MICROPLATE CARRIER

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/336,927 filed April 29, 2022, and U.S. Provisional Application No. 63/347,085, filed May 31, 2022, each of which are incorporated herein by reference in their entirety and for all purposes.

BACKGROUND

[0002] A tremendous interest in nucleic acid characterization tools was spurred by the mapping and sequencing of the human genome. Beyond reading genomic DNA and RNA, the combination of nucleic acid sequencing and detectable probes attached to antibodies introduces a wide range of multiomics applications, including imaging and measuring gene transcription and protein expression in individual cells and tissue pathology samples contained within a microplate. Typical microplates are made from plastic polymers (e.g., polypropylene). However, common plastic polymers, such as polypropylene and polyethylene, are susceptible to degradations issues. For example, biological analyses often require incubation with abrasive chemicals and/or significant thermal shifts (e.g., about 20°C to about 100°C). Thus, there is a need to develop stable microplates. Described herein, inter alia, are solutions to these and other problems in the art.

BRIEF SUMMARY

[0003] In an aspect, a microplate carrier, alternatively referred to herein as a microplate assembly, is provided. In embodiments, the microplate carrier includes one or more microplate insert(s), and the microplate carrier is configured to retain the microplate insert(s). The microplate insert includes a planar support.

[0004] The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 A illustrates a microplate with a plastic bottom or a glass bottom, wherein a primary well plate 101 is fused to a planar support piece 103 of a material such as glass or plastic 103 that is housed in a microplate carrier 102. FIG. IB illustrates the fully assembled microplate with a plastic bottom or a glass bottom 104.

[0006] FIGS. 2A-2B illustrates an embodiment of a microplate receiver frame 201 configured to retain one or more microplate inserts 202 therein. The microplate insert(s) includes a planar support (e.g., glass bottom) fused to a primary well plate. The microplate receiver frame 201 and microplate insert 202 may each include one or more respective biasing or retention elements 203 that bias and/or retain the microplate insert 202 relative to the microplate receiver frame 201. The dashed lines in FIG. 2 A are indicative of placement of the microplate insert(s) 202 into the microplate receiver frame 201. FIG. 2B illustrates the fully assembled microplate with a plastic bottom or a glass bottom (204). FIG. 2C illustrates an embodiment of the microplate receiver frame 201, including one or more biasing features 203, and a platform 205 configured to contact the planar support of the microplate insert.

[0007] FIGS. 3A-3D depicts an embodiment of the microplate insert 301. FIG. 3 A shows a top view of a 24-well microplate insert, FIG. 3B shows a perspective view of the 24-well microplate insert, and FIG. 3C shows a side view of the 24-well microplate insert. In embodiments, the microplate insert includes 24 wells, wherein each well is 7.3 mm wide and separated by 1.6 mm of interstitial space. In embodiments, the microplate insert is about 26.9+/-0.5 mm x 76.8+/-0.5 mm. FIG. 3C shows a planar support 302, wherein the planar support (e.g., the glass bottom) is about 0.7 mm thick. FIG. 3D depicts an assembled microplate carrier, including four microplate inserts.

[0008] FIGS. 4A-4D depicts an embodiment of the microplate insert. FIG. 4 A shows a top view of a 12-well microplate insert, FIG. 4B shows a perspective view of the 12-well microplate insert, and FIG. 4C shows a side view of the 12-well microplate insert. In embodiments, the microplate insert includes 12 wells, wherein each well is 15 mm wide and separated by 3 mm of interstitial space. In embodiments, the microplate insert is about 53.8+/-0.5 mm x 76.6+/-0.5 mm. FIG. 4C shows the planar support, wherein the planar support (e.g., the glass bottom) is about 0.7 mm thick. FIG. 4D depicts an assembled microplate carrier, including two microplate inserts. FIG. 4D illustrates a 24-well microplate assembly, wherein each well is separated by about 18 mm (edge-to-edge) distance.

[0009] FIG. 5 depicts an embodiment of a blank microplate insert. In embodiments, the blank insert is configured to not include any wells. [0010] FIG. 6 illustrates an embodiment of a microplate receiver frame 601 configured to retain one or more microplate inserts 602 and microplate gasket 604 therein. The microplate insert(s) includes a planar support 605 (e.g., glass bottom) attached to a primary well plate. The microplate receiver frame 601 and microplate insert 602 may each include one or more respective biasing or retention elements 603 that bias and/or retain the microplate insert 602 relative to the microplate receiver frame 601. The dashed lines in FIG. 6 are indicative of placement of the microplate insert(s) 602 into the microplate receiver frame 201. For example, microplate receiver frame 601 may contain four microplate inserts as described herein. The microplate gasket 604 is between the microplate insert 602 and the planar support 605. In embodiments, the gasket 604 is attached to the microplate insert 602 and the planar support 605 using an adhesive.

[0011] FIGS. 7A-C is an illustration of the different diameters and dimensions of the devices contemplated herein. For example, the diameters of the individual wells for 96-well, 48-well, 24-well, and 12-well plates may include circular, square, or rectangular dimensions of varying diameters. The shapes of the wells of the microplate insert may be cuboid, cylindrical, pyramidal, conical, or frustoconical as illustrated in FIG. 7B. FIG. 7C illustrates a microplate section 700. A portion of the microplate section is depicted showing a partial cross-section of four wells of the microplate section. Each well includes a top 701 section, and axial walls 702; the dashed lines of the axial walls refer to the wall being within the microplate section. Also illustrated is the gasket 703 that forms a fluidic barrier within each well (i.e., each well is fluidically isolated from each other) in contact with the planar support 704 (e.g., glass bottom).

[0012] FIG. 8 shows 5 wells of a 24-well microwell plate section 800 containing at least a portion of a transferred tissue section within each well.

[0013] FIG. 9 shows a perspective, bottom view of a microplate insert and microplate gasket coupled thereto.

[0014] FIG. 10 shows a cross-sectional view of the microplate insert and microplate gasket along line 10-10 of FIG. 9.

[0015] FIG. 11 shows a top view of an assembled microplate assembly with several microplate inserts positioned within a frame.

DETAILED DESCRIPTION [0016] The aspects and embodiments described herein relate to an integrated solution providing a panoply of information about a sample (e.g., a cell or tissue sample). For example, the device described herein is capable of i) in situ single cell analysis; ii) in situ tissue analysis; and iii) sequencing (e.g., RNA-seq or immune repertoire sequencing). In embodiments, the device is configured to provide RNA transcription analysis (e.g., counting RNA) for targeted panels; serological analyses, protein expression analysis (e.g., counting of proteins) for targeted panels; and/or sequencing variable regions in immune cells (e.g., B- cells or T-cells) or cancer cells. Utilizing the device and methods described herein, that is, combining cell morphology information with standard marker-based assessment provides improved specificity of detection, thereby enabling a lower limit of detection and improved diagnoses of diseases.

I. Definitions

[0017] The term “microplate” or “microplate carrier” as used herein, refers to a substrate comprising a surface, the surface including a plurality of reaction chambers separated from each other by interstitial regions on the surface. A microplate may include a microplate frame and one or more microplate inserts. In embodiments, the microplate has dimensions as provided and described by American National Standards Institute (ANSI) and Society for Laboratory Automation And Screening (SLAS); for example the tolerances and dimensions set forth in ANSI SLAS 1-2004 (R2012); ANSI SLAS 2-2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSI SLAS 4-2004 (R2012); and ANSI SLAS 6-2012, which are incorporated herein by reference. The dimensions of the microplate as described herein and the arrangement of the reaction chambers may be compatible with an established format for automated laboratory equipment. In embodiments, the device described herein provides methods for high-throughput screening. High-throughput screening (HTS) refers to a process that uses a combination of modem robotics, data processing and control software, liquid handling devices, and/or sensitive detectors, to efficiently process a large amount of (e.g., thousands, hundreds of thousands, or millions) samples in biochemical, genetic, or pharmacological experiments, either in parallel or in sequence, within a reasonably short period of time (e.g., days). Preferably, the process is amenable to automation, such as robotic simultaneous handling of 96 samples, 384 samples, 1536 samples or more. A typical HTS robot tests up to 100,000 to a few hundred thousand compounds per day. The samples are often in small volumes, such as no more than 1 mL, 500 pl, 200 pl, 100 pl, 50 pl or less. Through this process, one can rapidly identify active compounds, small molecules, antibodies, proteins or polynucleotides in a cell.

[0018] The wells (alternatively referred to as reaction chambers) of a microplate and/or microplate insert may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells. In embodiments, the 96 and 384 wells are arranged in a 2:3 rectangular matrix. In embodiments, the 24 wells are arranged in a 3:8 rectangular matrix. In embodiments, the 48 wells are arranged in a 3:4 rectangular matrix. In embodiments, the reaction chamber is a microscope slide (e.g., a glass slide about 75 mm by about 25 mm). In embodiments the slide is a concavity slide (e.g., the slide includes a depression). In embodiments, the slide includes a coating for enhanced cell adhesion (e.g., poly-L-lysine, silanes, carbon nanotubes, polymers, epoxy resins, or gold). In embodiments, the microplate receiver is about 5 inches by about 3.33 inches, and includes a plurality of 5 mm diameter wells. In embodiments, the microplate receiver is about 5 inches by about 3.33 inches, and includes a plurality of 6 mm diameter wells. In embodiments, the microplate receiver is about 5 inches by about 3.33 inches, and includes a plurality of 7 mm diameter wells. In embodiments, the microplate receiver is about 5 inches by about 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the microplate receiver is 5 inches by 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the microplate receiver is about 5 inches by about 3.33 inches, and includes a plurality of 8 mm diameter wells. In embodiments, the microplate insert is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples. In embodiments, the microplate is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples. In embodiments, the microplate has a rectangular shape that measures 127.7 mm±0.5 mm in length by 85.4 mm±0.5 mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each well has an average diameter of about 5-7 mm. In embodiments, the microplate has a rectangular shape that measures 127.7 mm±0.5 mm in length by 85.4 mm±0.5 mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each well has an average diameter of about 6 mm. In embodiments, the microplate includes an array of femtoliter wells, array of nanoliter wells, or array of microliter wells. In embodiments, the wells in an array may all have substantially the same volume. The array of wells may have a volume up to 100 e.g., about 0.1 femtoliter, 1 femtoliter, 10 femtoliter, 25 femtoliter, 50 femtoliter, 100 femtoliter, 0.1 pL, 1 pL, 10 pL, 25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL, 100 nL, 0.1 microliter, 1 microliter, 10 microliter, 25 microliter, 50 microliter, or 100 microliter.

[0019] The term “surface” is intended to mean an external part or external layer of a substrate. The surface can be in contact with another material such as a gas, liquid, gel, polymer, organic polymer, second surface of a similar or different material, metal, or coat. The surface, or regions thereof, can be substantially flat. The substrate and/or the surface can have surface features such as wells, pits, channels, ridges, raised regions, pegs, posts or the like.

[0020] The term “well” refers to a discrete concave feature in a substrate having a surface opening that is completely surrounded by interstitial region(s) of the surface. Wells can have any of a variety of shapes at their opening in a surface including but not limited to round, elliptical, square, polygonal, or star shaped (i.e., star shaped with any number of vertices). The cross section of a well taken orthogonally with the surface may be curved, square, polygonal, hyperbolic, conical, or angular. The wells of a microplate are available in different shapes, for example F-Bottom: flat bottom; C-Bottom: bottom with minimal rounded edges; V-Bottom: V-shaped bottom; or U-Bottom: U-shaped bottom. In embodiments, the well is substantially square. In embodiments, the well is square. In embodiments, the well is F- bottom. In embodiments, the microplate includes 24 substantially round flat bottom wells. In embodiments, the microplate includes 48 substantially round flat bottom wells. In embodiments, the microplate includes 96 substantially round flat bottom wells. In embodiments, the microplate includes 384 substantially square flat bottom wells. In embodiments, the well is a 3-dimensional structure. In some embodiments, the top view of a well is any suitable 2-dimensional shape, which when extended along the z-axis, produces a 3-dimensional structure capable of containing one or more samples and/or reagents. In embodiments, a well of the microwell insert shares at least one well wall (or a portion of the well wall) with an adjacent well. In some embodiments, a well does not share any walls or portion of a wall in common with another well. In some embodiments, the wells of a microwell insert is attached to a planar support, such that the wells are fluidically isolated from each other. In some embodiments, one end of the well is open (e.g., exposed), wherein the open end can be used to distribute samples and/or reagents into the well.

[0021] The discrete regions (i.e., features, wells) of the microplate may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like. In embodiments, the pattern of wells includes concentric circles of regions, spiral patterns, rectilinear patterns, hexagonal patterns, and the like. In embodiments, the pattern of wells is arranged in a rectilinear or hexagonal pattern A regular array of such regions is advantageous for detection and data analysis of signals collected from the arrays during an analysis. These discrete regions are separated by interstitial regions. As used herein, the term “interstitial region” refers to an area in a substrate or on a surface that separates other areas of the substrate or surface. For example, an interstitial region can separate one concave feature of an array from another concave feature of the array. The two regions that are separated from each other can be discrete, lacking contact with each other. In another example, an interstitial region can separate a first portion of a feature from a second portion of a feature. In embodiments the interstitial region is continuous whereas the features are discrete, for example, as is the case for an array of wells in an otherwise continuous surface. The separation provided by an interstitial region can be partial or full separation. In embodiments, interstitial regions have a surface material that differs from the surface material of the wells (e.g., the interstitial region contains a photoresist and the surface of the well is glass). In embodiments, interstitial regions have a surface material that is the same as the surface material of the wells (e.g., both the surface of the interstitial region and the surface of well contain a polymer or copolymer).

[0022] A “receiving substrate” or “planar support” is used according to its plain and ordinary meaning and generally refers to a substantially solid construct with a surface. A receiving substrate may be composed of any appropriate material such as metal, plastic, glass or polymer based materials. For example, a planar support can be a glass microscope slide (such as one that is 3 cm long by 1 cm wide by 0.25 cm thick). In embodiments, the planar support conforms to the dimensions of the microwell insert. In embodiments, the planar support is functionalized, resulting in positively charged surfaces.

[0023] As used herein, an “adhesive” generally refers to a substance used for sticking objects or materials together. Adhesives include, for example, glues, pastes, liquid tapes, epoxy, bioadhesives, gels, and mucilage. In some embodiments, an adhesive is liquid tape. In some embodiments, the adhesive is glue. In some embodiments, an adhesive is pressure senstive tape.

[0024] As used herein, the term “hydrogel” refers to a three-dimensional polymeric structure that is substantially insoluble in water, but which is capable of absorbing and retaining water (e.g. large quantities of water) to form a substantially stable, often soft and pliable, structure. In embodiments, water can penetrate in between polymer chains of a polymer network, subsequently causing swelling and the formation of a hydrogel. In embodiments, hydrogels are super-absorbent (e.g., containing more than about 90% water) and can be comprised of natural or synthetic polymers. Hydrogels can contain over 99% water and may include natural or synthetic polymers, or a combination thereof. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. A detailed description of suitable hydrogels may be found in published U.S. patent application 20100055733, herein specifically incorporated by reference. By “hydrogel subunits” or “hydrogel precursors” is meant hydrophilic monomers, prepolymers, or polymers that can be crosslinked, or “polymerized”, to form a three-dimensional (3D) hydrogel network.

[0025] Hydrogels may be prepared by cross-linking hydrophilic biopolymers or synthetic polymers. Thus, in some embodiments, the hydrogel may include a crosslinker. As used herein, the term “crosslinker” refers to a molecule that can form a three-dimensional network when reacted with the appropriate base monomers. Examples of the hydrogel polymers, which may include one or more crosslinkers, include but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO — PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N- vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethylene imine), polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. Thus, for example, a combination may include a polymer and a crosslinker, for example polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), or PEG/polypropylene oxide (PPO). In embodiments, the hydrogel includes chemical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a covalent bond) and may be referred to as a chemical hydrogel. In embodiments, the hydrogel includes physical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a non-covalent bond) and may be referred to as a physical hydrogel. In embodiments, the physical hydrogel include one or more crosslinks including hydrogen bonds, hydrophobic interactions, and/or polymer chain entanglements.

[0026] As used herein, the term “interfacial”, or “interfacial layer”, is used in accordance with its plain ordinary meaning and refers to the boundary between any two bulk phases (gas, liquid, or solid) in contact where the properties differ from the properties of the bulk phases. In embodiments, an interfacial layer includes water. Interfacial water differs from bulk water in a number of properties, for example, interfacial water has a higher heat capacity than bulk water because more energy is necessary to break its hydrogen bonds. The arrangement and structure of the interfacial water layer varies depending on the structure of the hydrophilic and/or hydrophobic surface(s) the water layer is in contact with. Additional properties of interfacial water may be found in, e.g., Mentre P. J. Biol. Phys, and Chem. 2004; 4: 115-123 and Tanaka M. Front. Chem. 2020; 8: 165, which are incorporated herein by reference in their entirety.

[0027] As used herein, the terms “solid support” and “substrate” and “substrate surface” and “solid surface” refers to discrete solid or semi-solid surfaces providing physical support to a material (e.g., a sample or object). A solid support may encompass any type of solid, porous, or hollow sphere, ball, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently). A solid support may include a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. A bead can be non-spherical in shape. A solid support may be used interchangeably with the term "bead." A solid support may further include a polymer or hydrogel on the surface to which the primers are attached. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefin copolymers, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, photopattemable dry film resists, UV-cured adhesives and polymers. Particularly useful solid supports for some embodiments have at least one surface located on a microplate. Solid surfaces can also be varied in their shape depending on the application in a method described herein. For example, a solid surface useful herein can be planar, or contain regions which are concave or convex. In embodiments, the geometry of the concave or convex regions (e.g., wells) of the solid surface conform to the size and shape of a substantially circular particle to maximize the contact between the particle. In embodiments, the wells of an array are randomly located such that nearest neighbor wells have random spacing between each other. Alternatively, in embodiments the spacing between the wells can be ordered, for example, forming a regular pattern. The term solid substrate is encompassing of a substrate (e.g., a microplate) having a surface including a polymer coating covalently attached thereto.

[0028] The term “image” is used according to its ordinary meaning and refers to a representation of all or part of an object. The representation may be an optically detected reproduction. For example, an image can be obtained from fluorescent, luminescent, scatter, or absorption signals. The part of the object that is present in an image can be the surface or other xy plane of the object. Typically, an image is a 2 dimensional representation of a 3 dimensional object. An image may include signals at differing intensities (i.e., signal levels). An image can be provided in a computer readable format or medium. An image is derived from the collection of focus points of light rays coming from an object (e.g., the sample), which may be detected by any image sensor.

[0029] As used herein, the term “signal” is intended to include, for example, fluorescent, luminescent, scatter, or absorption impulse or electromagnetic wave transmitted or received. Signals can be detected in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 391 to 770 nm), infrared (IR) range (about 0.771 to 25 microns), or other range of the electromagnetic spectrum. The term “signal level” refers to an amount or quantity of detected energy or coded information. For example, a signal may be quantified by its intensity, wavelength, energy, frequency, power, luminance, or a combination thereof. Other signals can be quantified according to characteristics such as voltage, current, electric field strength, magnetic field strength, frequency, power, temperature, etc. Absence of signal is understood to be a signal level of zero or a signal level that is not meaningfully distinguished from noise. [0030] The term “xy coordinates” refers to information that specifies location, size, shape, and/or orientation in an xy plane. The information can be, for example, numerical coordinates in a Cartesian system. The coordinates can be provided relative to one or both of the x and y axes or can be provided relative to another location in the xy plane (e.g., a fiducial). The term “xy plane” refers to a 2 dimensional area defined by straight line axes x and y. When used in reference to a detecting apparatus and an object observed by the detector, the xy plane may be specified as being orthogonal to the direction of observation between the detector and object being detected.

[0031] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g., chemical compounds including biomolecules, particles, solid supports, or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some embodiments contacting includes allowing a sample as described herein to interact with a microplate.

[0032] As used herein, the term “biomolecule” refers to an agent (e.g., a compound, macromolecule, or small molecule), and the like derived from a biological system (e.g., an organism). The biomolecule may contain multiple individual components that collectively construct the biomolecule, for example, in embodiments, the biomolecule is a polynucleotide wherein the polynucleotide is composed of nucleotide monomers. The biomolecule may be or may include DNA, RNA, organelles, carbohydrates, lipids, proteins, or any combination thereof. These components may be extracellular. In some examples, the biomolecule may be referred to as a clump or aggregate of combinations of components. In some instances, the biomolecule may include one or more constituents of a cell but may not include other constituents of the cell. In embodiments, a biomolecule is a molecule produced by a biological system (e.g., an organism). In embodiments, a biomolecule may be referred to as an analyte. Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In embodiments, the analytes within a cell can be localized to subcellular locations, including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In embodiments, analyte(s) can be peptides or proteins, including antibodies and/or enzymes. In embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.

[0033] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may include natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences. As may be used herein, the terms “nucleic acid oligomer” and “oligonucleotide” are used interchangeably and are intended to include, but are not limited to, nucleic acids having a length of 200 nucleotides or less. In some embodiments, an oligonucleotide is a nucleic acid having a length of 2 to 200 nucleotides, 2 to 150 nucleotides, 5 to 150 nucleotides or 5 to 100 nucleotides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. In some embodiments, an oligonucleotide is a primer configured for extension by a polymerase when the primer is annealed completely or partially to a complementary nucleic acid template. A primer is often a single stranded nucleic acid. In certain embodiments, a primer, or portion thereof, is substantially complementary to a portion of an adapter. In some embodiments, a primer has a length of 200 nucleotides or less. In certain embodiments, a primer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides. In some embodiments, an oligonucleotide may be immobilized to a solid support.

[0034] As used herein, the terms “polynucleotide primer” and “primer” refers to any polynucleotide molecule that may hybridize to a polynucleotide template, be bound by a polymerase, and be extended in a template-directed process for nucleic acid synthesis (e.g., amplification and/or sequencing). The primer may be a separate polynucleotide from the polynucleotide template, or both may be portions of the same polynucleotide (e.g., as in a hairpin structure having a 3’ end that is extended along another portion of the polynucleotide to extend a double-stranded portion of the hairpin). Primers (e.g., forward or reverse primers) may be attached to a solid support. A primer can be of any length depending on the particular technique it will be used for. For example, PCR primers are generally between 10 and 40 nucleotides in length. The length and complexity of the nucleic acid fixed onto the nucleic acid template may vary. In some embodiments, a primer has a length of 200 nucleotides or less. In certain embodiments, a primer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization procedure. The primer permits the addition of a nucleotide residue thereto, or oligonucleotide or polynucleotide synthesis therefrom, under suitable conditions. In an embodiment the primer is a DNA primer, i.e., a primer consisting of, or largely consisting of, deoxyribonucleotide residues. The primers are designed to have a sequence that is the complement of a region of template/target DNA to which the primer hybridizes. The addition of a nucleotide residue to the 3’ end of a primer by formation of a phosphodiester bond results in a DNA extension product. The addition of a nucleotide residue to the 3’ end of the DNA extension product by formation of a phosphodiester bond results in a further DNA extension product. In another embodiment the primer is an RNA primer. In embodiments, a primer is hybridized to a target polynucleotide. A “primer” is complementary to a polynucleotide template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the addition of covalently bonded bases linked at its 3' end complementary to the template in the process of DNA synthesis.

[0035] Nucleic acids, including e.g., nucleic acids with a phosphorothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0036] In some embodiments, a nucleic acid includes a label. As used herein, the term "label" or "labels" is used in accordance with their plain and ordinary meanings and refer to molecules that can directly or indirectly produce or result in a detectable signal either by themselves or upon interaction with another molecule. Non-limiting examples of detectable labels include fluorescent dyes, biotin, digoxin, haptens, and epitopes. In general, a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal. In embodiments, the label is a dye. In embodiments, the dye is a fluorescent dye. Non-limiting examples of dyes, some of which are commercially available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li -Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In embodiments, a particular nucleotide type is associated with a particular label, such that identifying the label identifies the nucleotide with which it is associated. In embodiments, the label is luciferin that reacts with luciferase to produce a detectable signal in response to one or more bases being incorporated into an elongated complementary strand, such as in pyrosequencing. In embodiment, a nucleotide includes a label (such as a dye). In embodiments, the label is not associated with any particular nucleotide, but detection of the label identifies whether one or more nucleotides having a known identity were added during an extension step (such as in the case of pyrosequencing). Examples of detectable agents (i.e., labels) include imaging agents, including fluorescent and luminescent substances, molecules, or compositions, including, but not limited to, a variety of organic or inorganic small molecules commonly referred to as “dyes,” “labels,” or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments, the detectable moiety is a fluorescent molecule e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). In embodiments, the detectable moiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). The term “cyanine” or “cyanine moiety” as described herein refers to a detectable moiety containing two nitrogen groups separated by a polymethine chain. In embodiments, the cyanine moiety has 3 methine structures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moiety has 5 methine structures (i.e., cyanine 5 or Cy5). In embodiments, the cyanine moiety has 7 methine structures i.e., cyanine 7 or Cy7).

[0037] In some embodiments, the label has a characteristic electromagnetic spectrum signal. As used herein, the “electromagnetic spectrum” refers to the range of frequencies of electromagnetic radiation. In embodiments, the label has a characteristic absorption spectrum. As used herein, the “absorption spectrum” refers to the range of frequencies of electromagnetic radiation that are absorbed. The “electromagnetic spectrum” or “absorption spectrum” can lead to different characteristic spectrum. In embodiments, the peak radiation or the peak absorption occurs at 380-450 nm (Violet), 450-485 nm (Blue), 485-500 nm (Cyan), 500-565 nm (Green), 565-590 nm (Yellow), 590-625 nm (Orange), or 625-740 nm (Red). In some embodiments, the peak radiation or the peak absorption occurs around 400 nm, 460 nm, or 520 nm.

[0038] As used herein, the terms “reversible blocking groups” and “reversible terminators” are used in accordance with their plain and ordinary meanings and refer to a blocking moiety located, for example, at the 3' position of a modified nucleotide and may be a chemically cleavable moiety such as an allyl group, an azidomethyl group or a methoxymethyl group, or may be an enzymatically cleavable group such as a phosphate ester. Non-limiting examples of nucleotide blocking moieties are described in applications WO 2004/018497, WO 96/07669, U.S. Pat. Nos. 7,057,026, 7,541,444, 5,763,594, 5,808,045, 5,872,244 and 6,232,465 the contents of which are incorporated herein by reference in their entirety. The nucleotides may be labelled or unlabeled. They may be modified with reversible terminators useful in methods provided herein and may be 3'-O-blocked reversible or 3 '-unblocked reversible terminators. In nucleotides with 3'-O-blocked reversible terminators, the blocking group -OR [reversible terminating (capping) group] is linked to the oxygen atom of the 3'- OH of the pentose, while the label is linked to the base, which acts as a reporter and can be cleaved. The 3'-O-blocked reversible terminators are known in the art, and may be, for instance, a 3'-ONH2 reversible terminator, a 3 '-O-allyl reversible terminator, or a 3'-O- azidomethyl reversible terminator. In embodiments, the reversible terminator moiety is _w herein the 3 ’ oxygen of the nucleotide is not shown in the formulae above.

The term “allyl” as described herein refers to an unsubstituted methylene attached to a vinyl group (i.e., -CH=CH2). In embodiments, the reversible terminator moiety is as described in U.S. Patent 10,738,072, which is incorporated herein by reference for all purposes. For example, a nucleotide including a reversible terminator moiety may be represented by the formula: Reversible Terminator moiety

, where the nucleobase is adenine or adenine analogue, thymine or thymine analogue, guanine or guanine analogue, or cytosine or cytosine analogue. In embodiments, the reversible terminator includes an azido moiety or a dithiol moiety. In embodiments, the reversible terminator is -NH2, -CN, -CH3, C2-C6 allyl (e.g., -CH ₂ -CH=CH ₂ ), methoxyalkyl (e.g., -CH ₂ - O-CH3), or -CH2N3. In embodiments, the reversible terminator includes a hydrocarbyl. In embodiments, the reversible terminator includes an ester (O-C(O)R ^Z ’ wherein R ^z ’ is any alkyl or aryl group which can include a formate, benzoyl formate, acetate, substituted acetate, propionate, and other esters as described in Green, T. W. (Protective Groups in Organic Chemistry, Wiley & Sons, New York, 1981)). In embodiments, the reversible terminator includes an ether (O-R ^zz wherein R ^zz can be substituted or unsubstituted alkyl such as methyl, substituted methyl, ethyl, substituted ethyl, allyl, substituted benzyl, silyl, or any other ether used to transiently protect hydroxyls and similar groups). In embodiments, the reversible terminator includes an O-CH2(OC2H5)MCH3 wherein M is an integer from 1-10. In embodiments, the reversible terminator includes a phosphate, phosphoramidate, phosphoramide, toluic acid ester, benzoic ester, acetic acid ester, or ethoxyethyl ether. In embodiments, the reversible terminator includes a disulfide moiety.

[0039] In some embodiments, a nucleic acid (e.g., an adapter or a primer) includes a molecular identifier or a molecular barcode. As used herein, the term "molecular barcode" (which may be referred to as a "tag", a "barcode", a "molecular identifier", an "identifier sequence" or a “unique molecular identifier” (UMI)) refers to any material (e.g., a nucleotide sequence, a nucleic acid molecule feature) that is capable of distinguishing an individual molecule in a large heterogeneous population of molecules. In embodiments, a barcode is unique in a pool of barcodes that differ from one another in sequence, or is uniquely associated with a particular sample polynucleotide in a pool of sample polynucleotides. In embodiments, every barcode in a pool of adapters is unique, such that sequencing reads including the barcode can be identified as originating from a single sample polynucleotide molecule on the basis of the barcode alone. In other embodiments, individual barcode sequences may be used more than once, but adapters including the duplicate barcodes are associated with different sequences and/or in different combinations of barcoded adaptors, such that sequence reads may still be uniquely distinguished as originating from a single sample polynucleotide molecule on the basis of a barcode and adjacent sequence information (e.g., sample polynucleotide sequence, and/or one or more adjacent barcodes). In embodiments, barcodes are about or at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75 or more nucleotides in length. In embodiments, barcodes are shorter than 20, 15, 10, 9, 8, 7, 6, or 5 nucleotides in length. In embodiments, barcodes are about 10 to about 50 nucleotides in length, such as about 15 to about 40 or about 20 to about 30 nucleotides in length. In a pool of different barcodes, barcodes may have the same or different lengths. In general, barcodes are of sufficient length and include sequences that are sufficiently different to allow the identification of sequencing reads that originate from the same sample polynucleotide molecule. In embodiments, each barcode in a plurality of barcodes differs from every other barcode in the plurality by at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions. In some embodiments, substantially degenerate barcodes may be known as random. In some embodiments, a barcode may include a nucleic acid sequence from within a pool of known sequences. In some embodiments, the barcodes may be pre-defined.

[0040] As used herein, the terms “incubate,” and “incubation refer collectively to altering the temperature of an object in a controlled manner such that conditions are sufficient for conducting the desired reaction. Thus, it is envisioned that the terms encompass heating a receptacle (e.g., a microplate) to a desired temperature and maintaining such temperature for a fixed time interval. Also included in the terms is the act of subjecting a receptacle to one or more heating and cooling cycles (i.e., “temperature cycling” or “thermal cycling”). While temperature cycling typically occurs at relatively high rates of change in temperature, the term is not limited thereto, and may encompass any rate of change in temperature.

[0041] Provided herein are methods, systems, devices, and compositions for analyzing a sample in situ. The term “z z situ” is used in accordance with its ordinary meaning in the art and refers to a sample surrounded by at least a portion of its native environment, such as may preserve the relative position of two or more elements. For example, an extracted human cell obtained is considered in situ when the cell is retained in its local microenvironment so as to avoid extracting the target (e.g., nucleic acid molecules or proteins) away from their native environment. An in situ sample (e.g., a cell) can be obtained from a suitable subject. An in situ cell sample may refer to a cell and its surrounding milieu, or a tissue. A sample can be isolated or obtained directly from a subject or part thereof. In embodiments, the methods described herein (e.g., sequencing a plurality of target nucleic acids of a cell in situ) are applied to an isolated cell (i.e., a cell not surrounded by least a portion of its native environment). For the avoidance of any doubt, when the method is performed within a cell (e.g., an isolated cell) the method may be considered in situ. In some embodiments, a sample is obtained indirectly from an individual or medical professional. A sample can be any specimen that is isolated or obtained from a subject or part thereof. A sample can be any specimen that is isolated or obtained from multiple subjects. Non-limiting examples of specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, platelets, buffy coats, or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), a biopsy sample, celocentesis sample, cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof. Non-limiting examples of tissues include organ tissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder, reproductive organs, intestine, colon, spleen, brain, the like or parts thereof), epithelial tissue, hair, hair follicles, ducts, canals, bone, eye, nose, mouth, throat, ear, nails, the like, parts thereof or combinations thereof. A sample may include cells or tissues that are normal, healthy, diseased (e.g., infected), and/or cancerous (e.g., cancer cells). A sample obtained from a subject may include cells or cellular material (e.g., nucleic acids) of multiple organisms (e.g., virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasite nucleic acid). A sample may include a cell and RNA transcripts. A sample can include nucleic acids obtained from one or more subjects. In some embodiments a sample includes nucleic acid obtained from a single subject. A subject can be any living or non-living organism, including but not limited to a human, non-human animal, plant, bacterium, fungus, virus, or protist. A subject may be any age (e.g., an embryo, a fetus, infant, child, adult). A subject can be of any sex (e.g., male, female, or combination thereof). A subject may be pregnant. In some embodiments, a subject is a mammal. In some embodiments, a subject is a plant. In some embodiments, a subject is a human subject. A subject can be a patient (e.g., a human patient). In some embodiments a subject is suspected of having a genetic variation or a disease or condition associated with a genetic variation.

[0042] As used herein, the term “disease state” is used in accordance with its plain and ordinary meaning and refers to any abnormal biological state or aberration of a cell. The presence of a disease state may be identified by the same collection of biological constituents used to determine the cell’s biological state. In general, a disease state will be detrimental to a biological system. A disease state may be a consequence of, inter alia, an environmental pathogen, for example a viral infection (e.g., HIV/AIDS, hepatitis B, hepatitis C, influenza, measles, etc.), a bacterial infection, a parasitic infection, a fungal infection, or infection by some other organism. A disease state may also be the consequence of some other environmental agent, such as a chemical toxin or a chemical carcinogen. As used herein, a disease state further includes genetic disorders wherein one or more copies of a gene is altered or disrupted, thereby affecting its biological function. Exemplary genetic diseases include, but are not limited to polycystic kidney disease, familial multiple endocrine neoplasia type I, neurofibromatoses, Tay-Sachs disease, Huntington's disease, sickle cell anemia, thalassemia, and Down's syndrome, as well as others (see, e.g., The Metabolic and Molecular Bases of Inherited Diseases, 7th ed., McGraw-Hill Inc., New York). Other exemplary diseases include, but are not limited to, cancer, hypertension, Alzheimer's disease, neurodegenerative diseases, and neuropsychiatric disorders such as bipolar affective disorders or paranoid schizophrenic disorders. Disease states are monitored to determine the level (e.g., the stage or progression) of one or more disease states of a subject and, more specifically, detect changes in the biological state of a subject which are correlated to one or more disease states (see, e.g., U.S. Pat. No. 6,218,122, which is incorporated by reference herein in its entirety). The methods of the present invention are also applicable to monitoring the disease state or states of a subject undergoing one or more therapies. Thus, the present invention also provides methods for determining or monitoring efficacy of a therapy or therapies (i.e., determining a level of therapeutic effect) upon a subject. In embodiment, the methods of the invention can be used to assess therapeutic efficacy in a clinical trial, e.g., as an early surrogate marker for success or failure in such a clinical trial. Within eukaryotic cells, there are hundreds to thousands of signaling pathways that are interconnected. For this reason, perturbations in the function of proteins within a cell have numerous effects on other proteins and the transcription of other genes that are connected by primary, secondary, and sometimes tertiary pathways. This extensive interconnection between the function of various proteins means that the alteration of any one protein is likely to result in compensatory changes in a wide number of other proteins. In particular, the partial disruption of even a single protein within a cell, such as by exposure to a drug or by a disease state which modulates the gene copy number (e.g., a genetic mutation), results in characteristic compensatory changes in the transcription of enough other genes that these changes in transcripts can be used to define a “signature” of particular transcript alterations which are related to the disruption of function, i.e., a particular disease state or therapy, even at a stage where changes in protein activity are undetectable.

