the hollow interior of the second set of tubing. A laser beam 101 emitted from a laser source is mounted on a movable element and allows the laser source to move in a clockwise or counterclockwise direction and / or in any position within a predetermined 180 degree plane, such that the beam can create a series of contact points that cut the biocompatible membrane in any predetermined path. In FIG. 1C, the path of the laser is predetermined by software instructions creating a perimeter (delineated by a dashed lime overlayed on the biocompatible membrane) defining a target area on the biocompatible membrane on a digital image captured by a camera mounted on the micrscope.

[0096] FIG. ID depicts a lateral view of the stacked system embodiment from FIG. IB. a glass slide is positioned horizontally or substantially horizontally relative to the ground of a user using the system. The biocompatible membrane 104 is positioned adjacent to and in contact with the glass slide that acts as a solid support. A box-shaped chip 103 is positioned underneath the biocompatible membrane 104, such that the glass slide and the chip form a compartment comprising a volume of space and a side of the biocompatible membrane upon which biomolecule and / or cells may be positioned. The volume within the compartment is in fluid communication with a fluid circuit (not shown), and a first set of tubing 105 is designed to bring fluid from the circuit into the compartment and a second set of tubing 106 is positioned at or proximate to the opposite side of the compartment, carrying fluid away from the compartment in the fluid circuit. An inlet at the intersection of the first set of tubing 105 with the compartment and an outlet positioned at the hollowed part of the second set of tubing 106 begins at the create lateral fluid flow over the biocompatible membrane 104 comprising one or a plurality of biomolecules. A fluid pump in fluid communication ith the compartment and the first and second set of tubing 105, 106 can be adjustable so that fluid flow rates can be controlled over the surface of the one or plurality of biomolecules. A laser source positioned above the biocompatible membrane 104 emits a laser 101 in a direction toward the membrane and around a predetermined path selected by the user. Movement of the laser 101 is depictedby the arrowhead. The laser 101 cuts out a portion of the biocompatible membrane 112 thereby exposing the cut portion or sample to the fluid flow underneath the membrane. The sample 112 can thereafter be carried in the fluid flow to another portion of the compartment or downstream to another component of the fluid circuit. In this embodiment, the chip 103 has ninety or approximately 90 degree square comers at either end of the underside comers.

[0097] FIG. IE depicts a lateral view of the stacked system embodiment from FIG. 1C. In some embodiments, a glass slide is positioned horizontally or substantially horizontally relative to the ground of a user using the system. The biocompatible membrane 104 is positioned adjacent to and in contact with the glass slide that acts as a solid support. A box-shaped chip with a hollow interior 103 is positioned underneath the biocompatible membrane 104, such that the glass slide and the chip form a compartment comprising a volume of space defined by a side of the biocompatible membrane upon which biomolecule and / or cells may be positioned. The volume within the compartment is in fluid communication with a fluid circuit (not shown), and a first set of tubing 105 is designed to bring fluid from the circuit into the compartment and a second set of tubing 106 is positioned at or proximate to the opposite side of the compartment, carrying fluid away from the compartment in the fluid circuit. An inlet at the intersection of the first set of tubing 105 with the compartment and an outlet positioned at the intersection of the second set of tubing 106 and the compartment create lateral fluid flow over the biocompatible membrane 104 comprising one or a plurality of biomolecules and / or cells. A fluid pump in fluid communication with the compartment and the first and second set of tubing 105, 106 can be adjustable so that fluid flow rates can be controlled over the surface of the one or plurality of biomolecules. A laser source positioned above the biocompatible membrane 104 emits a laser 101 in a direction toward the membrane and around a predetermined path selected by the user. Movement of the laser source 101 is depicted by the arrowhead. The laser 101 cuts out a portion of the biocompatible membrane 102 thereby exposing the cut portion or sample to the fluid flow underneath the membrane. The sample 102 can thereafter be carried in the fluid flow to another portion of the compartment or downstream to another component of the fluid circuit. In this embodiment, the chip 103 has ninety or approximately 90 degree square comers on its outside surface, however, the inside of the chip has stepped interior sidewalls that create two stacked rectangular prism shapes within the compartment. The first set and second set of tubing 105, 106 are positioned at the lateral -most portion of the compartment volume and the tubing extend upward through the glass slide. To create a closed fluid circuit, these embodiments comprise a seal or gasket positioned aroundthe first and second set of tubing at its contact point with the slide and membrane. In some embodiments, the seal or gasket comprises rubber.

[0098] FIG. 2B represents another embodiment of the disclosure. In FIG. 2B, a top view of a chip is depicted to show various microfluidic chambers within the device that are components of a microfluidic circuit. The disclosure relates to a chip or insert (the term “insert” used interchangeably with “chip” or “enclosure element”) that is configured for installation within a system for cutting a membrane comprising one or a plurality of biomolecules and / or cells. In some embodiments, the system comprises a chip such as the chip of FIG. 2B. comprising plastic, metal, or a combination of plastic and metal, wherein the chip or insert comprises a contiguous exterior surface and a hollow- interior that makes up all but one sidewall of a compartment into which a sample is collected.

[0099] The chip is intended to be brought into optical communication within a system comprising a solid support contacting the biocompatible membrane and fluid communication with a fluid circuit, the solid support and the chip forming a compartment containing a volume having suitable optical properties, for instance a volume of cell culture medium having a refractive index such as to minimize reflection losses at the interfaces. It is advantageous however, to employ a transparent elastic material, such as a suitable silicone rubber, having a matching refractive index, because this will eliminate the necessity7of handling oils which have to be wiped off every time a chip is replaced in the system. It is desirable to couple the chip to the optical system with the aid of a so-called “optointerface.” In some embodiments, the optointerface consists of a thin glass slide having at least one of its faces coated with the transparent elastic material, e.g., in the form of strings of square cross section. With this embodiment, air inclusions are avoided when the optointerface is mounted in contacting position with the optical unit and chip, respectively. Each string, of has a projection area covering the corresponding chip. In some embodiments, each elastic material piece forms a coating all over its underlying surface but has thickened portions at the positions of each of the corresponding chip exterior surfaces. To facilitate handling, the glass slide with its elastic material pieces thereon may suitably be mounted in a holder or stage, the holder when depressed to the chip during installation forms the compartment having an interior volume adjacent to the biocompatible membrane. In alternative embodiments, the optinterface comprises a seal or gasket between the glass slide and the chip, such that, upon installation of the removable and / or disposable chip, a clamp or other device suitable for holding the optointerface stationary while cutting and manipulation of the sample compresses the chip andthe slide such that the seal or gasket around the perimeter creates a closed fluid circuit in fluid communication with the rest of the fluid circuit.

[0100] In some embodiments, the chip, once installed, should be physically coupled, and fluidly sealed, to a block unit positioned within or on the chip compartment for liquid handling in such a manner that reagent and sample solutions in individual flow cells are caused to pass over the biocompatible membrane in either in series or in parallel. In some embodiments, a system comprising a microscope is positioned in alignment with a channel adjacent to a slide mount for a user to visualize a field of view above the compartment. In some embodiments, a laser source is positioned adjacent to or in alignment with the same channel such that, after visualization and identification of a predetermined perimeter that surrounds a target area within the biocompatible membrane, a laser is emitted from the laser source and directed to the predetermined perimeter.

[0101] In some embodiments, the chip is made in one solid piece of plastic, metal or combination of plastic and metal, for example, from a glass or plastic that has been made cut to form an interior cavity, optionally coated with a thin film of biomaterial which is acceptable in and conducive to laser cutting procedures. In some embodiments, the cavity has been made of an inorganic biocompatible polymer or a hydrogel forming a so-called an interior region of the interior surface which may contain functional groups for binding any desired ligands or biomaterials specific for the type of tissue or biomolecule within a target area of in the sample. In some embodiments, the interior region or compartment is free of hydrogel or functional groups. In some embodiments, the chip is a biocompatible plastic and the sidewalls of the compartment are defined by the interior surface of the chip and one length dimension defined by the solid support, such as a glass slide. After directly or indirectly physically abutting the surfaces to form a compartment, a closed fluid circuit is completed around a biocompatible membrane positioned on the glass slide and exposed to the compartment. In some embodiments, the solid support and the chip indirectly abut to form the compartment by one or a plurality of seals of gaskets positioned therebetween.

[0102] The embodiment disclosed in FIG. 2A depicts a top cross-sectional view of a system comprising a chip or single enclosure element 201 in fluid communication with a collection region 202. The enclosure element 201 comprises an interior surface with a height dimension that creates a volume within which the biocompatible membrane (not depicted) is exposed to fluid. A cross-section of the fluid inlet 203 is positioned on one end of the compartment and a cross section of an outlet 204 in fluid communication with the collection region is positioned between the enclosure element 201 and the collection region 202. Fluid flow from the inlet 203carries a cut sample from the compartment through the outlet 204 and into the collection region where a fluid conduit leads to a central distributor chamber 205. Multiple, additional fluid conduits are positioned in parallel begin most proximate to the distributor chamber and lead to individual collection traps 206 at aposition most distal end of the conduits. In this non-limiting embodiment, a microscope mounted with a camera in operable communication with a display is positioned above the collection traps such that a user of the system can visualize the contents of the collection traps and. if necessary, perform any interrogation techniques disclosed herein. In some embodiments, the distributor chamber 205 is also in operable connection with a microscope for the user such that the user may sort one or a plurality of samples into a conduit connecting the distributor to the collection trap 206. Sorting may be accomplished by observing a certain shape of cut membrane, measured density of sample of density of cells, or types of cells on the membrane. In some embodiments, sorting may also be accomplished by a user interrogating the sample within the distributor chamber by reading a QC code, barcode or other identifier preprinted on the side of the membrane opposite to the side upon which cells are adherent.

[0103] FIG. 2B depicts an embodiment similar to FIG. 2A, except that the collection region of the fluid circuit comprises an interrogation element 217 in operable fluid communication with the plurality of collection traps 216, such that samples positioned with the collection traps 216 may be fed by fluid flow into the interrogation element 217. In some embodiments, the interrogation element is a mass spectrometer, a spectrophotometer, a second microscope (or alternatively a second lens of a microscope creating the optinterface within the compartment) is in operable communication with a camera. Optionally, the interrogation element may be operably connected to a controller, display, and memory. Similar to FIG.2 A, cut biocompatible membranes fall into fluid flow within the compartment region depicted on the left-hand side of the page. Fluid flow from a fluid source is pumped into the compartment region through the inlet 213 and proceeds across the compartment volume and through the compartment outlet 214 and into the interrogation region (or, alternatively, the collection region). The interrogation region comprises a distributor chamber in operable alignment with a first interrogation device (depicted as a lens). The first interrogation device may be operably connected to a display, memory, and controller actively adaptable to view' one or a plurality of fields within the distributor chamber 215. The distribution chamber may comprise at least one collection vessel for viewing a membrane sample and, optionally, at least one constant or variable fluid flow control device, such as an impeller, to direct the speed and direction of flow of samples form the distributor chamber into parallel positioned collection traps 216. A firstseries of conduits positioned in the collection region carry fluid from the distributor chamber 215 to the collection traps 216. A second series of conduits similarly carry fluid from the collection traps 216 to a second interrogation element or device 217. While not depicted, a third series of conduits may be positioned in fluid communication with the second set of conduits or the second interrogation device, such that the fluid circuit returns and recycles culture media to a culture media reservoir (not pictured) and a pump.

[0104] FIG. 2D depicts a partial component of the fluid circuit that represents the collection region of the system. Whereas embodiments from FIGS. 2A through 2C depict a system in which two regions are adjacent within one, closed fluid circuit, FIG. 2D depicts an embodiment in which the compartment volume is formed upon installation of a replaceable consumable chip positioned upstream from the left edge of the image but not depicted. The collection region of FIG. 2D is depicted with an inlet 221 in fluid communication with the distributor chamber 222. Fluid from a detachable, consumable chip is received from the inlet and moves in a rightward direction relative to the top, cross-sectional image. The cross hair depicted on the left-hand edge of the image represents that the detachable chip is not installed, and the embodiments represent a first operable condition where fluid flow from the compartment is not actively flowing through the collection region. Once the chip is installed, fluid from the compartment is capable of entering the collection region through the cross-haired conduit and into the inlet221. At the distributor chamber 222 a first interrogation element or device (depicted as a lens graphically on the bottom of the image) is in operable alignment with the distributor chamber222. Fluid flow in the distributor chamber can be directed to parallel positioned collection traps 226 in fluid communication with the distributor chamber 222. In some embodiments, the step of sorting or characterizing the quantity, content, shape, color or other physical property' of the biomaterial on the biocompatible membrane can be performed while observing the sample in the distributor chamber 222. Fluid conduits from the collection traps 226 connect the collection traps to the second interrogation element 227. In this embodiment, adjustable valves down stream from the collection traps in electrical communication with the system elements independently open or close, or simultaneously open and close to release one or a plurality’ of samples serially or in parallel from the collection traps 226 to the second interrogation element 227.

[0105] FIG. 3A depicts an embodiment of the entire system for sample sorting, characterization, imaging and / or interrogation of other physical features of samples. The lefthand side of the image depicts a fluid reservoir with multiple fluid pumps in alignment with fluid receptacles. In this image, the receptables are rechargeable conical tubes withindependently addressable fluid cells operably connected to a user-operated controller. Fluid receptacles may comprise cell culture media or wash volumes that a user may decide to flow over through the compartment in fluid communication with the pump. Flow from the pump may be elected by the user to simultaneously feed (parallel feed) volume from the reservoir elements or serially feed volume from the reservoir elements into the compartment positioned in this image on the microscope stand after controlling adjustable valves beneath the fluid receptacles. A first set of tubes from the reservoir pump carries volume into the compartment in a closed system. The middle panel of the image depicts a microscope and microscope stand positioned proximate to a controllable laser source (not depicted). The inlay from the center panel shows a magnified image of, from top to bottom in vertical orientation, a movable laser source, a slide positioned between the laser source on a microscope stand, a biocompatible membrane positioned underneath the slide and a chip adjacent to the slide and membrane defining a volume within the compartment. A first set of tubing is in fluid communication with the reserv oir (not depicted) creates a closed fluid circuit from the lefthand side of the system and a second set of tubing leaves the righthand side of the compartment depicting the expectation of fluid flow through the compartment when the system is in operation. The inlay also depicts laser light directed from a laser source to a mirror (depicted as a slanted line) reflecting laser light through a movable optics package, the optics package movable in multiple planes to direct light 180 degrees across the biocompatible membrane. A display operably connected to the system is depicted on the top right inlay. The image on the display is a rendering of biomaterials such as cells growing on the biocompatible membrane. A controller, panel and computer interface (not shown) allows a user to manually adjust the microscope, digitally image the biomaterial on the biocompatible membrane, segment a the digital image, prepare a predetermined perimeter around a position or series of target areas for generation of cut samples, and observe cutting. The second inlay at the bottom righthand side of the image depicts a downstream collection chamber that receives fluid from the compartment volume. A small semicircular inlet receives fluid (and samples in the fluid) from the compartment volume and a conduit directs the fluid in a distributor chamber. A first interrogation device may be positioned in physical alignment with the distributor chamber such that methods of sorting, characterizing and interrogating the samples. Multiple conduits creating parallel fluid cells are in fluid communication with the distributor chamber and connected to collection traps. Collection traps, while depicted graphically by simple semicircles, may comprise multiple independently addressable conduits specific for that position and the individual collection trap. The collection trap may also comprise one or a plurality of storage cells in fluid communicationwith the collection trap itself. Storage cells may comprise reagents to label or modify biomaterial on a sample to facilitate visualization if viewed through another lens in optical alignment with the collection trap or to facilitate preparation of the sample before passage to a second interrogation device. In this case the second interrogation device is a mass spectrometer position downstream (her rightward) from the collection traps and in fluid communication with a fourth set of conduits. The second interrogation device is also in operable communication with the same controller and display enabling a user to visualize and manipulate samples through the depicted fluid circuit and operate the mass spectrometer.