[0043] As used herein, a “single cell” refers to one cell. Single cells useful in the methods described herein can be obtained from a tissue of interest, or from a biopsy, blood sample, or cell culture. Additionally, cells from specific organs, tissues, tumors, neoplasms, or the like can be obtained and used in the methods described herein. In general, cells from any population can be used in the methods, such as a population of prokaryotic or eukaryotic organisms, including bacteria or yeast.

[0044] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may optionally be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. A protein may refer to a protein expressed in a cell. A polypeptide, or a cell is “recombinant” when it is artificial or engineered, or derived from or contains an artificial or engineered protein or nucleic acid (e.g., non-natural or not wild type). For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example a variant of a naturally occurring gene, is recombinant.

[0045] The term “protein-specific binding agent” refers to an agent to a protein or polypeptide molecule, or portion thereof, capable of selectively binding or interacting with a protein. In embodiments, a protein-specific binding agent specifically binds a particular protein (e.g., a protein antigen or epitope thereof). In embodiments a protein-specific binding agent is an immunoglobulin (IgA, IgD, IgE, IgG, or IgM). Intact immunoglobulins, also known as antibodies, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each, and two heavy (H) chains of approximately 50 kDa each. In embodiments, the protein binding moiety is an antigen-specific antibody. Nonlimiting examples of protein-specific binding agent encompassed within the term “antigenspecific antibody” used herein include: (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) an F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be recombinantly joined by a synthetic linker, creating a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv)). The most commonly used linker is a 15-residue (Gly4Ser)3 peptide, but other linkers are also known in the art. Single chain antibodies are also intended to be encompassed within the terms “protein-specific binding agent,” of an antibody. The antibody can also be a polyclonal antibody, monoclonal antibody, chimeric antibody, antigen-binding fragment, Fc fragment, single chain antibodies, or any derivatives thereof. In embodiments, the protein-specific binding agent is the antigenbinding site (e.g., fragment antigen-binding (Fab) variable region) of an antibody. The term “antigen-binding site” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0046] An “antibody” (Ab) is a protein that binds specifically to a particular substance, known as an “antigen” (Ag). An “antibody” or “antigen-binding fragment” is an immunoglobulin that binds a specific “epitope.” The term encompasses polyclonal, monoclonal, and chimeric antibodies. In nature, antibodies are generally produced by lymphocytes in response to immune challenge, such as by infection or immunization. An “antigen” (Ag) is any substance that reacts specifically with antibodies or T lymphocytes (T cells). An antibody may include the entire antibody as well as any antibody fragments capable of binding the antigen or antigenic fragment of interest. Examples include complete antibody molecules, antibody fragments, such as Fab, F(ab')2, CDRs, VL, VH, and any other portion of an antibody which is capable of specifically binding to an antigen. Antibodies used herein are immunospecific for, and therefore specifically and selectively bind to, for example, proteins either detected (i.e., biological targets of interest) or used for detection (i.e., probes containing oligonucleotide barcodes) in the methods and devices as described herein.

[0047] The terms “bind” and “bound” as used herein are used in accordance with their plain and ordinary meanings and refer to an association between atoms or molecules. The association can be direct or indirect. For example, bound atoms or molecules may be directly bound to one another, e.g., by a covalent bond or non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). As a further example, two molecules may be bound indirectly to one another by way of direct binding to one or more intermediate molecules (e.g., as in a substrate, bound to a first antibody, bound to an analyte, bound to a second antibody), thereby forming a complex. As used herein, the term “attached” refers to the state of two things being joined, fastened, adhered, connected or bound to each other. For example, a sample such as a cell or tissue, can be attached to a material, such as a hydrogel, polymer, or solid support, by a covalent or non-covalent bond. In embodiments, attachment is a covalent attachment.

[0048] “Specific binding” is where the binding is selective between two molecules. A particular example of specific binding is that which occurs between an antibody and an antigen. Typically, specific binding can be distinguished from non-specific when the dissociation constant (KD) is less than about 1 * IO ^-5 M or less than about 1 * IO ^-6 M or 1 * IO ^-7 M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical crosslinking, two-hybrid assays and the like. In embodiments, specific binding can refer to hybridization of two complementary nucleic acid sequences.

[0049] The terms “cellular component” is used in accordance with its ordinary meaning in the art and refers to any organelle, nucleic acid, protein, or analyte that is found in a prokaryotic, eukaryotic, archaeal, or other organismic cell type. Examples of cellular components (e.g., a component of a cell) include RNA transcripts, proteins, membranes, lipids, and other analytes. In embodiments, a cellular component is a biomolecule derived from a cell.

[0050] A “gene” refers to a polynucleotide that is capable of conferring biological function after being transcribed and/or translated.

[0051] As used herein, the term “DNA polymerase” and “nucleic acid polymerase” are used in accordance with their plain ordinary meanings and refer to enzymes capable of synthesizing nucleic acid molecules from nucleotides (e.g., deoxyribonucleotides). Exemplary types of polymerases that may be used in the compositions and methods of the present disclosure include the nucleic acid polymerases such as DNA polymerase, DNA- or RNA-dependent RNA polymerase, and reverse transcriptase. In some cases, the DNA polymerase is 9°N polymerase or a variant thereof, E. Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Taq DNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0 DNA polymerase, 9°N polymerase (exo- )A485L/Y409V, Phi29 DNA Polymerase (q>29 DNA Polymerase), T7 DNA polymerase, DNA polymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNA polymerase V, VentR DNA polymerase, Therminator™ II DNA Polymerase, Therminator™ III DNA Polymerase, or Therminator™ IX DNA Polymerase. In embodiments, the polymerase is a protein polymerase. Typically, a DNA polymerase adds nucleotides to the 3'- end of a DNA strand, one nucleotide at a time. In embodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol P DNA polymerase, Pol p DNA polymerase, Pol X DNA polymerase, Pol c DNA polymerase, Pol a DNA polymerase, Pol 6 DNA polymerase, Pol s DNA polymerase, Pol r| DNA polymerase, Pol r DNA polymerase, Pol K DNA polymerase, Pol C, DNA polymerase, Pol y DNA polymerase, Pol 9 DNA polymerase, Pol u DNA polymerase, or a thermophilic nucleic acid polymerase (e.g. Therminator y, 9°N polymerase (exo-), Therminator II, Therminator III, or Therminator IX). In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a reverse transcriptase. In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO 2020/056044). In embodiments, the polymerase is an enzyme described in US 2021/0139884.

[0052] A nucleic acid can be amplified by a suitable method. The term “amplified” as used herein refers to subjecting a target nucleic acid in a sample to a process that linearly or exponentially generates amplicon nucleic acids having the same or substantially the same (e.g., substantially identical) nucleotide sequence as the target nucleic acid, or segment thereof, and/or a complement thereof. In some embodiments an amplification reaction includes a suitable thermal stable polymerase. Thermal stable polymerases are known in the art and are stable for prolonged periods of time, at temperature greater than 80° C. when compared to common polymerases found in most mammals. In certain embodiments the term “amplified” refers to a method that includes a polymerase chain reaction (PCR). Conditions conducive to amplification (i.e., amplification conditions) are well known and often include at least a suitable polymerase, a suitable template, a suitable primer or set of primers, suitable nucleotides (e.g., dNTPs), a suitable buffer, and application of suitable annealing, hybridization and/or extension times and temperatures. In certain embodiments an amplified product (e.g., an amplicon) can contain one or more additional and/or different nucleotides than the template sequence, or portion thereof, from which the amplicon was generated (e.g., a primer can contain “extra” nucleotides (such as a 5’ portion that does not hybridize to the template), or one or more mismatched bases within a hybridizing portion of the primer).

[0053] As used herein, the term “rolling circle amplification (RCA)” refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (e.g., singlestranded DNA circles) via a rolling circle mechanism. Rolling circle amplification reaction is initiated by the hybridization of a primer to a circular, often single-stranded, nucleic acid template. The nucleic acid polymerase then extends the primer that is hybridized to the circular nucleic acid template by continuously progressing around the circular nucleic acid template to replicate the sequence of the nucleic acid template over and over again (rolling circle mechanism). The rolling circle amplification typically produces concatemers including tandem repeat units of the circular nucleic acid template sequence. The rolling circle amplification may be a linear RCA (LRCA), exhibiting linear amplification kinetics (e.g., RCA using a single specific primer), or may be an exponential RCA (ERCA) exhibiting exponential amplification kinetics. Rolling circle amplification may also be performed using multiple primers (multiply primed rolling circle amplification or MPRCA) leading to hyperbranched concatemers. For example, in a double-primed RCA, one primer may be complementary, as in the linear RCA, to the circular nucleic acid template, whereas the other may be complementary to the tandem repeat unit nucleic acid sequences of the RCA product. Consequently, the double-primed RCA may proceed as a chain reaction with exponential (geometric) amplification kinetics featuring a ramifying cascade of multiple-hybridization, primer-extension, and strand-displacement events involving both the primers. This often generates a discrete set of concatemeric, double-stranded nucleic acid amplification products. The rolling circle amplification may be performed in-vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase. RCA may be performed by using any of the DNA polymerases that are known in the art (e.g., a Phi29 DNA polymerase, a Bst DNA polymerase, or SD polymerase).

[0054] A nucleic acid can be amplified by a thermocycling method or by an isothermal amplification method. In some embodiments a rolling circle amplification method is used. In some embodiments amplification takes place on a solid support (e.g., within a flow cell) where a nucleic acid, nucleic acid library or portion thereof is immobilized. In certain sequencing methods, a nucleic acid library is added to a flow cell and immobilized by hybridization to anchors under suitable conditions. This type of nucleic acid amplification is often referred to as solid phase amplification. In some embodiments of solid phase amplification, all or a portion of the amplified products are synthesized by an extension initiating from an immobilized primer. Solid phase amplification reactions are analogous to standard solution phase amplifications except that at least one of the amplification oligonucleotides (e.g., primers) is immobilized on a solid support.

[0055] In some embodiments solid phase amplification includes a nucleic acid amplification reaction including only one species of oligonucleotide primer immobilized to a surface or substrate. In certain embodiments solid phase amplification includes a plurality of different immobilized oligonucleotide primer species. In some embodiments solid phase amplification may include a nucleic acid amplification reaction including one species of oligonucleotide primer immobilized on a solid surface and a second different oligonucleotide primer species in solution. Multiple different species of immobilized or solution-based primers can be used. Non-limiting examples of solid phase nucleic acid amplification reactions include interfacial amplification, bridge PCR amplification, emulsion PCR, WildFire amplification (e.g., US patent publication US20130012399), the like or combinations thereof.

[0056] As used herein, the terms “sequencing”, “sequence determination”, and “determining a nucleotide sequence”, are used in accordance with their ordinary meaning in the art, and refer to determination of partial as well as full sequence information of the nucleic acid being sequenced, and particular physical processes for generating such sequence information. That is, the term includes sequence comparisons, fingerprinting, and like levels of information about a target nucleic acid, as well as the express identification and ordering of nucleotides in a target nucleic acid. The term also includes the determination of the identification, ordering, and locations of one, two, or three of the four types of nucleotides within a target nucleic acid. Sequencing produces one or more sequencing reads.

[0057] As used herein, the term “sequencing read” is used in accordance with its plain and ordinary meaning and refers to an inferred sequence of nucleotide bases (or nucleotide base probabilities) corresponding to all or part of a single polynucleotide fragment. A sequencing read may include 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more nucleotide bases. In embodiments, a sequencing read includes reading a barcode and a template nucleotide sequence. In embodiments, a sequencing read includes reading a template nucleotide sequence. In embodiments, a sequencing read includes reading a barcode and not a template nucleotide sequence.

[0058] As used herein, the term “sequencing cycle” is used in accordance with its plain and ordinary meaning and refers to incorporating one or more nucleotides (e.g., a compound described herein) to the 3’ end of a polynucleotide with a polymerase, and detecting one or more labels that identify the one or more nucleotides incorporated. The sequencing may be accomplished by, for example, sequencing by synthesis, pyrosequencing, and the like. In embodiments, a sequencing cycle includes extending a complementary polynucleotide by incorporating a first nucleotide using a polymerase, wherein the polynucleotide is hybridized to a template nucleic acid, detecting the first nucleotide, and identifying the first nucleotide. In embodiments, to begin a sequencing cycle, one or more differently labeled nucleotides and a DNA polymerase can be introduced. Following nucleotide addition, signals produced (e.g., via excitation and emission of a detectable label) can be detected to determine the identity of the incorporated nucleotide (based on the labels on the nucleotides). Reagents can then be added to remove the 3’ reversible terminator and to remove labels from each incorporated base. Reagents, enzymes and other substances can be removed between steps by washing. Cycles may include repeating these steps, and the sequence of each cluster is read over the multiple repetitions.

[0059] As used herein, the term “extension” or “elongation” is used in accordance with their plain and ordinary meanings and refer to synthesis by a polymerase of a new polynucleotide strand complementary to a template strand by adding free nucleotides (e.g., dNTPs) from a reaction mixture that are complementary to the template in the 5'-to-3' direction. Extension includes condensing the 5'-phosphate group of the dNTPs with the 3'- hydroxy group at the end of the nascent (elongating) DNA strand.

[0060] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of that range, and any other stated or unstated intervening value in, or smaller range of values within, that stated range is encompassed within the invention. The upper and lower limits of any such smaller range (within a more broadly recited range) may independently be included in the smaller ranges, or as particular values themselves, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0061] The term “multiplexing" as used herein refers to an analytical method in which the presence and/or amount of multiple targets, e.g., multiple nucleic acid target sequences, can be assayed simultaneously by using the methods and devices as described herein, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic.

[0062] As used herein, the term "associated" or "associated with" can mean that two or more species are identifiable as being co-located at a point in time. An association can mean that two or more species are or were within a similar container. An association can be an informatics association, where for example digital information regarding two or more species is stored and can be used to determine that one or more of the species were co-located at a point in time. An association can also be a physical association. In some instances two or more associated species are "tethered", "coated”, "attached", or "immobilized" to one another or to a common solid or semisolid support (e.g., the well of a microplate). An association may refer to a relationship, or connection, between two entities. Associated may refer to the relationship between a sample and the DNA molecules, RNA molecules, or polynucleotides originating from or derived from that sample. These relationships may be encoded in oligonucleotide barcodes, as described herein. A polynucleotide is associated with a sample if it is an endogenous polynucleotide, i.e., it occurs in the sample at the time the sample is obtained, or is derived from an endogenous polynucleotide. For example, the RNAs endogenous to a cell are associated with that cell. cDNAs resulting from reverse transcription of these RNAs, and DNA amplicons resulting from PCR amplification of the cDNAs, contain the sequences of the RNAs and are also associated with the cell. The polynucleotides associated with a sample need not be located or synthesized in the sample, and are considered associated with the sample even after the sample has been destroyed (for example, after a cell has been lysed). Barcoding can be used to determine which polynucleotides in a mixture are associated with a particular sample.

[0063] As used herein, the term “polymer” refers to macromolecules having one or more structurally unique repeating units. The repeating units are referred to as “monomers,” which are polymerized for the polymer. Typically, a polymer is formed by monomers linked in a chain-like structure. A polymer formed entirely from a single type of monomer is referred to as a “homopolymer.” A polymer formed from two or more unique repeating structural units may be referred to as a “copolymer.” A polymer may be linear or branched, and may be random, block, polymer brush, hyperbranched polymer, bottlebrush polymer, dendritic polymer, or polymer micelles. The term “polymer” includes homopolymers, copolymers, tripolymers, tetra polymers and other polymeric molecules made from monomeric subunits. Copolymers include alternating copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, linear copolymers and branched copolymers. The term "polymerizable monomer" is used in accordance with its meaning in the art of polymer chemistry and refers to a compound that may covalently bind chemically to other monomer molecules (such as other polymerizable monomers that are the same or different) to form a polymer. [0064] Polymers can be hydrophilic, hydrophobic or amphiphilic, as known in the art. Thus, “hydrophilic polymers” are substantially miscible with water and include, but are not limited to, polyethylene glycol and the like. “Hydrophobic polymers” are substantially immiscible with water and include, but are not limited to, polyethylene, polypropylene, polybutadiene, polystyrene, polymers disclosed herein, and the like. “Amphiphilic polymers” have both hydrophilic and hydrophobic properties and are typically copolymers having hydrophilic segment(s) and hydrophobic segment(s). Polymers include homopolymers, random copolymers, and block copolymers, as known in the art. The term “homopolymer” refers, in the usual and customary sense, to a polymer having a single monomeric unit. The term “copolymer” refers to a polymer derived from two or more monomeric species. The term “random copolymer” refers to a polymer derived from two or more monomeric species with no preferred ordering of the monomeric species. The term “block copolymer” refers to polymers having two or homopolymer subunits linked by covalent bond. Thus, the term “hydrophobic homopolymer” refers to a homopolymer which is hydrophobic. The term “hydrophobic block copolymer” refers to two or more homopolymer subunits linked by covalent bonds and which is hydrophobic.

[0065] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0066] As used herein, the term “tissue” is used in accordance with its plain and ordinary meaning and refers to an organization of cells in a structure, where the structure generally functions as a unit in an organism (e.g., mammals) and may carry out specific functions. In some examples, cells in a tissue are configured in a mass and may not be free from one another. This disclosure describes methods of obtaining single biological samples (e.g., cells or nuclei) from tissues that can be used in various single biological samples (e.g., single- cell/nucleus) workflows. In some examples, blood cells (e.g., lymphocytes) can be considered a tissue. However, blood cells, like lymphocytes, generally are free from one another in the blood. The methods disclosed herein can be used to process those cells to obtain cells and/or nuclei, although dissociation steps may not be necessary when using those types of tissues. Generally, any type of tissue can be used in the methods described herein. Examples of tissues that may be used in the disclosed methods include, but are not limited to connective, epithelial, muscle and nervous tissue. In some examples, the tissues are from mammals. Tissues that contain any type of cells may be used. For example, tissues from abdomen, bladder, brain, esophagus, heart, intestine, kidney, liver, lung, lymph node, olfactory bulb, ovary, pancreas, skin, spleen, stomach, testicle, and the like. The tissue may be normal or tumor tissue (e.g., malignant). This example is not meant to be limiting. Although the conditions used in the disclosed may not be identical for different types of tissue, the methods may be applied to any tissue. The tissues used in the disclosed methods may be in various states. In some examples, the tissues used in the disclosed methods may be fresh, frozen, or fixed.

[0067] As used herein, the term “tissue section” refers to a piece of tissue that has been obtained from a subject, optionally fixed and attached to a surface, e.g., a microscope slide.

[0068] As used herein, the term “fresh,” generally in the context of a fresh tissue means that the tissue has recently been obtained from an organism, generally before any subsequent fixation steps, for example, flash freezing or chemical fixation. In embodiments, a fresh tissue is obtained from an organism about 1 second up to about 20 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 second up to about 60 seconds before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 30 seconds up to about 60 seconds before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 minutes up to about 20 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 minutes up to about 10 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 minutes up to about 5 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes before any fixation steps are performed.

[0069] As used herein, the term “fix,” refers to formation of covalent bonds, such as crosslinks, between biomolecules or within molecules. The process of fixing tissue samples or biological samples (e.g., cells and nuclei) for example, is called “fixation.” The agent that causes fixation is generally referred to as a “fixative” or “fixing agent.” “Fixed biological samples” (e.g., fixed cells or nuclei) or “fixed tissues” refers to biological samples (e.g., cells or nuclei) or tissues that have been in contact with a fixative under conditions sufficient to allow or result in formation of intra- and inter-molecular crosslinks between biomolecules in the biological sample. Fixation may be reversed and the process of reversing fixation may be referred to as “un-fixing” or “decrosslinking.” Unfixing or decrosslinking refers to breaking or reversing the formation of covalent bonds in biomolecules formed by fixatives. In some examples, the tissue fixed is fresh tissue. In some examples, the tissue fixed may be frozen tissue. In some examples, the tissue fixed may not be dissociated. In some examples, the tissue fixed may be dissociated or partially dissociated (e.g., chopped, cut). In some examples, tissue that has been rapidly frozen and, perhaps, cut or chopped into pieces (e.g., small enough to fit into a tube or container used for fixation) may be used. In some examples, tissue may be dissociated or partially dissociated (e.g., cut, chopped) before or during fixation. In some examples, tissue that is fixed may not be dissociated. The frozen biological tissue can be fixed using a fixing agent, which is suitably an organic fixing agent. Suitable organic fixing agents include without limitation alcohols, ketones, aldehydes (e.g., glutaraldehyde), cross-linking agents, disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, dimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), ethylene glycol bis (succinimidyl succinate) (EGS), bis(sulfosuccinimidyl)suberate (BS3) and combinations thereof. A particularly suitable fixing agent is a formaldehyde-based fixing agent such as formalin, which is a mixture of formaldehyde and water. The formalin may include about 1% to about 15% by weight formaldehyde and about 85% to about 99% by weight water, suitable about 2% to about 8% by weight formaldehyde and about 92% to about 98% by weight water, or about 4% by weight formaldehyde and about 96% by weight water. In some examples, tissues may be fixed in 4% paraformaldehyde. Other suitable fixing agents will be appreciated by those of ordinary skill in the art (e.g., International PCT App. No. PCT/US2020/066705, which is incorporated herein by reference in its entirety).

[0070] As used herein, the term “permeable” refers to a property of a substance that allows certain materials to pass through the substance. “Permeable” may be used to describe a biological sample, such as a cell or nucleus, in which analytes in the biological sample can leave the biological sample. “Permeabilize” is an action taken to cause, for example, a biological sample (e.g., a cell) to release its analytes. In some examples, permeabilization of a biological sample is accomplished by affecting the integrity (e.g., compromising) of a biological sample membrane (e.g., a cellular or nuclear membrane) such as by application of a protease or other enzyme capable of disturbing a membrane allowing analytes to diffuse out of the biological sample. In some embodiments, permeabilizing a biological sample does not release the biomolecules (e.g., proteins and/or nucleic acids) contained within the sample.

[0071] As used herein, the term “single biological sample”, such as a single cell or a single nucleus generally refers to a biological sample that is not present in an aggregated form or clump. Single biological samples, such as cells and/or nuclei may be the result of dissociating a tissue sample.

[0072] As used herein, the term “tissue freezing” is used in accordance with its plain and ordinary meaning and refers to different methods for freezing tissues. In some examples, the methods used may be rapid methods (e.g., “flash freezing” or “snap freezing”). In some examples, tissues may be lowered to temperatures below about -70° C using these methods. In some examples, rapid freezing may use ultracold media. In some examples, an ultracold medium may be liquid nitrogen. In some examples, this type of freezing may preserve tissue integrity, in part by preventing the formation of ice crystals that would affect the tissue morphology. In some examples, an ultracold medium may be dry ice.

[0073] The terms “bioconjugate group,” “bioconjugate reactive moiety,” and “bioconjugate reactive group” refer to a chemical moiety which participates in a reaction to form a bioconjugate linker (e.g., covalent linker). Non-limiting examples of bioconjugate reactive groups and the resulting bioconjugate reactive linkers may be found in the Bioconjugate Table below:

Bioconjugate reactive group 1 Bioconjugate reactive group 2 Resulting Bioconjugate

(e.g., electrophilic (e.g., nucleophilic bioconjugate reactive linker bioconjugate reactive moiety) reactive moiety) activated esters amines/anilines carboxamides Bioconjugate reactive group 1 Bioconjugate reactive group 2 Resulting Bioconjugate (e.g., electrophilic (e.g., nucleophilic bioconjugate reactive linker bioconjugate reactive moiety) reactive moiety) acrylamides thiols thioethers acyl azides amines/anilines carboxamides acyl halides amines/anilines carboxamides acyl halides alcohols/phenols esters acyl nitriles alcohols/phenols esters acyl nitriles amines/anilines carboxamides aldehydes amines/anilines imines aldehydes or ketones hydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkyl halides amines/anilines alkyl amines alkyl halides carboxylic acids esters alkyl halides thiols thioethers alkyl halides alcohols/phenols ethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylic acids esters alkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenols esters anhydrides amines/anilines carboxamides aryl halides thiols thiophenols aryl halides amines aryl amines aziridines thiols thioethers boronates glycols boronate esters carbodiimides carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxides thiols thioethers haloacetamides thiols thioethers haloplatinate amino platinum complex haloplatinate heterocycle platinum complex haloplatinate thiol platinum complex halotriazines amines/anilines aminotriazines halotriazines alcohols/phenols triazinyl ethers halotriazines thiols triazinyl thioethers imido esters amines/anilines amidines isocyanates amines/anilines ureas isocyanates alcohols/phenols urethanes isothiocyanates amines/anilines thioureas maleimides thiols thioethers phosphoramidites alcohols phosphite esters silyl halides alcohols silyl ethers sulfonate esters amines/anilines alkyl amines sulfonate esters thiols thioethers sulfonate esters carboxylic acids esters sulfonate esters alcohols ethers sulfonyl halides amines/anilines sulfonamides sulfonyl halides phenols/alcohols sulfonate esters

[0074] As used herein, the te “bioconjugate reactive moiety” and “bioconjugate reactive group” refers to a moiety or group capable of forming a bioconjugate (e.g., covalent linker) as a result of the association between atoms or molecules of bioconjugate reactive groups. The association can be direct or indirect. For example, a conjugate between a first bioconjugate reactive group (e.g., -NH2, -COOH, -N-hydroxysuccinimide, or -maleimide) and a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate) provided herein can be direct, e.g., by covalent bond or linker (e.g., a first linker of second linker), or indirect, e.g., by non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e., the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels- Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., an amine). In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -sulfo-N- hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., an amine).

[0075] Useful bioconjugate reactive groups used for bioconjugate chemistries herein include, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.; (c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophile groups which are capable of participating in Diels- Alder reactions such as, for example, maleimido or maleimide groups; (e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or react with maleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized;(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc.; (j) epoxides, which can react with, for example, amines and hydroxyl compounds; (k) phosphoramidite and other standard functional groups useful in nucleic acid synthesis; (1) metal silicon oxide bonding; (m) metal bonding to reactive phosphorus groups (e.g., phosphines) to form, for example, phosphate diester bonds.; (n) azides coupled to alkynes using copper catalyzed cycloaddition click chemistry; (o) biotin conjugate can react with avidin or streptavidin to form a avidin-biotin complex or streptavidin-biotin complex.

[0076] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the specification as a whole. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by those of skill in the art. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0077] As used herein, the singular terms “a”, “an”, and “the” include the plural reference unless the context clearly indicates otherwise. Reference throughout this specification to, for example, "one embodiment", "an embodiment", "another embodiment", "a particular embodiment", "a related embodiment", "a certain embodiment", "an additional embodiment", or "a further embodiment" or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0078] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about means the specified value.

[0079] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0080] In the description, relative terms such as “before,” “after,” “above,” “below,” “up,” “down,” “top” and “bottom” as well as derivative thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then described or as shown in the drawing or figure under discussion. These relative terms are for convenience of description and do not require that the system be constructed or operated in a particular orientation. [0081] As used herein, the term “modified nucleotide” refers to nucleotide modified in some manner. Typically, a nucleotide contains a single 5-carbon sugar moiety, a single nitrogenous base moiety and 1 to three phosphate moieties. In embodiments, a nucleotide can include a blocking moiety and/or a label moiety. A blocking moiety on a nucleotide prevents formation of a covalent bond between the 3' hydroxyl moiety of the nucleotide and the 5' phosphate of another nucleotide. A blocking moiety on a nucleotide can be reversible, whereby the blocking moiety can be removed or modified to allow the 3' hydroxyl to form a covalent bond with the 5' phosphate of another nucleotide. A blocking moiety can be effectively irreversible under particular conditions used in a method set forth herein. In embodiments, the blocking moiety is attached to the 3’ oxygen of the nucleotide and is independently -NH2, -CN, -CH3, C2-C6 allyl (e.g., -CH2-CH=CH2), methoxyalkyl (e.g., - CH2-O-CH3), or -CH2N3. In embodiments, the blocking moiety is attached to the 3’ oxygen label moiety of a modified nucleotide can be any moiety that allows the nucleotide to be detected, for example, using a spectroscopic method. Exemplary label moieties are fluorescent labels, mass labels, chemiluminescent labels, electrochemical labels, detectable labels and the like. One or more of the above moieties can be absent from a nucleotide used in the methods and compositions set forth herein. For example, a nucleotide can lack a label moiety or a blocking moiety or both. Examples of nucleotide analogues include, without limitation, 7-deaza-adenine, 7-deaza-guanine, the analogues of deoxynucleotides shown herein, analogues in which a label is attached through a cleavable linker to the 5-position of cytosine or thymine or to the 7-position of deaza-adenine or deaza-guanine, and analogues in which a small chemical moiety is used to cap the OH group at the 3 '-position of deoxyribose. Nucleotide analogues and DNA polymerase-based DNA sequencing are also described in U.S. Patent No. 6,664,079, which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of detectable labels include labels including fluorescent dyes, biotin, digoxin, haptens, and epitopes. In general, a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal. In embodiments, the dye is a fluorescent dye. Non-limiting examples of dyes, some of which are commercially available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In embodiments, the label is a fluorophore.

[0082] In some embodiments, a nucleic acid includes a label. As used herein, the term "label" or "labels" is used in accordance with their plain and ordinary meanings and refer to molecules that can directly or indirectly produce or result in a detectable signal either by themselves or upon interaction with another molecule. Non-limiting examples of detectable labels include fluorescent dyes, biotin, digoxin, haptens, and epitopes. In general, a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal. In embodiments, the label is a dye. In embodiments, the dye is a fluorescent dye. Non-limiting examples of dyes, some of which are commercially available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In embodiments, a particular nucleotide type is associated with a particular label, such that identifying the label identifies the nucleotide with which it is associated. In embodiments, the label is luciferin that reacts with luciferase to produce a detectable signal in response to one or more bases being incorporated into an elongated complementary strand, such as in pyrosequencing. In embodiment, a nucleotide includes a label (such as a dye). In embodiments, the label is not associated with any particular nucleotide, but detection of the label identifies whether one or more nucleotides having a known identity were added during an extension step (such as in the case of pyrosequencing). Examples of detectable agents (i.e., labels) include imaging agents, including fluorescent and luminescent substances, molecules, or compositions, including, but not limited to, a variety of organic or inorganic small molecules commonly referred to as “dyes,” “labels,” or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments, the detectable moiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). In embodiments, the detectable moiety is a fluorescent molecule e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). The term “cyanine” or “cyanine moiety” as described herein refers to a detectable moiety containing two nitrogen groups separated by a polymethine chain. In embodiments, the cyanine moiety has 3 methine structures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moiety has 5 methine structures (i.e., cyanine 5 or Cy5). In embodiments, the cyanine moiety has 7 methine structures (i.e., cyanine 7 or Cy7).

[0083] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

II. Systems, Devices, and Kits

[0084] In an aspect is provided a microplate assembly. In embodiments, the microplate assembly includes a microplate receiver frame defining a pocket. In embodiments, the microplate assembly includes at least one microplate section and a planar support positioned on a bottom of the at least one microplate section. In embodiments the microplate section and the planar support have a similar coefficient of linear thermal expansion. In embodiments, the pocket of the microplate receiver frame is sized and shaped to receive a plurality of microplate sections (e.g., two, three, four, five, six, seven, or eight sections). In embodiments, the microplate assembly includes integrated unit, wherein the frame and microplate section are fused together or otherwise inseparable. For example, the microplate assembly may include a microwell insert, wherein a plurality of wells are bored directly into the microwell insert. The integrated unit may have dimensions as provided and described by American National Standards Institute (ANSI) and Society for Laboratory Automation And Screening (SLAS); for example the tolerances and dimensions set forth in ANSI SLAS 1-2004 (R2012); ANSI SLAS 2-2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSI SLAS 4-2004 (R2012); and ANSI SLAS 6-2012, which are incorporated herein by reference.

[0085] In another aspect is provided a microplate section including a plurality of wells, including a solid support including a plurality of openings; a planar support attached to the solid support. In embodiments, the solid support includes a coefficient of linear thermal expansion of about 1.0xl0' ⁶ /K to about 9.0xl0' ⁶ /K and the planar support includes a coefficient of linear thermal expansion of about 0.5xl0' ⁶ /K to about 8.0xl0' ⁷ /K. In embodiments, the solid support includes a coefficient of linear thermal expansion of about 2.0X10' ⁴ /K to about 1.0xl0' ⁵ /K and the planar support includes a coefficient of linear thermal expansion of about 0.5xl0' ⁵ /K to about 8.0xl0' ⁷ /K. In embodiments, the microplate section includes a plurality of wells formed by attaching the planar support to the solid support (e.g., wherein the openings form the wells). In embodiments, the solid support includes 96, 384, 1536, 2, 4, 6, 12, 24, or 48 wells. In embodiments, the solid support includes 2 wells. In embodiments, the solid support includes 4 wells. In embodiments, the solid support includes 6 wells. In embodiments, the solid support includes 12 wells. In embodiments, the solid support includes 24 wells. In embodiments, the solid support includes 48 wells. In embodiments, the solid support includes 96 wells. In embodiments, the solid support includes 384 wells. In embodiments, the solid support includes 1536 wells. In embodiments, each well is about 5 mm to about 8 mm in diameter. In embodiments, each well is about 5 mm, about 6 mm, about 7 mm, or about 8 mm in diameter. In embodiments, when the solid support including a plurality of openings is attached to the planar support, the plurality of openings form the plurality of wells.

[0086] In embodiments, the microplate assembly includes one microplate section. In embodiments, the microplate assembly includes two microplate sections. In embodiments, the microplate assembly includes three microplate sections. In embodiments, the microplate assembly includes four microplate sections. In embodiments, the microplate assembly includes five microplate sections. In embodiments, the microplate assembly includes six microplate sections. In embodiments, the microplate assembly includes seven microplate sections. In embodiments, the microplate assembly includes eight microplate sections. In embodiments, the microplate assembly includes one or more microplate sections.

[0087] The planar support may be composed of any suitable material. Exemplary substrates include, but are not limited to, glass, modified and/or functionalized glass, hydrogels, films, membranes, plastics (including e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, cyclic olefins, polyimides etc.), nylon, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene polycarbonate, or combinations thereof. In embodiments, the planar support is a conductive substrate. For example, an electrophoretic field can be applied to facilitate migration and or immobilization of the biological sample to the planar support. In embodiments, the conductive substrate can include glass (e.g., a glass slide) coated with a substance or otherwise modified to confer conductive properties to the glass. In embodiments, the glass slide can be coated with a conductive coating. In embodiments, the conductive coating includes tin oxide (TO) or indium tin oxide (ITO). In embodiments, the conductive coating includes a transparent conductive oxide (TCO). In embodiments, the conductive coating includes aluminum doped zinc oxide (AZO). In embodiments, the conductive coating includes fluorine doped tin oxide (FTO).

[0088] In embodiments, the planar support includes a functionalized glass surface or a functionalized plastic surface. Functionalization, as used herein, refers to a modification of the original surface. For example, functionalization may include topographical modifications (e.g., groves, posts, etching), chemical modifications (e.g., binding one or more compounds to the surface to alter the surface charge or bioconjugate reactive moieties on the surface), biological modifications (e.g., immobilizing one or more heparin proteins, heparin sulfate binding proteins, peptide sequences, growth factors, fibronectin, laminin, or collagen), or plasma treatment on reactive glass to generate bioconjugate reactive moieties on the surface. In embodiments, the planar support is functionalized with an RGD peptide or YIGSR peptide. RGD peptide is one of the most physiologically ubiquitous binding motifs commonly used, which is found in many natural adhesive proteins such as fibronectin, vitronectin, laminin and collagen type I.

[0089] In embodiments, the planar support includes a cell-permissive coating to permit adherence of live cells. A “cell-permissive coating” is a coating that allows or helps cells to maintain cell viability (e.g., remain viable) on the planar support. For example, a cell- permissive coatings may enhance cell attachment, cell growth, and/or cell differentiation, e.g., a cell-permissive coating can provide nutrients to the live cells. A cell-permissive coating can include a biological material and/or a synthetic material. Non-limiting examples of a cell-permissive coating include coatings that feature one or more extracellular matrix (ECM) components (e.g., proteoglycans and fibrous proteins such as collagen, elastin, fibronectin and laminin), poly-lysine, poly(L)-ornithine, and/or a biocompatible silicone (e.g., CYTOSOFT®). For example, a cell-permissive coating that includes one or more extracellular matrix components can include collagen Type I, collagen Type II, collagen Type IV, elastin, fibronectin, laminin, and/or vitronectin. In some embodiments, the cell- permissive coating includes a solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma (e.g., MATRIGEL®). In embodiments, the cell-permissive coating includes collagen. In embodiments, the planar support includes immobilized oligonucleotides capable of capturing target polynucleotides of interest.

[0090] In embodiments, the planar support is functionalized with one or more synthetic chemical molecules. In embodiments, the planar support includes dimethyl sulfoxide (DMSO), all-trans retinoic acid (RA), dynorphin B, ascorbic acid. In embodiments, the planar support includes one or more bioconjugate reactive moieties (e.g., carboxyl or amine groups) on the surface of the planar support. In embodiments, the planar support includes a glass solid support that is functionalized by contacting the glass solid support in triethanolamine buffer containing glutaraldehyde and 1-hydroxbenzol (HOBt), followed by contacting with 1 -ethyl - 3 -(3 -dimethylaminopropyl) carbodiimide (EDC) and/or N-hydroxysuccinimide (NHS). In embodiments, the functionalized glass surface includes (3 -aminopropyl )tri ethoxysilane (APTES), (3 -Aminopropyl )trimethoxysilane (APTMS), y- Aminopropyl silatrane (APS), N-(6- aminohexyl)aminomethyltriethoxysilane (AHAMTES), polyethylenimine (PEI), 5,6- epoxyhexyltriethoxysilane, or triethoxysilylbutyraldehyde, or a combination thereof. In embodiments, the functionalized glass surface includes (3 -aminopropyl )tri ethoxysilane (APTES). In embodiments, the functionalized glass surface includes (3- Aminopropyl)trimethoxysilane (APTMS). In embodiments, the functionalized glass surface includes y-Aminopropylsilatrane (APS). In embodiments, the functionalized glass surface includes N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES). In embodiments, the functionalized glass surface includes polyethylenimine (PEI). In embodiments, the functionalized glass surface includes 5,6-epoxyhexyltriethoxysilane. In embodiments, the functionalized glass surface includes triethoxysilylbutyraldehyde. In embodiments, the planar support is a functionalized glass surface or a functionalized plastic surface. In embodiments, the functionalized glass surface is functionalized with APTES, APTMS, APS, or AHAMTES. In embodiments, the functionalized glass surface includes (3- aminopropyl)tri ethoxy silane (APTES), (3 -Aminopropyl )trimethoxysilane (APTMS), y- Aminopropylsilatrane (APS), N-(6-aminohexyl)aminom ethyl tri ethoxysilane (AHAMTES), polyethylenimine (PEI), 5,6-epoxyhexyltriethoxysilane, or triethoxysilylbutyraldehyde, or a combination thereof.

[0091] In embodiments, the planar support includes a polymer coating or a polymer layer. In embodiments, the polymer coating or a polymer layer. [0092] In embodiments, the planar support includes a photoresist, alternatively referred to herein as a resist. A “resist” as used herein is used in accordance with its ordinary meaning in the art of lilthography and refers to a polymer matrix (e.g., a polymer network). In embodiments, the photoresist is a silsesquioxane resist, an epoxy-based polymer resist, poly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist, an Off-stoichiometry thiol-enes (OSTE) resist, amorphous fluoropolymer resist, a crystalline fluoropolymer resist, polysiloxane resist, or a organically modified ceramic polymer resist. In embodiments, the photoresist is a silsesquioxane resist. In embodiments, the photoresist is an epoxy-based polymer resist. In embodiments, the photoresist is a poly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist. In embodiments, the photoresist is an Off-stoichiometry thiol-enes (OSTE) resist. In embodiments, the photoresist is an amorphous fluoropolymer resist. In embodiments, the photoresist is a crystalline fluoropolymer resist. In embodiments, the photoresist is a polysiloxane resist. In embodiments, the photoresist is an organically modified ceramic polymer resist. In embodiments, the photoresist includes polymerized alkoxysilyl methacrylate polymers and metal oxides (e.g., SiCh, ZrO, MgO, AI2O3, TiCh or Ta2C>5). In embodiments, the photoresist includes polymerized alkoxysilyl acrylate polymers and metal oxides (e.g., SiCh, ZrO, MgO, AI2O3, TiO2 or Ta2Os). In embodiments, the photoresist includes metal atoms, such as Si, Zr, Mg, Al, Ti or Ta atoms. In embodiments, the planar support is a glass slide about 75 mm by about 25 mm. In embodiments, the planar support includes a resist (e.g., a photoresist or nanoimprint resist including a crosslinked polymer matrix attached to the planar support).

[0093] In embodiments, the planar support is subjected to lithographic patterning methods (e.g., nanolithographic to microlithographic patterning). Typically, features smaller than 10 micrometers are considered microlithographic, and features smaller than 100 nanometers are considered nanolithographic. Lithographic techniques make use of masks or templates to transfer patterns over a large area simultaneously. A powerful microfabrication technique is photolithography, i.e. the lithography using a UV light source and a photosensitive material as resist. As the name suggests, the photoresist (alternatively referred to as a resist) is an active material layer that can be patterned by selective exposure and must “resist” chemical/physical attach of the underlying substrate. In embodiments, the resist is a crosslinked polymer matrix. In embodiments, the resist includes silsesquioxane molecules. In embodiments, the resist includes polymerized epoxy-containing monomers, or polymerized poly(vinylpyrrolidone-vinyl acrylic acid) copolymers. In embodiments, the planar support includes a glass substrate having a surface coated in silsesquioxane resist (e.g., polyhedral oligosilsesquioxanemethacrylate (POSS)), an epoxy-based polymer resist (e.g., SU-8 as described in U.S. 4,882,245), poly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist (e.g., as described in U.S. 7,467,632), or novolaks resist, bisazides resist, or a combination thereof (e.g., as described in U.S. 4,970,276).

[0094] In embodiments, the planar support includes a photoresist. A photoresist is a lightsensitive polymer material used to form a patterned coating on a surface. The process begins by coating a substrate (e.g., a glass substrate) with a light-sensitive organic material. A mask with the desired pattern is used to block light so that only unmasked regions of the material will be exposed to light. In the case of a positive photoresist, the photo-sensitive material is degraded by light and a suitable solvent will dissolve away the regions that were exposed to light, leaving behind a coating where the mask was placed. In the case of a negative photoresist, the photosensitive material is strengthened (either polymerized or cross-linked) by light, and a suitable solvent will dissolve away only the regions that were not exposed to light, leaving behind a coating in areas where the mask was not placed. In embodiments, the planar support includes an epoxy-based photoresist (e.g., SU-8, SU-8 2000, SU-8 3000, SU-8 GLM2060). In embodiments, the planar support includes a negative photoresist. Negative refers to a photoresist whereby the parts exposed to UV become cross-linked (i.e., immobilized), while the remainder of the polymer remains soluble and can be washed away during development. In embodiments, the planar support includes an Off-stoichiometry thiolenes (OSTE) polymer (e.g., an OSTE resist). In embodiments, the planar support includes a Hydrogen Silsesquioxane (HSQ) polymer (e.g., HSQ resist). In embodiments, the planar support includes a crosslinked polymer matrix on the surface of the wells.

[0095] In embodiments, the planar support includes a nanoimprint resist. In embodiments, the planar support includes a photoresist and polymer layer, wherein the photoresist is between the planar support and the polymer layer. Suitable photoresist compositions are known in the art, such as, for example the compositions and resins described in U.S.

6,897,012; U.S. 6,991,888; U.S. 4,882,245; U.S. 7,467,632; U.S. 4,970,276, each of which is incorporated herein by reference in their entirety. In embodiments, the planar support includes a photoresist and polymer layer, wherein the photoresist is covalently attached to the planar support and covalently attached to the polymer layer. In embodiments, the resist is an amorphous (non-crystalline) fluoropolymer (e.g., CYTOP® from Bellex), a crystalline fluoropolymer, or a fluoropolymer having both amorphous and crystalline domains. In embodiments, the resist is a suitable polysiloxane, such as polydimethylsiloxane (PDMS).

[0096] In embodiments, the planar support includes a resist (e.g., a nanoimprint lithography (NIL) resist). Nanoimprint resists can include thermal curable materials (e.g., thermoplastic polymers), and/or UV-curable polymers. In embodiments, the planar support is generated by pressing a transparent mold possessing the pattern of interest (e.g., the pattern of wells) into photo-curable liquid film, followed by solidifying the liquid materials via a UV light irradiation. Typical UV-curable resists have low viscosity, low surface tension, and suitable adhesion to the glass substrate. For example, the planar support surface, but not the surface of the wells, is coated in an organically modified ceramic polymer (ORMOCER®, registered trademark of Fraunhofer-Gesellschaft zur Fbrderung der angewandten Forschung e. V. in Germany). Organically modified ceramics contain organic side chains attached to an inorganic siloxane backbone. Several ORMOCER® polymers are now provided under names such as “Ormocore”, “Ormoclad” and “Ormocomp” by Micro Resist Technology GmbH. In embodiments, the planar support includes a resist as described in Haas et al Volume 351, Issues 1-2, 30 August 1999, Pages 198-203, US 2015/0079351A1, US 2008/0000373, or US 2010/0160478, each of which is incorporated herein by reference. In embodiments, the planar support surface, and the surface of the wells, is coated in an organically modified ceramic polymer (ORMOCER®, registered trademark of Fraunhofer-Gesellschaft zur Fbrderung der angewandten Forschung e. V. in Germany).

[0097] In embodiments, the planar support includes a hydrophobic polymer layer. In embodiments, the planar support includes a perfluorinated polymer. In embodiments, the planar support includes a polyfluorinated polymer. In embodiments, the planar support includes polymerized units of a fluorine-containing methacrylate (e.g., CH2=C(CH3)COOC- (CF3)2CF2CF2CF3). Non-limiting examples and synthetic protocols of fluorine-containing methacrylate monomers may be found in Zhang, D., (2018). Materials (Basel, Switzerland), 11(11), 2258 (2018), which is incorporated herein by reference. In embodiments, the fluorinated polymer is an amorphous (non-crystalline) fluoropolymer (e.g., CYTOP® from Bellex), a crystalline fluoropolymer, or a fluoropolymer having both amorphous and crystalline domains.

[0098] In embodiments, the planar support includes a polymer layer. In embodiments, the polymer layer includes polymerized units of alkoxysilyl methacrylate, alkoxysilyl acrylate, alkoxysilyl methyl acrylamide, alkoxysilyl methylacrylamide, or a copolymer thereof. In embodiments, the polymer layer includes polymerized units of alkoxysilyl methacrylate. In embodiments, the polymer layer includes polymerized units of alkoxysilyl acrylate. In embodiments, the polymer layer includes polymerized units of alkoxysilyl methylacrylamide. In embodiments, the polymer layer includes polymerized units of alkoxysilyl methylacrylamide. In embodiments, the polymer layer includes glycidyloxypropyl- trimethyloxysilane. In embodiments, the polymer layer includes methacryloxypropyltrimethoxysilane. In embodiments, the polymer layer includes polymerized units of copolymer thereof, embodiments, the polymer layer is an organically-modified ceramic polymer. In embodiments, the polymer includes polymerized monomers of alkoxysilyl polymers, such as embodiments, the polymer layer includes one or more ceramic particles, (e.g., silicates, aluminates, and titanates). In embodiments, the polymer layer includes titanium dioxide, zinc oxide, and/or iron oxide.

[0099] In embodiments, the planar support includes a hydrogel. Non-limiting examples of hydrogels include polymerized monomers of acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethylene glycol) and derivatives thereof (e.g., PEG-acrylate (PEG- DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, or combinations thereof.

[0100] In embodiments, the solid support (e.g., the planar support) includes a polymer coating on the surface. In embodiments, the polymer coating includes polymerized units of polyacrylamide (AAm), poly-N-isopropylacrylamide, poly N-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate, methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, glicydyl methacrylate (GMA), glicydyl methacrylate (GMA) azide, hydroxy ethylmethacryl ate (HEMA), hydroxyethyl acrylate (ELEA), hydroxypropylmethacrylate (HPMA), polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof. In embodiments, the polymer coating includes polymerized units of polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate (GMA). In embodiments, the polymer coating includes polymerized units of polyethylene glycol methacrylate (PEGMA) and isocyanatoethyl methacrylate (IEM). In embodiments, the polymer coating includes polymerized units of glicydyl methacrylate azide (GMA azide) and polyethylene glycol methacrylate (PEGMA).

[0101] In embodiments, the polymer coating includes polymerized units of 3-azido-2- hydroxypropyl methacrylate, 2-azido-3 -hydroxypropyl methacrylate, 2-(((2- azidoethoxy)carbonyl)amino)ethyl methacrylate, 3-azido-2-hydroxypropyl acrylate, 2-azido- 3 -hydroxypropyl acrylate, or 2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, the polymer coating includes polymerized units of 3-azido-2-hydroxypropyl methacrylate, 2-azido-3 -hydroxypropyl methacrylate, or 2-(((2- azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments, the polymer coating includes polymerized units of 3-azido-2-hydroxypropyl methacrylate. In embodiments, the polymer coating includes polymerized units of 3-azido-2-hydroxypropyl methacrylate 2- azido-3 -hydroxypropyl methacrylate. In embodiments, the polymer coating includes polymerized units of 3-azido-2-hydroxypropyl methacrylate 2-(((2- azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments, the polymer coating includes polymerized units of a) polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate (GMA), b) polyethylene glycol methacrylate (PEGMA) and isocyanatoethyl methacrylate (IEM), or c) polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate (GMA) azide.

[0102] In embodiments, the polymer coating provides bioconjugate reactive moieties (e.g., azides). In embodiments, the polymer coating includes polymerized units of glicydyl methacrylate azide (GMA azide) and polyethylene glycol methacrylate (PEGMA) in the ratio of 1 : 1. In embodiments, the ratio of GMA azide to PEGMA is 1 :2. In embodiments, the ratio of GMA azide to PEGMA is 1 :3. In embodiments, the ratio of GMA azide to PEGMA is 1 :4. In embodiments, the ratio of GMA azide to PEGMA is 1 :5. In embodiments, the ratio of GMA azide to PEGMA is 1 :6. In embodiments, the ratio of GMA azide to PEGMA is 1 :7. In embodiments, the ratio of GMA azide to PEGMA is 1 :8. In embodiments, the polymer coating includes a plurality of bioconjugate reactive moieties. In embodiments, a bioconjugate reactive moiety includes an amine moiety, aldehyde moiety, alkyne moiety, azide moiety, carboxylic acid moiety, dibenzocyclooctyne (DBCO) moiety, norbomene moiety, tetrazine moiety, epoxy moiety, isocyanate moiety, furan moiety, maleimide moiety, thiol moiety, or transcyclooctene (TCO) moiety. In embodiments, the particle includes a plurality of azide moieties, alkyne moieties, dibenzocyclooctyne (DBCO) moieties, norbomene moieties, epoxy moieties, or isocyanate moieties.

[0103] In embodiments, the polymer coating includes polymerized units of polyacrylamide (AAm), glicydyl methacrylate (GMA), glicydyl methacrylate (GMA) azide, polyethylene glycol methacrylate (PEGMA), polyethylene glycol methacrylate (PEGMA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof.

[0104] In embodiments, a bottom region of the microplate section is glass. In embodiments, a bottom region of the microplate section is a functionalized solid support (i.e., a planar support). In embodiments, the planar support is attached to the microplate section. In embodiments, the planar support is bonded to the microplate section. In embodiments, the planar support is bonded to a gasket, and the gasket is bonded to the microplate section.

[0105] In embodiments, the planar support includes one or more markings on a surface of the planar support, for example to provide guidance for correlating spatial information with the characterization of the analyte of interest. For example, the planar support can be marked with a grid of lines (e.g., to allow the size of objects seen under magnification to be easily estimated and/or to provide reference areas for counting objects). In embodiments, fiducial markers can be included on a substrate. Such markings can be made using techniques including, printing, sand-blasting, and depositing on the surface of the planar support. In embodiments, imaging can be performed using one or more fiducial markers, i.e., objects placed in the field of view of an imaging system which appear in the image produced. Fiducial markers are typically used as a point of reference or measurement scale. Fiducial markers can include, but are not limited to, detectable labels such as fluorescent, radioactive, chemiluminescent, and colorimetric labels. The use of fiducial markers to stabilize and orient biological samples is described, for example, in Carter et al., Applied Optics 46:421-427, 2007), which is incorporated herein by reference. In some embodiments, a fiducial marker can be a physical particle (e.g., a nanoparticle, a microsphere, a nanosphere, a bead). In embodiments, a fiducial marker can be an particle attached to the planar support. For example, a fiducial marker can be a nanoparticle, e.g., a nanorod, a nanowire, a nanocube, a nanopyramid, or a spherical nanoparticle. In embodiments, the nanoparticle can be made of a heavy metal (e.g., gold). In embodiments, the nanoparticle can be made from diamond. In embodiments, the fiducial marker can be visible by eye. In embodiments, the fiducial markers described herein are be located at positions within the well of a microplate assembly corresponding to a specific pattern or design to aid in orientation of a biological sample.

[0106] In embodiments, the pocket of the frame is sized and shaped to receive the plurality of the microplate sections arranged in a side-by-side configuration. For example, FIG. 2B illustrates the microplate assembly 200 containing four microplate sections.

[0107] In embodiments, the microplate section includes one or more retention elements for retaining the microplate section within the microplate receiver. In embodiments, the receiver includes one or more bores, slots, seats, retention mechanisms, or other structures sized and shaped to receive, align, and secure a microplate section within the receiver (e.g., wherein the microplate section includes a complementary structure to the bores, slots, seats, retention mechanisms, or other structures of the receiver). In embodiments, the microplate section includes a first retention element and the microplate receiver includes a second retention element, and wherein the first retention element mechanically interacts with the second retention element to retain the microplate section within the pocket. In embodiments, the first retention element is biased toward the second retention element when the microplate section is positioned in the pocket. In embodiments, the first retention element is a prong and the second retention element is a slot. In embodiments, the microplate receiver includes one or more retention elements for retaining the microplate section within the microplate receiver. In embodiments, the microplate receiver includes one or more biasing features. The biasing feature can be a spring finger. In embodiments, the biasing feature includes a metal or rigid polymer. The biasing feature can be a tab. It should be appreciated the structure of the spring fingers can vary and can be any type of spring or biasing member that exerts a force onto the microplate section to retain or position the microplate section. For example, the microplate section 202 can be positively located on the frame by biasing the microplate section 202 toward a short edge of the pocket of the microplate receiver.

[0108] In embodiments, the microplate assembly includes at least one well. In embodiments, the microplate section includes at least one well. In embodiments, the microplate assembly includes a plurality of wells. In embodiments, the microplate section includes a plurality of wells. In embodiments, the microplate assembly includes 6, 8, 10, 12, 16, 18, 24, 30, 36, 40, 42, 46, 48, 96, 144, or 192 wells in total. In embodiments, the microplate section includes 6, 8, 10, 12, 16, 18, 24, 30, 36, 40, 42, 46, 48, 96, 144, or 192 wells. In embodiments, substantially all of the wells are identical in size. In embodiments, the one or more of the wells are substantially non-identical in size. In embodiments, the wells are separated from each other by interstitial regions.

[0109] In embodiments, the solid support includes a plurality of wells (e.g., a billion or more wells). In embodiments, the wells (e.g., each well) is separated from each other by about 0.2 pm to about 2.0 pm. In embodiments, the wells (e.g., each well) is separated from each other by about 0.3 pm to about 2.0 pm. In embodiments, the wells (e.g., each well) is separated from each other by about 0.4 pm to about 2.0 pm. In embodiments, the wells (e.g., each well) is separated from each other by about 0.5 pm to about 2.0 pm. In embodiments, the wells (e.g., each well) is separated from each other by about 1.0 pm to about 2.0 pm. In embodiments, the wells (e.g., each well) is separated from each other by about 1.0 pm to about 1.5 pm. In embodiments, the wells of the solid support are all substantially the same size (e.g., within a tolerance). In embodiments, the solid support includes wells that are from about 0.1 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.2 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.3 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.4 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.5 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.6 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.7 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.8 m to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.9 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 1.0 pm to about 3 pm in diameter. In embodiments, the solid support includes wells that are from about 0.1 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.2 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.3 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.4 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.5 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.6 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.7 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.8 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 0.9 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 1.0 pm to about 2 pm in diameter. In embodiments, the solid support includes wells that are from about 1.0 pm to about 1.5 pm in diameter. In embodiments, the solid support includes wells that are about 0.1 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.2 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.3 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.4 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.5 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.6 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.7 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.8 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.9 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 1.0 pm to about 2 pm in depth. In embodiments, the solid support includes wells that are about 0.1 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.2 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.3 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.4 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.5 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.6 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.7 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.8 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 0.9 pm to about 1.5 pm in depth. In embodiments, the solid support includes wells that are about 1.0 pm to about 1.5 pm in depth. In embodiments, one or more wells are different sizes (e.g., one population of wells are 1.0 pm in diameter, and a second population are 0.5 pm in diameter). In embodiments, the solid support is a glass slide about 75 mm by about 25 mm.

[0110] In embodiments, the interior of the well includes a coating. In embodiments, the coating is non-stick. In embodiments, the coating may be used to reduce or prevent the ability of other materials to stick to the surface of the well. Properties of such non-stick coatings may include a low surface energy value. A surface energy value may be used to quantify the disruption of intermolecular bonds that occur when a surface is created. The surface energy may therefore be considered as the excess energy at the surface of a material compared to the bulk, or the work required to build an area of a particular surface. Another way to view the surface energy is to relate it to the work required to cut a bulk sample, thereby creating two surfaces. For purposes of comparison, stainless steel may typically have a surface energy value of about 700 mJ/m ² (dyne/cm) to about 1000 mJ/m ² (dyne/cm) which promotes low adhesion (low adhesion results in lower sticking). Non-stick materials, however, may have surface energy values of about 50 mJ/m ² to about 40 mJ/m ² . Non-limiting examples of such surface energy values for non-stick materials may include values of about 50 mJ/m ² , about 48 mJ/m ² , about 46 mJ/m ² , about 44 mJ/m ² , about 42 mJ/m ² , about 40 mJ/m ² , and any value or range of values therebetween including endpoints. Such non-stick materials may include common polymers, such as aliphatic or semi-aromatic polyamides (for example Nylon) and polyimides (for example, Kapton®). It may be recognized that the surface energy values of such polymers are much lower than that of, for example, stainless steel, and thus may be less prone to sticking. Other materials, having even lower surface energy values — for example, in the range of about 40 mJ/m ² to about 12 mJ/m ² — may be even more resistant to sticking. Non-limiting examples of such surface energy values for non-stick materials may include values of about 40 mJ/m ² , about 36 mJ/m ² , about 32 mJ/m ² , about 28 mJ/m ² , about 24 mJ/m ² , about 20 mJ/m ² , about 16 mJ/m ² , about 12 mJ/m ² , and any value or range of values therebetween including endpoints. Polytetrafluoroethylene (PTFE) is one such material, having a surface energy of about 18 mJ/m ² . Still other materials may have even lower values of surface free energy, such as those materials having surface micro- and/or nano-structures that may take advantage of the “lotus leaf effect.” The surface free energy of such materials, natural or man-made, may have a value of about 5 mJ/m ² . In some non-limiting aspects, therefore, a non-stick material may be one having a non-zero, positive-valued surface energy less than that of stainless steel. For example, a non-stick material may have a surface energy of less than about 1100 mJ/m ² to about 5 mJ/m ² . Thus, in embodiments, such surface energy values of a non-stick material includes values of about 1100 mJ/m ² , about 1000 mJ/m ² , about 900 mJ/m ² , about 800 mJ/m ² , about 700 mJ/m ² , about 600 mJ/m ² , about 500 mJ/m ² , about 400 mJ/m ² , about 300 mJ/m ² , about 200 mJ/m ² , about 100 mJ/m ² , about 50 mJ/m ² , about 40 mJ/m ² , about 30 mJ/m ² , about 20 mJ/m ² , about 10 mJ/m ² , about 5 mJ/m ² , and any value or range of values therebetween including endpoints.

[OHl] In embodiments, the coating includes vinyl functionalized organopolysiloxane, polytetrafluoroethylene, fluorinated ethylene propylene, perfluoroalkoxy, tetrafluoroethylene- hexafluoropropylene (FEP), polyetheretherketone (PEEK), polyetherketone (PEK), tetrafluoroethylene perfluoromethyl vinyl ether copolymer (MFA), and/or a polydimethylsiloxane. In embodiments, the vinyl functionalized organopolysiloxane can be for example, Momentive® Product Code No. MSC2631 silicone manufactured by Momentive® Performance Materials of Waterford, N.Y. In embodiments the thickness of the coating is from about 1 to about 100 pm, e.g., about 1 to about 5 pm, and the variation in the thickness along the coating is within 0.5 pm, within 0.25 pm, within 0.1 pm or within 10% of the total thickness of the coating, within 5% of the total thickness of the coating, or within 2.5% of the total thickness of said coating. In embodiments the thickness of the coating is from about 0.1 to about 10.0 pm.

[0112] In some embodiments, the wells of the microplate section are separated from each other by about 1 mm to about 10 mm. In embodiments, the well is about 3 mm in diameter. In embodiments, the well is about 3.6 mm in diameter. In embodiments, the well is about 4 mm in diameter. In embodiments, the well is about 5 mm in diameter. In embodiments, the well is about 6 mm in diameter. In embodiments, the well is about 6.5 mm in diameter. In embodiments, the well is about 7 mm in diameter. In embodiments, the well is about 7.5 mm in diameter. In embodiments, the well is about 8 mm in diameter. In embodiments, the well is 5 mm in diameter. In embodiments, the well is 6 mm in diameter. In embodiments, the well is 6.5 mm in diameter. In embodiments, the well is 7 mm in diameter. In embodiments, the well is 7.5 mm in diameter. In embodiments, the well is 8 mm in diameter. In embodiments, the well is about 6 to 12 mm in depth. It is also understood that the size of the wells on the microplate section can be of various sizes and will ultimately depend on the systems and/or apparatus used to analyze later reactions. In embodiments, the wells may further include a removable sleeve or sheathe inserted within each well. For example, CellCrown™ inserts from SigmaAldrich, or Transwell® Permeable Supports from Corning Life Sciences.

[0113] In embodiments, the microplate (e.g., the microwell insert) includes a thermoplastic. In embodiments, the microplate includes a thermoplastic polyetherimide (PEI), for example ULTEM® PEI PolyEtherlmide (PEI). Ultem® resin is an amorphous thermoplastic polytherimide used in medical, aircraft, aerospace, electronics manufacturing, and communications due to outstanding high heat resistance, high strength and rigidity at elevated temperatures, and long-term heat resistance. In embodiments, the microplate is glass. In embodiments, the microplate is ceramic. In embodiments, the microplate includes steel attached to a glass bottom. In embodiments, the microplate is glass, wherein a plurality of wells are bored directly into the glass. In embodiments, the microplate does not degrade at temperatures greater than 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, or 120°C. In embodiments, the microplate does not degrade at temperatures greater than 50°C, 60°C, 70°C, 80°C, 90°C, or 100°C. In embodiments, the microplate does not degrade at 100°C. In embodiments, the microplate bonded to the planar support does not degrade or result in sample contamination at elevated temperatures (e.g., 80°C -120°C). The microplate may be used to detect biomolecules (e.g., nucleic acids and/or proteins). Typically, the nucleic acids need to be amplified. In embodiments the term “amplified” refers to a method that includes a polymerase chain reaction (PCR). Conditions conducive to amplification (i.e., amplification conditions) are well known and often include at least a suitable polymerase, a suitable template, a suitable primer or set of primers, suitable nucleotides e.g., dNTPs), a suitable buffer, and application of suitable annealing, hybridization and/or extension times and temperatures. Amplification conditions may cycle between different temperatures, often involving a large temperature gradient (e.g., 20°C -40°C). Additionally, samples embedded in formalin may require additional protocols to render biomolecules available. Heat induced epitope retrieval (HIER) uses heat coupled with buffered solutions to recover antigen reactivity in formalin fixed paraffin embedded tissue samples. Typical HIER methods include increasing the temperature from 25°C to 95°C-120°C, if utilizing a water bath or pressure enhanced temperature device (e.g., a pressure cooker). In embodiments, the microplate includes a microplate insert and a planar support attached to the microplate insert. In embodiments, a the planar support can include glass (e.g., a glass slide) that has been coated with a substance or otherwise modified to confer conductive properties to the glass. In some embodiments, a glass slide can be coated with a conductive coating. In some embodiments, a conductive coating includes tin oxide (TO) or indium tin oxide (ITO). In some embodiments, a conductive coating includes a transparent conductive oxide (TCO). In some embodiments, a conductive coating includes aluminum doped zinc oxide (AZO). In some embodiments, a conductive coating includes fluorine doped tin oxide (FTO).

[0114] In embodiments, the microplate includes a plurality of wells. In embodiments, each well includes about 10,000 to 100,000 cells per well. In embodiments, each well includes at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or at least 10,000 cells per well. In embodiments, each well includes about 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or at least 100,000 cells per well.

[0115] In embodiments, one or more wells include a tissue sample. In embodiments, one or more wells include a cell. In embodiments, one or more wells include a biological sample. In some embodiments, the biological sample is stained using a detectable label (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes). In embodiments, staining includes biological staining techniques such as H&E staining. In embodiments, staining includes identifying analytes using fluorescently- conjugated antibodies. In embodiments, the biological sample is stained using two or more different types of stains, or two or more different staining techniques. For example, a biological sample can be prepared by staining and imaging using one technique (e.g., H&E staining and brightfield imaging), followed by staining and imaging using another technique (e.g., IHC/IF staining and fluorescence microscopy) for the same biological sample. The biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In embodiments, the biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAP I, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In embodiments, the biological sample is derived from a formalin-fixation and paraffin-embedding (FFPE) sample. In embodiments, FFPE samples are stained (e.g., using H&E). The methods disclosed herein are compatible with H&E will allow for morphological context overlaid with transcriptomic analysis. However, depending on the need some samples may be stained with only a nuclear stain, such as staining a sample with only hematoxylin and not eosin, when location of a cell nucleus is needed. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy. In some instances, the biological sample can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. In some instances, the biological sample includes cancer or tumor cells.

[0116] In embodiments, the microplate and wells are comprised of the same material. Though typically glass, suitable microplate materials may include polymeric materials, plastics, silicon, quartz (fused silica), Borofloat® glass, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, sapphire, or plastic materials such as COCs and epoxies. The material can be selected based on properties desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will utilize radiation of the desired wavelength. Conversely, it may be desirable to select a material that does not pass radiation of a certain wavelength (e.g., being opaque, absorptive, or reflective). In embodiments, at least a portion of the bottom of the wells is transparent and the sides (i.e., walls) of the wells are opaque. In embodiments, the material of the microplate is selected due to the ability to conduct thermal energy. In embodiments, the microplate and wells as used herein may be collectively referred to herein as the receiving substrate.

[0117] In embodiments, the well of the microwell insert includes an internal cavity. In embodiments, the well includes a top end and a bottom end. In embodiments, the diameter of the internal cavity at the top end is greater than the diameter of the internal cavity at the bottom end. In embodiments, when the well is a cylindrical or frustoconical, the cross section may provide a useful metric for determining the size. In embodiments, the internal cavity may have a cross-section which is circular, triangular, square, rectangular, or a combination thereof. In embodiments, the cross section of the internal cavity at the top end is greater than the cross section of the internal cavity at the bottom end of the punch device. The cross- sectional area is simply the area of the circle (area=pi*r ² , where r is the radius). In embodiments, when the cross-sectional area varies throughout the well, e.g., having a frustoconical shape, the average cross-sectional area is an average of the cross-sectional areas. As a good approximation, the average cross-sectional area of a frustoconical-shaped device is the average of the circular cross-sections at each end. In embodiments, the internal diameter of the well is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 mm. In embodiments, the diameter of the internal cavity at the top end is about 4.5 mm to about 6.5 mm and the diameter of the internal cavity at the bottom end is about 5.0 mm to about 5.5 mm.

[0118] In embodiments, the microplate assembly does not include any wells. For example, FIG. 5 shows an embodiment of a blank microplate insert 501 that does not include any wells. For example, the microplate insert may be configured to retain a microscope slide.

[0119] FIG. 6 illustrates an embodiment of a microplate receiver frame 601 configured to retain one or more microplate inserts 602 and microplate gasket 604 therein, as described more fully below. Each microplate insert(s) includes a planar support 605 (e.g., glass bottom) attached to a primary well plate. The microplate receiver frame 601 and microplate insert 602 may each include one or more respective biasing or retention elements 603 that bias and/or retain the microplate insert 602 relative to the microplate receiver frame 601. The dashed lines in FIG. 6 are indicative of placement of the microplate insert(s) 602 into the microplate receiver frame 201. For example, microplate receiver frame 601 may contain four microplate inserts as described herein. The microplate gasket 604 is between the microplate insert 602 and the planar support 605. In embodiments, the gasket 604 is attached to the microplate insert 602 and the planar support 605 using an adhesive. In embodiments, the adhesive is a UV curable silicone pottant or sealant. In embodiments, the well includes a pocket to retain a portion of the gasket.

[0120] FIGS. 7A-C is an illustration of examples of the different diameters and dimensions of the devices contemplated herein. For example, the diameters of the individual wells for 96- well, 48-well, 24-well, and 12-well plates may include circular, square, or rectangular dimensions of varying diameters. The shapes of the wells of the microplate insert may be cuboid, cylindrical, pyramidal, conical, or frustoconical as illustrated in FIG. 7B. FIG. 7C illustrates a microplate section 700. A portion of the microplate section is depicted showing a partial cross-section of four wells of the microplate section. Each well includes a top 701 section, and axial walls 702; the dashed lines of the axial walls refer to the wall being within the microplate section. Also illustrated is the gasket 703 that forms a fluidic barrier within each well (i.e., each well is fluidically isolated from each other) in contact with the planar support 704 (e.g., glass bottom).

[0121] FIG. 8 shows 5 wells of a 24-well microwell plate section 800 containing at least a portion of a transferred tissue section within each well.

[0122] FIG. 9 shows a perspective, bottom view of a microplate insert 602 (or microplate section) and microplate gasket 604 coupled thereto. The microplate gasket 604 is juxtaposed with a bottom surface of the microplate insert 602 such that the microplate gasket 604 effectively forms a bottom-most surface of the microplate insert 602 when the two are attached. The microplate gasket 604 is sealingly engaged with the microplate insert 602 across the entire area of attachment such that the microplate gasket 604 can seal off each of the wells from one another. The microplate gasket 604 can also be juxtaposed with the planar support 605 when mounted thereon such that the microplate gasket 604 is positioned between the planar support 605 and the microplate insert 602. The microplate gasket 604 can have a shape that corresponds to the shape of the microplate insert 602 including a collection of openings that align with and complement bottom openings of the wells. In this manner, the microplate gasket 604 can form an enclosed, bottom surface of the wells. The microplate gasket 604 forms a uniform seal with the planar support 605 when coupled thereto.

[0123] FIG. 10 shows a cross-sectional view of the microplate insert 602 and microplate gasket 604 along line 10-10 of FIG. 9. The microplate insert 602 and microplate gasket 604 can couple to one another in a variety of manners. For example, the microplate gasket 604 can define one or more pockets that receive complementary shaped prongs of the microplate insert 602 in a sealing relationship. Other manners of coupling the microplate insert 602 and the microplate gasket are within the scope of this disclosure.

[0124] FIG. 11 shows a top view of an assembled microplate assembly 200 with several microplate inserts or sections 201 positioned within a frame 202. One or more bumpers 1105 are positioned between at least one section 201 and the frame 202. Each bumper 1105 is configured to provide a biasing or compression force between the section 201 and the frame such as to prevent shaking or rattling. The bumper(s) 1105 can be made of various materials including a deformable or malleable material such as rubber or silicone. The bumpers 1105 can be positioned at any of a variety of locations around the sections 201 to assist in fixing the position and orientation of the sections 201 within the frame 202. [0125] In embodiments, the planar support is glass. In embodiments, the planar support is attached via an adhesive to the microplate section (e.g., via a black adhesive). In embodiments, the microwell insert is attached to a planar solid support. In embodiments, the microwell insert is bonded to the planar solid support via an adhesive. In embodiments, the microwell insert is bonded to the planar solid support via a pressure sensitive adhesive. Pressure sensitive adhesives, e.g., a PSA tape, is self-stick tape or sticky tape including a pressure sensitive adhesive coated onto a scaffold support (e.g., backing material such as paper, plastic film, cloth, or metal foil). The pressure sensitive adhesive is sticky (i.e., tacky) without any heat or solvent for activation and adheres with minimal pressure. In embodiments, the adhesive includes an epoxy, poly(methyl methacrylate) (PMMA), or cyanoacrylate resin. For example, the adhesive may include Paraloid® B-72, Paraloid® B-67, Primal® AC 35, Acrifix® 116, HXTAL®-NYL-1, Araldite® 2020, or Loctite® Super Attack Precision. In embodiments, the adhesive is capable of absorbing light. In embodiments, the adhesive is transparent. In embodiments, the adhesive is capable of reflecting light. In embodiments, the adhesive includes a scaffold support (e.g., a tape). In embodiments, the scaffold support includes polyethylene terephthalate or cellulose. In embodiments, the adhesive includes a carboxylated styrene-butadiene binder. In embodiments, the adhesive has a thickness of about 0.05 to about 0.5 pm (i.e., the adhesive contacts the planar support and the microwell insert and is about 0.05 to about 0.5 pm thick). In embodiments, the adhesive is about 0.1, 0.2, 0.3, 0.4 or about 0.5 pm thick. The adhesive may include a pressure sensitive adhesive (PSA), a glue, a tape, magnets, mechanical connectors, or the like.