[0106] FIG. 3B depicts an alternative arrangement in which six, independently adjustable reagent reservoirs 301 are connected in fluid communication to a reservoir pump 304 by way of tubing 302 connecting the reservoirs to the pump. Individual and independently addressable valve, not shown, are in operable connection to a controller available to the user for directed pump flow-, drawing reagents into the fluid circuit and controlling rate of fluid flow. A revolver valve 303 is adjustable for threading the tubing and propelling fluid within the fluid circuit. The pump 304 can be adjusted for flow rate of the fluid though a first set of tubing 305 that sends fluid into the compartment region 312 of the system. The compartment region comprises a slide positioned above a membrane positioned above a hollowed chip, w hi ch, when aligned w ith the slide and biocompatible membrane, form a volume within the compartment in fluid communication with the rest of the fluid circuit. A laser source 316 is positioned in operable alignment with a mirror 307 that reflect laser light from the laser source 316 to the compartment biocompatible membrane. Detection or interrogation equipment 309 is positioned in alignment with light reflected from the compartment region 312, such that optical measurement can be measured (and, in some embodiments, visualization of the membrane) can be performed by the user. Amovable optical interface 308 comprises a lens which is movable in a plurality of positions to direct light from the mirror to the biocompatible membrane. The movable series of angles at which the lens can be positioned allows for cutting of the membrane in multiple continuous positions in a selected, longitudinal plane of the membrane. A solid line from the laser source depicts the path of laser light to the compartment region while a dashed light indicate reflected laser light back to the detection equipment 309 but also light emanating from a light source 306 positioned beneath the compartment region 312. In some embodiments, the light source is functionally connected to the base of a microscope available to a user for monitoring the positioned and direction of laser light to the biocompatible membrane. Movement of the laser light through the moveable optical interface 308 around a predetermined path is depicted as a semicircular arrow 311. Laser light (the dotted line) isdepicted as actively cutting a small portion of the biocompatible membrane, or sample 311 away from its original position within the biocompatible membrane. A user may image the biomaterial adherent to the sample 311 as it is cut and drops within the fluid circuit by way of a microscope or other imaging equipment operably connected to the controller. The figure also depicts a second set of tubing connecting the compartment region 312 to the collection region 313 of the system. The collection region 313 comprises one or a plurality of collection traps where the sample may be directed and interrogated further by way of an interrogation device, in this case depicted as a lens, positioned in optical alignment with the one or plurality of collection traps. While the box representing the collection region is depicted as rectangular box, it should be understood that any embodiments, such as those depicted in FIG. 2A through 2D, may be present within the collection region 313. Fluid flow cells may comprise multiple collection traps through which reagents, such as probes or washing or fixing reagents may be directed across a sample 311 surface. A conduit also fluidly communicates the collection region 313 to a second interrogation device 315. Any interrogation device may be used, although it is contemplated that the user may operate (and visualize through the controller operably connected to a display) software stored within the second interrogation device 315. The second interrogation device may enable detection of a probe or other biomolecule, quantify certain biomolecules within a sample by mass spectrometry' or photometry devices, or detect and quantify the presence or absence of nucleic acids, for instance by way of DNA or RNA sequencing. In some embodiments, the second interrogation device 315 is capable of measuring the presence, absence or quantity of nucleic acids within cells adherent to the biocompatible membrane or is capable of sequencing the same.

[0107] FIG. 3C depicts another embodiment similar to the embodiments depicted in FIG.3B, except that a first set of tubing is positioned on the top of the compartment region 312. The inlet of the first set of tubing 325 is positioned beneath the slide and into the compartment volume and the first set tubing perforates the slide and biocompatible membrane. A closed fluid circuit is maintained by a gasket or seal (not depicted) around the portion of the tubing perforating the slide and membrane. A similar arrangement is depicted on the second set of tubing positioned on the opposite end of the compartment volume, where a fluid outlet is positioned ta the try point of the second set of tubing.

[0108] Various configurations of a first set of tubing and a second set of tubing, inlets, and outlets are illustrated in FIGS. 1A through 3C, 6A through 9E, and 12D. As described in Example 5, below, the embodiment of FIG. 12D is comprises a glass slide in place of a membrane. It is contemplated, however, as further embodiments herein, that any embodimentherein described with a glass slide could instead comprises a membrane and vice versa. For example, embodiments herein comprise any chip herein adapted to comprise the configuration of inlet and outlet illustrated in FIG. 12D, whether the chip includes a membrane or a glass slide.Laser

[0109] Systems of the disclosure relate to a laser position proximate to one side of a biocompatible membrane comprising cells, either live or fixed, that have been grown in culture on a solid support. In some embodiments, the laser emanates from a laser source positioned from about 0.1 millimeters to about 12,000 millimeters from the biocompatible membrane, slide or coverslip upon which one or a plurality’ of cells are grown, positioned or fixed. In some embodiments, the laser emanates from a laser source positioned from about 1 millimeters to about 1000 millimeters from the biocompatible membrane, slide or coverslip upon which one or a plurality of cells are grown, positioned or fixed. In some embodiments, the laser emanates from a laser source positioned from about 1 millimeters to about 150 millimeters from the biocompatible membrane, slide or coverslip upon which one or a plurality of cells are grown, positioned or fixed. In some embodiments, the laser emanates from a laser source positioned from about 20 millimeters to about 150 millimeters from the biocompatible membrane, slide or coverslip upon which one or a plurality of cells are grown, positioned or fixed. In some embodiments, the laser emanates from a laser source positioned from about 20 millimeters to about 75 millimeters from the biocompatible membrane, slide or coverslip upon which one or a plurality of cells are grown, positioned or fixed. In some embodiments, the laser emanates from a laser source positioned from about 25 millimeters to about 65 millimeters from the biocompatible membrane, slide or coverslip upon which one or a plurality of cells are grown, positioned or fixed. In some embodiments, the distance between the laser source and the biocompatible membrane, coverslip or slide may be adjustable in the x,y direction (relative to the membrane coverslip or slide) by positioning of the laser source on a movable mount. Additionally, in some embodiments, the laser source may be adjustable in the x direction relative to the membrane, such that the laser can be operated at one setting one distance from the biocompatible membrane, glass slide or coverslip and then operated at a second or additional settings at a second or more distances from biocompatible membrane, glass slide or coverslip. In some of the disclosed methods, biomaterials on a biocompatible membrane are cut by the laser ate one distance and then the cut membrane is pushed from the rest of the membrane to overcome electrostatic force by positioning the laser source at a second, distancefrom the membrane and operating the laser to push the cut membrane region from the rest of the biocompatible membrane into the fluid circuit adjacent to the side of the membrane opposite to the laser source.

[0110] The laser may be positioned (such as FIG. 1A) on a side of the biocompatible membrane opposite to the side of the membrane against which the enclosure element is positioned, such that laser light form the laser source hits the membrane and cuts the membrane under conditions sufficient to cut the membrane without disrupting the cell or cells within a predetermined perimeter or path around the cell or cells. In some embodiments, the conditions sufficient to cut the membrane are also sufficient to allow the cut membrane piece produced around the predetermined perimeter or path and comprising the cell or plurality of cells or interest to be collected within the enclosure element and exposed to fluid flow that is capable of carrying the cut membrane comprising the cell or cells to one or a plurality of collection chambers, at least one of the collection chambers proximate to an interrogation device calibrated to interrogate the cell or cells on the membrane once positioned within the chamber. In some embodiments, the disclosure relates to methods of cutting the membrane, isolating a portion of biomaterial (such as cell or cells), interrogating the biomaterial, sorting the biomaterial, and / or labeling the biomaterial to identify or measure physical features about the biomaterial useful for characterization. As a non-limiting example, mass spectrometry data or receptor density data may be obtained which can be correlated to a spatial position of the certain proteins within the biomaterials.For 8pm section after IF staining protocol (xylene, ethanol, and methanol exposure) - optimal conditions electrostatics can occur when cutting shapes in close proximityFor 8pm section after IF staining protocol (xylene, ethanol and methanol exposure) - medium risk for electrostaticFor 8pm section after IF staining protocol (xylene, ethanol and methanol exposure) - very high risk for electrostaticFor 8pm section after IF staining protocol (xylene, ethanol and methanol exposure) - high risk for electrostatic (power adjusted based on tissue (folds, edges))[OHl] In some embodiments, the laser is a UV laser. In some embodiments, the UV laser has a wavelength of 405 nm. In some embodiments the laser comprises a laser head having 0.5W Optical Power, 8K 2500DPI lOum spot, 405nm laser wavelength, and a power density of 1841 kW / cm2Enclosure element

[0112] Enclosure elements of the disclosure are depicted in FIGS. 6A - 6E, 7 and 8. Embodiments of the disclosure can be plastic chips in generally rectangular shape with partially hollowed interiors that form the continuous volume of a compartment and portion of the fluid circuit when the chip is installed within the disclosed systems. The disclosure relates to an enclosure element that serves as a consumable, detachable chip capable of being installed and uninstalled within any one of the disclosed systems to respectively form a, respectively, closed and open fluid circuit. In some embodiments, the consumable chip comprises an exterior and interior region with one contiguous surface, the interior region can be stepped to create a first, second and third height dimension. In some embodiments, that first height dimension defines the total height of the chip, the second height dimension defines a height of the compartmentwhen the chip is mated with a solid support to form a block unit, and the third height defines the height of a ridge around at least a portion of the interior perimeter of the chip responsible for creating a dimension of the chip responsible for mating, directly or indirectly by way of a gasket or seal, with a solid support. In some embodiments, the enclosure element comprises one or a combination of two or more of: Glass, PTFE, High density Polyethylene, polyurethane acrylate (PUA), of Cyclo olefin (co) polymer (COC / COP).

[0113] In some embodiments, the disclosure relates to a method of manufacturing the chip of any of the dimensions identified herein, including FIG. 3, comprising a step of 3D printing Generally, methods of manufacturing plastic or silica- based chips are known. 3D printers are commonly based on additive manufacturing that creates successive layers in order to fabricate 3D real objects. Each lay could be created according to a horizontal cross-section of a model of a real object to be printed. 3D printers are typically used to create new physical objects that do not exist before. Publications about #D printing are below and are incorporated by reference in their entireties.

[0114] In US 2011 / 0087350 Al, which is incorporated herein by reference in its entirety, there is provided a method and system enabling the transform of possibly corrupted and inconsistent virtual models into valid printable virtual models to be used for 3D printing devices.

[0115] U.S. Pat. No. 8.243,334 A, which is incorporated herein by reference in its entirety, generates a 3D virtual model for the use in 3D printing by automatically delineating object of interest in images and selecting a 3D wire-frame model of an object if interest as the virtual model. The 3D wire-frame model may be automatically calculated from stereoscopic set of images.

[0116] U.S. Pat. No. 7.343,216 A, which is incorporated herein by reference in its entirety, proposes a method of assembling two real physical objects to have a final physical object. The method discloses an architectural site model facilitating repeated placement and removal of foliage to the model. The site model is constructed as an upper shell portion and a lower base portion, while the model foliage is attached to the shell portion. The upper shell portion of the site model is configured for removable attachment to the lower base portion.

[0117] Methods of the making the chip disclosed herein comprise uploading a design image to a computer with a memory, wherein the computer is operably connected to a 3D printer and initiating instructions on a computer program product to create the object, in this case a chip with dimensions disclosed herein. In some embodiments the total height of the chip is from about 1 millimeters (mm) to about 5 mm. In some embodiments the total height of thechip is from about 1 millimeters (mm) to about 4 mm. In some embodiments the total height of the chip is from about 1 millimeters (mm) to about 3 mm. In some embodiments the total height of the chip is from about 1 millimeters (mm) to about 2 mm. In some embodiments the total height of the chip is from about 1 mm to about 3 mm. In some embodiments the total height of the chip is from about 1 mm to about 3 mm. In some embodiments the total height of the chip is from about 1.1 mm to about 3.0 mm. In some embodiments the total height of the chip is from about 1.2 mm to about 3.0 mm. In some embodiments the total height of the chip is from about 1.3 mm to about 4 mm. In some embodiments the total height of the chip is from about 1.4 mm to about 4 mm. In some embodiments the total height of the chip is from about 1.5 mm to about 4 mm. In some embodiments the total height of the chip is from about 1.6 mm to about 4 mm. In some embodiments the total height of the chip is from about 1.7 mm to about4 mm. In some embodiments the total height of the chip is from about 1.8 mm to about 4 mm.In some embodiments the total height of the chip is from about 1.9 mm to about 4 mm. In some embodiments the total height of the chip is from about 2.0 mm to about 4 mm. In some embodiments the total height of the chip is from about 1 mm to about 2 mm. In some embodiments the total height of the chip is from about 1.1 mm to about 2.0 mm. In some embodiments the total height of the chip is from about 1.2 mm to about 2.0 mm. In some embodiments the total height of the chip is from about 1.3 mm to about 2 mm. In some embodiments the total height of the chip is from about 1.4 mm to about 2 mm. In some embodiments the total height of the chip is from about 1.5 mm to about 2 mm. In some embodiments the total height of the chip is from about 1.6 mm to about 2 mm. In some embodiments the total height of the chip is from about 1.7 mm to about 2 mm. In some embodiments the total height of the chip is from about 1.8 mm to about 2 mm. In some embodiments the total height of the chip is from about 1.9 mm to about 2 mm. In some embodiments the total height of the chip is from about 2.0 mm to about 2 mm.