[0126] In embodiments, the adhesive is extruded (i.e., is a liquid) onto the microplate section and cures to form rigid substance and bonds to both the microplate section and the planar support. For example, the adhesive may include an epoxy resin, polyurethane, or silicone, and is resistant to extreme temperatures, chemicals, and mechanical stress. In embodiments, the adhesive forms a seal thereby preventing contamination from adjacent wells. As used herein, the term “curing” and related terms can refer to treating a substance with an agent and/or condition that transforms the substance from a liquid state to a solid state (e.g., transformed into a matrix), wherein the substance retains a three dimensional shape after the curing process. Suitable examples of curing techniques and/or conditions include ultraviolet (UV) radiation, infrared (IR) radiation, thermal radiation, microwave radiation, visible radiation, narrow- wavelength radiation, laser light, natural light, humidity, or combinations thereof. Suitable examples of a curing source include, for example, a UV light, a heating device, a radiation device, a microwave device, a plasma device, or combinations thereof. The adhesive may be applied via needle dispensingjetting, hand dispensing, flow coating, or brushing. Typical curing may include exposure for 1-2 seconds at 500 mW/cm ² or 3-5 seconds at a of 250 mW/cm ² .

[0127] In embodiments, the microwell insert is attached to a gasket, and the gasket is attached to the planar solid support. In embodiments, the gasket includes substantially the same pattern as the microwell insert (e.g., such that the bottom of each well of the microwell insert is in direct contact with the planar support). In embodiments, the gasket is bonded to the planar support. For example, the gasket may be irreversibly bound to a glass support when the surfaces of the gasket and the planar support are oxidized in an air plasma and then brought together. In embodiments, the gasket improves the autofocus stability.

[0128] In embodiments, the gasket is composed of a chemically inert material such that it does not substantially interact or interfere with the sample. In embodiments, the gasket does not substantially change shape when exposed to solvents or different analytical temperatures (e.g., 4°C to 150°C). For example, the gasket may include poly(dimethylsiloxane) (PDMS) material, which is known to minimally swell when exposed to solvents such as water, nitromethane, dimethyl sulfoxide, ethylene glycol, per-fluorotributylamine, perfluorodecalin, acetonitrile, and/or propylene carbonate. In embodiments, the gasket is composed of polydimethylsiloxane (PDMS), polystyrene (PS), or polycarbonate (PC). In embodiments, the gasket is composed of a thermoplastic fluoroelastomer, for example as described in McMillan et al. Nano Select. 2021; 2: 1385- 1402, which is incorporated herein by reference. In embodiments, the gasket is a poly(TFE-ter-E-ter HFP) gasket, wherein TFE = tetrafluoroethylene, E = ethylene, HFP = hexafluoropropylene. In embodiments, the gasket is melt processable, optically transparent, and includes self-sealing properties.

[0129] In embodiments, the gasket is 250 pm, 750 pm, 1200 pm and 2000 pm thick. In embodiments, the gasket is 2000 pm thick. In embodiments, the gasket is 1200 pm pm thick. In embodiments, the gasket is 750 pm, thick. In embodiments, the gasket is 250 pm thick. In embodiments, the gasket substantially conforms to the shape and pattern of the microplate insert. In embodiments, the gasket is between the microplate insert and the planar support, wherein the gasket does not cover the bottom of the well. In embodiments, the gasket provides a seal between each well, such that no liquid from a first well may contact or contaminate a second well. [0130] In embodiments, the gasket is substantially transparent to UV and/or visible light. In embodiments, the gasket is chemically inert. In embodiments, the gasket does not substantially swell (i.e., increase in volume) upon incubation in water. In embodiments, the gasket does not substantially swell (i.e., increase in volume) upon incubation in an organic solvent (e.g., xylene or hexane). In embodiments, the gasket does not dissolve in an organic solvent. In embodiments, the gasket does not dissolve in an a strong acid (e.g., HC1). In embodiments, the gasket does not substantially increase its volume upon incubation in xylene at room temperature. In embodiments, the gasket does not substantially increase its volume upon incubation in xylene for 10 minutes.

[0131] In embodiments, the planar solid support is a borosilicate glass (e.g., D263 glass). In embodiments, the planar solid support is Borofloat glass (e.g., SiCh: 81%; B2O3: 13%; Na2O/K2O: 4%; AI2O3: 2%), B270 glass, D263 glass, Eagle XG glass, Schott Supremax glass, Schott Xensation glass, or Gorilla glass. In embodiments, the planar solid support is a glass described in US 8,598,056; US 9,440,875; or US 2020/0339468, each of which are incorporated by reference. In embodiments, the planar solid support is a glass including 67 SiO ₂ = 70; 11 ^A1 ₂ O ₃ ^ 13.5; 3 B2O3 ^6; 3.5 0.5

BaO =3; 0.02 SnO ₂ i 0.3; CeO ₂ ^0.3; 0.00 As ₂ O ₃ i 0.5; 0.00 Sb ₂ O ₃ i 0.5; 0.01 Fe2O3 = 0.08; and F+Cl+Br 0.4; wherein all oxides are in mol %. In embodiments, the planar solid support is a borosilicate glass including 78% SiCh, 10% B2O2, 7% Na2O, 3% AI2O3, and 2% ZrCh. In embodiments, the planar solid support is a borosilicate glass including 80% SiCh, 13% B2O2, 4% Na2O, 2% AI2O3, and 1% K2O. In embodiments, the planar solid support is optically transparent. In embodiments, the planar solid support is 100 pm to 900 pm thick. In embodiments, the planar solid support is 500 pm to 900 pm thick. In embodiments, the planar solid support is 600 pm to 800 pm thick. In embodiments, the planar solid support is about 700 pm thick.

[0132] In embodiments, the planar support includes a functionalized glass surface or a functionalized plastic surface. In embodiments, the planar support includes (3- aminopropyl)tri ethoxy silane (APTES), (3 -Aminopropyl )trimethoxysilane (APTMS), y- Aminopropylsilatrane (APS), N-(6-aminohexyl)aminom ethyl tri ethoxysilane (AHAMTES), polyethylenimine (PEI), 5,6-epoxyhexyltriethoxysilane, or triethoxysilylbutyraldehyde, or a combination thereof. In embodiments, the planar support includes (3- aminopropyl)tri ethoxysilane (APTES). In embodiments, the planar support includes (3- Aminopropyl)trimethoxysilane (APTMS). In embodiments, the planar support includes y- Aminopropylsilatrane (APS). In embodiments, the planar support includes N-(6- aminohexyl)aminomethyltriethoxysilane (AHAMTES). In embodiments, the planar support surface includes polyethylenimine (PEI). In embodiments, the planar support includes 5,6- epoxyhexyltriethoxysilane. In embodiments, the planar support includes triethoxysilylbutyraldehyde. In embodiments, the functionalized glass surface is functionalized with APTES, APTMS, APS, or AHAMTES.

[0133] In embodiments, the microwell insert is resistant to chemical degradation. Chemical durability is measured according to known methods in the art, for example via measuring weight loss per surface area following contact with a chemical (e.g., HC1). In embodiments, the microwell insert is capable of contacting xylene without significant degradation (e.g., without significant weight loss). In embodiments, the microwell insert is capable of contacting HC1, HN03, HF, and/or NaOH, without significant degradation (e.g., without significant weight loss). In embodiments, the microwell insert is capable of contacting organic solvents, such as hexanes or xylenes. Such chemicals can react with the microplate polymers (i.e., oxidization, reaction with functional groups, catalyze de-polymerization), or be absorbed into the bulk microplate material and soften/swell the microplate.

[0134] In embodiments, the tissue is immobilized to the planar support by covalently binding the tissue to one or more bioconjugate reactive moieties of the planar support. In embodiments, the tissue is immobilized to the planar support by non-covalently binding the tissue to the planar support. For non-covalent binding, the tissue sections attach to the planar support surface due to surface interactions, such as Van der Waal forces, electrostatic forces, hydrophobic interactions and hydrogen bonds. The physical adsorption efficiency can be enhanced by treating the material with air plasma to increase its hydrophilicity.

[0135] In embodiments, the tissue section is attached to the bottom of a well, wherein the surface of the well is provided by the planar support. In embodiments, the tissue section is attached to the planar support via a bioconjugate reactive linker. In embodiments, the tissue section is attached to the planar support via a specific binding reagent. In embodiments, the specific binding reagent includes an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer. In embodiments, the specific binding reagent includes an antibody, or antigen binding fragment, an aptamer, affimer, or nonimmunoglobulin scaffold. In embodiments, the specific binding reagent is a peptide, a cell penetrating peptide, an aptamer, a DNA aptamer, an RNA aptamer, an antibody, an antibody fragment, a light chain antibody fragment, a single-chain variable fragment (scFv), a lipid, a lipid derivative, a phospholipid, a fatty acid, a triglyceride, a glycerolipid, a glycerophospholipid, a sphingolipid, a saccharolipid, a polyketide, a polylysine, polyethyleneimine, diethylaminoethyl (DEAE)-dextran, cholesterol, or a sterol moiety. Substrates may be prepared for selective capture of particular cells of the tissue section. For example, a substrate containing a plurality of bioconjugate reactive moi eties or a plurality of specific binding reagents, optionally in an ordered pattern, contacts a plurality of cells of the tissue section. Only cells of the tissue section containing complementary bioconjugate reactive moieties or complementary specific binding reagents are capable of reacting, and thus adhering, to the substrate.

[0136] In embodiments, the planar support includes a coating for enhanced biomolecule adhesion. Coatings for enhanced biomolecule adhesion are known, for example extracellular matrix proteins such as collagen type I, fibronectin, and laminin mediate specific binding of the cell to the protein. Poly-D-Lysine (PDL), a synthetically produced biomolecule, belongs to the non-specific adhesion-promoting polypeptides. PDL is typically used to promote cell adhesion, especially during washing steps, as well as to enhance cell vitality and proliferation during serum-reduced or serum-free cultivation. In embodiments, the planar support includes a plurality of avidin or streptavidin molecules. In embodiments, the planar support includes a plurality of bioconjugate reactive moieties.

[0137] In an aspect, provided herein are systems and devices that can detect the quantity of a biological target, detect biological activity indicative of a biological target, and perform nucleic acid sequencing. In an aspect is provided a device including the microplate assembly as described herein. In embodiments, the device is an integrated system of one or more chambers, ports, and channels that are directly or indirectly interconnected and in fluid communication and configured for carrying out an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides support functions, such as sample introduction, fluid and/or reagent driving means, temperature control, detection systems, data collection and/or integration systems, at least for the purpose of profiling a cell and/or determining the nucleic acid sequence of a template polynucleotide. In embodiments, the device as described herein is capable of multiplex analysis of a sample.

[0138] In an aspect is provided a device including: a sample stage configured to be coupled to a microplate receiver; a microplate receiver configured to be coupled to a microplate as described herein; at least one heating element thermally coupled to the microplate receiver; a fluidics dispenser configured to dispense one or more reagents into the microplate; and an imaging system configured to detect at least one feature (e.g., one or more features) in the microplate; and a structure physically coupled to the sample stage, the heating element, the fluidics dispenser, and the imaging system. In embodiments, the sample stage retains the microplate. In embodiments, the structure includes a support structure formed of a base platform or a table. In embodiments, the one or more features include a reaction chamber and its contents. In embodiments, the one or more features includes a target (e.g., a nucleic acid, protein, or biomarker), a cell, or a tissue sample. In embodiments, the feature is a nucleotide (e.g., a fluorescently labeled nucleotide). In embodiments, the feature is a nucleic acid. In embodiments, the feature is a protein. In embodiments, the feature is a biomolecule.

[0139] In embodiments, a lid is movably attached to the microplate receiver. The lid is sized and shaped to be able to enclose or cover a microplate positioned on the microplate receiver. In embodiments, the lid 410 is attached to the microplate receiver via a hinge assembly, which is configured to enable the lid to move between a raised or open position (relative to the microplate receiver) and closed position relative to the microplate receiver. In an embodiment, the lid transitions between the open and closed positions via a rotational or pivoting movement although the type of movement can vary. The lid encloses the microplate relative to the platform when the lid is closed. The lid may define a containment structure that defines an enclosed, temperature-controlled region. In this regard, the lid is configured to be heated or to provide heat. The lid can be thermally coupled to or can contain one or more heating elements for providing heat to and controlling the temperature-controlled region. In embodiments, the containment structure is an enclosed, temperature-controlled region with a defined humidity. In embodiments, the device includes at least one heating element, wherein the heating element is thermally coupled to the sample stage or microplate receiver. The heating element is configured to provide heat to a microplate coupled to the microplate receiver. In embodiments, a lid is configured to provide heat.

[0140] In an aspect is provided a microplate assembly (e.g., a microplate assembly as described herein) including 2 or more wells, wherein each well includes a cell including a polymerase complex, wherein the polymerase complex is bound (e.g., non-covalently bound) to a modified nucleotide. In embodiments, the microplate assembly includes a cell. In embodiments, the cell includes a polymerase complex, wherein the polymerase complex includes a polymerase bound to a double stranded nucleic acid molecule, wherein one strand of the double-stranded nucleic acid molecule comprises a modified nucleotide. In embodiments, the cell is within the well of the microplate assembly. In embodiments, the modified nucleotide includes a label. In embodiments, the modified nucleotide includes a reversible terminator (e.g., a reversible terminator as described herein. In embodiments, the microplate assembly includes 12, 24, 36, 48, 60, 72, 84, or 96 wells, wherein one or more wells includes a cell. In embodiments, each well includes a cell. In embodiments, one or more wells includes a plurality of cells, wherein each cell includes a polymerase complex.

[0141] In embodiments, the DNA polymerase is 9°N polymerase or a variant thereof, E. Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Taq DNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0 DNA polymerase, 9°N polymerase (exo-)A485L/Y409V, Phi29 DNA Polymerase (cp29 DNA Polymerase), T7 DNA polymerase, DNA polymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNA polymerase V, VentR DNA polymerase, Therminator™ II DNA Polymerase, Therminator™ III DNA Polymerase, or Therminator™ IX DNA Polymerase. In embodiments, the polymerase is a protein polymerase. Typically, a DNA polymerase adds nucleotides to the 3'- end of a DNA strand, one nucleotide at a time. In embodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol P DNA polymerase, Pol p DNA polymerase, Pol X DNA polymerase, Pol c DNA polymerase, Pol a DNA polymerase, Pol 6 DNA polymerase, Pol a DNA polymerase, Pol q DNA polymerase, Pol r DNA polymerase, Pol K DNA polymerase, Pol C, DNA polymerase, Pol y DNA polymerase, Pol 9 DNA polymerase, Pol u DNA polymerase, or a thermophilic nucleic acid polymerase (e.g. Therminator y, 9°N polymerase (exo-), Therminator II, Therminator III, or Therminator IX). In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a reverse transcriptase. In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO 2020/056044). In embodiments, the polymerase is an enzyme described in US 2021/0139884. In embodiments, the polymerase is phi29 polymerase, phi29 mutant polymerase, or a thermostable phi29 mutant polymerase. In embodiments, the polymerase is a DNA polymerase. In embodiments, the DNA polymerase is a thermophilic nucleic acid polymerase. In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a modified terminal deoxynucleotidyl transferase (TdT) enzyme. [0142] In an aspect is provided a kit. In embodiments, the kit is to support analysis of single cells and tissue sections on the device described herein. In embodiments, the kits enable multiomics analysis, including RNA transcription, protein expression, and targeted gene sequencing. In embodiments, the kits include specialized well-plates (e.g., a microplate assembly as describe herein), and reagents for sample preparation, and sequencing readout. In embodiments, the kits for protein detection include DNA-conjugated antibodies.

[0143] In embodiments, the microplate assembly as described herein is sterile prior to immobilizing the tissue section. Methods of sterilization include, but are not limited to, steam autoclaving (e.g., sterilization in an autoclave under a standard condition at 121 °C for 30 min), ethanol sterilization, and gamma irradiation, as described further in Han X. Biointerphases. 2017; 12(2): 02C411 and Galante R et al., J. Biomed. Mater. Res. B Appl. Biomater. 2018; 106(6): 2472-2492, each of which is incorporated herein by reference.

[0144] The term “kit” includes both fragmented and combined kits. In embodiments, the kit includes, without limitation, nucleic acid primers, probes, adapters, enzymes, and the like, and are each packaged in a container, such as, without limitation, a vial, tube or bottle, in a package suitable for commercial distribution, such as, without limitation, a box, a sealed pouch, a blister pack and a carton. The package typically contains a label or packaging insert indicating the uses of the packaged materials. As used herein, “packaging materials” includes any article used in the packaging for distribution of reagents in a kit, including without limitation containers, vials, tubes, bottles, pouches, blister packaging, labels, tags, instruction sheets and package inserts.

[0145] In embodiments, the kit can further include one or more biological stain(s) (e.g., any of the biological stains as described herein). For example, the kit can further include eosin and hematoxylin. In other examples, the kit can include a biological stain such as acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, safranin, or any combination thereof.

[0146] In embodiments the kits are for use in accordance with any of the devices, systems, or methods disclosed herein, and including one or more elements thereof. In embodiments, a kit includes labeled nucleotides including differently labeled nucleotides, enzymes, buffers, oligonucleotides, and related solvents and solutions. In embodiments, the kit includes an oligonucleotide primer (e.g., an oligonucleotide primer as described herein). The kit may also include a template nucleic acid (DNA and/or RNA), one or more primer polynucleotides, nucleoside triphosphates (including, e.g., deoxyribonucleotides, dideoxynucleotides, ribonucleotides, labeled nucleotides, and/or modified nucleotides), buffers, salts, and/or labels (e.g., fluorophores). In embodiments, the kit includes components useful for circularizing template polynucleotides using a ligation enzyme (e.g., Circligase enzyme, Taq DNA Ligase, HiFi Taq DNA Ligase, T4 ligase, or Ampligase DNA Ligase). For example, such a kit further includes the following components: (a) reaction buffer for controlling pH and providing an optimized salt composition for a ligation enzyme (e.g., Circligase enzyme, Taq DNA Ligase, HiFi Taq DNA Ligase, T4 ligase, or Ampligase DNA Ligase), and (b) ligation enzyme cofactors. In embodiments, the kit further includes instructions for use thereof. In embodiments, kits described herein include a polymerase. In embodiments, the polymerase is a DNA polymerase. In embodiments, the DNA polymerase is a thermophilic nucleic acid polymerase. In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the kit includes a sequencing solution. In embodiments, the sequencing solution include labeled nucleotides including differently labeled nucleotides, wherein the label (or lack thereof) identifies the type of nucleotide. For example, each adenine nucleotide, or analog thereof; a thymine nucleotide; a cytosine nucleotide, or analog thereof; and a guanine nucleotide, or analog thereof may be labeled with a different fluorescent label. In embodiments, the kit includes a modified terminal deoxynucleotidyl transferase (TdT) enzyme.

[0147] In embodiments, the kit includes a sequencing polymerase, and one or more amplification polymerases. In embodiments, the sequencing polymerase is capable of incorporating modified nucleotides. In embodiments, the polymerase is a DNA polymerase. In embodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol P DNA polymerase, Pol p DNA polymerase, Pol X DNA polymerase, Pol c DNA polymerase, Pol a DNA polymerase, Pol 6 DNA polymerase, Pol a DNA polymerase, Pol q DNA polymerase, Pol r DNA polymerase, Pol K DNA polymerase, Pol C, DNA polymerase, Pol y DNA polymerase, Pol 9 DNA polymerase, Pol u DNA polymerase, or a thermophilic nucleic acid polymerase (e.g., Therminator y, 9°N polymerase (exo-), Therminator II, Therminator III, or Therminator IX). In embodiments, the DNA polymerase is a thermophilic nucleic acid polymerase. In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a reverse transcriptase. In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO 2020/056044, each of which are incorporated herein by reference for all purposes). In embodiments, the kit includes a strand-displacing polymerase. In embodiments, the kit includes a strand-displacing polymerase, such as a phi29 polymerase, phi29 mutant polymerase or a thermostable phi29 mutant polymerase.

[0148] In embodiments, the kit includes a buffered solution. Typically, the buffered solutions contemplated herein are made from a weak acid and its conjugate base or a weak base and its conjugate acid. For example, sodium acetate and acetic acid are buffer agents that can be used to form an acetate buffer. Other examples of buffer agents that can be used to make buffered solutions include, but are not limited to, Tris, bicine, tricine, HEPES, TES, MOPS, MOPSO and PIPES. Additionally, other buffer agents that can be used in enzyme reactions, hybridization reactions, and detection reactions are known in the art. In embodiments, the buffered solution can include Tris. With respect to the embodiments described herein, the pH of the buffered solution can be modulated to permit any of the described reactions. In some embodiments, the buffered solution can have a pH greater than pH 7.0, greater than pH 7.5, greater than pH 8.0, greater than pH 8.5, greater than pH 9.0, greater than pH 9.5, greater than pH 10, greater than pH 10.5, greater than pH 11.0, or greater than pH 11.5. In other embodiments, the buffered solution can have a pH ranging, for example, from about pH 6 to about pH 9, from about pH 8 to about pH 10, or from about pH 7 to about pH 9. In embodiments, the buffered solution can include one or more divalent cations. Examples of divalent cations can include, but are not limited to, Mg ²⁺ , Mn ²⁺ , Zn ²⁺ , and Ca ²⁺ . In embodiments, the buffered solution can contain one or more divalent cations at a concentration sufficient to permit hybridization of a nucleic acid. In embodiments, the buffered solution can contain one or more divalent cations at a concentration sufficient to permit hybridization of a nucleic acid. In embodiments, the buffered solution includes about 10 mM Tris, about 20 mM Tris, about 30 mM Tris, about 40 mM Tris, or about 50 mM Tris. In embodiments the buffered solution includes about 50 mM NaCl, about 75 mM NaCl, about 100 mM NaCl, about 125 mM NaCl, about 150 mM NaCl, about 200 mM NaCl, about 300 mM NaCl, about 400 mM NaCl, or about 500 mM NaCl. In embodiments, the buffered solution includes about 0.05 mM EDTA, about 0.1 mM EDTA, about 0.25 mM EDTA, about 0.5 mM EDTA, about 1.0 mM EDTA, about 1.5 mM EDTA or about 2.0 mM EDTA. In embodiments, the buffered solution includes about 0.01% Triton X-100, about 0.025% Triton X-100, about 0.05% Triton X-100, about 0.1% Triton X-100, or about 0.5% Triton X-100. In embodiments, the buffered solution includes 20 mM Tris pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 0.025% Triton X-100. In embodiments, the buffered solution includes 20 mM Tris pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 0.025% Triton X-100. In embodiments, the buffered solution includes 20 mM Tris pH 8.0, 300 mM NaCl, 0.1 mM EDTA, 0.025% Triton X-100. In embodiments, the buffered solution includes 20 mM Tris pH 8.0, 400 mM NaCl, 0.1 mM EDTA, 0.025% Triton X-100. In embodiments, the buffered solution includes 20 mM Tris pH 8.0, 500 mM NaCl, 0.1 mM EDTA, 0.025% Triton X-100.

[0149] In embodiments, the kit includes one or more sequencing reaction mixtures. In embodiments, the sequencing reaction mixture includes a buffer. In embodiments, the buffer includes an acetate buffer, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, N-(2- Acetamido)-2-aminoethanesulfonic acid (ACES) buffer, phosphate-buffered saline (PBS) buffer, 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffer, N-(l,l- Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer, borate buffer (e.g., borate buffered saline, sodium borate buffer, boric acid buffer), 2-Amino-2- methyl- 1,3 -propanediol (AMPD) buffer, N-cy cl ohexyl -2 -hydroxyl-3 -aminopropanesulfonic acid (CAPSO) buffer, 2-Amino-2-methyl-l -propanol (AMP) buffer, 4-(Cyclohexylamino)-l- butanesulfonic acid (CABS) buffer, glycine-NaOH buffer, N-Cyclohexyl-2- aminoethanesulfonic acid (CHES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, or a N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. In embodiments, the buffer is a borate buffer. In embodiments, the buffer is a CHES buffer. In embodiments, the sequencing reaction mixture includes nucleotides, wherein the nucleotides include a reversible terminating moiety and a label covalently linked to the nucleotide via a cleavable linker. In embodiments, the sequencing reaction mixture includes a buffer, DNA polymerase, detergent (e.g., Triton X), a chelator (e.g., EDTA), and/or salts (e.g., ammonium sulfate, magnesium chloride, sodium chloride, or potassium chloride).

[0150] Adapters and/or primers may be supplied in the kits ready for use, as concentrates- requiring dilution before use, or in a lyophilized or dried form requiring reconstitution prior to use. If required, the kits may further include a supply of a suitable diluent for dilution or reconstitution of the primers and/or adapters. Optionally, the kits may further include supplies of reagents, buffers, enzymes, and dNTPs for use in carrying out nucleic acid amplification and/or sequencing. Further components which may optionally be supplied in the kit include sequencing primers suitable for sequencing templates prepared using the methods described herein.

[0151] In embodiments, the kit includes a microplate assembly. In embodiments, the microplate assembly includes a plurality of wells, wherein one or more wells include a functionalized glass surface or a functionalized plastic surface. In embodiments, the microplate assembly includes a container suitable for air- and moisture-sensitive components (e.g., the receiving substrate is packaged under nitrogen or argon). In embodiments, the kit includes a cutting device (e.g., a punch biopsy device). For example, a cutting device refers to a hollow, circular scalpel used to cut into portion of the tissue sample and/or the carrier substrate, which may be turned clockwise and counterclockwise to cut down about 4 millimeters (mm). In embodiments, the cutting device includes a circular hollow blade attached to a handle ranging, wherein the diameter of the circular hollow blade is about 0.5 mm to about 10 mm. In embodiments, the cutting device is disposable. In embodiments, the cutting device is reusable. In embodiments, the cutting device includes a plunger to aid in ejection of the cut section. In embodiments, the kit includes one or more detection agents (e.g., a detection agent as described herein, for example a fluorescent oligonucleotide probe and/or sequencing reagents).

[0152] The kits may include one or more of the following: fixative; carrier substrate (e.g., agarose, amylose, amylopectin, alginate, gelatin, cellulose, polyolefin, polyethylene glycol, polyvinyl alcohol, and/or acrylate polymers and copolymers); a surface including a plurality of wells separated from each other by interstitial regions on the surface, clearing reagents; nucleic acid probes, in situ hybridization buffer, labeled and/or un-labeled antibodies, buffers, e.g. buffer for fixing, washing, clearing, and/or staining specimens; mounting medium; embedding molds; dissection tools; etc. In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, digital storage medium, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the Internet to access the information at a removed site. Any convenient means may be present in the kits. II. Methods

[0153] In an aspect is provided a method of imaging a cell or tissue, the method including: obtaining a sample from a subject, wherein the sample includes a cell or tissue; contacting the microplate assembly of as described herein with the sample; and obtaining an image of the sample, thereby imaging the cell or tissue.

[0154] In another aspect is provided a method of imaging a cell, the method including obtaining a sample from a subject, wherein the sample comprises a cell (e.g., the sample is a tissue sample including a plurality of cells); contacting the microplate assembly as described herein with the sample; and obtaining an image of the sample, thereby imaging the cell. In embodiments, the method includes heating the cell. In embodiments, the method includes heating the cell to at least about 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., or higher. For example, the temperature can be about 37° C. In embodiments, the temperature is about 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C. to about 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C. 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., or higher. In embodiments, the microplate assembly is heated to about 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, or about 120°C. In embodiments, the microplate assembly is heated to 70° C., or higher. In embodiments, the microplate assembly is heated to 90° C., or higher.

[0155] In an aspect is provided a method of detecting a biomolecule in a tissue section, the method including: immobilizing the tissue section onto a microplate assembly as described herein (e.g., within a well), optionally permeabilizing the immobilized tissue section; and contacting the biomolecule in the tissue section with a detection agent thereby detecting the biomolecule in the tissue section. In embodiments, the tissue section is immobilized onto the microplate section. In embodiments, the tissue section is immobilized within one of the wells of the microplate section. In embodiments, the tissue section is 5 mm x 5 mm. In embodiments, the tissue section is 14 mm x 14 mm. In embodiments, the microplate assembly as described herein includes a tissue section in one or more wells. In embodiments, the tissue is 4-10 pm thick. In embodiments, the method detects one or more biomolecules per sample. In embodiments, the method detects 150-1000 different biomolecules per well. In embodiments, the method detects about 150 different biomolecules per well. In embodiments, the method detects about 150 to about 300 different biomolecules per well. [0156] In embodiments, the detection agent is a fluorescent oligonucleotide probe. In embodiments, the detection agent is an oligonucleotide, wherein the oligonucleotide may be detected (e.g., with a FISH probe). In embodiments, the probes can be labeled with a detectable label. In some embodiments, probes that do not specially bind (e.g., hybridize) to biomolecule can be washed away. In embodiments, the probes can be complementary to a single biomolecule (e.g., a single gene). In some embodiments, the probes can be complementary to one or more biomolecules (e.g., biomolecules in a family of genes). In some embodiments, the probes (e.g., detectable probes) can be for a panel of genes associated with a disease (e.g., cancer, Alzheimer's disease, Parkinson's disease).

[0157] In embodiments, the probe includes a fluorescent dye, a fluorescent protein, or combinations thereof. In embodiments, the probe includes a fluorescent dye that is selected from the group consisting of an acridine dye, a fluorone dye, a cyanine dye, a luciferin, an oxazine dye, a phenanthridine dye, a rhodamine dye, and combinations thereof. In embodiments, the probe includes a fluorescent protein that is selected from the group consisting of a green fluorescent protein (GFP), a Tag blue fluorescent protein (TagBFP), cerulean, a cyan fluorescent protein (CFP), venus, citrine, a yellow fluorescent protein (YFP), a monomeric enhanced green fluorescent protein (EGFP), mCherry, mKate2, a photoactivable green fluorescent protein (PA-GFP), a photoactivable mCherry (PA- mCherry), a fluorescent protein-fusion protein, and combinations thereof. In embodiments, the detection agent is an antibody. In embodiments, the biomolecule can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or a capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein. In embodiments, more than one biomolecule type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique.

[0158] In embodiments, detecting the biomolecule includes contacting the sample with a stain. In embodiments, a sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAP I, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. The sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In some embodiments, the biological sample can be stained using a detectable label (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes) as described elsewhere herein. In embodiments, a biological sample is stained using only one type of stain or one technique. In embodiments, staining includes biological staining techniques such as H&E staining. In embodiments, staining includes identifying analytes using fluorescently-conjugated antibodies. In some embodiments, a biological sample is stained using two or more different types of stains, or two or more different staining techniques. For example, a biological sample can be prepared by staining and imaging using one technique (e.g., H&E staining and brightfield imaging), followed by staining and imaging using another technique for the same biological sample.

[0159] A biomolecule can be identified in a tissue sample using a variety of different techniques, e.g., expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy, confocal microscopy, and visual identification (e.g., by eye), and combinations thereof. For example, the staining and imaging of a biomolecule can be performed to identify the presence of the biomolecule. In embodiments, the sample can be stained prior to visualization to provide contrast between the different regions of the sample. The type of stain can be chosen depending on the type of biological sample and the region of the cells to be stained. In embodiments, more than one stain can be used to visualize different aspects of the sample, e.g., different regions of the sample, specific cell structures (e.g., organelles), or different cell types. In other embodiments, the biological sample can be visualized or imaged without staining the biological sample.

[0160] In embodiments, biological samples can be destained. Methods of destaining or discoloring a biological sample are known in the art, and generally depend on the nature of the stain(s) applied to the sample. For example, H&E staining can be destained by washing the sample in HC1, or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid, benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid, succinic acid, salicylic acid, tartaric acid, sulfurous acid, trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid, orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citric acid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid, hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonic acid, hydrogen sulfide, or combinations thereof). In some embodiments, destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g., HC1). In some embodiments, destaining can include dissolving an enzyme used in the disclosed methods (e.g., pepsin) in an acid (e.g., HC1) solution. In some embodiments, after destaining hematoxylin with an acid, other reagents can be added to the destaining solution to raise the pH for use in other applications. For example, SDS can be added to an acid destaining solution in order to raise the pH as compared to the acid destaining solution alone. In embodiments, one or more immunofluorescence stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57:899-905, the entire contents of each of which are incorporated herein by reference.

[0161] In embodiments, substantially all of the tissue section is immobilized within a well of the microplate assembly. In embodiments, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the tissue section is immobilized within a well of the microplate assembly. In embodiments, greater than 90% of the tissue section is immobilized within a well of the microplate assembly. In embodiments, greater than 95% of the tissue section is immobilized within a well of the microplate assembly. In embodiments, greater than 96% of the tissue section is immobilized within a well of the microplate assembly. In embodiments, greater than 97% of the tissue section is immobilized within a well of the microplate assembly. In embodiments, greater than 98% of the tissue section is immobilized within a well of the microplate assembly. In embodiments, greater than 99% of the tissue section is immobilized within a well of the microplate assembly. In embodiments, about 100% of the tissue section is immobilized within a well of the microplate assembly. Methods for permeabilization are known in the art, as exemplified by Cremer et al., The Nucleus: Volume 1 : Nuclei and Subnuclear Components, R. Hancock (ed.) 2008; and Larsson et al., Nat. Methods (2010) 7:395-397, the content of each of which is incorporated herein by reference in its entirety. In embodiments, the tissue section is cleared (e.g., digested) of proteins, lipids, or proteins and lipids. In embodiments, permeabilizing the tissue section does not release the biomolecules (e.g., the one or more biomolecules) from within the tissue section. For example, after a fixation process (e.g. formaldehyde cross-linking), proteins and nucleic acids are immobilized within the cells of a tissue section, and are therefore not liberated into the environment following permeabilization of the cells.

[0162] In embodiments, the thickness of the tissue section is about 1 pm to about 20 pm. In embodiments, the thickness of the tissue section is about 5 pm to about 12 pm. In embodiments, the thickness of the tissue section is about 8 pm to about 15 pm. In embodiments, the thickness of the tissue section is about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, or about 15 pm. In embodiments, the thickness of the tissue section is about 1 pm. In embodiments, the thickness of the tissue section is about 2 pm. In embodiments, the thickness of the tissue section is about 3 pm. In embodiments, the thickness of the tissue section is about 4 pm. In embodiments, the thickness of the tissue section is about 5 pm. In embodiments, the thickness of the tissue section is about 6 pm. In embodiments, the thickness of the tissue section is about 7 pm. In embodiments, the thickness of the tissue section is about 8 pm. In embodiments, the thickness of the tissue section is about 9 pm. In embodiments, the thickness of the tissue section is about 10 pm. In embodiments, the thickness of the tissue section is about 11 pm. In embodiments, the thickness of the tissue section is about 12 pm. In embodiments, the thickness of the tissue section is about 13 pm. In embodiments, the thickness of the tissue section is about 14 pm. In embodiments, the thickness of the tissue section is about 15 pm. The thickness of the tissue section can be a fraction of (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximum cross-sectional dimension of a cell. However, tissue sections having a thickness that is larger than the maximum cross-section cell dimension can also be used. For example, cryostat sections can be used, which can be, e.g., 10-20 micrometers thick. For example, the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 micrometers. Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 micrometers or more. Typically, the thickness of a tissue section is between 1-100 micrometers, 1-50 micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1- 15 micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6 micrometers. Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by performing serial sectioning of the biopsy sample using a sectioning blade. Spatial information among the serial sections can be preserved in this manner, and the sections can be detected successively to obtain three- dimensional information about the biological sample.

[0163] In embodiments, the tissue section includes a tissue or a cell (e.g. plurality of cells such as blood cells). In embodiments, the tissue section includes one or more cells. In embodiments, one or more wells include a tissue or a cell.

[0164] Tissue sections include tissue or organ samples obtained from a subject, e.g., a mammal. In certain embodiments, the subject is diagnosed with a disease or disorder, such as a cancerous tumor, or considered at risk of having or developing the disease or disorder. Tissue sections may also be obtained from healthy donors, e.g., as normal control samples. In certain embodiments, both a disease tissue (e.g., a tumor tissue) sample and a normal sample are obtained from the same subject. In embodiments, the tissue section is obtained from a patient, e.g., a mammal such as a human. In other embodiments, a tissue section is obtained from an animal model of disease. Various animal models of disease are known and available in the art. Particular animal models of cancer include but are not limited to xenograft, syngeneic, and PDx models, e.g., in mice or rats. Animal models may also include human cells, cancerous or otherwise, introduced into animal models wherein tumor properties, progress, and treatment may be assessed. In vitro 3D tissue arrangements, organoids, and stem or iPS-cell-derived 3D compositions are also relevant models, and these may include human or other animal cells, for example.

[0165] In embodiments, the tissue section includes a tissue or a cell. Biological tissue samples suitable for use with the methods and systems described herein generally include any type of tissue samples collected from living or dead subjects, such as, for example, tumor tissue and autopsy samples. Tissue samples may be collected and processed using the methods and systems described herein and subjected to microscopic analysis immediately following processing, or may be preserved and subjected to microscopic analysis at a future time, e.g., after storage for an extended period of time. In some embodiments, the methods described herein may be used to preserve tissue samples in a stable, accessible and fully intact form for future analysis. For example, tissue samples, such as, e.g., human tumor tissue samples, may be processed as described herein and cleared to remove a plurality of cellular components, such as, e.g., lipids, and then stored for future analysis. In some embodiments, the methods and systems described herein may be used to analyze a fresh tissue section. In some embodiments, the methods and systems described herein may be used to analyze a previously-preserved (e.g., previously fixed) or stored tissue section (e.g., tissue sample). For example, in some embodiments a previously-preserved tissue sample that has not been subjected to a sample preparation process described herein may be processed and analyzed as described herein. In particular methods, a tissue sample is frozen prior to being processed as described herein.

[0166] In certain embodiments, tissue sections are tumor tissue samples. Tumor samples may contain only tumor cells, or they may contain both tumor cells and non-tumor cells. In particular embodiments, a tissue section includes only non-tumor cells. In particular embodiments, the tumor is a solid tumor. In particular embodiments, the tissue section is obtained from or includes an adrenal cortical cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumor, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, head or neck cancer, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, myelodysplasia syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal or squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancers caused by cancer treatment, is a tissue section obtained from a subject diagnosed with or suspected of having any of these tumors or cancers.

[0167] Tissue sections may be obtained from a subject by any means known and available in the art. In particular embodiments, a tissue section, e.g., a tumor tissue sample, is obtained from a subject by fine needle aspiration, core needle biopsy, stereotactic core needle biopsy, vacuum-assisted core biopsy, or surgical biopsy. In particular embodiments, the surgical biopsy is an incisional biopsy, which removes only part of the suspicious area. In other embodiments, the surgical biopsy is an excisional biopsy, which removes the entire diseased tissue (e.g., tumor) or abnormal area. In particular embodiments, an excisional tumor tissue sample is obtained from a tumor that has been excised with the intent to “cure” a patient in the case of early stage disease, wherein in other embodiments, the excisional tumor tissue sample is obtained from an excised bulk of primary tumor in later stage disease. Tumor tissue samples may include primary tumor tissue, metastastic tumor tissue and/or secondary tumor tissue. Tumor tissue samples may be cell cultures, e.g., cultures of tumor-derived cell lines. In certain embodiments, a tissue section is a cell line, e.g., a cell pellet of a cultured cell line, such as a tumor cell line. In particular embodiments, the cell line or cell pellet is frozen or was previously frozen. Such cell lines and pellets are useful, e.g., as positive or negative controls for imaging with various reagents. Tumor tissue samples may also be xenograft tumors, e.g., tumors obtained from animals administered with tumor cells, e.g., a human tumor cell line. In certain embodiments, a first tumor tissue sample from a subject is a primary tumor tissue sample obtained during an initial surgery intended to remove the entire tumor, and a second tumor tissue sample is obtained from the same subject is a metastatic tumor tissue sample or a secondary tumor tissue sample obtained during a later surgery.

[0168] Tissue sections, e.g., tumor tissue samples, may be obtained surgically or using a laparoscope. A tissue section may be a tissue sample obtained from any part of the body to examine it for disease or injury, e.g., presence of cancer tissue or cells, or the extent or characteristics thereof. In particular embodiments, the tissue section includes abdominal tissue, bone, bone marrow, breast tissue, endometrial tissue, kidney tissue, liver tissue, lung or chest tissue, lymph node, nerve tissue, skin, testicular tissue, head or neck tissue, or thyroid tissue. In certain embodiments, the tissue is obtained from brain, breast, skin, bone, joint, skeletal muscle, smooth muscle, red bone marrow, thymus, lymphatic vessel, thoracic duct, spleen, lymph node, nasal cavity, pharynx, larynx, trachea, bronchus, lung, oral cavity, esophagus, liver, stomach, small intestine, large intestine, rectum, anus, spinal cord, nerve, pineal gland, pituitary gland, thyroid gland, thymus, adrenal gland, pancreas, ovary, testis, heart, blood vessel, kidney, uterus, urinary bladder, urethra, prostate gland, penis, prostate, testis, scrotum, ductus deferens, mammary glands, ovary, uterus, vagina, or uterine tube.

[0169] In particular embodiments, a tissue section has a size greater than sections typically examined by traditional pathology thin section or immunohistochemical analysis, which are typically in the range of 4-10 microns thick. In certain embodiments, a tissue section is greater than 20 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, greater than 500 microns, greater than 1 mm, greater than 2 mm, greater than 5 mm, greater than 10 mm or greater than 20 mm in thickness and/or length. In particular embodiments, the tissue section has a length and/or a thickness between 20 microns and 20 mm, between 20 microns and 10 mm, or between 50 microns and 1 mm. In certain embodiments, a tissue section is a cubic sample with each side greater than 10 microns, greater than 20 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, greater than 500 microns, greater than 1 mm, greater than 2 mm, greater than 5 mm, greater than 10 mm, or greater than 2 mm in thickness and/or length. In some embodiments, a tissue section is thinner, e.g., from about 4-10 or 4-20 microns in thickness.

[0170] In embodiments, the tissue section includes an adherent cell (e.g., epithelial cell, endothelial cell, or neural cell). Adherent cells are usually derived from tissues of organs and attach to a substrate (e.g., epithelial cells adhere to an extracellular matrix coated substrate via transmembrane adhesion protein complexes). Adherent cells typically require a substrate, e.g., tissue culture plastic, which may be coated with extracellular matrix (e.g., collagen and laminin) components to increase adhesion properties and provide other signals needed for growth and differentiation. In embodiments, the tissue section includes a neuronal cell, an endothelial cell, epithelial cell, germ cell, plasma cell, a muscle cell, peripheral blood mononuclear cell (PBMC), a myocardial cell, or a retina cell. In embodiments, the tissue section includes a suspension cell (e.g., a cell free-floating in the culture medium, such a lymphoblast or hepatocyte). In embodiments, the tissue section includes a glial cell (e.g., astrocyte, radial glia), pericyte, or stem cell (e.g., a neural stem cell). In embodiments, the tissue section includes a neuronal cell. In embodiments, the tissue section includes an endothelial cell. In embodiments, the tissue section includes an epithelial cell. In embodiments, the tissue section includes a germ cell. In embodiments, the tissue section includes a plasma cell. In embodiments, the tissue section includes a muscle cell. In embodiments, the tissue section includes a peripheral blood mononuclear cell (PBMC). In embodiments, the tissue section includes a myocardial cell. In embodiments, the tissue section includes a retina cell. In embodiments, the tissue section includes a lymphoblast. In embodiments, the tissue section includes a hepatocyte. In embodiments, the tissue section includes a glial cell. In embodiments, the tissue section includes an astrocyte. In embodiments, the tissue section includes a radial glia. In embodiments, the tissue section includes a pericyte. In embodiments, the tissue section includes a stem cell. In embodiments, the tissue section includes a neural stem cell. Non-limiting examples of adherent cells include DU145 (prostate cancer) cells, H295R (adrenocortical cancer) cells, HeLa (cervical cancer) cells, KBM-7 (chronic myelogenous leukemia) cells, LNCaP (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breast cancer) cells, PC3 (prostate cancer) cells, SaOS- 2 (bone cancer) cells, SH-SY5Y (neuroblastoma, cloned from a myeloma) cells, T-47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, National Cancer Institute's 60 cancer cell line panel (NCI60), vero (African green monkey Chlorocebus kidney epithelial cell line) cells, MC3T3 (embryonic calvarium) cells, GH3 (pituitary tumor) cells, PC12 (pheochromocytoma) cells, dog MDCK kidney epithelial cells, Xenopus A6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insect epithelial cells.

[0171] In embodiments, the tissue section includes a cell bound to a known antigen. In embodiments, the cell is a cell that selectively binds to a desired target, wherein the target is an antibody, or antigen binding fragment, an aptamer, affimer, non-immunoglobulin scaffold, small molecule, or genetic modifying agent. In embodiments, the cell is a leukocyte (i.e., a white-blood cell). In embodiments, leukocyte is a granulocyte (neutrophil, eosinophil, or basophil), monocyte, or lymphocyte (T cells and B cells). In embodiments, the cell is a lymphocyte. In embodiments, the cell is a T cell, an NK cell, or a B cell.

[0172] In embodiments, the tissue section includes an immune cell. In embodiments, the immune cell is a granulocyte, a mast cell, a monocyte, a neutrophil, a dendritic cell, or a natural killer (NK) cell. In embodiments, the immune cell is an adaptive cell, such as a T cell, NK cell, or a B cell. In embodiments, the cell includes a T cell receptor gene sequence, a B cell receptor gene sequence, or an immunoglobulin gene sequence. In embodiments, the immune cell is a granulocyte. In embodiments, the immune cell is a mast cell. In embodiments, the immune cell is a monocyte. In embodiments, the immune cell is a neutrophil. In embodiments, the immune cell is a dendritic cell. In embodiments, the immune cell is a natural killer (NK) cell. In embodiments, the immune cell is a T cell. In embodiments, the immune cell is a B cell. In embodiments, the cell includes a T cell receptor gene sequence. In embodiments, the cell includes a B cell receptor gene sequence. In embodiments, the cell includes an immunoglobulin gene sequence. In embodiments, the plurality of target nucleic acids includes non-contiguous regions of a nucleic acid molecule. In embodiments, the non-contiguous regions include regions of a VDJ recombination of a B cell or T cell.

[0173] In embodiments, the tissue section includes a cancer cell. In embodiments, the cancer is lung cancer, colorectal cancer, skin cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer, lymphoma, leukemia, or a cancer associated with aberrant K-Ras, aberrant APC, aberrant Smad4, aberrant p53, or aberrant TGFp. In embodiments, the cancer cell includes & ERBB2, KRAS, TP53, PIK3CA, or FGFR2 gene. In embodiments, the cancer cell includes a cancer-associated gene (e.g., an oncogene associated with kinases and genes involved in DNA repair) or a cancer-associated biomarker. A “biomarker” is a substance that is associated with a particular characteristic, such as a disease or condition. A change in the levels of a biomarker may correlate with the risk or progression of a disease or with the susceptibility of the disease to a given treatment. In embodiments, the cancer is Acute Myeloid Leukemia, Adrenocortical Carcinoma, Bladder Urothelial Carcinoma, Breast Ductal Carcinoma, Breast Lobular Carcinoma, Cervical Carcinoma, Cholangiocarcinoma, Colorectal Adenocarcinoma, Esophageal Carcinoma, Gastric Adenocarcinoma, Glioblastoma Multiforme, Head and Neck Squamous Cell Carcinoma, Hepatocellular Carcinoma, Kidney Chromophobe Carcinoma, Kidney Clear Cell Carcinoma, Kidney Papillary Cell Carcinoma, Lower Grade Glioma, Lung Adenocarcinoma, Lung Squamous Cell Carcinoma, Mesothelioma, Ovarian Serous Adenocarcinoma, Pancreatic Ductal Adenocarcinoma, Paraganglioma & Pheochromocytoma, Prostate Adenocarcinoma, Sarcoma, Skin Cutaneous Melanoma, Testicular Germ Cell Cancer, Thymoma, Thyroid Papillary Carcinoma, Uterine Carcinosarcoma, Uterine Corpus Endometrioid Carcinoma, or Uveal Melanoma. In embodiments, the cancer-associated gene is a nucleic acid sequence identified within The Cancer Genome Atlas Program, accessible at www.cancer.gov/tcga.

[0174] In embodiments, the tissue section is obtained from a subject (e.g., human or animal tissue). Once obtained, the tissue section is placed in an artificial environment in plastic or glass containers supported with specialized medium containing essential nutrients and growth factors to support proliferation. In embodiments, the tissue section is permeabilized and immobilized to a solid support surface. In embodiments, the tissue section is permeabilized and immobilized to an array (i.e., to discrete locations arranged in an array). In embodiments, the tissue section is immobilized to a solid support surface. In embodiments, the surface includes a patterned surface (e.g., suitable for immobilization of a plurality of cells in an ordered pattern. The discrete regions of the ordered pattern may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like. These discrete regions are separated by interstitial regions. As used herein, the term “interstitial region” refers to an area in a substrate or on a surface that separates other areas of the substrate or surface. In embodiments, a plurality of cells are immobilized on a patterned surface that have a mean or median separation from one another of about 10-20 pm. In embodiments, a plurality of cells are immobilized on a patterned surface that have a mean or median separation from one another of about 10-20; 10-50; or 100 pm. In embodiments, a plurality of cells are arrayed on a substrate. In embodiments, a plurality of cells are immobilized in a 96-well microplate having a mean or median well-to- well spacing of about 8 mm to about 12 mm (e.g., about 9 mm). In embodiments, a plurality of cells are immobilized in a 384-well microplate having a mean or median well-to-well spacing of about 3 mm to about 6 mm (e.g., about 4.5 mm).

[0175] In embodiments, the tissue section is embedded in an embedding material including paraffin wax, polyepoxide polymer, polyacrylic polymer, agar, gelatin, celloidin, cryogel, optimal cutting temperature (OCT) compositions, glycols, or a combination thereof. In embodiments, the tissue section is embedded in an embedding material including paraffin wax. In embodiments, the OCT composition includes about 10% polyvinyl alcohol and about 4% polyethylene glycol. In embodiments, the OCT composition includes sucrose (e.g., 30% sucrose). In embodiments, the OCT composition is Tissue Freezing Medium (TFM) available from Leica Microsystems, Catalog #14020108926.

[0176] In embodiments, the tissue section is an artificial tissue section, wherein the artificial tissue section includes one or more cells suspended in a hydrogel. In embodiments, the artificial tissue section includes one or more cells suspended in a hydrogel that is embedded in an optimal cutting temperature (OCT) composition. In embodiments, the artificial tissue section is prepared according to the following method: the sample containing the biomolecule of interest (e.g., a cell or a particle) is embedded in a crosslinked hydrogel (e.g., a polymer composition including 3 to 20% acrylamide and N,N-dimethylacrylamide). Any suitable hydrogel may be used, for example a hydrogel including poly(2 -hydroxyethyl methacrylate) (PHEMA), optionally crosslinked with polyethylene glycol dimethacrylate; 2- hydroxyethyl methacrylate (HEMA) optionally crosslinked with TEGDMA (triethylene glycol dimethacrylate); polyethylene glycol methacrylate (PEGMA), optionally crosslinked with TEGDMA (triethylene glycol dimethacrylate); a copolymer of methacrylic acid (MAA) and polyethylene glycol methacrylate (PEGMA), optionally crosslinked with tetra(ethylene glycol) dimethacrylate; or poly(N-isopropyl acrylamide) (PNIPAM), optionally crosslinked with N,N-methylene bisacrylamide. Additional hydrogels include a polymer such as poly(hydroxyethyl methacrylate) (PHEMA), poly(glyceryl methacrylate) (PGMA), poly(hydroxypropyl methacrylate) (PHPMA), polyacrylamide (PAM), polymethacrylamide (PMAM), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyvinyl pyrrolidone (PVP), poly(s-caprolactone) (PCL), poly(ethyleneimine) (PEI), poly(N,N-dimethylacrylamide) (PDMAM), poly(2-methoxyethyl acrylate) (PMEA), or a copolymer thereof. Polymer chains in a hydrogel may be crosslinked with each other chemically via covalent bonds or physically via non-covalent interactions to produce the network structure. The physical cross-linking involves hydrogen bonding, hydrophobic interactions, crystallinity, and ionic interactions. In chemically cross-linked hydrogels, covalent bonds cross-link individual polymer chains. Any suitable crosslinker may be used, for example N,N-methylene bisacrylamide, N,N-ethylene bisacrylamide, 1,4-Bis(acryloyl)piperazine, tri ethylene glycol dimethacrylate (TEGDMA), 1,1,1 -trimethylolpropane trimethacrylate (TMPTMA), poly(ethylene glycol) dimethacrylate (PEGDMA), glyoxal, or tetramethylethylenediamineor N,N'-Bis(acryloyl)cystamine.

[0177] Following hydrogel embedding, the sample was frozen in OCT at -80°C. The frozen OCT-hydrogel complex was then sectioned (e.g., tissue sections of 5pm and 9pm thickness were derived). It is known that OCT compounds may impact PCR amplification, see for example Turbett and Sellner (Diagn Mol Pathol. 1997 Oct;6(5):298-303), so embedding the biological sample in a hydrogel first helps protect the sample from downstream effects from the OCT.

[0178] In embodiments, the biological sample includes cells (e.g., derived from a cell culture or a tissue sample). In a biological sample with a plurality of cells, individual cells can be naturally unaggregated. For example, cells can be derived from a suspension of cells and/or disassociated or disaggregated cells from a tissue or tissue section. Alternatively, the cells in the sample may be aggregated, and may be disaggregated into individual cells using, for example, enzymatic or mechanical techniques. Examples of enzymes used in enzymatic disaggregation include, but are not limited to, dispase, collagenase, trypsin, or combinations thereof. Mechanical disaggregation can be performed, for example, using a tissue homogenizer.

[0179] In embodiments, the tissue section is exposed to paraformaldehyde (i.e., by contacting the cell with paraformaldehyde). Any suitable permeabilization and fixation technologies can be used for making the cell available for the detection methods provided herein. In embodiments the method includes affixing single cells or tissues to a transparent substrate. Exemplary tissue include those from skin tissue, muscle tissue, bone tissue, organ tissue and the like. In embodiments, the method includes immobilizing the tissue section in situ to a substrate and permeabilized for delivering probes, enzymes, nucleotides and other components required in the reactions. In embodiments, the tissue section includes many cells from a tissue section in which the original spatial relationships of the cells are retained. In embodiments, the tissue section in situ is within a Formalin-Fixed Paraffin-Embedded (FFPE) sample. In embodiments, the tissue section is subjected to paraffin removal methods, such as methods involving incubation with a hydrocarbon solvent, such as xylene or hexane, followed by two or more washes with decreasing concentrations of an alcohol, such as ethanol. The tissue section may be rehydrated in a buffer, such as PBS, TBS or MOPs. In embodiments, the FFPE sample is incubated with xylene and washed using ethanol to remove the embedding wax, followed by treatment with Proteinase K to permeabilized the tissue. In embodiments, the paraffin-embedding material is removed from the tissue section (e.g., deparaffinization) by incubating the tissue section in an appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes). In embodiments, the tissue section is fixed with a chemical fixing agent. In embodiments, the chemical fixing agent is formaldehyde or glutaraldehyde. In embodiments, the chemical fixing agent is glyoxal or dioxolane. In embodiments, the chemical fixing agent includes one or more of ethanol, methanol, 2-propanol, acetone, and glyoxal. In embodiments, the chemical fixing agent includes formalin, Greenfix®, Greenfix® Plus, UPM, CyMol®, HOPE®, CytoSkelFix™, F-Solv®, FineFIX®, RCL2/KINFix, UMFIX, Glyo-Fixx®, Histochoice®, or PAXgene®. In embodiments, the tissue section is fixed within a synthetic three-dimensional matrix (e.g., polymeric material). In embodiments, the synthetic matrix includes polym eric-crosslinking material. In embodiments, the material includes polyacrylamide, poly-ethylene glycol (PEG), poly(acrylate-co-acrylic acid) (PAA), or Poly (N-i sopropyl acryl ami de) (NIP AM) .

[0180] In embodiments, the fixed tissue may be frozen tissue. The frozen biological tissue can be fixed using a fixing agent, which is suitably an organic fixing agent. In some embodiments, the fixing agent can be chilled and can be at a temperature of about 0° C to about 100° C, suitably about zero to about 50° C, or about 1° C to about 50° C. The fixing agent can be chilled by placing it over a bed of ice to maintain its temperature as close to 0° C as possible. The frozen biological tissue can be treated with the fixing agent using any suitable technique, suitably by immersing it in the fixing agent for a period of time. Depending on the type and size of the biological tissue sample, the treatment time can range from about 5 minutes to about 60 minutes, suitably about 10 minutes to about 30 minutes, or about 15 minutes to about 25 minutes, or about 20 minutes. In some embodiments, treatment time may be overnight. During fixing, the snap-frozen tissue will thaw but will suitably remain at a low temperature due to the low temperature environment of the fixing agent.

[0181] In some embodiments, the type/identity of a fixation agent, the amount/concentration of a fixation agent, the temperature at which it is used, the duration for which it is used, and the like, may be empirically determined or titrated. These parameters, and others, may need to be varied to obtain optimal results for different tissues, for different organisms, or for different days on which an experiment is performed. Insufficient fixation (e.g., too little fixing agent, too low temperature, too short duration) may not, for example, stabilize/preserve the cells/organelles/analytes of tissues. Excess fixation (e.g., too much fixing agent, too high temperature, too long duration) may result in the single biological samples (e.g., cells/nuclei) obtained from the methods not yielding good results in single biological sample (e.g., single-cell or single nucleus) workflows or assays in which the biological samples (e.g., cells or nuclei) are used. Generally, the quality of data obtained in these workflows/assays may be a good measure of the extent of the fixation process.

[0182] In some embodiments, the fixative can be diluted in a buffer, e.g., saline, phosphate buffer (PB), phosphate buffered saline (PBS), citric acid buffer, potassium phosphate buffer, etc., usually at a concentration of about 1-10%, e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10%, for example, 4% paraformaldehyde/0.1 M phosphate buffer; 2% paraformaldehyde/0.2% picric acid/0.1 M phosphate buffer; 4% paraformaldehyde/0.2% periodate/1.2% lysine in 0.1 M phosphate buffer; 4% paraformaldehyde/0.05% glutaraldehyde in phosphate buffer; etc. The type of fixative used and the duration of exposure to the fixative can depend on the sensitivity of the molecules of interest in the tissue section to denaturation by the fixative, and can be readily determined using conventional histochemical or immunohistochemical techniques, for example as described in Buchwalow and Bocker. Immunohistochemistry: Basics and Methods. Springer-Verlag Berlin Heidelberg 2010.

[0183] During the fixing, the biological tissue sample can be periodically cut into successively smaller segments while it is submerged in the fixation solution, to facilitate perfusion and fixation of the biological tissue sample by the organic fixing agent. For example, the tissue sample may have an initial length, width and/or diameter of about 0.25 cm to about 1.5 cm or may be initially cut into segments having such suitable dimensions. After a first periodic interval, the tissue sample or segments can be cut into smaller segments, and the smaller segments can remain immersed in the fixing agent. This process can be repeated after a second periodic interval, after a third periodic interval, after a fourth periodic interval, and so on. The periodic intervals can range from about 1 to about 10 minutes, or about 2 to about 8 minutes, or about 4 to about 6 minutes. The sum of the periodic intervals can equal the entire fixing time and can range from about 5 to about 60 minutes, or about 10 to about 30 minutes, or about 15 to about 25 minutes, for example. The resulting fixed tissue segments can have a length, width and/or diameter in a range of less than 1 mm to about 10 mm, by way of example. In some embodiments, the tissue is not cut into smaller segments during fixation. In some embodiments, this may be performed prior to fixation. In some embodiments, this may be performed after fixation.

[0184] In embodiments, the tissue section is embedded in an embedding material including a polyepoxide polymer. In embodiments, the tissue section is embedded in an embedding material including polyacrylic polymer. In embodiments, the tissue section is embedded in an embedding material including agar. In embodiments, the tissue section is embedded in an embedding material including gelatin. In embodiments, the tissue section is embedded in an embedding material including celloidin. In embodiments, the tissue section is embedded in an embedding material including a cryogel. In embodiments, the tissue section is embedded in an embedding material including an optimal cutting temperature (OCT) compositions. In embodiments, the tissue section is embedded in an embedding material including one or more glycols.

[0185] In embodiments, the method further includes removing the embedding material. In embodiments, the method further includes removing the embedding material prior to immobilizing the tissue section. For example, if the embedding material is paraffin wax, the embedding material is removed by contacting the tissue section with a hydrocarbon solvent, such as xylene or hexane, followed by two or more washes with decreasing concentrations of an alcohol, such as ethanol.

[0186] In embodiments, the methods disclosed herein also include a wash step. The wash step removes any unbound agents (e.g., probes or nucleotides). Wash steps could be performed between any of the steps in the methods disclosed herein. For example, a wash step can be performed after adding nucleotides and/or probes (e.g., antibodies or oligonucleotides) to the sample. As such, free/unbound probes are washed away, leaving only probes that have hybridized to a biomolecule. In some embodiments, multiple (i.e., at least 2, 3, 4, 5, or more) wash steps occur between the methods disclosed herein. Wash steps can be performed at times (e.g., 1, 2, 3, 4, or 5 minutes) and temperatures (e.g., room temperature; 4° C. known in the art and determined by a person of skill in the art. In embodiments, wash steps are performed using a wash buffer. In some instances, the wash buffer includes SSC (e.g., 1 x SSC). In some instances, the wash buffer includes PBS (e.g., 1 xPBS). In some instances, the wash buffer includes PBST (e.g., 1 xPBST).

[0187] Imaging deep into a tissue volume is problematic due to inherently fluorescent molecules present in the tissue or introduced during processing which give rise to autofluorescence that masks fluorescently labelled structures of interest. Additionally, some plastics (e.g., a plastic planar support) are a source of autofluorescence. Typically, autofluorescence decreases image quality by lowering the signal to noise ratio across multiple fluorescence channels and undermines sharp images. Autofluorescence may arise from endogenous fluorescent biomolecules (NADPH, collagen, flavins, tyrosine, and others) or be introduced by the formation of Schiffs bases during fixation with aldehydes (e.g., glutaraldehyde and paraformaldehyde). Additional light scattering is provided by various cellular components, such as ribosomes, nuclei, nucleoli, mitochondria, lipid droplets, membranes, myelin, cytoskeletal components, and extracellular matrix components such as collagen and elastin.

[0188] In embodiments, the tissue is cleared using a solvent-based clearing approach. Solvent-based clearing techniques typically includes two steps: 1) dehydration (e.g., contacting the sample with methanol with or without hexane or, tetrahydrofurane (THF) alone) and 2) clearing by refractive index matching to the remaining dehydrated tissue’s index (e.g., contacting the tissue sample with methylsalicilate, benzyl alcohol, benzyl benzoate, dichloromethane, or dibenzyl ether). Alternatively, the initial dehydration may be performed using phosphate buffered saline (PBS), detergent, and dimethyl sulfoxide (DMSO). In embodiments, the tissue is cleared by contacting the tissue sample with an aqueous solution containing sucrose, fructose, 2,2'-thiodiethanol (TDE), or formamide.

[0189] In embodiments, the tissue is cleared utilizing the 3D imaging of solvent-cleared organs (3DISCO) method as described in Erturk Aet al. Nat Protoc. 2012 Nov;7(l 1): 1983-95, which is incorporated herein by reference. For example, a sample is incubated overnight in 50% v/v tetrahydrofuran/FEO (THF), followed by incubation for at least one hour 80% THF/H2O and followed by incubation in a 100% THF solution. This is then followed by contacting the sample with dichloromethane (DCM) and an incubation in dibenzyl ether (DBE) until clear.

[0190] In embodiments, the tissue is cleared according to a known technique in the art, for example CLARITY (Chung K., et al. Nature 497, 332-337 (2013)), PACT-PARS (Yang Bet al. Cell 158, 945-958 (2014).), CUBIC (Susaki E. A. et al. Cell 157, 726-739 (2014)., 18), ScaleS (Hama H., et al. Nat. Neurosci. 18, 1518-1529 (2015)), OPTIClear (Lai H. M., et al. Nat. Commun. 9, 1066 (2018)), C _e 3D (Li W., et al. Proc. Natl. Acad. Sci. U.S.A. 114, E7321-E7330 (2017)), BABB (Dodt H.U. et al. Nat. Methods 4, 331-336 (2007)), iDISCO (Renier N., et al. Cell 159, 896-910 (2014)), uDISCO (Pan C., et al. Nat. Methods 13, 859- 867 (2016)), FluoClearBABB (Schwarz M. K., et al. PLOS ONE 10, e0124650 (2015)), Ethanol-ECi (Klingberg A., et al. J. Am. Soc. Nephrol. 28, 452-459 (2017)), and PEGASOS (Jing D. et al. Cell Res. 28, 803-818 (2018)).

[0191] In embodiments, the tissue section is contacted with an alkaline solution containing a combination of 2,2 '-thiodi ethanol (TDE), DMSO, D-sorbitol, and Tris. In embodiments, the tissue section is contacted with an aqueous solution including 20% (vol/vol) DMSO, 40% (vol/vol) TDE, 20% (wt/vol) sorbitol, and 6% (wt/vol, equal to 0.5 M) Tris base. In embodiments, the tissue section is contacted with an aqueous solution including 25% (wt/wt) urea, 25% (wt/wt) N,N,N',N'-Tetrakis (2 -hydroxypropyl) ethylenediamine, and 15% (wt/wt) Triton X-100. In embodiments, the tissue section is contact with an aqueous solution including 9.1 M urea, 22.5% (wt/vol) D-sorbitol, and 5% (wt/vol) Triton X-100. In embodiments, the tissue section is contact with an aqueous solution including 30% (wt/vol) urea, 20% (wt/vol) D-sorbitol, and 5% (wt/vol) glycerol dissolved in DMSO. In embodiments, the tissue section is contact with an aqueous solution according to the protocols described in Shan, QH., Qin, XY., Zhou, N. et al. BMC Biol 20, 77 (2022).

[0192] In embodiments, the biological sample can be permeabilized using any of the methods described herein (e.g., using any of the detergents described herein, e.g., SDS and/or N-lauroylsarcosine sodium salt solution) before or after enzymatic treatment (e.g., treatment with any of the enzymes described herein, e.g., trypin, proteases (e.g., pepsin and/or proteinase K)). In embodiments, the biological sample can be permeabilized by contacting the sample with a permeabilization solution. In some embodiments, the biological sample is permeabilized by exposing the sample to greater than about 1.0 w/v % (e.g., greater than about 2.0 w/v %, greater than about 3.0 w/v %, greater than about 4.0 w/v %, greater than about 5.0 w/v %, greater than about 6.0 w/v %, greater than about 7.0 w/v %, greater than about 8.0 w/v %, greater than about 9.0 w/v %, greater than about 10.0 w/v %, greater than about 11.0 w/v %, greater than about 12.0 w/v %, or greater than about 13.0 w/v %) sodium dodecyl sulfate (SDS) and/or N-lauroylsarcosine or N-lauroylsarcosine sodium salt. In some embodiments, the biological sample can be permeabilized by exposing the sample (e.g., for about 5 minutes to about 1 hour, about 5 minutes to about 40 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, or about 5 minutes to about 10 minutes) to about 1.0 w/v % to about 14.0 w/v % (e.g., about 2.0 w/v % to about 14.0 w/v %, about 2.0 w/v % to about 12.0 w/v %, about 2.0 w/v % to about 10.0 w/v %, about 4.0 w/v % to about 14.0 w/v %, about 4.0 w/v % to about 12.0 w/v %, about 4.0 w/v % to about 10.0 w/v %, about 6.0 w/v % to about 14.0 w/v %, about 6.0 w/v % to about 12.0 w/v %, about 6.0 w/v % to about 10.0 w/v %, about 8.0 w/v % to about 14.0 w/v %, about 8.0 w/v % to about 12.0 w/v %, about 8.0 w/v % to about 10.0 w/v %, about 10.0% w/v % to about 14.0 w/v %, about 10.0 w/v % to about 12.0 w/v %, or about 12.0 w/v % to about 14.0 w/v %) SDS and/or N-lauroylsarcosine salt solution and/or proteinase K (e.g., at a temperature of about 4% to about 35° C., about 4° C. to about 25° C., about 4° C. to about 20° C., about 4° C. to about 10° C., about 10° C. to about 25° C., about 10° C. to about 20° C., about 10° C. to about 15° C., about 35° C. to about 50° C., about 35° C. to about 45° C., about 35° C. to about 40° C., about 40° C. to about 50° C., about 40° C. to about 45° C., or about 45° C. to about 50° C ).

[0193] In embodiments, the biomolecule is a nucleic acid sequence, carbohydrate, or protein. In embodiments, the biomolecule is a nucleic acid sequence. In embodiments, contacting the biomolecule includes detecting the biomolecule by hybridizing one or more fluorescent probes to the biomolecule and detecting the one or more fluorescent probes. In embodiments, contacting the biomolecule includes hybridizing a sequencing primer to the biomolecule and sequencing the biomolecule. In embodiments, sequencing includes (a) extending a sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue and (b) detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue. Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, Conn., a Life Technologies subsidiary) or sequencing methods and systems described in US 2009/0026082; US 2009/0127589; US 2010/0137143; or US 2010/0282617, each of which is incorporated herein by reference. In embodiments, detecting includes sequencing.

[0194] In embodiments, the probe includes a sequence that is about 10 nucleotides to about 100 nucleotides (e.g., a sequence of about 10 nucleotides to about 90 nucleotides, about 10 nucleotides to about 80 nucleotides, about 10 nucleotides to about 70 nucleotides, about 10 nucleotides to about 60 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 20 nucleotides, about 20 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 30 nucleotides, about 30 nucleotides to about 100 nucleotides, about 30 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 30 nucleotides to about 70 nucleotides, about 30 nucleotides to about 60 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 40 nucleotides, about 40 nucleotides to about 100 nucleotides, about 40 nucleotides to about 90 nucleotides, about 40 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 40 nucleotides to about 60 nucleotides, about 40 nucleotides to about 50 nucleotides, about 50 nucleotides to about 100 nucleotides, about 50 nucleotides to about 90 nucleotides, about 50 nucleotides to about 80 nucleotides, about 50 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 60 nucleotides to about 100 nucleotides, about 60 nucleotides to about 90 nucleotides, about 60 nucleotides to about 80 nucleotides, about 60 nucleotides to about 70 nucleotides, about 70 nucleotides to about 100 nucleotides, about 70 nucleotides to about 90 nucleotides, about 70 nucleotides to about 80 nucleotides, about 80 nucleotides to about 100 nucleotides, about 80 nucleotides to about 90 nucleotides, or about 90 nucleotides to about 100 nucleotides).

[0195] In embodiments, prior to contacting the tissue section to the microplate section, a portion of the tissue section is removed. Removal of a portion of the tissue section may be performed, for example, with a cutting device. The cutting device may include a sharp blade, and the cutting may be performed manually, or may be automated. In other embodiments, removal of a portion of the tissue section may be performed, for example, through the use of photon or acoustic energy (see, e.g., U.S. Pat. Pubs. US2004/0247777 and US2016/0025604, each of which is incorporated herein by reference in its entirety). In embodiments, a portion of the tissue section is cut and removed from the total tissue section.

[0196] In embodiments, sequencing includes extending a sequencing primer to incorporate a nucleotide containing a detectable label that indicates the identity of a nucleotide in the target polynucleotide, detecting the detectable label, and repeating the extending and detecting of steps. In embodiments, the methods include sequencing one or more bases of a target nucleic acid by extending a sequencing primer hybridized to a target nucleic acid (e.g., an amplification product of a target nucleic acid). In embodiments, the sequencing includes sequencing-by-synthesis, sequencing-by-binding, sequencing by ligation, sequencing-by- hybridization, or pyrosequencing, and generates a sequencing read. In embodiments, generating a sequencing read includes executing a plurality of sequencing cycles, each cycle including extending the sequencing primer by incorporating a nucleotide or nucleotide analogue using a polymerase and detecting a characteristic signature indicating that the nucleotide or nucleotide analogue has been incorporated.