[0118] In some embodiments, the second height of the chip is from about 0.1 mm to about 1.0 mm. In some embodiments, the second height of the chip is from about 0.2 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.3 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.4 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.5 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.6 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.7 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.8 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.9 mm to about1.0 mm. In some embodiments, the second height of the chip is from about 0.4 mm to about 0.6 mm. In some embodiments, the second height of the chip is from about 0.5 mm to about 0.6 mm.

[0119] In some embodiments, the third height dimension of the chip is from about 1.0 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.0 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1. 1 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.2 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.3 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.4 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.5 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.6 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.7 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.8 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.9 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 2.0 mm to about 2.5 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.4 mm to about 1.7 mm as measured from the exterior surface. In some embodiments, the third height dimension of the chip is from about 1.1 mm to about 1.3 mm as measured from the exterior surface.

[0120] In some embodiments the first height dimension of the chip is from about 1.7 mm to 2.0 mm, the second height dimension of the chip is from about 0.40 mm to about 0.55 mm, and the third height dimension is from about 1.3 mm to about 1.6 mm.

[0121] FIG. 6A depicts a top, cross-sectional view of a chip 601 predominantly made of plastic. The chip itself is largely a rectangular prism in shape but the has a hollowed interior portion with a different height dimension to account for its mating with the slide within the compartment. The highest height dimension of this interior section creates a volume 660 of the compartment whereby a contiguous portion of the interior 620 mates with one side of the slide comprising the biocompatible membrane upon installation. A ridge 610 around the under sideof the chip 601 can optionally align with a seal, gasket or other rubber station creating contact points that form the compartment volume 660 and a closed fluid circuit. When aligned to form the fluid circuit a first set of tubing is positioned in fluid communication with the inlet points 640a, 640b, 640c, 640d on one end of the chip 601. Fluid flow can be directed from inlet points 640a, 640b, 640c, 640d toward outlet point 630. A ramp 650 is positioned downstream from the middle of the volume 660, such that the ramp has downward angular pitch relative to a mostly horizontal compartment volume 660. After cutting of a sample of biocompatible membrane by laser operation, the sample may flow in a mostly or substantially horizontal direction from the inlet points 640a, 640b, 640c and 640d toward the outlet point 630. The downward angular pitch of the ramp at the outlet 630 creates a drag on fluid within the compartment volume 660, such that samples can be drawn away and downward from the position of cutting with minimal or extremely limited electrostatic friction. Inlet points 640a, 640b, 640c and 640d provide evenly distributed fluid flow over the entire surface area of the biocompatible membrane. In some embodiments, fluid flow through the inlet points may be controlled by independently manipulatable valves positioned in parallel along each of or one or more of the parallel conduits. In some embodiments, the independently manipulatable valves also allow selective flow of culture media or other reagents on a desired portion of the biocompatible membrane. The outlet point 630 leads to a second set of conduits aligned with one or a plurality of collection chambers. A sample can be sent to the collection chamber and interrogated by any methods disclosed herein after cutting through the fluid dynamics prepared created within the compartments by dimensions of the compartment and the position of the inlet points 640a, 640b, 640c and 640d. In some embodiments, outlet point 630 is operably connected to a pump drawing fluid within the volume 660 out. In some embodiments, inlet points 640a, 640b. 640c and 640d are operable connected to a pump pushing fluid into the volume 660. In some embodiments, outlet point 630 is operably connected to a pump drawing fluid within the volume 660 out and the inlet points 640a, 640b, 640c and 640d are operable connected to a pump pushing fluid into the volume 660. The pump may be any suitable device to draw or push fluid. A pump may be a syringe that is manually, mechanically, or robotically operable.

[0122] In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 40 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 28 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 29 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 30 mm. In someembodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 31 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 32 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 33 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 34 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 35 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 36 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 37 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 38 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 20 mm to about 39 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 21 mm to about 25 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 22 mm to about25 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 23 mm to about 25 mm. In some embodiments, the total, narrowest width dimension of the chip is from about 24 mm to about 25 mm.

[0123] In some embodiments, the longest length dimension of the chip from about 25 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about26 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 27 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 28 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 28 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 30 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 31 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 32 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 33 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 34 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 35 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 36 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 37 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 38 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 39 mm to about 60 mm. In some embodiments, the longest length dimension of the chip from about 40 mm to about 60 mm. In some embodiments, the longest length dimension of thechip from about 41 mm to about 55 mm. In some embodiments, the longest length dimension of the chip from about 25 mm to about 50 mm. In some embodiments, the longest length dimension of the chip from about 25 mm to about 45 mm. In some embodiments, the longest length dimension of the chip from about 25 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 26 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 27 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 28 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 29 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 30 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 31 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 32 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 33 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 34 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 35 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 36 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 37 mm to about 40 mm. In some embodiments, the longest length dimension of the chip from about 37 mm to about 39 mm. In some embodiments, the longest length dimension of the chip is about 30. 31, 32, 33, 34, 35, 36, 37, 38. 39 or about 40 mm.

[0124] FIG 6B depicts an isometric orientation of another chip 611 whereby a volume 661 of the compartment is created after installation of the chip 611 into one of the disclosed systems against a solid support (not depicted). A portion of the chip 661 comprises a contiguous ridge 621 formed by a height dimension defining the interior sidewalls of the volume 661. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 400 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 410 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 420 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 430 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 440 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is fromabout 450 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 460 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 470 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 480 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 490 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 500 microns to about 520 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 500 microns to about 650 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 510 microns to about 700 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is from about 410 microns to about 700 microns. In some embodiments, the height dimension defining the interior sidewalls of the volume of the compartment is about 500. 505, 515. 516, 517, 518. 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 528 microns.

[0125] A second height dimension defines a second contiguous surface positioned around the ridge 621 that allows for mating of the chip 661 to the solid support or a gasket positioned around the solid support. Proper alignment of the chip 661 for installation also may allow that the closed fluid circuit be formed between the chip and the fluid reservoir (not depicted) at inlet 642 as well as between the chip and one or more collection chambers (not depicted) at the outlet 633. Similar to the orientation of the chip depicted in FIG. 6A, Fluid flow in this embodiment flows from the inlet 642 into parallel conduits 641a, 641b, 641c and 641d and into the compartment volume 661 via a ramp 651 positioned proximate to the outlet points of parallel conduits 641a, 641b, 641c and 641d. In some embodiments, the compartment is connected to the parallel conduits by one aperture (in the shape of a slot) the width of the entire width of the compartment or a series of apertures or slots. In some embodiments, the height of the slot compartment from the ramp to the opening of the compartment most adjacent to the ramp may be from about 0.1 mm to about 0.3 mm. In some embodiments, the height of the slot may be about 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or about 0.30 mm. Fluid flow proceeds across a biocompatible membrane and toward ramp 631 by way of a pump when the chip is installed in fluid communication with the system. After proceeding over the ramp 631, fluid flow is directed tothe outlet 633 by way of a single outlet conduit 632 connecting the compartment volume 661 to the outlet 633.

[0126] FIG. 6C and FIG. 6D depict a side views of an embodiment depicted in FIG. 6B wherein the compartment volume is shown between inlet ramp 653 and outlet ramp 631. Outlet conduit 632 is in fluid communication with the outlet ramp 631 and the outlet 633. Outlet conduits can have microfluidic dimensions. In some embodiments, the outlet conduit height is no more than the height of the compartment height. In some embodiments, the outlet conduit height is no more than 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55 mm. In some embodiments, the outlet conduit height is from about 350 to about 540 microns. In some embodiments, the outlet conduit height is from about 350 to about 530 microns. In some embodiments, the outlet conduit height is from about 350 to about 520 microns. In some embodiments, the outlet conduit height is from about 400 to about 550 microns. In some embodiments, the outlet conduit height is from about 410 to about 520 microns.

[0127] FIG. 6E depicts the bottom side isometric view of the chip 602 from FIG. 6A. Parallel conduits 670 connect the outlet point 675 to the compartment volume. Fluid ingress points 641a 641b, 641c, 64 Id are positioned adjacent to the ramp 653 that comprises a downward pitch to distribute fluid evenly across the surface area of the longitudinal plane subsuming the widest dimension of the cavity (which is the side of the compartment comprising the biocompatible membrane when the chip 602 mates with the solid support).

[0128] FIG. 7 depicts an exploded view of an embodiment of a chip 701 where parallel conduits are positioned on the left hand side of the chip 701 body at and near a fluid ingress point 720. In this embodiment, the parallel conduits open to a cavity the runs along the width of the chip compartment and positioned adjacent to hollow ramp 710. The hollow ramp creates a fluid conduit to connect the cavity with the region of the chip that defines the compartment volume when mated w ith the solid support (not depicted). The hollow- ramp passes through the a rectangular opening 720. The ramp 710 opens in the cavity of the chip 701 along the width of the chip 701, and the collective width and pitch of the ramp encourages fluid distribution over one side of the surface area of the cavity within the chip 701.

[0129] FIG. 8 depicts a magnified diagram of a portion of the compartment volume 805 within the chip 801 responsible for fluid egress away from the compartment and into a second region of the system through the outlet 804. Fluid from the righthand side of the diagram moves toward the lefthand side of the diagram when the chip 801 is mated to a solid support (not shown). Fluid flow' exits the compartment first through tapered sidewalls that are adjacent toan exit ramp 802 positioned at a connection point of the single outlet conduit 808 and the compartment volume 805. The exit ramp 802 can have an increasing upward slope toward the outlet conduit 808 or a decreasing downward slope toward the outlet conduit 808. The outlet conduit is in fluid connection to an outlet 804, which connects the cutting region of the system to a collection region of the system. Within the collection region, the sample may interrogated by one or a combination of interrogation devices in one or more collection vessels or traps.

[0130] Referring to FIGS. 9 A through 9E. an embodiment comprising chip 901 is illustrated. Inlet points 930a, 930b, 930c, and 930d are in fluid communication with an inlet 930 and a volume 960. Similar to chips 601, 611, and 602, the chip itself is largely a rectangular prism in shape but the has a hollowed interior portion with a different height dimension to account for its mating with a slide within the compartment. The highest height dimension of this interior section creates a volume 960 of the compartment whereby a contiguous portion of the interior 920 mates with one side of the slide comprising the biocompatible membrane upon installation. In some embodiments, a ridge around the under side of the chip 901 can optionally align with a seal, gasket or other rubber station creating contact points that form the compartment volume 960 and a closed fluid circuit. When aligned to form the fluid circuit a first set of tubing is positioned in fluid communication with the inlet points 930a, 930b, 930c, 930d on one end of the chip 901. Fluid flow can be directed from inlet points 930a, 930b, 930c, 940d toward outlet point 640a. A ramp 950 is positioned downstream from the middle of the volume 960, such that the ramp has downward angular pitch relative to a mostly horizontal compartment volume 960. After cutting of a sample of biocompatible membrane by laser operation, the sample may flow in a mostly or substantially horizontal direction from the inlet points 930a, 930b, 930c and 930d toward the outlet point 940a. Samples can be drawn away and downward from the position of cutting. Inlet points 930a, 930b, 930c and 930d provide evenly distributed fluid flow over the entire surface area of the biocompatible membrane. In some embodiments, fluid flow through the inlet points may be controlled by independently manipulatable valves positioned in parallel along each of or one or more of the parallel conduits. In some embodiments, the independently manipulatable valves also allow selective flow of culture media or other reagents on a desired portion of the biocompatible membrane. The outlet 640, fluidly connected to the output point 640a, leads to a second set of one or more conduits aligned with one or a plurality of collection chambers. A sample can be sent to the collection chamber and interrogated by any methods disclosed herein after cutting through the fluid dynamics prepared created within the compartments by dimensions of the compartment and the position of the inlet points 930a, 930b, 930c and 930d. In some embodiments, outletpoint 940a is operably connected to a pump drawing fluid within the volume 960 out. In some embodiments, inlet points 930a, 930b. 930c and 930d are operable connected to a pump pushing fluid into the volume 960. In some embodiments, outlet point 940a is operably connected to a pump drawing fluid within the volume 960 out and the inlet points 930a, 930b, 930c and 930d are operable connected to a pump pushing fluid into the volume 960. The pump may be any suitable device to draw or push fluid. A pump may be a syringe that is manually, mechanically, or robotically operable. Such an arrangement of pumps operably connected to one or both of the inlet points and the outlet point may be applied to any chip herein.

[0131] In some embodiments, a system herein may comprise any one of the chips disclosed herein. A system herein can be in one of at least three operative conditions. A first operative condition comprises a state where the system is implemented to cut membrane, or ablate matter from a glass surface, which is set in place of the membrane. For example, laser light can be directed at or around a point or series of points around one or a plurality of cells growing on the membrane resulting in a microdissected membrane comprising biomaterial or the one or plurality of cells. A second operative condition comprises a collection state where pieces of the membrane, or matter released from the glass surface, flow through the volume and are collected. For example, fluid flow across, under and / or above the surface of the membrane transfers the microdissected membrane to a collection chamber adjacent to and in fluid communication with the compartment. A third operative condition comprises a state in which a valve attached to the fluid circuit before and after the collection vessel (e.g. , a syringe), closed before the collection vessel and open at the syringe outlet to allow a pump on the outlet side to pump collected membranes or samples into an array of containment vessels; e.g., a 96 well plate. This configuration allows selective serial volume fluid flow for collected samples into selective containment vessels for analytical steps (e.g, mass spec, flow cytometry, etc.). For example, a user may interrogate the one or plurality of cells on the microdissected membrane in the collection chamber. Interrogation can include one or a combination of: imaging the biomaterial or one or plurality of cells, labeling the biomaterial or one or plurality of cells, classifying / stratifying the biomaterial or one or plurality of cells, and / or exposing the biomaterial or cells to mass spectrometry, and / or exposing the biomaterial or cells to cell sorting.

[0132] The volume of a chip herein; for example but not limited to volume 660 or volume 960, may be any suitable volume allowing for the operative conditions of a system herein. In some embodiments, the volume is from about 20 pl to about 250 pl. . In some embodiments, the volume is about 20 pl, about 50pl, about 75pl, about 100 pl, about 125 pl, about 150 pl,about 175 pl, about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 20 pl to about 50pl, about 75 pl. about 100 pl, about 125 .1, about 150 pl, about 175 pl, about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 50pl to about 75pl, about 100 pl, about 125 pl, about 150 pl, about 175 pl, about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 75pl to about 100 pl, about 125 pl, about 150 pl, about 175 pl, about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 100 pl to about 125 pl, about 150 pl, about 175 pl, about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 125 pl to about 150 pl, about 175 pl, about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 150 pl to about 175 pl, about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 175 pl to about 200 pl, about 225 pl, or about 250 pl. In some embodiments, the volume is from about 200 pl to about 225 pl, or about 250 pl. In some embodiments, the volume is from about 225 pl to about 250 pl.

[0133] The volume of a chip herein refers to the volume contained within the chip. In some embodiments, a second volume, referred to as a fluid transport volume, is the volume of liquid from a first liquid source, through the chip, and into the collection chamber. In some embodiments, the fluid transport volume is from about 30 pl to about 300 pl. In some embodiments, the volume is about 30 pl, about 50pl, about 75pl, about 100 pl, about 125 pl, about 150 pl. about 170 pl, about 200 pl, about 225 pl, about 250 pl, or about 300 pl. In some embodiments, the volume is from about 30 pl to about 50pl, about 75pl, about 100 pl, about 125 pl, about 150 pl, about 170 pl, about 200 pl, about 225 pl, about 250 pl, or about 300 pl. In some embodiments, the volume is from about 50pl to about 75pl, about 100 pl, about 125 pl, about 150 pl. about 170 pl, about 200 pl, about 225 pl, about 250 pl, or about 300 pl. In some embodiments, the volume is from about 75 pl to about 100 pl, about 125 pl, about 150 pl, about 170 pl, about 200 pl, about 225 pl, about 250 pl, or about 300 pl. In some embodiments, the volume is from about 100 pl to about 125 pl, about 150 pl, about 170 pl, about 200 pl, about 225 pl, about 250 pl, or about 300 pl. In some embodiments, the volume is from about 125 pl to about 150 pl, about 170 pl. about 200 pl, about 225 pl, about 250 pl. or about 300 pl. In some embodiments, the volume is from about 150 pl to about 170 pl, about 200 pl, about 225 pl, about 250 pl, or about 300 pl. In some embodiments, the volume is from about 170 pl to about 200 pl, about 225 pl, about 250 pl, or about 300 pl. In some embodiments, the volume is from about 200 pl to about 225 pl. about 250 pl, or about 300 pl.In some embodiments, the volume is from about 225 ja.1 to about 250 pl, or about 300 pl. In some embodiments, the volume is from about 250 l to about 300 pl.

[0134] Referring to FIG. IB, the biocompatible membrane 14 has a width and length that may be referred to as being aligned with X and Y axis of the system. The direction of the laser may then be referred to as being aligned with the Z axis of the system. In some embodiments, a system herein may comprise a laser adjustable along the Z axis, that is by the distance from the membrane. The laser head may be mounted with a lead screw allowing vertical adjustment along the Z axis. In some embodiments, the system is implemented at a first height in order to micro dissect a shape from the membrane. The shape may be one circumscribing a particular feature of the sample. For example, the feature may be a single cell or group of cells. In some embodiments, more than one micro dissection may be implemented, creating a plurality’ of micro dissected membrane pieces. The shape may be a square, a circle, an oval, etc. In some embodiments, the each of the plurality of micro dissected membrane pieces may be sorted based on shape. The sorting, in some embodiments, may be accomplished by selectively operating valves to direct an isolated shape to a specific containment vessel in a containment vessel array and thereby create an array of samples. The array may be set such that the position of each micro dissected piece within the original membrane is known. The array may be set such that the sample type on each micro dissected piece is known. For example, predetermined shapes may be utilized to indicated sample containing a particular type of cell(s), and segregation in the array by shape will indicated the type of cell. In some embodiments, the microdissection is performed with the laser set at a first distance and the micro dissected piece is liberated by the microdissection or further by flow of fluid underneath. In some embodiments, a “cut and push"’ method is utilized where the microdissection is performed with the laser set at a first distance. Then, the laser is set at a second distance farther from the membrane, upward along the Z axis, and one or more of the micro dissected pieces is liberated by a second pulse(s) from the laser at the second distance. See, for example, Example 4. The adaptive change in focus on the stage, in some embodiments, allows for a wider or tighter radius surface cuts. In some embodiments, the laser cuts the desired path around a cell sample to cut the piece of membrane, then the diameter and intensity of the laser is changed to push the cut membrane from the stage and into the fluid beneath the membrane.

[0135] In some embodiments, a glass slide replaces the membrane in a chip or system herein. Samples may then be ablated by operation of the laser in order to release the samples into the fluid volume for collection. The ablated sample may also be distributed into an array and subject to interrogation as otherwise described herein.

[0136] In some embodiments, a chip or system herein further comprises a fastening element. The fastening element, in some embodiments, is in physical contact with the sidewalls and / or the partition. In some embodiments the fastening element secures the membrane or the glass slide to in place. In some embodiments, the fastener is a clip or metal clip that clasps the membrane or glass slide in place.Kits

[0137] In some embodiments, kits in accordance with the present disclosure comprise a first container comprising an enclosure element; and a second container comprising instructions on its installation and use within the disclosed systems. The disclosure further provides for a kit comprising one or a plurality of containers that comprise one or a plurality of reagents that can quantify a physical feature of one or a plurality of cells adherent to the disclosed membrane and . In some embodiments, the kit comprises ae disposable enclosure element and / or a device disclosed herein. In some embodiments, the kit comprises a first container comprising an enclosure element, and, optionally, instructions for operating a laser in the system for cutting a membrane upon which cells adhere.

[0138] In some embodiments, the kit comprises: a system, any isolated enclosure element disclosed herein, and a computer program product disclosed herein optionally comprising instructions to perform any one or more steps of any method disclosed herein. In some embodiments, the kit is free of tissue culture cell media. In some embodiments, the kit comprises a solid support comprising a membrane disclosed herein and, optionally, embedded with at least one cell or a plurality of cells.

[0139] The disclosure also relates to a kit comprising a computer program product disclosed herein optionally comprising instructions to perform any one or more steps of any method disclosed herein.

[0140] The kit may contain two or more containers, packs, or dispensers together with instructions for preparation of an array. In some embodiments, the kit comprises at least one container comprising the enclosure element or system described herein and a second container comprising a solution for maintenance, use, and / or storage of the enclosure element such as storage buffer and / or tissue culture medium. In some embodiments, the kit comprises a composition comprising any molecule disclosed herein in solution or lyophilized or dried and accompanied by a rehydration mixture. In some embodiments, the molecules and rehydration mixture may be in one or more additional containers. In some embodiments, the kit comprises a composition comprising any one or combination of enclosure elements optionally with anon-transient computer program product comprising instructions for the steps of image masking of a image taken of a cell or plurality of cells adherent to a disclosed membrane before during and after dissection of the membrane. In some embodiments, the step of masking comprises defining a boundary around a cell of interest, masking the remainder of cells other than the cells of interest in the image and editing an unmasked region of the image using a cellular feature. In some embodiments, the cellular feature is the presence or quantity of one or a pattern of organelles, the volume of one or a plurality of organelles, the shape of a cell, the presence or quantity of expressed protein in a cell, the presence or distribution of protein in a microenvironment within a cell, the shape of a cell, or a combination thereof. In some embodiments, the kit comprises a composition comprising any one or combination of enclosure elements optionally with a non-transient computer program product comprising instructions for steps: (a) obtaining an image of one or plurality of cells adherent to a membrane disclosed within the system; (b) image masking of the image; (c) image clustering; (d) single region or single cell cluster selection from a region of an unmasked cell or plurality of cells in the image; and (e) mask alignment with physical sample. In some embodiments, the kit comprises a composition comprising any one or combination of enclosure elements optionally with a nontransient computer program product comprising instructions for steps: (a) obtaining an image of one or plurality of cells adherent to a membrane disclosed within the system; (b) image masking of the image; (c) image clustering; (d) single region or single cell cluster selection from a region of an unmasked cell or plurality of cells in the image; (e) mask alignment with physical sample; and (f) initiating laser cutting of the membrane. In some embodiments, the kit comprises a composition comprising any one or combination of enclosure elements optionally with a non-transient computer program product comprising instructions for steps: (a) obtaining an image of one or plurality of cells adherent to a membrane disclosed within the system; (b) image masking of the image; (c) image clustering; (d) single region or single cell cluster selection from a region of an unmasked cell or plurality of cells in the image; (e) mask alignment with physical sample; and (I contour import for dissection. In some embodiments, the kit comprises a composition comprising any one or combination of enclosure elements optionally with a non-transient computer program product comprising instructions for steps: (a) obtaining an image of one or plurality of cells adherent to a membrane disclosed within the system; (b) image masking of the image; (c) image clustering; (d) single region or single cell cluster selection from a region of an unmasked cell or plurality of cells in the image; (e) mask alignment with physical sample; (I) contour import for dissection; and (g) initiating laser cutting of the membrane within an enclosure element on a system disclosed herein. In someembodiments, one or a combination of (b). (c), (d), (e), and (f) result in establishing a predetermined point or series of points within the unmasked region of the image or the boundary between a masked and unmasked region of the image corresponding to a position or series of positions on the physical sample.

[0141] The compositions included in the kit may be supplied in containers of any sort such that the shelf-life of the different components are preserved and are not adsorbed or altered by the materials of the container. For example, suitable containers include simple bottles that may be fabricated from glass, organic polymers, such as polycarbonate, polystyrene, polypropylene, polyethylene, ceramic, metal or any other material typically employed to hold reagents such as probes or to hold ; envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, and syringes. The containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components of the compositions to mix. Removable membranes may be glass, plastic, rubber, or other inert material.

[0142] Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrates, and / or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zip disc, videotape, audio tape, or other readable memory storage device. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit. or supplied as electronic mail.Computer Program Product

[0143] In some embodiments, disclosed is a system comprising a disclosed computer program product, and one or more of (i) a processor operable to execute programs; and (ii) a memory associated with the processor.

[0144] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smart phone, or any other suitable portable or fixed electronic device.

[0145] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output.Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible format.

[0146] Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks, or fiber optic networks.

[0147] A computer employed to implement at least a portion of the functionality described herein may include a memory, coupled to one or more processing units (also referred to herein simply as ‘"processors”), one or more communication interfaces, one or more display units, and one or more user input devices. The memory may include any computer-readable media, and may store computer instructions (also referred to herein as “processor-executable instructions”) for implementing the various functionalities described herein. The processing unit(s) may be used to execute the instructions. The communication interface(s) may be coupled to a wired or wireless network, bus. or other communication means and may therefore allow the computer to transmit communications to and / or receive communications from other devices. The display unit(s) may be provided, for example, to allow a user to view various information in connection with execution of the instructions. The user input device(s) may be provided, for example, to allow the user to make manual adjustments, make selections, enter data or various other information, and / or interact in any of a variety of manners with the processor during execution of the instructions.

[0148] The various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. The disclosure also relates to a computer readable storage medium comprising executable instructions. Additionally, such software may be written using any of a number of suitable programming languages and / or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

[0149] In this respect, various inventive concepts may be embodied as a computer readable storage medium (or multiple computer readable storage media) (e.g., a computer memory. one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other non-transitory medium or tangible computer storage medium) encoded with one or moreprograms that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the invention disclosed herein. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the present invention as discussed above. In some embodiments, the system comprises cloud-based software that executes one or all of the steps of each disclosed method instruction.

[0150] The terms ‘'program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of embodiments as discussed above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present invention.

[0151] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0152] Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

[0153] Also, the disclosure relates to various embodiments in which one or more methods. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments. Computer-implemented embodiments of the disclosure relate to methods of cutting a membrane or isolating a sample comprising a sample or target area comprising directing a laser around a predetermined path encompassing the targetarea and allowing the cut membrane comprising the sample to be exposed to a fluid flow for carrying the sample to a location distally from the step of cutting. In some embodiments, the computer-implemented method comprises: (a) creating an image of a target area: (b) segmenting the image; (c) creating a mask for the segmented image that corresponds to a position or plurality7of positions within or around the target area. In some embodiments, the computer-implemented methods further comprises (d) selection of a position or plurality7of positions within the image to define where within the sample to quantify a feature or perform data capture. In some embodiments, data capture includes detecting a signal within a target area, normalizing that signal to a signal obtained from a control area within the image and assigning a numerical value to the signal based upon the signal intensity relative to a control area within the segmented image. In some embodiments, data capture within the disclosed computer-implemented methods further comprises calculating a presence, absence or quantity of a biomarker, protein, nucleic acid or other biomolecule within the target area based upon the normalized value of signal intensity measured within the target area and corresponding to the biomarker, protein, nucleic acid or other biomolecule. In some embodiments, the disclosure relates to a computer pro a non-transient computer program product comprising instructions for steps: (a) obtaining an image of one or plurality of cells adherent to a membrane disclosed within the system; (b) image masking of the image; (c) image clustering; (d) single region or single cell cluster selection from a region of an unmasked cell or plurality of cells in the image; (e) mask alignment with physical sample; and (f) contour import for dissection. In some embodiments, the kit comprises a composition comprising any one or combination of enclosure elements optionally with a non-transient computer program product comprising instructions for steps: (a) obtaining an image of one or plurality of cells adherent to a membrane disclosed within the system; (b) image masking of the image; (c) image clustering; (d) single region or single cell cluster selection from a region of an unmasked cell or plurality of cells in the image; (e) mask alignment with physical sample; (f) contour import for dissection; and (g) initiating laser cutting of the membrane within an enclosure element on a system disclosed herein. In some embodiments, one or a combination of (b), (c), (d), (e), and (f) result in establishing a predetermined point or series of points within the unmasked region of the image or the boundary betw een a masked and unmasked region of the image corresponding to a position or series of positions on the physical sample. Non-transient, computer program products of the disclosure comprise algorithms that perform one or a combination of two or more steps generally disclosed within FIG. 4. Digital imaging of a microscopic surface is generally known. Systems, kits and devices of the disclosure can comprise a computer program product withinstructions for digital image capture of a sample or biomaterial within the compartment volume. Analysis of the image segmentation and image clustering 401 can be performed serially or in parallel with mask generation followed by selection of single objects within the image 402. A user may select objects or groups of objects 402 within the segmented image to encompass a two dimensional surface area corresponding to the real-time cell culture. In some embodiments, a user may select a single cell or a portion of the image that covers one particular section of extracellular architecture around a single cell. Creating a cutting path for the laser can be accomplished by first aligning the mask with the physical sample 403 through the realtime microscopy and then importing the contour of the membrane into the image 404.