[0197] In embodiments, sequencing includes extending a first sequencing primer to generate a sequencing read. In embodiments, sequencing includes extending a first sequencing primer to generate a sequencing read comprising a first sequence, or a portion thereof, and extending a second sequencing primer to generate a sequencing read comprising a second sequence. In embodiments, sequencing includes sequentially extending a plurality of sequencing primers (e.g., sequencing a first barcode followed by sequencing a second barcode, followed by sequencing TV barcodes, where TV is the number of sequencing primers in the known sequencing primer set). In embodiments, sequencing includes generating a plurality of sequencing reads. In embodiment, the probes contain multiple different sequencing primer binding sites, and using the methods described herein multiple sequencing primers are sequentially introduced, and the target sequence is read out using the sequencing methods described herein. In embodiments, the sequencing method is SBS (Sequencing-by- Synthesis), with labeled and reversibly terminated nucleotides.

[0198] In embodiments, the method includes detecting at least two nucleic acid molecules in a cell, wherein the cell is in a well of a microplate assembly as described herein, the method including: contacting the cell with a first polynucleotide probe and binding the first polynucleotide probe to a first nucleic acid molecule, and contacting the cell with a second polynucleotide probe and binding the second polynucleotide probe to a second nucleic acid molecule, wherein the first polynucleotide probe includes a first primer binding sequence and a first barcode sequence, and wherein the second polynucleotide probe includes a second primer binding sequence and a second barcode sequence; amplifying the first and second polynucleotide probes to generate amplification products; sequentially sequencing the amplification products including the first and second barcode sequences, or the complement thereof, in situ, wherein sequentially sequencing includes hybridizing a first sequencing primer to a first amplification product or complement thereof, incorporating one or more modified nucleotides into the first sequencing primer with a polymerase to generate a first extension strand, and detecting the one or more incorporated nucleotides in a first optically resolvable feature; and, after generating the first extension strand, hybridizing a second sequencing primer to a second amplification product or complement thereof, incorporating one or more modified nucleotides into the second sequencing primer with a polymerase to generate a second extension strand, and detecting the one or more incorporated nucleotides in a second optically resolvable feature, wherein the first sequencing primer and the second sequencing primer are different sequences; and detecting the two or more nucleic acid targets by identifying the associated barcode sequences detected in the cell.

[0199] In embodiments, sequencing is performed by sequential fluorescence hybridization (e.g., sequencing by hybridization). Sequential fluorescence hybridization can involve sequential hybridization of probes including degenerate primer sequences and a detectable label. A degenerate primer sequence is a short oligonucleotide sequence which is capable of hybridizing to any nucleic acid fragment independent of the sequence of the nucleic acid fragment. For example, such a method could include the steps of (a) providing a mixture including four probes, each of which includes either A, C, G, or T at the 5 '-terminus, further including degenerate nucleotide sequence of 5 to 11 nucleotides in length, and further including a fluorescent molecule that is spectrally distinct for probes with A, C, G, or T at the 5 '-terminus; (b) associating the probes of step (a) to the target polynucleotide sequences, whose sequence will be determined by this method; (c) detecting the labels and recording the relative spatial location of the activities; (d) removing the reagents from steps (a)-(b) from the target polynucleotide sequences; and repeating steps (a)-(d) for n cycles, until the nucleotide sequence is determined, with modification that the oligonucleotides used in step (a) are complementary to part of the target polynucleotide sequences and the positions 1 through n flanking the part of the sequences. [0200] In SBS, extension of a nucleic acid primer along a nucleic acid template is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be catalyzed by a polymerase, wherein fluorescently labeled nucleotides are added to a primer (thereby extending the primer) in a template dependent fashion such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template. A plurality of different nucleic acid fragments that have been attached at different locations of an array can be subjected to an SBS technique under conditions where events occurring for different templates can be distinguished due to their location in the array. In embodiments, the sequencing step includes annealing and extending a sequencing primer to incorporate a detectable label that indicates the identity of a nucleotide in the target polynucleotide, detecting the detectable label, and repeating the extending and detecting steps. In embodiments, the methods include sequencing one or more bases of a target nucleic acid by extending a sequencing primer hybridized to a target nucleic acid (e.g., an amplification product produced by the amplification methods described herein). In embodiments, the sequencing step may be accomplished by a sequencing-by-synthesis (SBS) process. In embodiments, sequencing comprises a sequencing by synthesis process, where individual nucleotides are identified iteratively, as they are polymerized to form a growing complementary strand. In embodiments, nucleotides added to a growing complementary strand include both a label and a reversible chain terminator that prevents further extension, such that the nucleotide may be identified by the label before removing the terminator to add and identify a further nucleotide. Such reversible chain terminators include removable 3’ blocking groups, for example as described in U.S. Pat. Nos. 10,738,072, 7,541,444 and 7,057,026. Once such a modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced, there is no free 3'-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides. Once the identity of the base incorporated into the growing chain has been determined, the 3’ block may be removed to allow addition of the next successive nucleotide. By ordering the products derived using these modified nucleotides it is possible to deduce the DNA sequence of the DNA template. Non-limiting examples of suitable labels are described in U.S. Pat. No. 8,178,360, U.S. Pat. No. 5,188,934 (4,7-dichlorofluorscein dyes); U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes); U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine dyes); U.S. Pat. No. 4,318,846 (ethersubstituted fluorescein dyes); U.S. Pat. No. 5,800,996 (energy transfer dyes); U.S. Pat. No. 5,066,580 (xanthene dyes): U.S. Pat. No. 5,688,648 (energy transfer dyes); and the like. [0201] Sequencing includes, for example, detecting a sequence of signals. Examples of sequencing include, but are not limited to, sequencing by synthesis (SBS) processes in which reversibly terminated nucleotides carrying fluorescent dyes are incorporated into a growing strand, complementary to the target strand being sequenced. In embodiments, the nucleotides are labeled with up to four unique fluorescent dyes. In embodiments, the nucleotides are labeled with at least two unique fluorescent dyes. In embodiments, the readout is accomplished by epifluorescence imaging. A variety of sequencing chemistries are available, non-limiting examples of which are described herein.

[0202] In embodiments, sequencing includes a plurality of sequencing cycles. In embodiments, sequencing includes 10 to 100 sequencing cycles. In embodiments, sequencing includes 50 to 100 sequencing cycles. In embodiments, sequencing includes 50 to 300 sequencing cycles. In embodiments, sequencing includes 50 to 150 sequencing cycles. In embodiments, sequencing includes at least 10, 20, 30 40, or 50 sequencing cycles. In embodiments, sequencing includes at least 10 sequencing cycles. In embodiments, sequencing includes 10 to 20 sequencing cycles. In embodiments, sequencing includes 10, 11, 12, 13, 14, or 15 sequencing cycles. In embodiments, sequencing includes (a) extending a sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue and (b) detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue. In embodiments, detecting includes two-dimensional (2D) or three-dimensional (3D) fluorescent microscopy. Suitable imaging technologies are known in the art, as exemplified by Larsson et al., Nat. Methods (2010) 7:395-397 and associated supplemental materials, the entire content of which is incorporated by reference herein in its entirety. In embodiments of the methods provided herein, the imaging is accomplished by confocal microscopy. Confocal fluorescence microscopy involves scanning a focused laser beam across the sample, and imaging the emission from the focal point through an appropriately- sized pinhole. This suppresses the unwanted fluorescence from sections at other depths in the sample. In embodiments, the imaging is accomplished by multi-photon microscopy (e.g., two-photon excited fluorescence or two-photon-pumped microscopy). Unlike conventional single-photon emission, multi-photon microscopy can utilize much longer excitation wavelength up to the red or near-infrared spectral region. This lower energy excitation requirement enables the implementation of semiconductor diode lasers as pump sources to significantly enhance the photostability of materials. Scanning a single focal point across the field of view is likely to be too slow for many sequencing applications. To speed up the image acquisition, an array of multiple focal points can be used. The emission from each of these focal points can be imaged onto a detector, and the time information from the scanning mirrors can be translated into image coordinates. Alternatively, the multiple focal points can be used just for the purpose of confining the fluorescence to a narrow axial section, and the emission can be imaged onto an imaging detector, such as a CCD, EMCCD, or s-CMOS detector. A scientific grade CMOS detector offers an optimal combination of sensitivity, readout speed, and low cost. One configuration used for confocal microscopy is spinning disk confocal microscopy. In 2-photon microscopy, the technique of using multiple focal points simultaneously to parallelize the readout has been called Multifocal Two-Photon Microscopy (MTPM). Several techniques for MTPM are available, with applications typically involving imaging in biological tissue. In embodiments of the methods provided herein, the imaging is accomplished by light sheet fluorescence microscopy (LSFM). In embodiments, detecting includes 3D structured illumination (3DSIM). In 3DSIM, patterned light is used for excitation, and fringes in the Moire pattern generated by interference of the illumination pattern and the sample, are used to reconstruct the source of light in three dimensions. In order to illuminate the entire field, multiple spatial patterns are used to excite the same physical area, which are then digitally processed to reconstruct the final image. See York, Andrew G., et al. “Instant super-resolution imaging in live cells and embryos via analog image processing.” Nature methods 10.11 (2013): 1122-1126 which is incorporated herein by reference. In embodiments, detecting includes selective planar illumination microscopy, light sheet microscopy, emission manipulation, pinhole confocal microscopy, aperture correlation confocal microscopy, volumetric reconstruction from slices, deconvolution microscopy, or aberration-corrected multifocus microscopy. In embodiments, detecting includes digital holographic microscopy (see for example Manoharan, V. N. Frontiers of Engineering: Reports on Leading-edge Engineering from the 2009 Symposium, 2010, 5-12, which is incorporated herein by reference). In embodiments, detecting includes confocal microscopy, light sheet microscopy, or multi-photon microscopy.

[0203] Use of the sequencing method outlined above is a non-limiting example, as essentially any sequencing methodology which relies on successive incorporation of nucleotides into a polynucleotide chain can be used. Suitable alternative techniques include, for example, pyrosequencing methods, FISSEQ (fluorescent in situ sequencing), MPSS (massively parallel signature sequencing), or sequencing by ligation-based methods. [0204] In embodiments, generating a sequencing read includes determining the identity of the nucleotides in the template polynucleotide (or complement thereof). In embodiments, a sequencing read, e.g., a first sequencing read or a second sequencing read, includes determining the identity of a portion (e.g., 1, 2, 5, 10, 20, 50 nucleotides) of the total template polynucleotide. In embodiments the first sequencing read determines the identity of 5-10 nucleotides and the second sequencing read determines the identity of more than 5-10 nucleotides (e.g., 11 to 200 nucleotides). In embodiments the first sequencing read determines the identity of more than 5-10 nucleotides (e.g., 11 to 200 nucleotides) and the second sequencing read determines the identity of 5-10 nucleotides. In embodiments, following the generation of a sequencing read, subsequent extension is performed using a plurality of standard (e.g., non-modified) dNTPs until the complementary strand is copied. In other embodiments, following the generation of a sequencing read, subsequent extension is performed using a plurality of dideoxy nucleotide triphosphates (ddNTPs) to prevent further extension of the first sequencing read product during a second sequencing read. In embodiments, following the identification of at least 5-10 (e.g., 11 to 200 nucleotides, or up to 1000 nucleotides), subsequent extension is performed using a plurality of standard (e.g., non-modified) dNTPs until the complementary strand is copied. In embodiments, following the identification of at least 5-10 (e.g., 11 to 200 nucleotides, or up to 1000 nucleotides), subsequent extension is performed using a plurality of dideoxy nucleotide triphosphates (ddNTPs) to prevent further extension of the sequencing read product.

[0205] In embodiments, the detection agent includes a label. In embodiments, the detection agent includes a fluorescent label. In embodiments, the detection agent includes an oligonucleotide barcode (e.g., a 5 to 15 nucleotide sequence). In embodiments, the oligonucleotide barcode includes at least two primer binding sequences. In embodiments, the oligonucleotide barcode includes an amplification primer binding sequence. In embodiments, the oligonucleotide barcode includes a sequencing primer binding sequence. The amplification primer binding sequence refers to a nucleotide sequence that is complementary to a primer useful in initiating amplification (i.e., an amplification primer). Likewise, a sequencing primer binding sequence is a nucleotide sequence that is complementary to a primer useful in initiating sequencing (i.e., a sequencing primer). Primer binding sequences usually have a length in the range of between 3 to 36 nucleotides, also 5 to 24 nucleotides, also from 14 to 36 nucleotides. In embodiments, an amplification primer and a sequencing primer are complementary to the same primer binding sequence, or overlapping primer binding sequences. In embodiments, an amplification primer and a sequencing primer are complementary to different primer binding sequences. In embodiments, the primer binding sequence is complementary to a fluorescent in situ hybridization (FISH) probe. FISH probes may be custom designed using known techniques in the art, see for example Gelali, E., et al. Nat Commun 10, 1636 (2019). In embodiments, the detection probe is an oligonucleotide including a barcode sequence. In embodiments the oligonucleotide further includes a primer binding sequence.

[0206] In embodiments, contacting the biomolecule includes hybridizing a padlock probe to two adjacent nucleic acid sequences of the biomolecule, wherein the padlock probe is a single-stranded polynucleotide having a 5’ and a 3’ end, the padlock probe includes at least one oligonucleotide barcode, and wherein the padlock probe includes a primer binding sequence. In embodiments, the method further includes ligating the 5’ and 3’ ends of the padlock probe to form a circular polynucleotide.

[0207] In embodiments, contacting the biomolecule includes hybridizing a padlock probe to a nucleic acid sequence of the biomolecule, wherein the padlock probe is a single-stranded polynucleotide having a 5’ and a 3’ end, wherein the 3' end hybridizes to a first complementary region of the biomolecule and the 5' end hybridizes to a second complementary region of the biomolecule. In embodiments, the padlock probe includes a primer binding sequence. In embodiments, the method further includes extending the 3' end of the padlock probe along the nucleic acid sequence of the biomolecule to generate a complementary sequence and ligating the complementary sequence to the 5' end of the padlock probe thereby forming a circular oligonucleotide.

[0208] In embodiments, the method includes sequencing an endogenous nucleic acid of a cell, the method including: contacting the cell with a polynucleotide probe including a first region and a second region, hybridizing the first region of the polynucleotide probe to a first sequence of the endogenous nucleic acid, and hybridizing the second region of the polynucleotide probe to a second sequence of the endogenous nucleic acid, thereby forming a complex including the polynucleotide probe hybridized to the endogenous nucleic acid, wherein the endogenous nucleic acid includes a target sequence between the first sequence and the second sequence; extending the polynucleotide probe with nucleotides (e.g., deoxynucleotide triphosphates (dNTPs)) along the target sequence to generate a complement of the target sequence, and ligating the complement of the target sequence to the polynucleotide probe thereby forming a circular oligonucleotide; amplifying the circular oligonucleotide to form an extension product including one or more copies of the target sequence; and sequencing the one or more copies of the target sequence in the cell.

[0209] A variety of sequencing methodologies can be used such as sequencing-by- synthesis (SBS), pyrosequencing, sequencing by ligation (SBL), or sequencing by hybridization (SBH). Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568; and.

6,274,320, each of which is incorporated herein by reference in its entirety). In pyrosequencing, released Ppi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated can be detected via light produced by luciferase. In this manner, the sequencing reaction can be monitored via a luminescence detection system. In both SBL and SBH methods, target nucleic acids, and amplicons thereof, that are present at features of an array are subjected to repeated cycles of oligonucleotide delivery and detection. SBL methods, include those described in Shendure et al. Science 309: 1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated herein by reference in its entirety; and the SBH methodologies are as described in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which is incorporated herein by reference in its entirety.

[0210] In embodiments, sequencing is performed according to a “sequencing-by-binding” method (see, e.g., U.S. Pat. Pubs. US2017/0022553 and US2019/0048404, each of which is incorporated herein by reference in its entirety), which refers to a sequencing technique wherein specific binding of a polymerase and cognate nucleotide to a primed template nucleic acid molecule (e.g., blocked primed template nucleic acid molecule) is used for identifying the next correct nucleotide to be incorporated into the primer strand of the primed template nucleic acid molecule. The specific binding interaction need not result in chemical incorporation of the nucleotide into the primer. In some embodiments, the specific binding interaction can precede chemical incorporation of the nucleotide into the primer strand or can precede chemical incorporation of an analogous, next correct nucleotide into the primer. Thus, detection of the next correct nucleotide can take place without incorporation of the next correct nucleotide. As used herein, the “next correct nucleotide” (sometimes referred to as the “cognate” nucleotide) is the nucleotide having a base complementary to the base of the next template nucleotide. The next correct nucleotide will hybridize at the 3 '-end of a primer to complement the next template nucleotide. The next correct nucleotide can be, but need not necessarily be, capable of being incorporated at the 3' end of the primer. For example, the next correct nucleotide can be a member of a ternary complex that will complete an incorporation reaction or, alternatively, the next correct nucleotide can be a member of a stabilized ternary complex that does not catalyze an incorporation reaction. A nucleotide having a base that is not complementary to the next template base is referred to as an “incorrect” (or “non-cognate”) nucleotide.

[0211] In embodiments, the sequencing method relies on the use of modified nucleotides that can act as reversible reaction terminators. Once the modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced there is no free 3 ’-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides. Once the identity of the base incorporated into the growing chain has been determined, the 3’ reversible terminator may be removed to allow addition of the next successive nucleotide. These such reactions can be done in a single experiment if each of the modified nucleotides has attached a different label, known to correspond to the particular base, to facilitate discrimination between the bases added at each incorporation step. Alternatively, a separate reaction may be carried out containing each of the modified nucleotides separately.

[0212] In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs and/or one or more ddNTPs into the 3' end of the extension strand. In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs. In embodiments, the method further includes terminating extension by incorporating one or more ddNTPs into the 3' end of the extension strand.

[0213] The modified nucleotides may carry a label (e.g., a fluorescent label) to facilitate their detection. Each nucleotide type may carry a different fluorescent label. However, the detectable label need not be a fluorescent label. Any label can be used which allows the detection of an incorporated nucleotide. One method for detecting fluorescently labeled nucleotides includes using laser light of a wavelength specific for the labeled nucleotides, or the use of other suitable sources of illumination. The fluorescence from the label on the nucleotide may be detected (e.g., by a CCD camera, CMOS camera, or other suitable detection means).

[0214] In embodiments, the method includes detecting a protein in a cell, the method including: contacting a cell with a specific binding reagent (e.g., antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer) and binding the specific binding reagent to the protein, wherein the specific binding reagent includes an oligonucleotide; hybridizing a first sequence of a polynucleotide to the oligonucleotide, and hybridizing a second sequence of the polynucleotide to the oligonucleotide, thereby forming a complex including the polynucleotide hybridized to the oligonucleotide, wherein the oligonucleotide includes a barcode sequence between the first sequence and the second sequence; extending the polynucleotide along the barcode sequence to generate a complement of the barcode sequence, and ligating the complement of the barcode sequence to the polynucleotide thereby forming a circular oligonucleotide; amplifying the circular oligonucleotide to form an extension product including one or more copies of the barcode sequence; and sequencing the one or more copies of the barcode sequence in the cell, thereby detecting the protein.

[0215] In embodiments, contacting the biomolecule includes hybridizing a padlock probe to a nucleic acid sequence of the biomolecule, wherein the padlock probe is a single-stranded polynucleotide having a 5’ and a 3’ end, wherein the 3' end hybridizes to a first complementary region of the nucleic acid sequence and the 5' end hybridizes to a second complementary region of the RNA molecule. In embodiments, the padlock probe includes a primer binding sequence. In embodiments, the method further includes extending the 3' end of the padlock probe along the nucleic acid sequence of the biomolecule to generate a complementary sequence and ligating the complementary sequence to the 5' end of the padlock probe thereby forming a circular oligonucleotide.

[0216] In embodiments, the second complementary region is about 5 to about 75 nucleotides in the 5' direction with respect to the first complementary region. In embodiments, the second complementary region is about 10 to about 100 nucleotides in the 5' direction with respect to the first complementary region. In embodiments, the second complementary region is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides in the 5' direction with respect to the first complementary region. [0217] In embodiments, the detection agent includes a padlock probe. Padlock probes are specialized ligation probes, examples of which are known in the art, see for example Nilsson M, et al. Science. 1994;265(5181):2085-2088), and has been applied to detect transcribed RNA in cells, see for example Christian AT, et al. Proc Natl Acad Sci U S A.

2001;98(25): 14238-14243, both of which are incorporated herein by reference in their entireties. In embodiments, the padlock probe is approximately 50 to 200 nucleotides. In embodiments, a padlock probe has a first domain that is capable of hybridizing to a first target sequence domain, and a second ligation domain, capable of hybridizing to an adjacent second sequence domain. The configuration of the padlock probe is such that upon ligation of the first and second ligation domains of the padlock probe, the probe forms a circular polynucleotide, and forms a complex with the sequence (i.e., the sequence it hybridized to, the target sequence) wherein the target sequence is “inserted” into the loop of the circle. Padlock probes are useful for the methods provided herein and include, for example, padlock probes for genomic analyses, as exemplified by Gore, A. et al. Nature 471, 63-67 (2011); Porreca, G. J. et al. Nat Methods 4, 931-936 (2007); Li, J. B. et al. Genome Res 19, 1606- 1615 (2009), Zhang, K. et al. Nat Methods 6, 613-618 (2009); Noggle, S. et al. Nature 478, 70-75 (2011); and Li, J. B. et al. Science 324, 1210-1213 (2009), the content of each of which is incorporated by reference in its entirety.

[0218] In embodiments, the padlock probe is a single-stranded polynucleotide having a 5’ and a 3’ end, wherein the padlock probe includes at least one oligonucleotide barcode. In embodiments, the padlock probe includes a primer binding sequence. In embodiments, the padlock probe includes a primer binding sequence from a known set of primer binding sequences. In embodiments, the padlock probe includes only one primer binding sequence, wherein the primer binding sequence serves as the amplification primer binding sequence and sequencing primer binding sequence. In embodiments, the padlock probe includes at least two primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes two or more primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes up to 50 different primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes up to 10 different primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes up to 5 different primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes two or more sequencing primer binding sequences from a known set of sequencing primer binding sequences. In embodiments, the padlock probe includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes two or more different primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes 2 to 5 primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes 2 to 5 different primer binding sequences from a known set of primer binding sequences. In embodiments, the padlock probe includes 2 to 5 sequencing primer binding sequences from a known set of sequencing primer binding sequences. In embodiments, the padlock probe includes 2 to 5 different sequencing primer binding sequences from a known set of sequencing primer binding sequences.

[0219] In embodiments, the padlock probe includes one oligonucleotide barcode, and one primer binding sequence. In embodiments, the padlock probe includes at least two (optionally different) oligonucleotide barcodes, and at least two different primer binding sequences. In embodiments, the padlock probe includes at least two (optionally different) oligonucleotide barcodes, and at least two different sequencing primer binding sequences. In embodiments, the padlock probe includes two different oligonucleotide barcodes and two different sequencing primer binding sequences. In embodiments, the padlock probe includes identical oligonucleotide barcodes and two different sequencing primer binding sequences.

[0220] In embodiments, the method further includes ligating the 5’ and 3’ ends of the padlock probe to form a circular polynucleotide (i.e., a polynucleotide that is a continuous strand lacking free 5’ and 3’ ends). In embodiments, the method includes ligating the 5’ and 3’ ends of the padlock probe to form a circular polynucleotide, wherein the circular polynucleotide includes the target nucleic acid. In embodiments, the method includes ligating the 5’ and 3’ ends of the padlock probe to form a circular polynucleotide, wherein the circular polynucleotide includes the oligonucleotide barcode. In embodiments, the ligation includes enzymatic ligation. In embodiments, ligating includes enzymatic ligation including a ligation enzyme (e.g., Circligase enzyme, Taq DNA Ligase, HiFi Taq DNA Ligase, T4 ligase, PBCV- 1 DNA Ligase (also known as SplintR ligase) or Ampligase DNA Ligase). Non-limiting examples of ligases include DNA ligases such as DNA Ligase I, DNA Ligase II, DNA Ligase III, DNA Ligase IV, T4 DNA ligase, T7 DNA ligase, T3 DNA Ligase, E. coll DNA Ligase, PBCV-1 DNA Ligase (also known as SplintR ligase) or a Taq DNA Ligase. In embodiments, the ligase enzyme includes a T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2, T3 DNA ligase or T7 DNA ligase. In embodiments, the enzymatic ligation is performed by a mixture of ligases. In embodiments, the ligation enzyme is selected from the group consisting of T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2, RtcB ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, PBCV-1 DNA Ligase, a thermostable DNA ligase (e.g.,

5 'AppDNA/RNA ligase), an ATP dependent DNA ligase, an RNA-dependent DNA ligase (e.g., SplintR ligase), and combinations thereof.

[0221] In embodiments, ligating includes chemical ligation (e.g., enzyme-free, click- mediated ligation). In embodiments, the oligonucleotide primer includes a first bioconjugate reactive moiety capable of bonding upon contact with a second (complementary) bioconjugate reactive moiety. In embodiments, the oligonucleotide primer includes an alkynyl moiety at the 3’ and an azide moiety at the 5’ end that, upon hybridization to the target nucleic acid react to form a triazole linkage during suitable reaction conditions. Reaction conditions and protocols for chemical ligation techniques that are compatible with nucleic acid amplification methods are known in the art, for example El-Sagheer, A. H., & Brown, T. (2012). Accounts of chemical research, 45(8), 1258-1267; Manuguerra I. et al. Chem Commun (Camb). 2018;54(36):4529-4532; and Odeh, F., et al. (2019). Molecules (Basel, Switzerland) , 25(1), 3, each of which is incorporated herein by reference in their entirety.

[0222] In embodiments, the method includes amplifying the circular polynucleotide by extending an amplification primer with a strand-displacing polymerase, wherein the primer extension generates an extension product including multiple complements of the circular polynucleotide. In embodiments, the method of amplifying includes an isothermal amplification method. In embodiments, the method of amplifying includes rolling circle amplification (RCA) or rolling circle transcription (RCT). In embodiments, the method of amplifying is rolling circle amplification (RCA). In embodiments, amplifying includes exponential rolling circle amplification (eRCA). Exponential RCA is similar to the linear process except that it uses a second primer (e.g., one or more immobilized oligonucleotide(s)) having a sequence that is identical to at least a portion of the circular template (Lizardi et al. Nat. Genet. 19:225 (1998)). This two-primer system achieves isothermal, exponential amplification. Exponential RCA has been applied to the amplification of non-circular DNA through the use of a linear probe that binds at both of its ends to contiguous regions of a target DNA followed by circularization using DNA ligase (Nilsson et al. Science 265(5181):208 5(1994)).

[0223] Optionally, the rolling circle amplification reaction can be done with modified nucleotides that contain chemical groups that serve as attachment points to the cell or the matrix in which the cell is embedded (e.g. a hydrogel). The attachment of the amplified product to the matrix can help confine & fix the amplicon to a small volume. In embodiments, amplification reactions include standard dNTPs and a modified nucleotide (e.g., amino-allyl dUTP, 5-TCO-PEG4-dUTP, C8-Alkyne-dUTP, 5-Azidomethyl- dUTP, 5- Vinyl-dUTP, or 5-Ethynyl dLTTP). For example, during amplification a mixture of standard dNTPs and aminoallyl deoxyuridine 5 '-triphosphate (dUTP) nucleotides may be incorporated into the amplicon and subsequently cross-linked to the cell protein matrix by using a crosslinking reagent (e.g., an amine-reactive crosslinking agent with PEG spacers, such as (PEGylated bis(sulfosuccinimidyl)suberate) (BS(PEG)9)).

[0224] In embodiments, the method does not include ligation or amplification. For example, the method includes hybridizing a probe nucleic acid to the target (i.e., to a complementary region or gene of interest), wherein the probe nucleic acid is branched DNA or a concatemer and includes at least one sequencing primer binding sequence and a plurality of oligonucleotide barcodes. In embodiments, the probe nucleic acid includes a plurality of identical barcodes. In embodiments, associating an oligonucleotide barcode with each of the plurality of targets includes hybridizing a probe nucleic acid, wherein the probe nucleic acid includes branched DNA or a concatemer and includes at least one sequencing primer binding sequence and a plurality of oligonucleotide barcodes. In embodiments, the probe nucleic acid includes a plurality of identical oligonucleotide barcodes. In embodiments, the probe nucleic acid includes two or more complementary sequences to the target. In embodiments, the probe nucleic acid includes two or more different oligonucleotide barcodes.

[0225] In embodiments, the probe nucleic acid includes a two or more complementary sequences to the target. In embodiments, the probe nucleic acid includes two or more different oligonucleotide barcodes. In embodiments, the probe includes a primer binding sequence from a known set of primer binding sequences. In embodiments, the probe includes a sequencing primer binding sequence from a known set of sequencing primer binding sequences [0226] In embodiments, the detection agent includes a protein-specific binding agent. In embodiments, the detection agent includes a protein-specific binding agent bound to a nucleic acid sequence, bioconjugate reactive moiety, an enzyme, or a label. In embodiments, the protein-specific binding agent is an antibody, single domain antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), affimer, or an aptamer.

[0227] In embodiments, the method includes detecting a plurality of biomolecules. In embodiments, the biomolecules are proteins or carbohydrates. In embodiments, the biomolecules are proteins. In embodiments, the biomolecules are carbohydrates. In embodiments when the biomolecules are proteins and/or carbohydrates, the method includes contacting the proteins with a specific binding reagent, wherein the specific binding reagent includes an oligonucleotide barcode. In embodiments, the specific binding reagent includes an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer. In embodiments, the specific binding reagent is a peptide, a cell penetrating peptide, an aptamer, a DNA aptamer, an RNA aptamer, an antibody, an antibody fragment, a light chain antibody fragment, a single-chain variable fragment (scFv), a lipid, a lipid derivative, a phospholipid, a fatty acid, a triglyceride, a glycerolipid, a glycerophospholipid, a sphingolipid, a saccharolipid, a polyketide, a polylysine, polyethyleneimine, diethylaminoethyl (DEAE)-dextran, cholesterol, or a sterol moiety. In embodiments, the specific binding reagent interacts (e.g., contacts, or binds) with one or more specific binding reagents in or on the cell. Carbohydrate-specific antibodies are known in the art, see for example Kappler, K., Hennet, T. Genes Immun 21, 224-239 (2020).

[0228] In embodiments, the biomolecule is a nucleic acid sequence. In embodiments, the method further includes amplifying the nucleic acid sequence to generate amplification products. In embodiments, the method includes detecting the amplification products.

[0229] In embodiments, the barcode is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In embodiments, the barcode is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In embodiments, the barcode is 10 to 15 nucleotides in length. An oligonucleotide barcode is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. An oligonucleotide barcode can be at most about 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or fewer or more nucleotides in length. In embodiments, an oligonucleotide barcode includes between about 5 to about 8, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 10 to about 150 nucleotides. In embodiments, an oligonucleotide barcode includes between 5 to 8, 5 to 10, 5 to 15, 5 to 20, 10 to 150 nucleotides. In embodiments, an oligonucleotide barcode is less than 10 nucleotides. In embodiments, an oligonucleotide barcode is about 10 nucleotides. In embodiments, an oligonucleotide barcode is 10 nucleotides. An oligonucleotide barcode may include a unique sequence (e.g., a barcode sequence) that gives the oligonucleotide barcode its identifying functionality. The unique sequence may be random or non-random. Attachment of the barcode sequence to a nucleic acid of interest (i.e., the target) may associate the barcode sequence with the nucleic acid of interest. The barcode may then be used to identify the nucleic acid of interest during sequencing, even when other nucleic acids of interest (e.g., including different oligonucleotide barcodes) are present. In embodiments, the oligonucleotide barcode consists only of a unique barcode sequence. In embodiments, the 5' end of a barcoded oligonucleotide is phosphorylated. In embodiments, the oligonucleotide barcode is known (i.e., the nucleic sequence is known before sequencing) and is sorted into a basis-set according to their Hamming distance. Oligonucleotide barcodes can be associated with a target of interest by knowing, a priori, the target of interest, such as a gene or protein. In embodiments, the oligonucleotide barcodes further include one or more sequences capable of specifically binding a gene or nucleic acid sequence of interest. For example, in embodiments, the oligonucleotide barcode include a sequence capable of hybridizing to mRNA, e.g., one containing a poly-T sequence (e.g., having several T's in a row, e.g., 4, 5, 6, 7, 8, or more T's). In embodiments, the padlock probe is at least about 50, 60, 70, 80, 90, 100, 110, 120, 130 or more nucleotides in length. In embodiments, the padlock probe is at most about 300, 200, 100, 90, 80, or fewer or more nucleotides in length. In embodiments, the total length of the padlock probe is about 80, 90, 100, 110, 120, 130, or 140 nucleotides in length.

[0230] In embodiments, the oligonucleotide barcode is taken from a “pool” or “set” or “basis-set” of potential oligonucleotide barcode sequences. The set of oligonucleotide barcodes may be selected using any suitable technique, e.g., randomly, or such that the sequences allow for error detection and/or correction, or having a particular feature, such as by being separated by a certain distance (e.g., Hamming distance). In embodiments, the method includes selecting a basis-set of oligonucleotide barcodes having a specified Hamming distance (e.g., a Hamming distance of 10; a Hamming distance of 5). The pool may have any number of potential barcode sequences, e.g., at least 100, at least 300, at least 500, at least 1,000, at least 3,000, at least 5,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 300,000, at least 500,000, or at least 1,000,000 barcode sequences. [0231] In embodiments, the method further includes digesting the tissue section by contacting the sample- carrier construct with an endopeptidase. In embodiments, the endopeptidase is pepsin.

[0232] In embodiments, the method further includes subjecting the tissue section to expansion microscopy methods and techniques (e.g., prior to detection). Expansion allows individual targets (e.g., mRNA or RNA transcripts) which are densely packed within a cell, to be resolved spatially in a high-throughput manner. Expansion microscopy techniques are known in the art and can be performed as described in US 2016/0116384 and Chen et al., Science, 347, 543 (2015), each of which are incorporated herein by reference in their entirety. The increased resolution in spatial profiling can be determined by comparison of an isometrically expanded sample with a sample that has not been isometrically expanded. In embodiments, the biological sample is isometrically expanded to a volume at least 2*, 2.1 *, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3. lx, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4. l x, 4.2 x, 4.3 x, 4.4x, 4.5 x, 4.6x, 4.7x, 4.8x, or 4.9 x its non-expanded volume. In some embodiments, the biological sample is isometrically expanded to at least 2x and less than 20 x of its non-expanded volume.

[0233] In embodiments, the method does not include subjecting the tissue section to expansion microscopy. Typically, expansion microscopy techniques utilize a swellable polymer or hydrogel (e.g., a synthetic matrix-forming material) which can significantly slow diffusion of enzymes and nucleotides. Matrix (e.g., synthetic matrix) forming materials include polyacrylamide, cellulose, alginate, polyamide, cross-linked agarose, cross-linked dextran or cross-linked polyethylene glycol. The matrix forming materials can form a matrix by polymerization and/or crosslinking of the matrix forming materials using methods specific for the matrix forming materials and methods, reagents and conditions known to those of skill in the art. Additionally, expansion microscopy techniques may render the temperature of the cell sample difficult to modulate in a uniform, controlled manner. Modulating temperature provides a useful parameter to optimize amplification and sequencing methods. In embodiments, the method does not include an exogenous matrix.

[0234] In embodiments, the method includes sequencing an endogenous nucleic acid of a cell in a microplate assembly as described herein (e.g., wherein the cell is within a well of the microplate assembly), the method including: contacting the cell with a polynucleotide probe including a first region and a second region, hybridizing the first region of the polynucleotide probe to a first sequence of the endogenous nucleic acid, and hybridizing the second region of the polynucleotide probe to a second sequence of the endogenous nucleic acid, thereby forming a complex including the polynucleotide probe hybridized to the endogenous nucleic acid, wherein the endogenous nucleic acid includes a target sequence between the first sequence and the second sequence; extending the polynucleotide probe with nucleotides (e.g., deoxynucleotide triphosphates (dNTPs)) along the target sequence to generate a complement of the target sequence, and ligating the complement of the target sequence to the polynucleotide probe thereby forming a circular oligonucleotide; amplifying the circular oligonucleotide to form an extension product including one or more copies of the target sequence; and sequencing the one or more copies of the target sequence in the cell.