[0154] In some embodiments, Raw image files are captured by using LAS X for imaging from Leica. exporting raw images in tiff format https: / / www.leica- microsystems.com / products / microscope-software / p / leica-las-x-id / . In some embodiments, Image segmentation of the digital images captured by the systems is performed by executing Stardist, Al model to detect cell nuclei.

[0155] In some embodiments, the method of defining boundaries or the perimeter path of the laser within a the sample comprising a cell, using cell membrane markers is performed using the following: https: / / docs.opencv.Org / 4.x / d3 / d47 / group imgproc segmentation.html#ga3267243e4d3f95 165d55a618c65ac6el, the contents of which are incorporated by reference in its entirety.

[0156] In some embodiments, the image analysis is performed on the mask generated by PIPEX giving each cell a unique ID. After clusters have been identified using cluster algorithms such as k means. Cluster information is stored in the dataframe.

[0157] The image of the tissue and the generated mask from PIPEX is also imported into BIAS which gives each contour a software specific ID. In some embodiments, methods include a feature matrix containing all BIAS cell IDs and matching the feature matrix them PIPEX ID containing the cluster information. Then we reimport this matched dataframe into BIAS and extract cluster by cluster via a query method in the BIAS software. (https: / / single-cell- technologies.com / bias-2 / )

[0158] In some embodiments, the methods of the disclosure relate to a method of imaging a biomaterial (such as a cell plurality of cells, nucleic acid expression and protein expression) or any of the above disclosed methods comprising a step of digital imaging the biomaterial in the compartment or within a collection region of the systems. In some embodiments, the step of digital imaging comprises modifying contours slightly to enhance cutting performance by closing them to avoid sharp edges and dilating them to protect the outer edges of the cell.Finally, in some embodiments, methods of the disclosure further comprise choosing three alignment points over the digital image comprising the cell, plurality of cells or biomaterials by creating three digital shapes on the image. A cluster can then be selected and exported as an xml file and imported in the system computer program product by importing the shape to the images. The same three alignment points that have been selected in the BIAS software have to be selected on the physical sample allowing the contours of the image to be aligned with the tissue.

[0159] In some embodiments, the computer program product and computer-implemented methods using the computer program product comprise the following steps to generate an image and prepare the a predetermined path for laser cutting of the biocompatible membrane.1. Load reference points from file - No2. Place the calibration cross over the 1. Calibration mark in the slide by moving the stage and press Ok. Or cancel the import. - OK3. Place the calibration cross over the 2. Calibration mark in the slide by moving the stage and press Ok. Or cancel the import. - OK4. Place the calibration cross over the 3. Calibration mark in the slide by moving the stage and press Ok. Or cancel the import. - OK5. Save this reference point to file -NO6. Insert the sample slide belonging to the selected xml file and focus on the specimen. Click OK when ready or cancel the import. - OK7. Use the magnification for all imported shapes? - Yes

[0160] In some embodiments, the computer program product executes a method of cutting a membrane, sorting samples, interrogating samples further comprising a step of (e) determining the count and / or proportion of cells and their activation levels in response to different stimulants or different concentrations of a single stimulant (simultaneously or in sequence with the same dataset of a sample). In some embodiments step (e) comprising steps of: (x) comparing the activation level relative to controls at one or more concentrations of the stimulant in a target area to identify or calculate the maximum activation level across all concentrations, the area under the dose response curve, the concentration eliciting the halfmaximum activation (ECso), or other similar metrics; and (y) classifying the sample as having an disorder based upon results of comparing of step (x) relative to a control threshold; wherein each of steps (x) and (y) are performed after step (d). In some embodiments the methods further comprise a step of determining an expression pattern of a probe associated with a biomarker of interest, wherein the biomarker is associated with a protein, nucleic acid or small moleculewithin a target area and correlating the presence, absence or quantity with the target area to the presence, absence or quantity within a control sample.

[0161] Computer-implemented methods also relate to methods of analyzing a sample comprising a target area (a) creating an image of a target area; (b) segmenting the image; and (c) creating a mask for the segmented image that corresponds to a position or plurality of positions within or around the target area. In some embodiments, the methods of analyzing further comprise (d) selecting a cluster of segmentation; (e) aligning the mask on the image of the sample; and (f) importing a contour such that each of steps (a) through (f) create a predetermined path at or around a target area by prior dissection. In some embodiments, the methods relate to methods of dissecting a sample from a cell culture comprising: (a) growing one or a plurality of cells in culture on a biocompatible membrane in the presence of a system comprising a microscope, camera, display and controller all in electronic communication with a processor and memory. In some embodiments, the methods further comprise (b) segmenting an image of the cell culture; (c) creating a mask for the segmented image that corresponds to a position or plurality of positions within or around a target area of the cell culture, wherein the target area may comprise a cell, plurality of cells, biomolecule, extracellular matrix material, or other compound targeted for interrogation, where the presence, absence, morphology and / or quantity of such cells, biomolecule, extracellular matrix material or other compound may be used as a physical feature to characterize the sample or target area. In some embodiments, the methods of analyzing comprise a step of sequencing a nucleic acid or protein identified or isolated on the membrane. In some embodiments, the step of sequencing is performed either within the collection vessel, collection trap or down stream from the collection vessel or collection trap when exposed to an interrogation device.

[0162] In some embodiments, the disclosure relates to a system that comprises at least one processor, a program storage, such as memory, for storing program code executable on the processor, and one or more input / output devices and / or interfaces, such as data communication and / or peripheral devices and / or interfaces. In some embodiments, the user device and computer system or systems are communicably connected by a data communication network, such as a Local Area Network (LAN), the Internet, or the like, which may also be connected to a number of other client and / or server computer systems. The user device and client and / or server computer systems may further include appropriate operating system software. In some embodiments, the system comprises a processor comprising a computer program product for calculating the alignment of known telomeric repeat sequences to the data from a sample registered with the computer program product. In some embodiments, the system comprises adevice that interacts with one or more communication channels or mediums or links, such that alignment processes for telomere sequences of a sample are compared to control sequences stored on a memory and shared with a network in operable communication with the device.

[0163] In some embodiments, components and / or units of the devices described herein may be able to interact through one or more communication channels or mediums or links, for example, a shared access medium, a global communication network, the Internet, the World Wide Web. a wired network, a wireless network, a combination of one or more wired networks and / or one or more wireless networks, one or more communication networks, an a-synchronic or asynchronous wireless network, a synchronic wireless network, a managed wireless network, a non-managed wireless network, a burstable wireless network, a non-burstable wireless network, a scheduled wireless network, a non-scheduled wireless network, or the like.

[0164] Discussions herein utilizing terms such as, for example, “processing,” “computing,” “calculating,” “determining,” or the like, may refer to operation(s) and / or process(es) of a computer, a computing platform, a computing system, or other electronic computing device, that manipulate and / or transform data represented as physical (e.g., electronic) quantities within the computer’s registers and / or memories into other data similarly represented as physical quantities within the computer’s registers and / or memories or other information storage medium that may store instructions to perform operations and / or processes. In some embodiments, the operation comprises one or a plurality of

[0165] Some embodiments may take the form of an entirely hardw are embodiment, an entirely software embodiment, or an embodiment including both hardware and software elements. Some embodiments may be implemented in software, which includes but is not limited to firmware, resident softw are, microcode, or the like.

[0166] Furthermore, some embodiments may take the form of a computer program product accessible from a computer-usable or computer-readable medium providing program code for use by or in connection with a computer or any instruction execution system. For example, a computer-usable or computer-readable medium may be or may include any apparatus that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

[0167] In some embodiments, the medium may be or may include an electronic, magnetic, optical, electromagnetic, InfraRed (IR), or semiconductor system (or apparatus or device) or a propagation medium. Some demonstrative examples of a computer-readable medium may include a semiconductor or solid state memory. magnetic tape, a removable computer diskette, a Random Access Memory (RAM), a Read-Only Memory (ROM), a rigid magnetic disk, anoptical disk, or the like. Some demonstrative examples of optical disks include Compact Disk- Read-Only Memory (CD-ROM), Compact Disk-Read / Write (CD-R / W), DVD. or the like.

[0168] In some embodiments, a data processing system suitable for storing and / or executing program code may include at least one processor coupled directly or indirectly to memory elements, for example, through a system bus. The memory elements may include, for example, local memory employed during actual execution of the program code, bulk storage, and cache memories which may provide temporary storage of at least some program code in order to reduce the number of times code must be retrieved from bulk storage during execution.

[0169] In some embodiments, input / output or I / O devices (including but not limited to keyboards, displays, pointing devices, etc.) may be coupled to the system either directly or through intervening I / O controllers. In some embodiments, network adapters may be coupled to the system to enable the data processing system to become coupled to other data processing systems or remote printers or storage devices, for example, through intervening private or public networks. In some embodiments, modems, cable modems and Ethernet cards are demonstrative examples of t pes of network adapters. Other suitable components may be used.

[0170] Some embodiments may be implemented by software, by hardware, or by any combination of software and / or hardware as may be suitable for specific applications or in accordance with specific design requirements. Some embodiments may include units and / or sub-units, which may be separate of each other or combined together, in whole or in part, and may be implemented using specific, multi-purpose or general processors or controllers. Some embodiments may include buffers, registers, stacks, storage units and / or memory units, for temporary or long-term storage of data or in order to facilitate the operation of particular implementations.

[0171] Some embodiments may be implemented, for example, using a machine-readable medium or article which may store an instruction or a set of instructions that, if executed by a machine, cause the machine to perform method steps and / or operations described herein. Such machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, electronic device, electronic system, computing system, processing system, computer, processor, or the like, and may be implemented using any suitable combination of hardware and / or software. The machine-readable medium or article may include, for example, any suitable type of memory unit, memory' device, memory' article, memory' medium, storage device, storage article, storage medium and / or storage unit; for example, memory, removable or non-removable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk drive, floppy disk, CompactDisk Read Only Memory' (CD-ROM). Compact Disk Recordable (CD-R), Compact Disk Rewriteable (CD-RW), optical disk, magnetic media, various types of Digital Versatile Disks (DVDs), a tape, a cassette, or the like. The instructions may include any suitable type of code, for example, source code, compiled code, interpreted code, executable code, static code, dynamic code, or the like, and may be implemented using any suitable high-level, low-level, object-oriented, visual, compiled and / or interpreted programming language, e.g., C, C++, Java™. BASIC, Pascal, Fortran. Cobol, assembly language, machine code, or the like.

[0172] Many of the functional units described in this specification have been labeled as circuits, in order to more particularly emphasize their implementation independence. For example, a circuit may be implemented as a hardware circuit comprising custom very -large- scale integration (VLSI) circuits or gate arrays, off-the-shelf semiconductors such as logic chips, transistors, or other discrete components. A circuit may also be implemented in programmable hardware devices such as field programmable gate arrays, programmable array logic, programmable logic devices or the like.

[0173] In some embodiment, the circuits may also be implemented in machine-readable medium for execution by various types of processors. An identified circuit of executable code may, for instance, comprise one or more physical or logical blocks of computer instructions, which may, for instance, be organized as an object, procedure, or function. Nevertheless, the executables of an identified circuit need not be physically located together, but may comprise disparate instructions stored in different locations which, when joined logically together, comprise the circuit and achieve the stated purpose for the circuit. Indeed, a circuit of computer readable program code may be a single instruction, or many instructions, and may even be distributed over several different code segments, among different programs, and across several memory devices. Similarly, operational data may be identified and illustrated herein within circuits, and may be embodied in any suitable form and organized within any suitable type of data structure. The operational data may be collected as a single data set, or may be distributed over different locations including over different storage devices, and may exist, at least partially, merely as electronic signals on a system or network.

[0174] The computer readable medium (also referred to herein as machine-readable media or machine-readable content) may be a tangible computer readable storage medium storing the computer readable program code. The computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, holographic, micromechanical, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. As alluded to above, examples of the computer readable storagemedium may include but are not limited to a portable computer diskette, a hard disk, a random access memory’ (RAM), a read-only memory’ (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a portable compact disc read-only memory7(CD-ROM), a digital versatile disc (DVD), an optical storage device, a magnetic storage device, a holographic storage medium, a micromechanical storage device, or any suitable combination of the foregoing. In the context of this document, a computer readable storage medium may be any tangible medium that can contain, and / or store computer readable program code for use by and / or in connection with an instruction execution system, apparatus, or device.

[0175] The computer readable medium may also be a computer readable signal medium. A computer readable signal medium may include a propagated data signal with computer readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such a propagated signal may take any of a variety of forms, including, but not limited to, electrical, electro-magnetic, magnetic, optical, or any suitable combination thereof. A computer readable signal medium may be any computer readable medium that is not a computer readable storage medium and that can communicate, propagate, or transport computer readable program code for use by or in connection with an instruction execution system, apparatus, or device. As also alluded to above, computer readable program code embodied on a computer readable signal medium may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, Radio Frequency (RF). or the like, or any suitable combination of the foregoing. In one embodiment, the computer readable medium may comprise a combination of one or more computer readable storage mediums and one or more computer readable signal mediums. For example, computer readable program code may be both propagated as an electro-magnetic signal through a fiber optic cable for execution by a processor and stored on RAM storage device for execution by the processor.

[0176] Computer readable program code for carry ing out operations for aspects of the present disclosure may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C++ or the like and conventional procedural programming languages, such as the "C programming language or similar programming languages. The computer readable program code may execute entirely on a user’s computer, partly on the user’s computer, as a stand-alone computer-readable package, partly on the user’s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user’s computer through any type of network, including a local area network (LAN) or awide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider).

[0177] The program code may also be stored in a computer readable medium that can direct a computer, other programmable data processing apparatus, or other devices to function in a particular manner, such that the instructions stored in the computer readable medium produce an article of manufacture including instructions which implement the function / act specified in the schematic flowchart diagrams and / or schematic block diagrams block or blocks.