[0235] In embodiments, the method includes imaging the immobilized tissue section. In embodiments, the method further includes an imaging modality, immunofluorescence (IF), or immunohistochemistry modality (e.g., immunostaining). In embodiments, the method includes ER staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the endoplasmic reticula), Golgi staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the Golgi), F-actin staining (e.g., contacting the tissue section with a phalloidin-conjugated dye that binds to actin filaments), lysosomal staining (e.g., contacting the tissue section with a cell-permeable dye that accumulates in the lysosome via the lysosome pH gradient), mitochondrial staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the mitochondria), nucleolar staining, or plasma membrane staining. For example, the method includes live cell imaging (e.g., obtaining images of the tissue section) prior to or during fixing, immobilizing, and permeabilizing the tissue section. Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, commonly detected and examined with the light microscope. Known IHC modalities may be used, such as the protocols described in Magaki, S., Hojat, S. A., Wei, B., So, A., & Yong, W. H. (2019). Methods in molecular biology (Clifton, N.J.), 1897, 289-298, which is incorporated herein by reference. In embodiments, the additional imaging modality includes bright field microscopy, phase contrast microscopy, Nomarski differential-interference- contrast microscopy, or dark field microscopy. In embodiments, the method further includes determining the cell morphology of the tissue section (e.g., the cell boundary or cell shape) using known methods in the art. For example, to determining the cell boundary includes comparing the pixel values of an image to a single intensity threshold, which may be determined quickly using histogram -based approaches as described in Carpenter, A. et al Genome Biology 7, R100 (2006) and Arce, S., Sci Rep 3, 2266 (2013)). By “microscopic analysis” is meant the analysis of a specimen using techniques that provide for the visualization of aspects of a specimen that cannot be seen with the unaided eye, i.e., that are not within the resolution range of the normal human eye. Such techniques may include, without limitation, optical microscopy, e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflection, epifluorescence, confocal microscopy, CLARITY-optimized light sheet microscopy (COLM), light field microscopy, tissue expansion microscopy, etc., laser microscopy, such as, two photon microscopy, electron microscopy, and scanning probe microscopy. By “preparing a biological specimen for microscopic analysis” is generally meant rendering the specimen suitable for microscopic analysis at an unlimited depth within the specimen. In embodiments, the immobilized tissue section is imaged using “optical sectioning” techniques, such as laser scanning confocal microscopes, laser scanning 2-Photon microscopy, parallelized confocal (i.e. spinning disk), computational image deconvolution methods, and light sheet approaches. Optical sectioning microscopy methods provide information about single planes of a volume by minimizing contributions from other parts of the volume and do so without physical sectioning. The resulting “stack” of such optically sectioned images, represents a full reconstruction of the 3-dimensional features of a tissue volume. A typical confocal microscope includes a 10x/0.5 objective (dry; working distance, 2.0 mm) and/or a 20*/0.8 objective (dry; working distance, 0.55 mm), with a s z-step interval of 1 to 5 pm. A typical light sheet fluorescence microscope includes an sCMOS camera, a 2*/0.5 objective lens, and zoom microscope body (magnification range of *0.63 to ^x 6.3). For entire scanning of whole samples, the z-step interval is 5 or 10 pm, and for image acquisition in the regions of interest, an interval in the range of 2 to 5 pm may be used.

[0236] To microscopically visualize tissue sections prepared by the subject methods, in some embodiments the tissue section is embedded in a mounting medium. Mounting medium is typically selected based on its suitability for the reagents used to visualize the cellular biomolecules, the refractive index of the tissue section, and the microscopic analysis to be performed. For example, for phase-contrast work, the refractive index of the mounting medium should be different from the refractive index of the specimen, whereas for bright- field work the refractive indexes should be similar. As another example, for epifluorescence work, a mounting medium should be selected that reduces fading, photobleaching or quenching during microscopy or storage. In certain embodiments, a mounting medium or mounting solution may be selected to enhance or increase the optical clarity of the cleared tissue specimen. Nonlimiting examples of suitable mounting media that may be used include glycerol, CC/Mount™, Fluoromount™ Fluoroshield™, ImmunHistoMount™, Vectashield™, Permount™, Acrytol™, CureMount™, FocusClear™, or equivalents thereof.

[0237] The biological targets or molecules to be detected can be any biological molecules including but not limited to proteins, nucleic acids, lipids, carbohydrates, ions, or multicomponent complexes containing any of the above. Examples of subcellular targets include organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. Exemplary nucleic acid targets can include genomic DNA of various conformations (e.g., A-DNA, B-DNA, Z-DNA), mitochondria DNA (mtDNA), mRNA, tRNA, rRNA, hRNA, miRNA, and piRNA. For example, following immobilization on the receiving substrate, the sections may be fixed with methanol, permeabilized with 0.025% Triton in PBS solution, and stained with primary antibodies directed against vimentin (fibroblasts) and macrophages, followed by secondary antibody labeling (e.g., Alexa-594 conjugated secondary antibodies). Additional counterstaining may be performed, for example using 4,6-diamidino-2-phenylindole (DAPI) mounting media to counterstain nuclei.

[0238] In embodiments, the collection of information (e.g., sequencing information and cell morphology) is referred to as a signature. The term “signature” may encompass any gene or genes, protein or proteins, or epigenetic element(s) whose expression profile or whose occurrence is associated with a specific cell type, subtype, or cell state of a specific cell type or subtype within a population of cells. It is to be understood that also when referring to proteins (e.g., differentially expressed proteins), such may fall within the definition of “gene” signature. Levels of expression or activity or prevalence may be compared between different cells in order to characterize or identify for instance signatures specific for cell (sub)populations. Increased or decreased expression or activity of signature genes may be compared between different cells in order to characterize or identify for instance specific cell (sub)populations.

[0239] In embodiments, the methods described herein may further include constructing a 3- dimensional pattern of abundance, expression, and/or activity of each target from spatial patterns of abundance, expression, and/or activity of each target of multiple samples. In embodiments, the multiple samples can be consecutive tissue sections of a 3-dimensional tissue sample.

[0240] In embodiments, following permeabilization, the tissue section is contacted with one or more imaging reagents or stains. In embodiments, the tissue section is contacted with one or more imaging reagents or stains without permeabilization. In embodiments, the imaging reagents or stains include hematoxylin and eosin (H&E) staining reagents. In embodiments, the imaging (e.g., step E)) includes phase-contrast microscopy, bright-field microscopy, Nomarski differential-interference-contrast microscopy, dark field microscopy, electron microscopy, or cryo-electron microscopy. In embodiments, the imaging reagents or stains include phase-contrast microscopy, bright-field microscopy, Nomarski differential- interference-contrast microscopy, or dark field microscopy imaging reagents. In embodiments, the light transmittance of the sample is measured. For example, light transmittance may be measured with a visible near-infrared optical fiber spectrometer, wherein a circular spot of light (e.g., diameter, 5 mm) is irradiated on the central part a sample and the transmitted light is collected using an optical sensor.

[0241] In embodiments, the imaging reagents or stains include electron microscopy (e.g., transmission electron microscopy or scanning electron microscopy) or cryo-electron microscopy imaging reagents. Examples of electron microscopy contrast agents may include one or more heavy metals (e.g., gold particles, colloidal gold particles, uranium, lead, platinum, and/or osmium) and/or antibodies bound to one or more types of heavy metals (e.g., gold particles, colloidal gold particles, uranium, lead, platinum, and/or osmium). For example, immunogold labels that may be used to contact the tissue section include may include different antibodies bound to gold particles of different sizes to image different molecules of interest. Optionally, the method may include contacting the tissue section with heavy metals. Heavy metals that may be used to stain additional features of interest and/or provide contrast between different structures in the tissue section may include uranium, lead, platinum, and/or osmium (see, e.g., U.S. Pat. Pubs. 2019/0355550 and 2013/0344500, each of which is incorporated herein by reference in its entirety).

[0242] In embodiments, additional methods may be performed to further characterize the sample. For example, in addition to sequencing, the method includes protein analysis, lipid analysis, metabolite analysis (e.g., glucose analysis), or measuring the transcriptomic profile, gene expression activity, genomic profile, protein expression activity, proteomic profile,

Ill protein interaction activity, cellular receptor expression activity, lipid profile, lipid activity, carbohydrate profile, microvesicle activity, glucose activity, and combinations thereof.

[0243] In another aspect is provided a method of making a microwell insert as described herein. In embodiments, the method includes bonding a planar support to a solid support including a plurality of openings, thereby forming a microwell insert.

EXAMPLES

[0244] Microtiter plates, referred to as or microplates, are a mainstay in high-throughput, often automated, bioanalytical research. The first known microplate originated around 1950, where a Hungarian scientist, Gyula Takatsy, machined six rows of 12 wells out of poly(methyl methacrylate (PMMA) intended for serology analysis of influenza. This initial 72 well microplate served as a useful platform in research, and advances in biotechnology, drug research, microscopy, imaging, and liquid handling, gave rise to a variety of alternatives platforms. Researchers can select application-specific microplates from a range of products that differ in format, design, base material, color, and surface properties. For example, 96 well microplates are widely used in basic research, whereas drug discovery and bioanalytical assays typically use 384 well or 1536 well microplates. Microplates with different well designs are available for these formats.

[0245] Due to the efforts of the Microplate Standards Development Committee of the Society for Biomolecular Sciences, microplate dimensions (e.g., length, width, and height) and tolerances are standardized according to the American National Standards Institute (ANSI), which greatly facilitates automation and cross-platform analyses. Efforts to maximize throughput by increasing the number of available reaction vessels within the standardized footprint of the microplate continue. Volume reduction, ease of testing, and cost reduction are some of the priorities driving research in this area. For example, a microplate containing 384 wells relative to a 96 well microplate, quadrupled the available reaction vessels (i.e., wells), and reduced the required volume consumption from 382 pl up to 28 pl.

[0246] Commercial microplates are typically made from plastic polymers (e.g., polypropylene). However, common plastic polymers, such as polypropylene and polyethylene, are susceptible to degradations issues. Thermal degradation, photodegradation, oxidative degradation, and UV degradation can occur, limiting the service life of a plastic microplate. Moreover, solvent compatibility with a range of solvents is required for certain types of analyses. Degradation generally involves changes to the molecular weight and/or structure of the plastic. Other property changes include a reduction in ductility and embrittlement, chalking, color changes, cracking, and a general reduction in desirable physical properties. Biological analyses often require incubation with abrasive chemicals and/or significant thermal shifts (e.g., about 20°C to about 100°C). Thus, there is a need to develop stable microplates. Described herein, inter alia, are solutions to these and other problems in the art.

Example 1. Microplate Receiver and Microplate Inserts

[0247] Advanced molecular technologies are required to enable a high-resolution view of DNA, RNA, and proteins in individual cells, along with their spatial arrangement, shedding greater insight into the function of both cells and tissues. Multiomics is a biological analysis approach that unifies the study of traditionally separate and distinct data sets derived from different “omes” (e.g., genomes, proteomes, transcriptomes, epigenomes, metabolomes, and microbiomes). By combining these “omes” it is possible to analyze complex biological processes to find novel associations between biological entities, identify relevant biomarkers, and build revelatory signatures of disease and physiology. Single-cell technologies have emerged to enable profiling the composition of the genome, epigenome, transcriptome, or proteome of a single cell. Uncovering the distribution, heterogeneity, spatial gene and protein co-expression patterns within cells and tissues is vital for understanding how cell colocalization influences tissue development and the spread of diseases such as cancer, which could lead to important new discoveries and therapeutics. Quantifying gene and protein expression and obtaining precise sequencing information enables precise identification, monitoring, and possible treatment at the molecular level.

[0248] It is generally advantageous when microplates have i) dimensional stability under multiple temperature and humidity conditions; ii) flatness; iii) chemical and biological compatibility with reagents (e.g., DMSO-stable, does not denature proteins, or non- specifically adsorb biological materials); and iv) low autofluorescence. The devices provided herein satisfy one or more of the above criteria.

[0249] Biological analyses often require subjecting the sample to significant thermal changes. For example, nucleic acid amplification and/or epitope expression may require cycling between room temperatures (e.g., 20°C to 25°C) to an elevated temperature (e.g., 90°C to 120°C). Plastic microplates (e.g., polystyrene, polypropylene, cyclic olefin copolymer, or cyclic olefin plastic microplates) are susceptible to warping and thermal degradation. Experiments with plastic microplates fused to an optically clear (COC/COP, glass, or quartz) bottom supports over these temperature ranges resulted in significant sample contamination. Without wishing to be bound by any theory, the different thermal expansion between the planar support (i.e., the glass bottom) and the fused well frame resulted in shearing, separating the well frame from the planar support, resulting in well-to-well leakage. It is advantageous to select a well frame and planar support that have a similar coefficient of linear thermal expansion (CLTE). The CLTE is provided as the change in length of the material due heating or to cooling, divided by the original length multiplied by the temperature change, or CLTE = AL / (Lo * AT). Methods for measuring CLTE are known in the art, for example using dilatometry, interferometry, and/or thermomechanical analysis. For example, the most widely used standards to measure coefficient of linear thermal expansion in plastics are ASTM D696, ASTM E831, ASTM E228 and ISO 11359. Additives may be used to modulate the thermal expansion of the material, for example fibers and other fillers significantly reduce thermal expansion.

[0250] Depending on the type of experiment warranted, microplates are typically manufactured with a plastic bottom or a glass bottom, as illustrated in FIG. 1, wherein the primary well plate is fused to a planar support piece of glass or plastic. Microplates with clear-bottom wells facilitate optical measurements from the bottom, e.g., inverted high- resolution microscopy and imaging. For optical detection modalities, an optically transparent planar support is useful. Microplate color may be tuned to maximize the signal-to- background ratio. Black microplates are well-suited for fluorescence-based readouts; the black color can reduce well-to-well crosstalk, while also reducing background autofluorescence.

[0251] In an aspect is provided a microplate assembly. In embodiments, the microplate assembly includes a microplate receiver frame defining a pocket. In embodiments, the microplate assembly includes at least one microplate section and a planar support positioned on a bottom of the at least one microplate section. In embodiments the microplate section and the planar support have a similar coefficient of linear thermal expansion. In embodiments, the pocket of the microplate receiver frame is sized and shaped to receive a plurality of microplate sections (e.g., two, three, four, five, six, seven, or eight sections). In embodiments, the microplate assembly includes integrated unit, wherein the frame and microplate section are fused together or otherwise inseparable. For example, the microplate assembly may include a microwell insert, wherein a plurality of wells are bored directly into the microwell insert. The integrated unit may have dimensions as provided and described by American National Standards Institute (ANSI) and Society for Laboratory Automation And Screening (SLAS); for example the tolerances and dimensions set forth in ANSI SLAS 1-2004 (R2012); ANSI SLAS 2-2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSI SLAS 4-2004 (R2012); and ANSI SLAS 6-2012, which are incorporated herein by reference.

[0252] As shown in FIG. 1 A, a microplate assembly 100 includes a primary well plate 101 that is fused or otherwise mechanically coupled (e.g., via an adhesive) to a planar support piece 103, which can be for example glass or plastic. The planar support piece 103 is sized and shaped to support the primary well plate 101. The planar support piece 103 is also housed in a microplate carrier 102 that is sized and shaped to receive the planar support piece 103. The microplate carrier 102 can include a portion, such as a cavity, seat, volume, or other structure sized and shaped to receive or contain the microplate carrier 102. FIG. IB illustrates the fully assembled microplate assembly with a plastic bottom or a glass bottom 104.

[0253] The cost of a microplate is typically determined by several factors, for example the material, type of planar support (e.g., glass or plastic bottom), molds for the wells, well density, and any surface treatments/coatings. Diamond has the lowest known thermal expansion coefficient of all naturally occurring materials and thus is unlikely to deform or warp during large temperature gradients. However, the significant costs associated with a diamond microplate render it unpractical. Glass supports typically have very low average coefficient of linear expansion, 3.3xl0' ⁶ /K to 6.7xlO' ⁷ /K, depending on the additive and manufacturing process. On the contrary, typical thermoplastics have a coefficient of linear expansion around 0.6xl0' ⁴ /K to 2.3xlO' ⁴ /K. To minimize confusion, the coefficient of linear expansion reported herein are in Kelvin. Known conversion techniques for translating commercially reported coefficient of linear expansion in Fahrenheit and Celsius may be used.

[0254] FIGS. 2 A and 2B illustrate an embodiment of a microplate assembly 200 that includes a plurality of microplate inserts 202 that collectively fit within or on a microplate receiver 201. FIG. 2A shows the microplate assembly in an exploded or disassembled state while FIG. 2B shows the microplate assembly 200 in an assembled state with the microplate inserts 202 coupled to the microplate receiver 201. The microplate assembly 200 includes the microplate receiver 201 that is sized and shaped to receive and/or retain one or more of the microplate inserts 202 therein or thereon. The microplate assembly 200 is made up of multiple sections including the multiple microplate inserts 202. Dividing the microplate into sections (such as the microplate inserts 202) enables additional customization and reduces the amount of expansion of the microplate inserts either collectively or individually. Because the change in length of the material due heating or to cooling is directly proportional to the length, reducing the length limits the amount of overall expansion. Each microplate insert 202 includes a planar support (e.g., glass bottom) fused to the primary well plate. The microplate receiver 201 and the microplate insert 202 may include one or more biasing features 203 and/or retention features, as described more fully below. The dashed lines in FIG. 2A are indicative of placement of the microplate insert(s) 202 into the microplate receiver 201. FIG. 2B illustrates the fully assembled microplate assembly 200 with a plastic bottom or a glass bottom (204).

[0255] FIG. 2C shows a perspective view of the microplate receiver 201. The microplate receiver 201, alternatively referred to herein as the microplate frame or microplate receiver frame, may include one or more biasing or retention elements 203. The biasing or retention elements mechanically interact with corresponding biasing or retention elements 203 of the microplate inserts 202 (FIG. 2A). When the microplate inserts 202 are inserted into or onto the microplate receiver 201, the respective biasing or retention elements 203 interact with one another to retain the microplate insert(s) 202 in the microplate receiver 201. The biasing or retention elements 203 can be a combination of biased prongs on one device that mate with corresponding slots or openings on the other device for example. The microplate receiver 201 can further include a pocket 207 that receives one or more of the microplate inserts 202 as shown in FIG. 2B. The pocket is defined by sidewalls of the microplate receiver 201 and is sized and shaped to receive a collection of microplate inserts 202. The pocket 207 can include at least one of the biasing or retention elements 203. The microplate receiver 20 lean be configured to retain the microplate insert 202 within the pocket 207. The microplate receiver 20 lean be configured to retain the microplate insert 202 within the pocket 207 such that an increased or maximal surface area of the microplate insert 202 can be available to be exposed to an optical lens (e.g., the optical lens of a nucleic acid sequencing or imaging device). The optical lens (e.g., the optical lens of the sequencing device) can be configured to detect excitation, emission, or other signals present on the microplate insert. The microplate receiver 20 lean be configured to retain the microplate insert 202 within the pocket 207 such that a maximal surface area of the microplate insert 202 can be available to be in contact with the receiver of a nucleic acid sequencing device. For example, the microplate insert 202 can be positively located on the frame by biasing the microplate insert 202 toward a corresponding biasing or retention element 203 of the microplate receiver 201, such as a proximal banking tab and/or a distal banking tab. The retaining of the microplate insert 202 within the pocket 207 further can include constraining a first, a second, a third, a fourth, a fifth, and a sixth degree of freedom of the microplate insert 202. The microplate receiver 20 lean be an injection molded frame. The frame can be anodized aluminum. The microplate receiver 201 further can include at least one pin such as a ferromagnetic pin. The at least one biasing or retention element 203 can be a spring finger for example. The at least one biasing or retention element 203 can be a tab. The microplate insert 202 can further include a microchip. The microplate receiver frame can further include a microchip.

[0256] The pocket 207 of the microplate receiver frame 201 defines multiple contact points (such as three contact points) for the plane defined by the microplate insert 202. The pocket 207 can constrain the microplate insert 202 in multiple degrees of freedom, such as six degrees of freedom. Constraining is used in accordance with its ordinary meaning in the art and refers to partially restricted movement or complete immobilization. Six degrees of freedom refers to the freedom of movement of the microplate insert in a three-dimensional space. Specifically, the microplate insert is free to move along and rotate around three perpendicular X-, Y-, and Z-axes. A first degree of freedom can be defined as moving left and right along the X-axis. A second degree of freedom can be defined as moving backward and forward along the Y-axis. A third degree of freedom can be defined as moving up and down along the Z-axis. A fourth degree of freedom can be defined as rotating around the X- axis, or “roll.” A fifth degree of freedom can be defined as rotating around the Y-axis, or “pitch.” A sixth degree of freedom can be defined as rotating around the Z-axis, or “yaw.” The pocket or other component of the frame can constrain microplate insert in one or more of these degrees of freedom.

[0257] FIGS. 3 A-3D depict various views of an example embodiment of the microplate insert 301 wherein the microplate insert 301 has 24 wells 305. FIG. 3A shows a top view of the 24-well insert 301, FIG. 3B shows a perspective view of the 24-well insert 301, and FIG. 3C shows a side view of the 24-well insert 301. In this example embodiments, the microplate insert includes 24 wells, wherein each well is 7.3 mm wide and separated by 1.6 mm of interstitial space. In embodiments, the microplate insert is about 26.9+/-0.5 mm x 76.8+/-0.5 mm. The microplate insert 301 is shown having an outer prismatic shape wherein a plurality of microplate inserts 301 are sized and shaped to be placed side-by-side to one another and collectively fit within the pocket 207 of the microplate receive 201. As mentioned, the microplate insert 301 can include a biasing or retention element 203 that interacts with a corresponding biasing or retention element 203 of the microplate receiver when the microplate insert is positioned in the pocket 207 to thereby bias and/or retain the microplate insert 301 therein. FIG. 3C shows the planar support 302, wherein the planar support (e.g., the glass bottom) is positioned on a bottom surface of the microplate insert 301 and is about 0.7 mm thick. FIG. 3D depicts an assembled microplate carrier, including four microplate inserts 301a, 301b, 301c, and 301d, which are collectively positioned within the pocket of the microplate receive 201 in a side-by-side configuration. In embodiments, the microplate sections are provided as a single column of wells (e.g., 8 wells). In embodiments, a 5 mm x 5 mm tissue sample may be in one or more wells.

[0258] FIGS. 4A-4D depicts another embodiment of the microplate insert 401 wherein the microplate insert includes 12 wells 405. FIG. 4A shows a top view of the 12-well microplate insert 401, FIG. 3B shows a perspective view of the 12-well microplate insert 401, and FIG. 4C shows a side view of the 12-well microplate insert 402. In embodiments, the microplate insert includes 12 wells, wherein each well is 15 mm wide and separated by 3 mm of interstitial space. In embodiments, the microplate insert is about 53.8+/-0.5 mm x 76.6+/-0.5 mm. FIG. 4C shows a bottom view of the microplate insert 401 and also shows the planar support 402, wherein the planar support 402 (e.g., the glass bottom) is about 0.7 mm thick. FIG. 4D depicts an assembled microplate carrier, including two microplate inserts 401a and 401b. In embodiments, a 14mm x 14 mm tissue sample may be in one or more wells.

[0259] The microplate insert does not necessarily include any wells. For example, FIG. 5 shows an embodiment of a blank microplate insert 501 that does not include any wells. For example, the microplate insert may be configured to retain a microscope slide.

[0260] The microplate inserts (also referred to as a microwell insert) may be comprised of any suitable material. In embodiments, the microwell insert includes a low average coefficient of linear expansion which is similar to the planar support, e.g., 3.3xl0' ⁶ /K to 6.7X10' ⁷ /K. In embodiments, the microwell insert has a coefficient of linear thermal expansion (e.g., a maximum coefficient of linear thermal expansion) of about 1.0xl0' ⁵ /K, 2.0X10' ⁵ /K, 3.0X10' ⁵ /K, 4.0X10' ⁵ /K, 5.0X10'7K, 6.0X10' ⁵ /K, 7.0X10' ⁵ /K, 8.0X10' ⁵ /K, or about 9.0X10' ⁵ /K. In embodiments, the microwell insert has a coefficient of linear thermal expansion of about 1.0xl0' ⁶ /K, 2.0xl0' ⁶ /K, 3.0xl0' ⁶ /K, 4.0xl0' ⁶ /K, 5.0xl0' ⁶ /K, 6.0xl0' ⁶ /K, 7.0X10' ⁶ /K, 8.0X10' ⁶ /K, or about 9.0xl0' ⁶ /K. In embodiments, the microwell insert has a coefficient of linear thermal expansion less than about 1.0xl0' ⁵ /K, 2.0xl0' ⁵ /K, 3.0xl0' ⁵ /K, 4.0xl0' ⁵ /K, 5.0X10' ⁵ /K, 6.0X10' ⁵ /K, 7.0X10'7K, 8.0X10' ⁵ /K, or about 9.0xl0' ⁵ /K. In embodiments, the average coefficient of linear expansion of the microwell insert is the same as the planar support. In embodiments, the average coefficient of linear expansion of the microwell insert is less about 95% of the average coefficient of linear expansion of the planar support. In embodiments, the average coefficient of linear expansion of the microwell insert is about 80%-99% of the average coefficient of linear expansion of the planar support. In embodiments, the average coefficient of linear expansion of the microwell insert is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% of the average coefficient of linear expansion of the planar support. In embodiments, the maximum coefficient of linear expansion of the microwell insert is about 2-250 times the maximum coefficient of linear expansion of the planar support. In embodiments, the maximum coefficient of linear expansion of the microwell insert is about 2-100 times the maximum coefficient of linear expansion of the planar support. In embodiments, the maximum coefficient of linear expansion of the microwell insert is about 2-10 times the maximum coefficient of linear expansion of the planar support. In embodiments, the maximum coefficient of linear expansion of the microwell insert is about 2-5 times the maximum coefficient of linear expansion of the planar support. In embodiments, the maximum coefficient of linear expansion of the microwell insert is about 2, 3, or 4 times the maximum coefficient of linear expansion of the planar support.

[0261] In embodiments, the maximum coefficient of linear thermal expansion of the planar support is about 0.5xl0' ⁶ /K to about 8.0xl0' ⁷ /K and the maximum coefficient of linear thermal expansion of the microwell insert is about 1.0xl0' ⁶ /K to about 9.0xl0' ⁶ /K. In embodiments, the solid support includes a coefficient of linear thermal expansion of about 2.0X10' ⁴ /K to about 1.0xl0' ⁵ /K and the planar support includes a coefficient of linear thermal expansion of about 0.5xl0' ⁵ /K to about 8.0xl0' ⁷ /K. In embodiments, the solid support includes a coefficient of linear thermal expansion of about 3.0xl0' ⁴ /K to about 1.0xl0' ⁵ /K and the planar support includes a coefficient of linear thermal expansion of about 0.5xl0' ⁵ /K to about 8.0X10' ⁷ /K. In embodiments, the solid support includes a coefficient of linear thermal expansion of about 5.0xl0' ⁴ /K to about 1.0xl0' ⁵ /K and the planar support includes a coefficient of linear thermal expansion of about 1.0xl0' ⁵ /K to about 8.0xl0' ⁷ /K.

[0262] In embodiments, the coefficient of linear thermal expansion of the planar support is 0.5 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar support is 1.0 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 1.5 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 2.0 xlO' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 2.5 xlO' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 3.0 xlO' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 3.5 xlO' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 4.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 4.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 5.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 5.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 6.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 6.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 7.0 xlO" ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 7.5 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 8.0 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar support is 8.5 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 9.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 9.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 10.0 xl0' ⁵ /K.

[0263] In embodiments, the coefficient of linear thermal expansion of the planar supportis 0.5 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar support is 1.0 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 1.5 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 2.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 2.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 3.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 3.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 4.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 4.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 5.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 5.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 6.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 6.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 7.0 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 7.5 X 10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 8.0 X 10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar support is 8.5 X 10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 9.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 9.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 10.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 0.5 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 1.0 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 1.5 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 2.0 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 2.5 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 3.0 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 3.5 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 4.0 xlO" ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 4.5 X 10' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 5.0 X 10' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar support is 5.5 X 10' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 6.0 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 6.5 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 7.0 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 7.5 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 8.0 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 8.5 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 9.0 xlO' ⁷ /K. In embodiments, the coefficient of linear thermal expansion of the planar supportis 9.5 xlO' ⁷ /K.

[0264] In embodiments, the coefficient of linear thermal expansion of the microwell insert (e.g., alternatively referred to herein as the solid support) is 2.0 xlO' ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insert is 2.5 xlO' ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 3.0 xlO' ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insert is 3.5 x10" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insert is 4.0 X10" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insert is 4.5 X10" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 5.0 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 5.5 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 6.0 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 6.5 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 7.0 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 7.5 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 8.0 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 8.5 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 9.0 xlO" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 9.5 X10" ⁴ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 10.0 X10" ⁴ /K.

[0265] In embodiments, the coefficient of linear thermal expansion of the microwell insert is 0.5 X10" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 1.0 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 1.5 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 2.0 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 2.5 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 3.0 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 3.5 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 4.0 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 4.5 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 5.0 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 5.5 X10" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 6.0 X10" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 6.5 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 7.0 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 7.5 xlO" ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 8.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 8.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 9.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 9.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 10.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 0.5 xlO" ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 1.0 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 1.5 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 2.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 2.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 3.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 3.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 4.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 4.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 5.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 5.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 6.0 xlO" ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 6.5 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 7.0 X10' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 7.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 8.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 8.5 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 9.0 xl0' ⁵ /K. In embodiments, the coefficient of linear thermal expansion of the microwell insertis 9.5 xl0' ⁵ /K.

[0266] In embodiments, the microwell insert and the planar support both include a similar coefficient of linear thermal expansion (e.g., within 1 or 2 orders of magnitude). In embodiments, the coefficient of linear thermal expansion is 0.1 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.3 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.5 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.7 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 0.9 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.1 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.3 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.7 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 1.9 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.1 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.3 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.7 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 2.9 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.1 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.3 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.5 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.7 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 3.9 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.1 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.3 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.7 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 4.9 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.1 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.3 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.7 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 5.9 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.1 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.3 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.5 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.7 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 6.9 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.1 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.3 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.7 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 7.9 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.1 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.3 X10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.5 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.7 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 8.9 X 10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.0 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.1 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.2 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.3 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.4 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.5 X 10' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.6 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.7 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.8 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 9.9 xlO' ⁶ /K. In embodiments, the coefficient of linear thermal expansion is 10.0 xl0' ⁶ /K.

[0267] In embodiments, the coefficient of linear thermal expansion of the microwell insert is 1.1 x 10' ⁵ /K, 2.2 x 10' ⁵ /K, 9.0 x 10' ⁵ /K, or 4.0 x 10' ⁵ /K. In embodiments, the planar support includes a coefficient of linear thermal expansion of 3.2 xlO' ⁶ /K or 7.2xlO' ⁶ /K. In embodiments, the microplate section includes a maximum coefficient of linear thermal expansion of about 2.0 xlO' ⁴ /K to about 1.0 xl0' ⁵ /K. In embodiments, the microplate section includes a maximum coefficient of linear thermal expansion of about 2.5 xlO' ⁴ /K to about 1.0 X 10' ⁵ /K. In embodiments, the microplate section includes a maximum coefficient of linear thermal expansion of about 3.0 xlO' ⁴ /K to about 1.0 xl0' ⁵ /K. In embodiments, the microplate section includes a maximum coefficient of linear thermal expansion of about 1.0 xl0' ⁵ /K, about 1.5 X 10' ⁵ /K, about 2.0 xl0' ⁵ /K, about 2.5 xl0' ⁵ /K, about 3.0 xl0' ⁵ /K, about 3.5 xl0' ⁵ /K, about 4.0 X 10' ⁵ /K, about 4.5 xl0' ⁵ /K, about 5.0 xl0' ⁵ /K, about 5.5 xl0' ⁵ /K, about 6.0 xl0' ⁵ /K, about 6.5 X 10' ⁵ /K, about 7.0 xl0' ⁵ /K, about 7.5 xl0' ⁵ /K, about 8.0 xl0' ⁵ /K, about 8.5 xl0' ⁵ /K, about 9.0 X 10' ⁵ /K, about 9.5 xl0' ⁵ /K. In embodiments, the microplate section includes a maximum coefficient of linear thermal expansion of about 1.0 X 10' ⁵ /K to about 3.0xl0' ⁵ /K. In embodiments, the microplate section includes a maximum coefficient of linear thermal expansion of about 1.8 xl0' ⁵ /K, 1.9 xlO' ⁵ /K, 2.0 xl0' ⁵ /K, 2.1 xl0' ⁵ /K, or about 2.2 xl0' ⁵ /K. In embodiments, the planar support includes a maximum coefficient of linear thermal expansion less than or equal to the maximum coefficient of linear thermal expansion of the microplate section. In embodiments, the planar support includes a maximum coefficient of linear thermal expansion less than the maximum coefficient of linear thermal expansion of the microplate section. In embodiments, the planar support includes a maximum coefficient of linear thermal expansion of about 0.5xl0' ⁵ /K to about 8.0xl0' ⁷ /K. In embodiments, the planar support includes a maximum coefficient of linear thermal expansion of about 1.0xl0' ⁵ /K to about 8.0X10' ⁷ /K. In embodiments, the planar support includes a maximum coefficient of linear thermal expansion of about 3.0xl0' ⁶ /K to about 8.0xl0' ⁶ /K.

[0268] In embodiments, the microwell insert includes a thermoplastic. In embodiments, the microwell insert includes a thermoplastic polyetherimide (PEI), for example ULTEM™ PEI PolyEtherlmide (PEI). In embodiments, the microwell insert is glass. In embodiments, the microwell insert is ceramic. In embodiments, the microwell insert is steel. In embodiments, the microwell insert is glass, wherein the plurality of wells are bored directly into the glass. In embodiments, the microwell insert includes a glass fiber-reinforced polymer (e.g., polyether ether ketone, polyacrylamide, or polyphthalamide).

[0269] In embodiments, the microplate includes a plurality of wells. In embodiments, the microplate array includes 2, 4, 6, 12, 24, 48, 96, 384 or 1536 wells. In embodiments, the microplate array includes 24, 48, 96, or 384 wells. In embodiments, the microplate array includes 24 wells. In embodiments, the microplate array includes 48 wells. In embodiments, the microplate array includes 96 wells. In embodiments, the microplate array includes 384 wells. In embodiments, each well includes about 10,000 to 100,000 cells per well. In embodiments, each well includes at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or at least 10,000 cells per well. In embodiments, each well includes about 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or at least 100,000 cells per well. In some embodiments, the wells of the array are separated from each other by about 1 mm to about 10 mm. In embodiments, the well is about 3 mm in diameter. In embodiments, the well is about 3.6 mm in diameter. In embodiments, the well is about 4 mm in diameter. In embodiments, the well is about 5 mm in diameter. In embodiments, the well is about 6 mm in diameter. In embodiments, the well is about 6.5 mm in diameter. In embodiments, the well is about 7 mm in diameter. In embodiments, the well is about 7.5 mm in diameter. In embodiments, the well is about 8 mm in diameter. In embodiments, the well is 5 mm in diameter. In embodiments, the well is 6 mm in diameter. In embodiments, the well is 6.5 mm in diameter. In embodiments, the well is 7 mm in diameter. In embodiments, the well is 7.5 mm in diameter. In embodiments, the well is 8 mm in diameter. In embodiments, the well is about 6 to 12 mm in depth. It is also understood that the size of the wells on the array can be of various sizes and will ultimately depend on the systems and/or apparatus used to analyze later reactions. [0270] In embodiments, the microwell insert includes a liquid crystal polymer. A liquid crystal polymer is a highly crystalline, thermotropic (melt-orienting), thermoplastic that typically includes rigid, rod-like macromolecules that are ordered in the melt phase to form liquid crystal structures.

[0271] In embodiments, the microwell insert includes a thermoplastic with glass fiber reinforcements and/or carbon fiber reinforcements. Reinforcement with glass fibers increases stiffness, mechanical strength, and heat resistance, while decreasing the degree of anisotropy. In embodiments, the microwell insert includes 15%, 30%, 40% or 50% glass fiber content. Reinforcement with carbon fibers gives even higher stiffness than with glass fibers. At the same time, carbon fiber reinforced compounds have a lower density than glass fiber grades with the same additive content. In embodiments, the microwell insert includes mica-group minerals (e.g., phyllosilicates such as muscovite, paragonite, margarite, biotite, lepidolite, phlogopite, zinnwaldite, or clintonite) or titanium dioxide. The mineral filled thermoplastics typically have high impact strength relative to the glass fiber reinforced compositions. In embodiments, the thermoplastic includes 10%, 20%, 30%, or 40% mineral content.