[0178] Turning to Figure 4, the disclosure relates to a computer program product. Instruction for a computer program product comprise:(a) receiving detection data from the device corresponding to the presence, absence or quantity of probes in each compartment;(b) quantifying the amount of probe in each compartment by normalizing the amount of probe in the compartment with the amount of probe in a control compartment; and(c) correlating the amount of probe in each compartment with the number of activated immune cells from a sample in each compartment.Methods

[0179] Methods of the disclosure relate to methods of labeling a cell, interrogating a single cell or biomaterial within a sample, or dissecting a sample from a membrane comprising biomaterial or one or a plurality of cells. Methods of the disclosure also relate to methods of sequencing one or more nucleic acid sequences or amino acid sequences on a sample disclosed herein comprising (i) growing a cell on a biocompatible membrane comprising biomaterials, which comprise one or a plurality of cells; (ii) dissecting the biocompatible membrane operating the laser source component of the disclosed system; and (iii) exposing the one or plurality of cells to one or a plurality of probes. In some embodiments, the methods further comprise a step of (iv) detecting a presence, absence or quantify of probe associated to the cell or cells. A user of the device or system disclosed herein may operate the system to further collect a sample (optionally in the collection region or collection compartment of the system disclosed herein) and then perform step (iv). In some embodiments, the step (ii) may be performed at least partially by operating the computer program product disclosed herein to create a target area on the biocompatible membrane and modify ing settings of the laser source through a controller to them emit a laser for cutting the biocompatible membrane creating a sample. In some embodiments, one or a combination of two or more probes from Table X is used to label the cell or cells are exposed to the cells prior to detecting the probes.

[0180] As noted, methods in some embodiments herein comprise interrogating a single cell or biomaterial within a sample, or dissecting a sample from a membrane comprising biomaterial or one or a plurality of cells. Such a method, in some embodiments, comprises (i) dissecting the biocompatible membrane operating the laser source component of the disclosed system; (ii) collecting individual dissected biocompatible membrane pieces or discrete groups thereof; and (iii) interrogating the collected individual dissected biocompatible membrane pieces or discrete groups thereof. The dissecting may be accomplished as described herein. The dissecting may comprise a cut and push method as described herein. The collecting may be accomplished as described herein and utilizing any system or chip herein. Collecting may include segregating different micro dissected pieces of the membrane based on selected features. The segregating may comprise depositing collected sample, different micro dissected pieces of the membrane, into discrete collection vessels. The different collection vessels may form an array, and the array, in some embodiments, enables tracking of the physical location and / or type of material for each micro dissected pieces (or collection of groups thereof) in each collection vessel of the array. The array, in some embodiments, would then allow assigning the results of downstream interrogation to be applied to the physical location and / or type of material from the membrane. Thus, in some embodiments, methods herein comprise assigning the results if interrogation to a physical location and / or type of material from the membrane. The selected features, in some embodiment, comprise one or more of staining characteristics, physical size, physical shape, etc. The interrogation, in some embodiments, comprises analysis by mass Spec., impedance sorting, or flow cytometry. The analysis, in some embodiments, comprises flow cytometry, impedance sorting, microscopy, mass cytometry, imaging, digital imaging, mass spectrometry, photo spectrometry, nucleic acid sequencing, infrared scanning, or immunohistochemistry, or combinations of two or more thereof.

[0181] Methods of the disclosure include embodiments comprising a step of (i) growing a cell on a biocompatible membrane; (ii) fixing cells on the biocompatible membrane by exposing the cells to a fixing agent such as about 70% ethanol; and then (iii) dissecting the biocompatible membrane to form a sample; (iv) exposing the sample to one or more probes; and (v) washing excess probe form the sample by exposing the sample to a wash solution, such as IX phosphate buffered saline; and then (vi) detecting the presence, absence or quantity of the probe associated to the one or plurality of cells. It should be noted that any biomaterial, such as extracellular matrix protein, may also be stained or alternatively be stained instead of a cell, but the methods of detection or quantifying the probe associated with the biomaterials can be performed in a similar fashion to those steps involving the one or plurality of cells. Afterdata collection, a user may correlate the presence of the probe to the presence or quantity of biomarker in the sample. Users of the disclosed systems may then catalogue or characterize a sample as containing one or more biomaterials, such as cells, by correlating the quantity of a probe in the sample relative to the amount of probe in a control sample. As an example, if the sample contains an unknown number of immune cells or lymphocytes, a user may count the number of immune cells or lymphocytes in the sample by cutting the biocompatible membrane comprising cells to form a sample, collecting the sample, exposing the sample to probes specific for immune biomarkers (such as CD4 or CD8), and quantifying or detecting the presence of the biomarkers in the sample, and then correlating the presence or quantity’ of immune biomarkers in the sample to a known amount of immune biomarkers on a lymphocyte.

[0182] Exposure to probes may comprise exposure to antibodies or antibody fragments. As used herein, an antibody is an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. In some embodiments an antibody molecule is a multispecific antibody molecule, e.g, it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0183] The term “antibody fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g, by binding, steric hinderance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide brudge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies. triabodies, tetrabodies, v-NAR and bis-scFv (see. e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragmentscan also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No.: 6.703,199, which describes fibronectin polypeptide minibodies). The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g, via a synthetic linker, e.g, a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity’ of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker- VL.

[0184] The term “complementarity determining region” or “CDR,” as used herein, refers to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat etal. (1991), “Sequences of Proteins of Immunological Interest.” 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani etal., (1997) JMB 273, 927-948 (“Chothia” numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7. 132-136 (1999); Lefranc, M.-P. et al.. Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme). For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1 ), 52- 56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1 ), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1 ). 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT, the CDR ammo acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to “IMGT”). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0185] The probes of the disclosure may comprise an antibody or antibody fragment thereof and may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al.. 1988. Proc. Natl. Acad. Sci. USA 85:5879-5883: Bird et al.. 1988, Science 242:423-426). In some embodiments, the antigen binding domain of a probe comprises an antibody fragment. In a further embodiment, the probe comprises an antibody fragment that comprises a scFv. In some embodiments, the antibody or antibody fragment is positioned in solution within a receptacle of the reservoir which is in fluid communication with the collection vessel and / or the compartment. In some embodiments, methods of the disclosure comprises exposing the sample to an antibody specific to a biomarker corresponding to a cell identity. In some embodiments, the probe is an antibody comprising a CDR that associates with a tumor associated antigen, a cellular receptor specific for lymphocytes, friboblasts, epithelial cells, endothelial cells, stem cells, mesenchymal cells, neurons, muscle cells, or the like. And detection of the quantity of such a probe would be indicative of the presence of cells specific to the biomarker. In some embodiments, the methods of the disclosure relate to a method of determining cell type or identifying cell type, the method comprising dissecting a sample from the biocompatible membrane, exposing the sample to one or a plurality of probes; and identifying or counting the cell or cells with a character associated with the presence of the probe.

[0186] Methods herein described with reference to a membrane and dissection of the membrane may be adapted to chips and systems comprising a glass slide in place of the membrane. In such methods, steps of microdissection are replaced with steps of ablation. The ablation, in some embodiments, comprises exposure of a portion of the biomaterial, a cell or group of cells on the glass slide to the laser such that the portion of the biomaterial, a cell or group of cells is ablated from the glass and into the fluid within the volume of the chip. Downstream steps of collecting and interrogating the ablated material, in some embodiments, are then carried out similarly to those described with reference to chips and systems including membranes.Table X - ProbesImmunohistochemistry stainings:(horseradish peroxidase or alkaline phosphatase, are used in conjunction with specific antibodies to provide color reactions at sites of antigen-antibody interactions.)Keratin (skin)VimentinS100HMB-45 estrogen and progesterone receptors in breast cancers, protein products of oncogenes (such as HER-2 / neu in breast cancers), antigens associated with tumor cell proliferation such as Ki-67 and the P-glycoprotein product of the multiple drug resistance (MDR) gene recognizing consistent chromosome abnormalities in a growing list of leukemias, lymphomas, and solid tumors, karyotype, FISH or RT-PCR. All of these technologies are labor-intensive and costly which has limited their application. However, higher throughput technologies, such as realtime PCR, are becoming a reality making more routine application realistic. 162 RT-PCR assays also have the advantage of being sensitive as well as specific. As few as 1 abnormal cell in 100,000 can be detected, making this an effective technique for following minimal residual disease posttherapy. avidin-biotin: an unlabeled primary antibody, a biotin-labeled anti-immunoglobulin secondary antibody, and, finally, preformed avidin or streptavidin-biotin-peroxidase complexesVan Gieson’s stain or the Masson trichrome method for distinguishing collagen and muscle, the Weigert’s stain for elastic tissue, silver stains for reticulin fibers, and special stains for mucins, amyloid, lipids, myelin and glycogen — all substances whose identification may aid in the diagnosis of one or another type of tumor. In other instances, enzy me histochemistry may be essential for defining cell lineage, as in certain types of leukemia; for example, chloroacetate esterase or endogenous peroxidase staining for cells of myelomonocytic lineage, alpha naphthyl butyrate esterase (so-called “nonspecific” esterase) staining for monocytes and macrophages. https: / / www.ncbi.nlm.nih.gov / books / NBK557663 / . which is incorporated by reference in its entirety.Hematoxylin and EosinAs the name implies, it is two stains done in subsequent steps. The hematoxylin is a basic dye that stains acidic structures. The resulting color is a purple / blue hue, and structures that are targeted with this dye are named Basophilic. Basophilic structures include DNAin cell nuclei, RNAin ribosomes, and the rough endoplasmic reticulum.fi] Eosin is a counterstain done after hematoxylin and is an acidic dye that targets basic structures. The resulting color is a pink / red hue, and structures that attract eosin are called eosinophilic.fi] The cytoplasm is an example of an eosinophilic structure.Gram StainThe gram stain is a sequential staining technique invented for differentiating bacterial species. Its major utility lies in determining the causative organism of bacterial infection by staining the cell wall. [2] While not all bacteria have a cell wall and thus cannot be stained with this method, it is still a very useful and commonly performed stain. A bacterial sample can be heat-fixed and undergo gram stain with these four steps: Primary staining with crystal violet, secondary staining with grams iodine, decolorized with alcohol or acetone, and counterstained with safranin. Gram-positive bacteria are those that contain a thick layer of peptidoglycan, making them retain the violet stain and appear purple. Alternatively, the gram-negative bacteria have a thin layer of peptidoglycan and more lipids in the cell wall, so the decolorizing step washes out the violet more, and the sample appears pink. [2]Giemsa StainThe Giemsa stain is commonly used in hematology’ for its superior ability to stain bone marrow, plasma cells, and mast cells. It is also very popular for identifying blood parasites. [3] The Giemsa stain can also help to visualize chromosome abnormalities through ‘'Giemsa-Based Banding,” or observing the alternating darker and lighter nucleotide portions on chromosomes during mitosis. [3]Periodic Acid Schiff Reaction StainThe periodic acid Schiff Reaction Stain, often called the PAS stain, is a way to examine structures containing high amounts of carbohydrate molecules, such as the intestinal brush border, renal tubular cells, mucus, and reticular fibers of connective tissue. [4] The glycogen, glycoprotein, glycolipids, and mucins stain red or magenta color when the stain is complete.The periodic acid, a highly oxidized iodine, oxidizes the hydroxyl groups of adjacent sugar molecules to produce aldehydes. After this step, the Schiff reagent attaches to the aldehyde and forms a red magenta color for visualization. [5] [6]Masson’s TrichromeMasson’s Trichrome Stain is a stain that can yield a multicolor result on the tissue. Even though it has red counterstains, it is popular for its ability to stain collagen fibers blue. Masson’s Trichrome can identify cardiac fibrosis, pulmonary fibrosis, chronic kidney disease, and muscular dystrophy. [7]Congo RedCongo red is a water-soluble blue dye that produces a red solution at a pH of 3.0-5.0. Its many aromatic rings can stack together through hydrophobic interactions and collect in tissue. Most notably, Congo red can stain amyloid fibers red and orange color, making it a useful study in amyloidosis. When viewed under polarized light in a microscope, Congo-red-stained tissues high in amyloid will show with bright “apple” green birefringence. PMID: 181Prussian BlueThe Prussian blue stain is useful for identifying iron stores in the body. [7] [9] The stain works by first staining the tissue with hydrochloric acid and then seeing the ferric ions react to form the insoluble bright blue pigment. It is useful in diagnosing iron accumulation states like hemochromatosis or hemosiderosis through staining liver tissue and seeing the build-up of iron near the peri-portal hepatocytes or along the sinusoidal lining.

[0010] [l 1] An overabundance in iron stores within bone marrow could signal ineffective erythropoiesis, like in anemia of chronic disease. Alternatively, absent reaction to the Prussian blue stain could indicate low iron levels, like in iron deficiency anemia.MucicarmineMucicarmine stains mucin, a secretion produced in epithelial and connective tissue cells.

[0012] The aluminum and carmine combine to form a positively charged chelating complex. The newly positive charge binds the mucin, stains it red, and allows visualization. It is useful in identifying potential carcinomas and inflammatory' conditions, where there is excessive mucin production.

[0012] In surgery, mucicarmine staining can also determine a primary tumor locationby staining the mucus-secreting epithelium in a site not containing mucin-producing cells.

[0013] Mucicarmine also stains the gelatinous capsule of fungi Cryptococcus.

[0012] Sudan BlackThe Sudan Black dye stains lipid-containing structures like triglycerides and lipoproteins, a dark black or brown color.[7][6]The tissue preparation for Sudan Black and Oil Red O skips the alcohol dehydration step to avoid washing away the lipids to be stained. It may be used to diagnose atherosclerosis by staining atherosclerotic plaques and autosomal dominant leukodystrophy by staining macrophages in white matter after a post-mortem brain biopsy. [7]Oil Red OSimilar to the Sudan Black dye, Oil Red O is the most common dye used on hydrophobic fat or lipids, substances that are traditionally difficult to stain. Oil Red O has high utility in visualizing atherosclerotic plaques and hepatic and muscular lipid accumulation. [7] [6]

[0014] Silver StainSilver stains are a larger category of stains used for the histopathological study of accumulation-based diseases in neurology. There are several methods to silver staining, including Bielschowsky, Galiyas, Bodian, and Campbell-Switzer. The staining method chosen is dependent on the neurological lesion in question, as each method speaks to a differing sensitivity and specificity. In general, the methods attach silver ions or salt complexes to the target tissue. Then, they must be reduced in situ, and the subsequent silver particles accumulate and can be analyzed.

[0015] Recently, there has been the use of fluorolabeling, where a fluorogenic semiconductor releases small 6nm nanoparticles at the silver depositions and produces colors. The diameter range of the silver particles that form correlates to different colors. For example, ranges in the 10 to 20 nm yield range of yellow colors whereas diameters exceeding lOOnm yield a black color.

[0016] The silver stains are very known for detecting amyloid beta-protein (AB) in Alzheimer’s disease and Pick bodies in Picks Disease. When dyed, the amyloid plaques become darker. They can range from yellow to black, depending on the size or amount of amyloid plaques.

[0015] Nissl StainThe Nissl Stain, also called the Cresyl Violet Stain, uses basic aniline dye to study neuronal structure in the brain and spinal cord. Neuropil stains blueish purple and granular. The Nissl substance has a high amount of ribosomal RNA, thus attracting the dye, appearing dark blue, and making the cytoplasm appear mottled. The advantage of using a Nissl stain for evaluating neuronal pathology is that it will recognizably stain the neuronal cytoplasm without staining the perikarya of other cell structures, like astrocytes.

[0017] Papanicolaou StainThe Papanicolaou stain colloquially referred to as the Pap smear is a cytological staining technique best known for detecting cervical cancer in female patients. The cells to be stained are collected from gynecological smears, sputum samples, brushings, fine needle aspiration materials, and washings. The multichromatic stain involves five dyes: Hematoxylin for the nucleus, Orange G for keratin, eosin for superficial structures, Light Green SF for cytoplasm, and Bismarck Brown.

[0018] In the setting of a cervical cancer screening Pap smear, the resulting stain of the epithelial cells from the transitional zone of the cervix undergo analysis for precancerous and cancerous processes. Often a second slide will be prepared for the immunostaining with the biomarker p!6INK4a for identifying dysplasia.

[0019]

[0187] The disclosure also relates to a method of operating the aforementioned systems (including all disclosed methods such as methods of labeling, methods of interrogating a sample and methods of sequencing nucleic acid or protein in a sample) comprising a step of calibrating the laser source and microscope in the system prior to a step of cutting the biocompatible membrane. One illustrative method of calibrating is depicted in FIG. 5G, in which large bubbles of sizes greater than about 100 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area (left panel). Settings on the laser source can be adjusted in real time by simultaneously lowering the laser power level and monitoring the production of bubbles in the compartment by way of the realtime imaging of the camera mounted microscope, such that the pow er is decreased until the number and size of bubbles are significantly reduced or eliminated prior to the step of cutting. In some embodiments, the method of calibrating the laser source comprises activating and directing a laser light positioned at or near the compartment into a solution of the compartment that is present in the compartment, or at or near a peripheral position on the biocompatible membrane distal from a target area of a sample at a power sufficient to produce large bubblesin the solution in the volume, and subsequently reducing the power levels such that the large bubbles are significantly reduced or not produced. In some embodiments, the step of calibrating is performed until fewer than about 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or about 1 large bubble is produced upon operating the laser in or on top of the fluid in the fluid circuit. In some embodiments, large bubbles of sizes greater than about 100 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 80 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 90 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 70 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 60 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 50 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the powder of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 40 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 30 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 20 microns in diameter are formed in the solution when the laser hits the membrane at a positioned aw ay from a target area, and the powder of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 10 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and thepower of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 5 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 1 micron in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field. In some embodiments, large bubbles of sizes greater than about 0.5 microns in diameter are formed in the solution when the laser hits the membrane at a positioned away from a target area, and the power of the laser source is adjusted until the large bubbles are eliminated in a visual field.

[0188] Methods of the disclosure relate to a method of automatically calibrating a laser source prior to its use with the systems disclosed herein comprising a laser source, electrically and operably connected to: a controller operating a display, a memory7, a microscope optically- aligned with a compartment comprising a biocompatible membrane in a fluid circuit, and a camera, wherein the controller and accompanying non-transient computer program product or products that control and adjust setting of the laser source are available to a user on a computer; the method comprising (i)operating the laser source comprises activating and directing a laser light positioned at or near the compartment into a solution of the compartment that is present in the compartment, or at or near a peripheral position on the biocompatible membrane distal from a target area of a sample, at a power sufficient to produce large bubbles in the solution in the volume, and subsequently (ii) reducing the power levels of the laser source such that the large bubbles are significantly reduced in number or not produced when the laser light is in contact with a portion of the biocompatible membrane or fluid in the fluid circuit.EXAMPLESEXAMPLE 1Materials and Methods

[0189] 5pm FFPE tonsil section was picked up from a 42°C water bath with a 4pm polyphenylene sulfide membrane on a metal frame slide (MicroDissect GmbH). The section was deparaffmized, rehydrated, and stained with Toluidine blue (Merck) following standard protocols. The flow cell was attached to the membrane by coating the attachment surface with cyanoacrylate (Starbond) and pressing it on the membrane enclosing the tissue. Inlet and outlet tubing with metal connectors (Elveflow) are connected to the inlet and outlet of the flow cellby perforating the membrane and inserting the metal connectors. LC / MS grade Ethanol (LiChrosolv®) is pushed into the chamber with a syringe connected to the tubing until it is filled.

[0190] A frame slide with the attached flow cell is mounted on the LMD (Leica) with the tissue facing down. The laser was calibrated in the software (LMD Version 8.3) by setting focus on the membrane and cutting and clicking on 4 crosses. The laser settings were set to standard values (Power: 6, 7; Aperture 8; Head current: >85%, Middle Pulse: 3; Pulse Frequency: 3000. Speed: 3-5, Final pulse: 8) used for dry cutting of 5pm tissue on a PPS membrane with the 63x objective. The power and middle pulse were adjusted on a peripheral tissue part to test cutting and push down into Ethanol.

[0191] The contours were drawn around the Toluidine-stained cells and cutting was started. The contours were pushed into Ethanol by the middle pulse. After cutting pressure was applied via the inlet syringe and contours accumulated at the outlet of the flow cell.Summary A:

[0192] 5pm FFPE tonsil section was picked up from a 42°C water bath with a 4pm polyphenylene sulfide membrane on a metal frame slide (MicroDissect GmbH). The section was deparaflfinized, rehydrated, and stained with Toluidine blue (Merck) following standard protocols. The flow cell was attached to the membrane by coating the attachment surface with cyanoacrylate (Starbond) and pressing it on the membrane enclosing the tissue. Inlet and outlet tubing with metal connectors (Elveflow) are connected to the inlet and outlet of the flow cell by perforating the membrane and inserting the metal connectors. LC / MS grade Ethanol (LiChrosolv®) is pushed into the chamber with a syringe connected to the tubing until it is filled. The frame slide with the attached flow cell is mounted on the LMD (Leica) with the tissue facing down. The laser was calibrated in the software (LMD Version 8.3) by setting focus on the membrane and cutting and clicking on 4 crosses. The laser settings were set to standard values (Power: 6, 7; Aperture 8; Head current: >85%, Middle Pulse: 3; Pulse Frequency: 3000, Speed: 3-5, Final pulse: 8) used for dry cutting of 5pm tissue on a PPS membrane with the 63x objective. The power and middle pulse were adjusted on a peripheral tissue part to test cutting and push down into Ethanol. The contours were drawn around the Toluidine-stained cells and cutting was started. The contours were pushed into Ethanol by the middle pulse. After cutting pressure was applied via the inlet syringe and contours accumulated at the outlet of the flow cell.EXAMPLE 2 (PROPHETIC EXAMPLE)

[0193] 5pm FFPE tonsil tissue section will be picked up from a 42°C water bath with a 4pm polyphenylene sulfide membrane on a metal frame slide (MicroDissect GmbH). The flow cell will be attached to the membrane immediately after sectioning by peeling the foil to expose the adhesive layer of the acrylic double adhesive (Horae New Material Co., Ltd.) and pressing it on the membrane enclosing the tissue. The slide will be places on a heat plate set 55°C for 20min. Inlet and outlet tubing will be connected to the inlet and outlet of the flow cell by inserting the metal connectors (Elveflow) and perforating the membrane. The slide with the flow cell will be mounted on the LMD (Leica) microscope stage. The tissue is deparaffinized and rehydrated by flowing 2mL Xylene at 10-100pL / min followed by 2mL Ethanol at 10- lOOpL / min in decreasing concentration (100%, 90%, 80%, 70%, 50%, 30%, 0%) with an increasing concentration of distilled water. Antigen retrieval is carried out by flowing Citrate buffer pH6 over a heated tubing line set to 90°C for 30min at lOOpL / min followed by a PBS wash. The tissue will be stained afterward with Hoechst Ipg / mL, a cell membrane marker, and proteins of interest at room temperature following standard indirect immunofluorescence staining protocol. The whole tissue section will be imaged at 20x magnification according to fluorophore wavelengths. The sample will be stored at 4°C until dissection. Inlet / outlet will be disconnected and sealed with tape. Images will be segmented with Stardist and watershed in real time and identified cells will be clustered based on selected features (e.g. shape, signal intensity, protein coexpression, cell location). Three alignment points are selected on distant edges of the tissue image covering the whole section. Cells matching a cluster are saved in xml format and imported into the LMD software. For import, the same alignment points selected on the image are selected on the physical sample.

[0194] Alternative to the above method with instant segmentation and clustering no storage of sample needed since 4°C cooled enclosure around stage: The whole tissue section will be imaged at 20x magnification according to fluorophore wavelengths. Images with stored stage position will be segmented in real-time with Stardist and Watershed and identified cells will be clustered based on predefined features (e.g. shape, signal intensity, protein coexpression, cell location). All cell IDs with clusters are saved in one xml file and reimported into the imaging / cutting softw are. The contours of all clusters are automatically aligned based on the stored stage position. Starting from standard laser settings at 63x magnification (Power: 10; Aperture 8; Head current: >85%, Middle Pulse: 3; Pulse Frequency: 3000, Speed: 3-5, Final pulse: 8) laser calibration is first done on an empty membrane part with three crosses to adjustfocus, second the power is calibrated on a peripheral part of the tissue cutting a straight line. The power is decreasingly adjusted while cutting until no plasma bubbles are detected (using a pretrained model for bubble detection). Laser cutting will be initiated by cutting cluster after cluster starting with contours that are closest to the outlet. Contours will be excised by the laser and pushed into the Ethanol layer moving out the contour at constant flow at 200pL / min. The contour will be registered by the detector in the distributor chamber and moved to the first collection chamber. After cutting the first cluster the first collection chamber releases the contours towards the secondary detector workflow. In some embodiments, after one full cluster will have been cut the contours will be moved out of the flow cell through the outlet at about 200pL / min into the second chip system.EXAMPLE 3

[0195] Experiments varying the rate of flow at the inlet and outlet were conducted on a chip of FIGS. 9A through 9E. In these experiments, outlet point 940a was operably connected to a pump drawing fluid within the volume 960 out and the inlet points 930a, 930b, 930c and 930d were operable connected to a pump pushing fluid into the volume p960. That is, the system of this experiment included a dual-pump system.

[0196] After microdissection of a shape from the membrane, fluid flow was controlled in order to collect a shape, or membraned contour, into a collection vessel. FIG. 10A shows deposit of the membraned contour in the collection chamber. In this example, the collection chamber is a well of the 96-well plate.

[0197] The dual-pump system operably connected to the inlet and outlet allows for fluid to enter the chamber under control of pumps to balance the fluid flow rate. When aligned to form the fluid circuit a first set of tubing is positioned in fluid communication with inlet 930. Fluid flow can be directed from inlet 930 to outlet points 940a and then to outlet 940. In this example, the width of the interior chamber was about 20.30 millimeters as depicted in FIG. 9D and the length of the interior chamber was about 13. 197 millimeters as depicted in FIG. 9E. The volume 960 in this example was about 134 pl, while the fluid transport volume of w as about 170 pl, which is the liquid volume to transport fluid through the system.

[0198] FIG. 10B shows the synchronization of both the inlet and outlet pumps to achieve proper flow through the chip system. The pumps generate fluid pressure within the fluid circuit to control the fluid flow' rate of tissue culture medium through the system. The cell tissue medium entered the system under control of the inlet pump and through the inlet opening, flowed through the cell at a flow controlled by the pumps, exiting the interior of the chip through the outlet tubing controlled by the outlet pump, which then and empties into acollection vessel, which was a syringe operably connected to a valve. Referring to FIG. 10B, the membrane was cut as it would be during use with laser, leaving holes indicated by black arrowheads. As depicted in FIG. 1 OB, the synchronization of the inlet and outlet pumps resulted in retention of fluid inside the chamber rather than escape through cut holes on the membrane. This control of the fluid flow allows for repeating cut and capture on a single membrane multiple times without losing fluid through cut holes. Further, the dual-pump control avoids ballooning of the membrane, as illustrated in FIG. IOC, which depicts liquid overflow induced by overpressure of the inlet pump. Proper synchronization of the inlet and outlet pumps controls fluid flow such that ballooning of the membrane is avoided.EXAMPLE 4

[0199] Experiments covering deposition of a cut membrane fragment into the fluid flow were performed. FIG. 11 illustrates a cut membrane fragment released into the fluid flow. The arrowhead in FIG. 11 indicates a locus of damage, which was caused by a centered pulse of the laser used to push the cut membrane fragment out of its position in the membrane and into the fluid flow.

[0200] A “cut and push” technique was employed in this example in order to avoid or minimize such a locus of damage. The cut and push technique is a two-step method. The first step of the method uses the laser source to cut into the perimeter of a cell or plurality of cells. The laser settings were set to standard values (Power: 6, 7; Aperture 8; Head current >85%, Middle Pulse: 3; Pulse Frequency: 3000, Speed: 3-5. Final pulse: 8). The height of the laser over the membrane was then adjusted upwardly to push the cut membrane piece out of the membrane with a laser push of lower intensity and wider diameter as compared to the initial cutting setting.EXAMPLE 5

[0201] As noted above, some embodiments include a glass surface in place of the membrane. In these embodiments, sample is harvested similarly, but relies on release of sample from the surface of the glass and into the fluid as a result of the laser. In this example, a standard, non-treated microscope slide was used to demonstrate successful ablation and deposition into the collection chamber.

[0202] The glass slide is the solid support used in this example on which the cell, plurality of cells, or membrane rests. The glass slide is positioned over a target surface containing a sample of interest, which can be a cell or a plurality of cells. A laser source (not depicted) generates a laser beam directed through the glass slides and at a point on the cell, plurality of cells, or membrane. FIG. 12A depicts multiple spots of different sizes and shapes that can beablated from the glass slide. These shapes depicted in the area represent the perimeter around target areas of the cell, plurality’ of cells, or membrane within which a user is interested to magnify, interrogate, and / or examine further. The target area is removed through ablation from the glass. FIG. 12B shows that multiple shapes can be repeatedly ablated from a single glass slide. FIG. 12C shows ablated tissue in the liquid of the collection chamber. FIG. 12D illustrates a schematic of the system used in this example, which constitutes another embodiment herein. Advantages of glass ablation include the following: (1) Versatile ablation shapes, unlike prior systems limited to squares. (2) Glass ablation allows for single-cell capability to target single cells, unlike prior systems. (3) Improved tissue capture with the ability’ to capture ablated tissue in liquid and transport to collection chambers, which is in contrast to prior systems ablating dried samples. (4) No holes or perforation in the membrane, which eliminates issues related to membrane perforation. (5) Less precision needed for pumps: glass ablation also allows for reduced alignment complexify as compared to flexible membranes. (5) Definite ablation and disposal. Clear ablation processing and disposal into the collection chamber is achieved. (6) No plastics.EXAMPLE 6

[0203] Cell Analysis

[0204] A system herein was implemented to isolate and then pool ten HeLa cells, which were then interrogated via mass Spec, to determine proteins present in the sample. The below table shows proteins detected and their target class from this experiment. The proteins detected are verified as signature of HeLa cells. These results validate the use of a system herein to isolate and analyze cells.Protein Target ClassPSMA1 Proteasome subunitPSMA2 Proteasome subunitPSMA3 Proteasome subunitPSMA4 Proteasome subunitPSMA5 Proteasome subunitPSMA6 Proteasome subunitPSMA7 Proteasome subunitPSMB1 Proteasome subunitPSMB2 Proteasome subunitPSMB3 Proteasome subunitPSMB4 Proteasome subunitPSMB5 Proteasome subunitPSMB6 Proteasome subunit EGFR Tyrosine Kinase MAP2K1 MAPK Kinase PARP1 DNA Repair XRCC5 DNA Repair XRCC6 DNA Repair IDH1 TCA Cycle VIM Cytoskeleton PKM Glycolysis LDHA Lactate Metabolism LDHB Lactate Metabolism HK1 Glycolysis PFKM Glycolysis PFKP Glycolysis GAPDH Glycolysis EN01 Glycolysis G6PD Pentose Phosphate SLC7A5 Amino Acid TransportHSP90AA1 Molecular Chaperone HSP90AB1 Molecular Chaperone HSP90B1 ER Chaperone HSPA4 Heat Shock Protein HSPA5 ER Stress Response HSPA8 Constitutive HSP HSPA9 Mitochondrial HSP HSPB1 Small HSP HSPD1 Mitochondrial Chaperone HSPE1 Chaperonin Co-factor

Claims

CLAIMS1. A sy stem compri sing :(a) a biocompatible membrane comprising a top surface and a bottom surface;(b) one or a plurality of cells positioned on the top surface of the biocompatible membrane;(c) a laser positioned beneath the membrane and at a distance of from about 0.1 millimeters to about 12,000 millimeters from the bottom surface of the biocompatible membrane;(d) one or a plurality' of enclosure elements positioned above the top surface of the biocompatible membrane that define a compartment and a compartment volume around the biocompatible membrane and the one or plurality of cells.

2. The system of claim 1, wherein the one or plurality7of enclosure elements comprise an inlet and an outlet on at least one side of the compartment, the inlet and outlet in fluid communication with a microfluidic circuit.

3. The system of claims 1 or 2, wherein the membrane and one or plurality7of cells have a height of from about 2 microns to about 10 microns.

4. The system of any of claims 1 through 3 further comprising a microscope operably connected to a controller, display and a camera; wherein the microscope positioned at or proximate to the biocompatible membrane at a distance sufficient for magnification and visualization of the one or plurality of cells in a visual field.

5. The system of claim 2, wherein the microfluidic circuit comprises a pump, a cell culture media reservoir and heating element, the pump adjustable for flow rate of cell culture medium through the microfluidic circuit.

6. The system of any of claims 1 through 5 further comprising a vessel positioned distally from the compartment and in fluid communication with the compartment volume by a fluid conduit.

7. The system of any of claims 1 through 6, wherein the vessel and conduit are free of air of greater than about 6% within the compartment.

8. The system of any of claims 1 through 7, wherein the system comprising a first operational mode in which light from the laser contacts the biocompatible membrane at an intersection point; wherein the electrostatic friction at the intersection point is less than about 2 nC.

9. The system of any of claims 1 through 8. wherein the system is free of oil positioned at or proximate to the membrane surface.

10. The system of any of claims 1 through 9, wherein the biocompatible membrane comprises polypropylene. PC (polycarbonate), PCL (poly(e-caprolactone)). PDMS (polydimethylsiloxane), PE (polyester), SiN (Silicon nitride), SiO2 (silicon dioxide), or a combination thereof.

11. The system of claim 6 further comprising a flow cytometer, a mass spectrometer, a digital scanner or a combination thereof operably connected to and in fluid connection with the vessel.

12. The system of any of claims 1 thorough 11. wherein the biocompatible membrane comprises one or a plurality of UPCs, shapes or probes printed on the bottom surface at one or a plurality of discrete positions beneath the one or plurality of cells.

13. The system of claim 4 wherein the controller comprises a computer program product with instructions for:(a) pixelating an image of the one or plurality of cells and the biocompatible membrane;(b) determining one or a plurality of positions in the image around a cell that correspond to the pixels of the image and that define a point or series of points of a perimeter around a single cell or plurality of cells;(c) directing the laser to emit a pulse of light at or proximate to the point or series of points defining the perimeter, such that the cell or plurality of cells are cut from the biocompatible membrane at the point or series of points with a single emission from the laser.

14. The system of any of claims 1 through 13, wherein the cell or plurality of cells comprise one or more DNA barcodes on their surface.

15. The system of claim 5, wherein the microfluidic circuit is a closed system with one or more adjustable valves configured to increase or decrease the fluid flow through the microfluidic circuit and the compartment.

16. A method of isolating a cell in a system comprising:(a) a biocompatible membrane comprising atop surface and a bottom surface;(b) one or a plurality of cells positioned on the top surface of the biocompatible membrane;(c) a laser source positioned beneath the membrane and at a distance of from about 10 millimeters to about 9,000 millimeters from the bottom surface of the biocompatible membrane; and(d) one or a plurality of enclosure elements positioned above the top surface of the biocompatible membrane that define a compartment and a compartment volume around the biocompatible membrane and the one or plurality of cells; the method comprising:(i) directing a light beam from the laser into the membrane and around a perimeter of one or a plurality of cells.(b) The method of claim 16 further comprising the step of predetermining a set of positions on the membrane corresponding to a perimeter around one or plurality of cells, wherein (x) is performed prior to step (i).(c) The method of claim either of claims 16 or 17, wherein the system comprises a collection vessel positioned distally from the membrane and the compartment, the collection vessel in fluid communication with the compartment by at least one fluid conduit.(d) The method of any of claims 16 through 18 further comprising the step of imaging the cells prior to step (i).(e) The method of any of claims 16 through 19, wherein the one or plurality of enclosure elements comprise an inlet and an outlet on at least one side of the compartment, the inlet andoutlet in fluid communication with a microfluidic circuit; and wherein the method further comprises a step of allowing the fluid flow in the microfluidic circuit to carry the one or plurality of cells on the cut by step (i) through the outlet and into a collection vessel.

21. The method of claim 20 wherein the fluid flow rate is from about 10 microliters per second to about 50 microliters per second.

22. The method of any of claims 16 through 21, wherein the method further comprises a step of imaging the one or pl urality of cells after step (i).

23. The method of any of claim 16 through 22. wherein the method further comprises exposing the cell to one or a plurality of antibodies or antibody fragments.

24. The method of any of claims 16 through 23, wherein the biocompatible membrane comprises one or a plurality of UPCs. barcodes, shapes, probes, or combinations thereof, printed on the bottom surface at one or a plurality of discrete positions beneath the one or plurality of cells; and wherein the method further comprises a step of detecting the one or plurality of UPCs, barcodes, shapes, probes, or combinations thereof.

25. The method of claim 24 further comprising the step of characterizing or isolating the cell based upon the presence, absence or quantity of UPCs, barcodes, shapes, probes, or combinations thereof.

26. A method of interrogating a cell or plurality of cells in a system of any of claims 1 through 15, the method comprising:(i) directing laser light from the laser source into the membrane and around a perimeter of one or a plurality of cells.

27. The method of claim 26 further comprising the step of predetermining a set of positions on the membrane corresponding to a perimeter around one or plurality of cells, wherein (x) is performed prior to step (i).

28. The method of claim either of claims 26 or 27, wherein the system comprises a collection vessel positioned distally from the membrane and the compartment, the collection vessel in fluid communication with the compartment by at least one fluid conduit.

29. The method of any of claims 26 through 28 further comprising the step of imaging the cells prior to step (i).

30. The method of any of claims 26 through 29, wherein the one or plurality of enclosure elements comprise an inlet and an outlet on at least one side of the compartment, the inlet and outlet in fluid communication with a microfluidic circuit; and wherein the method further comprises a step of allowing the fluid flow in the microfluidic circuit to carry the one or plurality of cells on the cut by step (i) through the outlet and into a collection vessel.

31. The method of claim 30 wherein the fluid flow rate is from about 10 microliters per second to about 50 microliters per second, optionally in a volume of tissue culture medium32. The method of any of claims 26 through 31, wherein the method further comprises a step of imaging the one or plurality of cells after step (i).

33. The method of any of claim 26 through 32, wherein the method further comprises exposing the cell to one or a plurality of antibodies or antibody fragments.

34. The method of any of claims 26 through 33, wherein the biocompatible membrane comprises one or a plurality of UPCs, barcodes, shapes, probes, or combinations thereof, printed on the bottom surface at one or a plurality’ of discrete positions beneath the one or plurality’ of cells; and wherein the method further comprises a step of detecting the one or plurality of UPCs, barcodes, shapes, probes, or combinations thereof.

35. A method of detecting a probe on one or a plurality’ of cells in a system of any of claims 1 through 15, the method comprising:(i) directing a light beam from the laser into the membrane and around a perimeter of one or a plurality of cells.

36. The method of claim 35 further comprising the step of predetermining a set of positions on the membrane corresponding to a perimeter around one or plurality of cells, wherein (x) is performed prior to step (i).

37. The method of claim either of claims 35 or 36, wherein the system comprises a collection vessel positioned distally from the membrane and the compartment, the collection vessel in fluid communication with the compartment by at least one fluid conduit.

38. The method of any of claims 35 through 37 further comprising the step of imaging the cells prior to step (i).

39. The method of any of claims 35 through 38, wherein the one or plurality of enclosure elements comprise an inlet and an outlet on at least one side of the compartment, the inlet and outlet in fluid communication with a microfluidic circuit; and wherein the method further comprises a step of allowing the fluid flow in the microfluidic circuit to carry the one or plurality of cells on the cut by step (i) through the outlet and into a collection vessel.

40. The method of claim 39 wherein the fluid flow rate is from about 10 microliters per second to about 50 microliters per second, optionally in a volume of tissue culture medium41. The method of any of claims 35 through 40, wherein the method further comprises a step of imaging the one or plurality of cells after step (i).

42. The method of any of claim 35 through 41, wherein the method further comprises exposing the cell to one or a plurality' of probes.

43. The method of claim 42, wherein the probe comprises an antibody or antibody fragment.

44. The method of any of claims 42 or 43, and further comprising a step of detecting the one or plurality of probes.

45. The method of any of claims 35 through 41, further comprising a step of detecting the one or plurality of UPCs. barcodes, shapes, probes, or combinations thereof at or positioned on the biocompatible membrane.

46. A method of labeling a cell in a system of any of claims 1 through 15, the method comprising:(i) directing a light beam from the laser into the membrane and around a perimeter of one or a plurality of cells.

47. The method of claim 46 further comprising the step of predetermining a set of positions on the membrane corresponding to a perimeter around one or plurality of cells, wherein (x) is performed prior to step (i).

48. The method of claim either of claims 46 or 47, wherein the system comprises a collection vessel positioned distally from the membrane and the compartment, the collection vessel in fluid communication with the compartment by at least one fluid conduit.

49. The method of any of claims 46 through 48 further comprising the step of imaging the cells prior to step (i).

50. The method of any of claims 46 through 49, wherein the one or plurality' of enclosure elements comprise an inlet and an outlet on at least one side of the compartment, the inlet and outlet in fluid communication with a microfluidic circuit; and wherein the method further comprises a step of allowing the fluid flow in the microfluidic circuit to carry the one or plurality of cells on the cut by step (i) through the outlet and into a collection vessel.

51. The method of claim 50 wherein the fluid flow rate is from about 10 microliters per second to about 50 microliters per second, optionally in a volume of tissue culture medium52. The method of any of claims 46 through 51, wherein the method further comprises a step of imaging the one or plurality' of cells after step (i).

53. The method of any of claim 46 through 52, wherein the method further comprises exposing the cell to one or a plurality of probes.

54. A method of sorting one or a plurality of cells in a system comprising:(a) a biocompatible membrane comprising atop surface and a bottom surface;(b) one or a plurality of cells positioned on the top surface of the biocompatible membrane;(c) a laser positioned beneath the membrane and at a distance of from about 10 millimeters to about 9,000 millimeters from the bottom surface of the biocompatible membrane; and(d) one or a plurality of enclosure elements positioned above the top surface of the biocompatible membrane that define a compartment and a compartment volume around the biocompatible membrane and the one or plurality of cells; the method comprising:(i) directing a light beam from the laser into the membrane and around a perimeter of one or a plurality of cells, resulting in a fragment of biocompatible membrane comprising the one or plurality of cells;(ii) allowing a time period to elapse for transfer of the fragment of biocompatible membrane to a collection vessel;(iii) labeling the one or plurality of cells with one or more probes; and(iv) performing cell sorting based upon the detection of the one or more probes.

55. The method of claim 54, wherein the cell sorting is fluorescently activated cell sorting and the probes are antibodies targeting one or more cell surface proteins expressed by the one or plurality of cells.

56. A method of microdissecting one or a plurality of cells from a tissue culture in a system of any of claims 1 through 15 comprising:(i) directing a light beam from the laser into the membrane and around a perimeter of one or a plurality of cells adherent to the biocompatible membrane, thereby obtaining a portion of the biocompatible membrane comprising the one or plurality of cells.

57. The method of claim 56 further comprising the step of predetermining a set of positions on the membrane corresponding to a perimeter around one or plurality of cells, wherein (x) is performed prior to step (i).

58. The method of claim either of claims 56 or 57, wherein the system comprises a collection vessel positioned distally from the membrane and the compartment, the collection vessel in fluid communication with the compartment by at least one fluid conduit.

59. The method of any of claims 56 through 58 further comprising the step of imaging the cells prior to step (i).

60. The method of any of claims 56 through 59 further comprising the step of (ii) allowing a time period for the portion of biocompatible membrane to separate from the biocompatible membrane; and (iii) collecting the portion of the biocompatible membrane in a collection vessel.

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