[0272] In embodiments, the microwell insert is resistant to gamma radiation, steam autoclaving, and chemical sterilization methods. In embodiments, the microwell insert includes a liquid crystal polymer selected from the following: VECTRA® Al 15, VECTRA® A l 30, VECTRA® A230, VECTRA® A430, VECTRA® A435, VECTRA® A515, VECTRA® A530, VECTRA® A625, VECTRA® A700, VECTRA® A725, VECTRA® A950, VECTRA® B230, VECTRA® C130, VECTRA® El 15i, VECTRA® E130G, VECTRA® E130i, VECTRA® E135i, VECTRA® E150i, VECTRA® E440i, VECTRA® E463i, VECTRA® E471i, VECTRA® E473i, VECTRA® E488i, VECTRA® E53 li, VECTRA® E540i, VECTRA® E820iPd, VECTRA® E830iPd, VECTRA® E840i LDS, VECTRA® E845i LDS, VECTRA® FIT30, VECTRA® FIT50, VECTRA® J540, VECTRA® MT®1300, VECTRA® MT® 1305, VECTRA® MT®1310, VECTRA® MT®1335, VECTRA® MT®1345, VECTRA® MT®4310, VECTRA® MT®4350, VECTRA® S135, VECTRA® S471, VECTRA® S475, VECTRA® S540, VECTRA® S625, VECTRA® V143XL, or VECTRA® V400P.

[0273] In embodiments, the microwell insert includes an epoxy resin. In embodiments, the microwell insert is a glass epoxy (e.g., G10, FR-4) thermoset epoxy resin. FR-4 is a composite material composed of woven fiberglass cloth with an epoxy resin binder. G- 10 is a fiberglass laminate, created by stacking multiple layers of glass cloth soaked in epoxy resin, and compressing the resulting material under heat.

[0274] In embodiments, the microwell insert does not degrade at temperatures greater than 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, or 120°C. In embodiments, the microwell insert does not degrade at temperatures greater than 50°C, 60°C, 70°C, 80°C, 90°C, or 100°C. In embodiments, the microwell insert does not degrade at temperatures between 50°C and 100°C. In embodiments, the microwell insert does not degrade at 100°C. In embodiments, the microwell insert bonded to the planar support does not degrade or result in sample contamination at elevated temperatures (e.g., 80°C -120°C). The microplate may be used to detect biomolecules (e.g., nucleic acids). Typically, the nucleic acids need to be amplified. In embodiments the term “amplified” refers to a method that comprises a polymerase chain reaction (PCR). Conditions conducive to amplification (i.e., amplification conditions) are well known and often comprise at least a suitable polymerase, a suitable template, a suitable primer or set of primers, suitable nucleotides e.g., dNTPs), a suitable buffer, and application of suitable annealing, hybridization and/or extension times and temperatures. Amplification conditions may cycle between different temperatures, often involving a large temperature gradient (e.g., 20°C -40°C). Additionally, samples embedded in formalin may require additional protocols to render biomolecules available. Heat induced epitope retrieval (HER) uses heat coupled with buffered solutions to recover antigen reactivity in formalin fixed paraffin embedded tissue samples. Typical HER methods include increasing the temperature from 25°C to 95°C-120°C, if utilizing a water bath or pressure enhanced temperature device (e.g., a pressure cooker). In embodiments, the microplate includes a microplate insert and a planar support attached to the microplate insert. In embodiments, a the planar support can include glass (e.g., a glass slide) that has been coated with a substance or otherwise modified to confer conductive properties to the glass. In some embodiments, a glass slide can be coated with a conductive coating. In some embodiments, a conductive coating includes tin oxide (TO) or indium tin oxide (ITO). In some embodiments, a conductive coating includes a transparent conductive oxide (TCO). In some embodiments, a conductive coating includes aluminum doped zinc oxide (AZO). In some embodiments, a conductive coating includes fluorine doped tin oxide (FTO).

[0275] As mentioned, the microplate receiver frame 201 can include or be coupled to one or more biasing or retention elements (also referred to as retaining or positioning components) that are configured to retain the microplate insert 202 in place when the insert is positioned in the frame. The retaining components are configured to interact with the insert to retain and/or position the insert in place or otherwise reduce the likelihood that the insert will disengage from the microplate frame.

[0276] In embodiments, the microwell insert includes two or more wells. In embodiments, the microwell insert may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells. In embodiments, the 96 and 384 wells are arranged in a 2:3 rectangular matrix. In embodiments, the wells includes a coating for enhanced cell adhesion (e.g., poly-L-lysine, silanes, carbon nanotubes, polymers, epoxy resins, or gold). Coatings for enhanced biomolecule adhesion are known, for example extracellular matrix proteins such as collagen type I, fibronectin, and laminin mediate specific binding of the cell to the protein. Poly-D-Lysine (PDL), a synthetically produced biomolecule, belongs to the non-specific adhesion-promoting polypeptides. PDL is typically used to promote cell adhesion, especially during washing steps, as well as to enhance cell vitality and proliferation during serum-reduced or serum-free cultivation.

[0277] In embodiments, the wells of the microwell insert includes one or more polymers. In embodiments, the wells of the microwell insert include a hydrogel. As used herein, the term “hydrogel” refers to a three-dimensional polymeric structure that is substantially insoluble in water, but which is capable of absorbing and retaining large quantities of water to form a substantially stable, often soft and pliable, structure. In embodiments, water can penetrate in between polymer chains of a polymer network, subsequently causing swelling and the formation of a hydrogel. In embodiments, hydrogels are super-absorbent (e.g., containing more than about 90% water) and can be comprised of natural or synthetic polymers. Hydrogels can contain over 99% water and may comprise natural or synthetic polymers, or a combination thereof. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. A detailed description of suitable hydrogels may be found in published U.S. patent application 2010/0055733, herein specifically incorporated by reference. By “hydrogel subunits” or “hydrogel precursors” is meant hydrophilic monomers, prepolymers, or polymers that can be crosslinked, or “polymerized”, to form a three-dimensional (3D) hydrogel network.

[0278] Hydrogels may be prepared by cross-linking hydrophilic biopolymers or synthetic polymers. Thus, in some embodiments, the hydrogel may include a crosslinker. As used herein, the term “crosslinker” refers to a molecule that can form a three-dimensional network when reacted with the appropriate base monomers. Examples of the hydrogel polymers, which may include one or more crosslinkers, include but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO — PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N- vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethylene imine), polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. Thus, for example, a combination may include a polymer and a crosslinker, for example polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), or PEG/polypropylene oxide (PPO). In embodiments, the hydrogel includes chemical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a covalent bond) and may be referred to as a chemical hydrogel. In embodiments, the hydrogel includes physical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a non-covalent bond) and may be referred to as a physical hydrogel. In embodiments, the physical hydrogel include one or more crosslinks including hydrogen bonds, hydrophobic interactions, and/or polymer chain entanglements.

[0279] In embodiments, the well contains a gel. The term “gel” in this context refers to a semi-rigid solid that is permeable to liquids and gases. Exemplary gels include, but are not limited to, those having a colloidal structure, such as agarose; polymer mesh structure, such as gelatin; or cross-linked polymer structure, such as polyacrylamide or a derivative thereof. Analytes, such as polynucleotides, can be attached to a gel or polymer material via covalent or non-covalent means. Exemplary methods and reactants for attaching nucleic acids to gels are described, for example, in US 2011/0059865 which is incorporated herein by reference. The analytes, sample, tissue, or cell can include nucleic acids and the nucleic acids can be attached to the gel or polymer via their 3' oxygen, 5' oxygen, or at other locations along their length such as via a base moiety of the 3' terminal nucleotide, a base moiety of the 5' nucleotide, and/or one or more base moieties elsewhere in the molecule. In embodiments, the microplate includes a polymer layer (alternatively referred to as a polymer coating). In embodiments, the microplate includes a polymer layer, wherein the polymer layer includes an amphiphilic copolymer. The term “amphiphilic copolymer” is used in accordance with its ordinary meaning and refers to a copolymer composed of polymerized hydrophilic (e.g., PEG monomers) and hydrophobic monomers (e.g., alkoxysilyl or (polypropylene oxide) monomers). Amphiphilic copolymers can have both hydrophilic and hydrophobic properties. In embodiments, the polymer layer includes an amphiphilic acrylate copolymer or amphiphilic methacrylate copolymer. In embodiments, the amphiphilic polymer includes a poloxamer. In some embodiments, the solid support includes a poloxamer layer. In some embodiments, the poloxamer is a polyoxyethyl ene-polyoxypropylene copolymer. In embodiments, the wells include a hydrogel. In embodiments, the wells include a sample (e.g., a biological sample such as a cell and/or tissue). In embodiments, the wells include a sample, wherein the sample includes an analyte of interest. Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non- nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In embodiments, the analytes within a cell can be localized to subcellular locations, including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In embodiments, analyte(s) can be peptides or proteins, including antibodies and/or enzymes. In embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.

[0280] In embodiments, the microwell insert is attached to a planar solid support. In embodiments, the microwell insert is bonded to the planar solid support via an adhesive. In embodiments, the microwell insert is bonded to the planar solid support via a pressure sensitive adhesive. Pressure sensitive adhesives, e.g., a PSA tape, is self stick tape or sticky tape including a pressure sensitive adhesive coated onto a scaffold support (e.g., backing material such as paper, plastic film, cloth, or metal foil). The pressure sensitive adhesive is sticky (i.e., tacky) without any heat or solvent for activation and adheres with minimal pressure. In embodiments, the adhesive includes an epoxy, poly(methyl methacrylate) (PMMA), or cyanoacrylate resin. For example, the adhesive may include Paraloid® B-72, Paraloid® B-67, Primal® AC 35, Acrifix® 116, HXTAL®-NYL-1, Araldite® 2020, or Loctite® Super Attack Precision. In embodiments, the adhesive is capable of absorbing light. In embodiments, the adhesive is transparent. In embodiments, the adhesive is capable of reflecting light. In embodiments, the adhesive includes a scaffold support (e.g., a tape). In embodiments, the scaffold support includes polyethylene terephthalate or cellulose. In embodiments, the adhesive includes a carboxylated styrene-butadiene binder. In embodiments, the adhesive has a thickness of about 0.05 to about 0.5 pm (i.e., the adhesive contacts the planar support and the microwell insert and is about 0.05 to about 0.5 pm thick). In embodiments, the adhesive is about 0.1, 0.2, 0.3, 0.4 or about 0.5 pm thick.

[0281] In embodiments, the microwell insert is attached to a gasket, and the gasket is attached to the planar solid support. In embodiments, the gasket includes substantially the same pattern as the microwell insert (e.g., such that the bottom of each well of the microwell insert is in direct contact with the planar support). In embodiments, the gasket is bonded to the planar support. For example, the gasket may be irreversibly bound to a glass support when the surfaces of the gasket and the planar support are oxidized in an air plasma and then brought together.

[0282] In embodiments, the gasket is composed of a chemically inert material such that it does not substantially interact or interfere with the sample. In embodiments, the gasket does not substantially change shape when exposed to solvents or different analytical temperatures (e.g., 4°C to 150°C). For example, the gasket may include poly(dimethylsiloxane) (PDMS) material, which is known to minimally swell when exposed to solvents such as water, nitromethane, dimethyl sulfoxide, ethylene glycol, per-fluorotributylamine, perfluorodecalin, acetonitrile, and/or propylene carbonate. In embodiments, the gasket is composed of polydimethylsiloxane (PDMS), polystyrene (PS), or polycarbonate (PC). In embodiments, the gasket is composed of a thermoplastic fluoroelastomer, for example as described in McMillan et al. Nano Select. 2021; 2: 1385- 1402, which is incorporated herein by reference. In embodiments, the gasket is a poly(TFE-ter-E-ter HFP) gasket, wherein TFE = tetrafluoroethylene, E = ethylene, HFP = hexafluoropropylene. In embodiments, the gasket is melt processable, optically transparent, and includes self-sealing properties.

[0283] In embodiments, the gasket is 250 gm, 750 gm, 1200 gm and 2000 gm thick. In embodiments, the gasket is 2000 gm thick. In embodiments, the gasket is 1200 gm gm thick. In embodiments, the gasket is 750 gm, thick. In embodiments, the gasket is 250 gm thick. In embodiments, the gasket substantially conforms to the shape and pattern of the microplate insert. In embodiments, the gasket is between the microplate insert and the planar support, wherein the gasket does not cover the bottom of the well. In embodiments, the gasket provides a seal between each well, such that no liquid from a first well may contact or contaminate a second well.

[0284] In embodiments, the gasket is substantially transparent to UV and/or visible light. In embodiments, the gasket is chemically inert. In embodiments, the gasket does not substantially swell (i.e., increase in volume) upon incubation in water. In embodiments, the gasket does not substantially swell (i.e., increase in volume) upon incubation in an organic solvent (e.g., xylene or hexane). In embodiments, the gasket does not dissolve in an organic solvent. In embodiments, the gasket does not dissolve in an a strong acid (e.g., HC1). In embodiments, the gasket does not substantially increase its volume upon incubation in xylene at room temperature. In embodiments, the gasket does not substantially increase its volume upon incubation in xylene for 10 minutes.

[0285] In embodiments, the planar solid support is a borosilicate glass (e.g., D263 glass). In embodiments, the planar solid support is Borofloat glass (e.g., SiCh: 81%; B2O3: 13%; Na2O/K2O: 4%; AI2O3: 2%), B270 glass, D263 glass, Eagle XG glass, Schott Supremax glass, Schott Xensation glass, or Gorilla glass. In embodiments, the planar solid support is a glass described in US 8,598,056; US 9,440,875; or US 2020/0339468, each of which are incorporated by reference. In embodiments, the planar solid support is a glass including 67 SiO ₂ = 70; 11 ^A1 ₂ O ₃ ^ 13.5; 3 B2O3 ^6; 3.5 0.5

BaO =3; 0.02 SnO ₂ i 0.3; CeO ₂ ^0.3; 0.00 As ₂ O ₃ i 0.5; 0.00 Sb ₂ O ₃ i 0.5;

0.01 Fe2O3 = 0.08; and F+Cl+Br 0.4; wherein all oxides are in mol %. In embodiments, the planar solid support is a borosilicate glass including 78% SiCh, 10% B2O2, 7% Na2O, 3% AI2O3, and 2% ZrCh. In embodiments, the planar solid support is a borosilicate glass including 80% SiCh, 13% B2O2, 4% Na2O, 2% AI2O3, and 1% K2O. In embodiments, the planar solid support is optically transparent. In embodiments, the planar solid support is 100 pm to 900 pm thick. In embodiments, the planar solid support is 500 pm to 900 pm thick. In embodiments, the planar solid support is 600 pm to 800 pm thick. In embodiments, the planar solid support is about 700 pm thick.

[0286] In embodiments, the planar support includes a functionalized glass surface or a functionalized plastic surface. In embodiments, the planar support includes (3- aminopropyl)tri ethoxy silane (APTES), (3 -Aminopropyl )trimethoxysilane (APTMS), y- Aminopropylsilatrane (APS), N-(6-aminohexyl)aminom ethyl tri ethoxysilane (AHAMTES), polyethylenimine (PEI), 5,6-epoxyhexyltriethoxysilane, or triethoxysilylbutyraldehyde, or a combination thereof. In embodiments, the planar support includes (3- aminopropyl)tri ethoxysilane (APTES). In embodiments, the planar support includes (3- Aminopropyl)trimethoxysilane (APTMS). In embodiments, the planar support includes y- Aminopropylsilatrane (APS). In embodiments, the planar support includes N-(6- aminohexyl)aminomethyltriethoxysilane (AHAMTES). In embodiments, the planar support surface includes polyethylenimine (PEI). In embodiments, the planar support includes 5,6- epoxyhexyltriethoxysilane. In embodiments, the planar support includes triethoxysilylbutyraldehyde. In embodiments, the functionalized glass surface is functionalized with APTES, APTMS, APS, or AHAMTES.

[0287] In embodiments, the microwell insert is resistant to chemical degradation. Chemical durability is measured according to known methods in the art, for example via measuring weight loss per surface area following contact with a chemical (e.g., HC1). In embodiments, the microwell insert is capable of contacting xylene without significant degradation (e.g., without significant weight loss). In embodiments, the microwell insert is capable of contacting HC1, HN03, HF, and/or NaOH, without significant degradation (e.g., without significant weight loss). In embodiments, the microwell insert is capable of contacting organic solvents, such as hexanes or xylenes. Such chemicals can react with the microplate polymers (i.e., oxidization, reaction with functional groups, catalyze de-polymerization), or be absorbed into the bulk microplate material and soften/swell the microplate.

Example 2. Integrated Device with Microplate Receiver and Microplate Inserts

[0288] Nature has evolved an elegant solution where four nucleotides - adenine (A), cytosine (C), guanine (G), thymine (T) - form the primary code upon which all of life and biologic diversity is built. Next-generation sequencing (NGS) directly reads the four nucleotides (or DNA bases) and thus can read out a limitless number of possible sequences. This contrasts with alternative genetic detection technologies that require a priori knowledge of the DNA sequence of interest, such as DNA microarrays, targeted probe hybridization and polymerase chain reaction (PCR). These technologies require prior knowledge of the sequence of a DNA target, and in many cases are also limited by detection with a small number of fluorescent dyes. The capability of NGS to read the vast combinatorial repertoire of DNA, even without prior knowledge of the DNA target, makes it a uniquely powerful platform technology and universal detection method to read and interrogate biology. Beyond reading genomic DNA and RNA, the combination of NGS and designed DNA probes attached to antibodies introduce a wide range of multiomics applications, including imaging and measuring gene transcription and protein expression in individual cells and tissue pathology samples.

[0289] NGS has been a transformational technology for the life sciences industry and has been critical to ushering in the genomics age and accelerating our understanding of biology. Since its introduction in the mid-2000s, NGS technology has advanced greatly, which has increased the power of the technology and enabled its broad adoption by the life sciences community. The first NGS platform in the mid-2000s enabled a 50,000-fold drop in cost of sequencing the human genome as compared to the initial first genome sequenced as part of the Human Genome Project at a cost of $300 million. By 2015, the cost of sequencing a human genome reached $1,000 (at approximately 30X coverage, to achieve sufficient accuracy).

[0290] NGS can serve as an extremely versatile molecular tool in biology, extending well beyond its current applications. Today, NGS is used to sequence DNA and RNA to identify inherited and acquired mutations, measure gene expression by counting RNA transcripts, detect and identify pathogens, and when combined with certain sample preparation techniques, determine the epigenetic state of the DNA. Despite the advances that NGS has enabled in the genomics space, we believe the power of sequencing has not been fully realized and that innovation across the core elements of a sequencer can drive further improvement in the technology. Described herein are devices, compositions, and kits that enable sequencing to be extended beyond genomics and leveraged as a multiomics reader of biology. The devices, compositions, and kits described herein will transform genomics, epigenetics, transcriptomics, and proteomics (i.e., multiomics). [0291] In an aspect, provided herein are systems and devices that can detect the quantity of a biological target, detect biological activity indicative of a biological target, and perform nucleic acid sequencing. In an aspect is provided a device including the microplate assembly as described herein. In embodiments, the device is an integrated system of one or more chambers, ports, and channels that are directly or indirectly interconnected and in fluid communication and configured for carrying out an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides support functions, such as sample introduction, fluid and/or reagent driving means, temperature control, detection systems, data collection and/or integration systems, at least for the purpose of profiling a cell and/or determining the nucleic acid sequence of a template polynucleotide. In embodiments, the device as described herein is capable of multiplex analysis of a sample.

[0292] In an aspect is provided a device including: a sample stage configured to be coupled to a microplate receiver; a microplate receiver configured to be coupled to a microplate as described herein; at least one heating element thermally coupled to the microplate receiver; a fluidics dispenser configured to dispense one or more reagents into the microplate; and an imaging system configured to detect at least one feature (e.g., one or more features) in the microplate; and a structure physically coupled to the sample stage, the heating element, the fluidics dispenser, and the imaging system. In embodiments, the sample stage retains the microplate. In embodiments, the structure includes a support structure formed of a base platform or a table. In embodiments, the one or more features include a reaction chamber and its contents. In embodiments, the one or more features includes a target (e.g., a nucleic acid, protein, or biomarker), a cell, or a tissue sample. In embodiments, the feature is a nucleotide (e.g., a fluorescently labeled nucleotide). In embodiments, the feature is a nucleic acid. In embodiments, the feature is a protein. In embodiments, the feature is a biomolecule.

[0293] In another aspect is provided a method of imaging a cell, the method including obtaining a sample from a subject, wherein the sample comprises a cell (e.g., the sample is a tissue sample including a plurality of cells); contacting the microplate assembly as described herein with the sample; and obtaining an image of the sample, thereby imaging the cell.

[0294] In embodiments, the sample includes one or more detection agents. In embodiments, the sample includes one or more barcodes. In embodiments, the detection agent includes a label. In embodiments, the detection agent includes a fluorescent label. In embodiments, the detection agent includes an oligonucleotide barcode (e.g., a 5 to 15 nucleotide sequence). In embodiments, the oligonucleotide barcode includes at least two primer binding sequences. In embodiments, the oligonucleotide barcode includes an amplification primer binding sequence. In embodiments, the oligonucleotide barcode includes a sequencing primer binding sequence. The amplification primer binding sequence refers to a nucleotide sequence that is complementary to a primer useful in initiating amplification (i.e., an amplification primer). Likewise, a sequencing primer binding sequence is a nucleotide sequence that is complementary to a primer useful in initiating sequencing (i.e., a sequencing primer). Primer binding sequences usually have a length in the range of between 3 to 36 nucleotides, also 5 to 24 nucleotides, also from 14 to 36 nucleotides. In embodiments, an amplification primer and a sequencing primer are complementary to the same primer binding sequence, or overlapping primer binding sequences. In embodiments, an amplification primer and a sequencing primer are complementary to different primer binding sequences. In embodiments, the primer binding sequence is complementary to a fluorescent in situ hybridization (FISH) probe. FISH probes may be custom designed using known techniques in the art, see for example Gelali, E., et al. Nat Commun 10, 1636 (2019). In embodiments, the detection agent includes a padlock probe. Padlock probes are specialized ligation probes, examples of which are known in the art, see for example Nilsson M, et al. Science. 1994;265(5181):2085-2088), and has been applied to detect transcribed RNA in cells, see for example Christian AT, et al. Proc Natl Acad Sci U S A. 2001;98(25): 14238- 14243, both of which are incorporated herein by reference in their entireties. In embodiments, the padlock probe is approximately 50 to 200 nucleotides. In embodiments, a padlock probe has a first domain that is capable of hybridizing to a first target sequence domain, and a second ligation domain, capable of hybridizing to an adjacent second sequence domain. The configuration of the padlock probe is such that upon ligation of the first and second ligation domains of the padlock probe, the probe forms a circular polynucleotide, and forms a complex with the sequence (i.e., the sequence it hybridized to, the target sequence) wherein the target sequence is “inserted” into the loop of the circle. Padlock probes are useful for the methods provided herein and include, for example, padlock probes for genomic analyses, as exemplified by Gore, A. et al. Nature 471, 63-67 (2011); Porreca, G. J. et al. Nat Methods 4, 931-936 (2007); Li, J. B. et al. Genome Res 19, 1606-1615 (2009), Zhang, K. et al. Nat Methods 6, 613-618 (2009); Noggle, S. et al. Nature 478, 70-75 (2011); and Li, J. B. et al. Science 324, 1210-1213 (2009), the content of each of which is incorporated by reference in its entirety. [0295] In embodiments, the detection agent includes a protein-specific binding agent. In embodiments, the detection agent includes a protein-specific binding agent bound to a nucleic acid sequence, bioconjugate reactive moiety, an enzyme, or a label. In embodiments, the protein-specific binding agent is an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), affimer, or an aptamer.

[0296] In embodiments, the systems, devices, methods, and compositions can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in a region of the sample or within an individual well of the substrate.

[0297] In embodiments, the imaging system is configured to detect one or more fluorescent features in the microplate. In embodiments, the imaging system is configured to detect one or more fluorescent nucleotides in the microplate. In embodiments, the imaging system configured to detect one or more features in each reaction chamber (e.g., well) of the microplate. In embodiments, the imaging system configured to detect the cellular morphology of a cell. In embodiments, the imaging system configured to image the cell and characterize the cell morphology (e.g., the cell boundary, granularity, or cell shape). In embodiments, the imaging system configured to obtain images of a histologically-stained cell. In embodiments, the imaging system is configured to detect fluorescently-labeled nucleotides and obtain structural information about a cell on the microplate.

[0298] In embodiments, the imaging system includes at least one of a three-dimensional imager, a TDI scanner, a laser, a camera, an autofocus, and a transilluminator. In embodiments, the imaging system is configured to perform conventional immunohistochemical (IHC) imaging and immunofluorescence (IF) imaging. Examples of suitable imaging systems include optical waveguides, microscopes, diodes, light stimulating devices (e.g., lasers), photo multiplier tubes, processors (e.g., computers and software), and combinations thereof, which cooperate to detect a signal representative of a characteristic, marker, or target. In embodiments, the imaging system includes a CCD, EMCCD, or s- CMOS detector. In embodiments, the imaging system includes a light source that illuminates a sample, an objective lens, and a sensor array (e.g., complementary metal-oxide- semiconductor (CMOS) array or a charge-coupled device (CCD) array). In embodiments, the illuminator or light source is a radiation source (i.e., an origin or generator of propagated electromagnetic energy) providing incident light to the sample. A radiation source can include an illumination source producing electromagnetic radiation in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 390 to 770 nm), or infrared (IR) range (about 0.77 to 25 microns), or other range of the electromagnetic spectrum. In embodiments, the illuminator or light source is a lamp such as an arc lamp or quartz halogen lamp. In embodiments, the illuminator or light source is a coherent light source. In embodiments, the light source is a laser, LED (light emitting diode), a mercury or tungsten lamp, or a super-continuous diode. In embodiments, the light source provides excitation beams having a wavelength between 200 nm to 1500 nm. In embodiments, the laser provides excitation beams having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm. In embodiments, the laser provides excitation beams having a wavelength of 405 nm, 488 nm, 532 nm, or 633 nm. In embodiments, the illuminator or light source is a light-emitting diode (LED). The LED can be, for example, an Organic Light Emitting Diode (OLED), a Thin Film Electroluminescent Device (TFELD), or a Quantum dot based inorganic organic LED. The LED can include a phosphorescent OLED (PHOLED). In embodiments, the device is configured to obtain images at various depths (e.g., different z depths) of a sample. For example, the device is capable of obtaining a image at a first depth and a second depth, wherein the first and second depth are separated by about 1.5 pm. In embodiments, the device is capable of obtaining multiple images at a plurality of depths (e.g., depths separated by about 1.5 pm intervals). In embodiments, the device obtains an image at different focus depths. In embodiments, the focus depths are at 0.0 (i.e., on the surface of the sample), -0.5, - 1.0, -1.5, and -2.0 pm, wherein the negative sign indicates the length from the surface. In embodiments, the focus depths are separated at interval depths. In embodiments, the interval depths (e.g., delta z) are 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, or 2.0 pm relative to the surface. For example, when the imaging system has an interval depth of 1.0 pm, wherein the total depth imaged is 5.0 pm relative to the surface of the sample. In embodiments, the imaging system is an imaging system as described in WO 2022/056385, which is incorporated herein by reference in its entirety for all purposes. In non-limiting example embodiments, the imaging system includes a light source that illuminates a sample, an objective lens, and a sensor array (e.g., complementary metal-oxide-semiconductor (CMOS) array or a charge-coupled device (CCD) array), wherein the sample is in a microplate, and the sensor array is on a detection stage.

[0299] In embodiments, the light source is a laser (e.g., a laser such as a solid state laser or a gas laser). In embodiments, the light source includes one or more vertical cavity surface emitting lasers (VCSELs), vertical external cavity surface emitting lasers (VECSELs), or diode pumped solid state (DPSS) lasers. In embodiments, the light source is a continuous wave (CW) laser or a pulsed laser. In embodiments, the light source is a pulsed laser. In embodiments, the light source is an ultrashort pulsed laser. An ultrashort laser is a laser capable of producing excitation beams for a time duration of a picosecond or less. An ultrashort laser typically includes additional components, such as a pulse controller, pulse shaper, and spatial light modulator, and the like for controlling the pulse of excitation beams. In embodiments, the ultrashort laser provides excitation beams for femtoseconds or picoseconds. In embodiments, the light source is a pulsed femtosecond or picosecond laser. In embodiments, the laser is a Ti-sapphire laser, a dye-laser, or a fiber laser. In embodiments, the system includes two or more light sources (e.g., lasers). In embodiments, the first light source configured to emit light in red wavelengths, and a second light source configured to emit light in green wavelengths. In embodiments, the device includes two or more lasers. In embodiments, the device includes two lasers. The system 305 may include safety features configured to limit unintentional exposure of the laser. For example, the system may include one or more kill switches that automatically turns off the laser when a predetermined condition is satisfied.

[0300] In embodiments, the imaging system includes components necessary to perform bright field microscopy, phase contrast microscopy, Nomarski differential-interference- contrast microscopy, or dark field microscopy. In embodiments, the imaging system includes either 2D or 3D fluorescent imaging modalities can be used. An advantage of 3D imaging is that a larger number of individual targets (e.g., proteins or nucleic acids) can be resolved within a single reaction chamber (e.g., a well). 3D fluorescent imaging methods include confocal microscopy, light sheet microscopy, and multi-photon microscopy. Suitable imaging technologies are known in the art, as exemplified by Larsson et al., Nat. Methods (2010) 7:395-397 and US Provisional application US/63/077,852; associated supplemental materials, the entire content of each is incorporated by reference herein in its entirety. In embodiments of the methods provided herein, the imaging is accomplished by confocal microscopy. Confocal fluorescence microscopy involves scanning a focused laser beam across the sample and imaging the emission from the focal point through an appropriately-sized pinhole. This suppresses the unwanted fluorescence from sections at other depths in the sample. In embodiments, the imaging is accomplished by multi-photon microscopy (e.g., two-photon excited fluorescence or two-photon-pumped microscopy). Unlike conventional single-photon emission, multi-photon microscopy can utilize much longer excitation wavelength up to the red or near-infrared spectral region. This lower energy excitation requirement enables the implementation of semiconductor diode lasers as pump sources to significantly enhance the photostability of materials. Scanning a single focal point across the field of view is likely to be too slow for many sequencing applications. To speed up the image acquisition, an array of multiple focal points can be used. The emission from each of these focal points can be imaged onto a detector, and the time information from the scanning mirrors can be translated into image coordinates. Alternatively, the multiple focal points can be used just for the purpose of confining the fluorescence to a narrow axial section, and the emission can be imaged onto an imaging detector, such as a CCD, EMCCD, or s-CMOS detector. A scientific grade CMOS detector offers an optimal combination of sensitivity, readout speed, and low cost. One configuration used for confocal microscopy is spinning disk confocal microscopy. In 2-photon microscopy, the technique of using multiple focal points simultaneously to parallelize the readout has been called Multifocal Two-Photon Microscopy (MTPM). Several techniques for MTPM are available, with applications typically involving imaging in biological tissue. In embodiments of the methods provided herein, the imaging is accomplished by light sheet fluorescence microscopy (LSFM). In embodiments, detecting includes 3D structured illumination (3DSIM). In 3DSIM, patterned light is used for excitation, and fringes in the Moire pattern generated by interference of the illumination pattern and the sample, are used to reconstruct the source of light in three dimensions. In order to illuminate the entire field, multiple spatial patterns are used to excite the same physical area, which are then digitally processed (e.g., aligned relative to other images) to reconstruct the final image. See York, Andrew G., et al. “Instant super-resolution imaging in live cells and embryos via analog image processing.” Nature methods 10.11 (2013): 1122- 1126 which is incorporated herein by reference. In embodiments, detecting includes selective planar illumination microscopy, light sheet microscopy, emission manipulation, pinhole confocal microscopy, aperture correlation confocal microscopy, volumetric reconstruction from slices, deconvolution microscopy, or aberration-corrected multifocus microscopy. In embodiments, detecting includes digital holographic microscopy (see for example Manoharan, V. N. Frontiers of Engineering: Reports on Leading-edge Engineering from the 2009 Symposium, 2010, 5-12, which is incorporated herein by reference). In embodiments, detecting includes confocal microscopy, light sheet microscopy, or multi-photon microscopy.

[0301] In embodiments, the imaging system is configured to perform histochemistry analysis (e.g., imaging a stained cell, wherein the stain is hematoxylin and eosin stain (or haematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain)). H&E is the combination of two histological stains: hematoxylin and eosin. The hematoxylin typically stains cell nuclei blue, and eosin typically stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors. In embodiments, the device is configured to image a cell (e.g., one or more cells within each reaction chamber of the microplate). Alternative histological stains are known in the art, for example 4', 6-diamidino-2-phenylindole (DAPI), acid fast, alkaline phosphatase, Bielschowsky Stain, Congo Red, Gram Stain, Grocott-Gomori's (or Gbmbri) Methenamine Silver, Hoechst stain, Luxol Fast Blue, Methylene Blue, Oil Red O, Periodic-acid Schiff, Perl’s Prussian Blue, Sudan Black B, Toluidine Blue, Trichrome, Verhoeff-van Gieson Stain, or Warthin-Starry.

[0302] In embodiments, the imaging system may also include other components, including a collection of lenses (such as a collimating lens, a beam shaping lens (e.g., Powell lens), and a cylindrical lens), mirrors (e.g., a dichromatic mirror), beam splitter, one or more pinhole apertures, excitation filter, or combinations thereof. For example, the direction, size, and/or polarization of the light source may be adjusted by using lenses, mirrors, and/or polarizers. In embodiments, one or more of the components of the system may be adjusted or manipulated automatically. Automatic control devices may include a motorized translation stage, an actuation device, one or more piezo stages, and/or one or more automatic switch and flip mirrors and lenses. In embodiments, the system includes one or more optical components (e.g., a beam shaping lens) configured to shape the light emitted from the one or more light sources into desired patterns. For example, in some embodiments, the optical components may shape the light into line patterns (e.g., by using one or more Powell lenses, or other beam shaping lenses, diffractive, or scattering components). In embodiments, the optical component includes a line generator. A “line generator” as used herein refers to an optical component that is configured to generate a diffraction-limited or near diffraction-limited excitation beam in the plane perpendicular to the optical axis of propagation with a substantially uniform intensity distribution along the horizontal axis of the line. Exemplary line generators include, but are not limited to, a one dimensional diffuser having angular uniformity, cylindrical micro-lens array, diffractive element or aspheric refractive lens such as a Powell lens. In embodiments, the optical components include a Powell lens, a microlens, or micro-lens array. In embodiments, the optical component includes a micro-lens fabricated on glass, metal, or plastic. In embodiments, the excitation beams may be directed through a beam shaping lens or lenses. In some embodiments, a single beam shaping lens may be used to shape the excitation beams output from a plurality light sources (e.g., 2 light sources). In some embodiments, a separate beam shaping lens may be used for each light beam. In embodiments, the beam shaping lens is a Powell lens, alternatively referred to as a Powell prism. The shape of the beam may be shaped into an appropriate geometry according to known techniques, e.g., a line, conical, super-Gaussian, ring, doughnut, Bessel-Gauss, Hermite-Gaussian, Laguerre-Gaussian, Hypergeometric-Gaussian, Ince-Gaussian, and the like. In embodiments, the beam is uniform within acceptable limits (e.g., less than 30% intensity variation across the beam). In embodiments, the beam is profiled or includes a gradient.

[0303] In embodiments, the image system includes an image sensor. In embodiments, the image sensor is a CMOS array. A CMOS array, alternatively referred to as a CMOS camera, typically use an active-pixel sensor (APS) that is an image sensor comprising of an integrated circuit containing an array of pixels, where each pixel includes a photodetector and an active amplifier. In embodiments, the image sensor includes a PIN photodiode, a CCD array, a CMOS array, a line scanner, a photodiode, a phototransistor, a photomultiplier, or an avalanche photodiode. In embodiments, the image sensor is a CCD array. In embodiments, the image sensor includes a confocal time delay and integration (TDI) line scan imaging system that has high S/N ratio and high confocality for producing high resolution images of a sample. In embodiments, the image system is described in US 11,415,515, which is incorporated herein by reference. The image sensor may be or include a complementary metal-oxide-semiconductor (CMOS) array, a charge-coupled device (CCD) array, an array of photodiodes, an array of avalanche photodiodes, an array of photomultiplier tubes (PMTs), or an array of optical fibers. In embodiments, the image sensor is at least one of a complementary metal-oxide-semiconductor (CMOS) array and a charge-coupled device (CCD) array. In an embodiment, the image sensor is a camera. In an embodiment, the image sensor is a plurality of cameras. In an embodiment, the image sensor includes four cameras. In an embodiment, the image sensor is two cameras. In an embodiment, the image sensor is a single camera. In embodiments, the image sensor is an array of optical fibers. Each camera is configured to move independently from each other to increase or maximize the coincidence of the image plane to minimize second order aberrations as the sample moves in the scan dimension. In embodiments, the cameras include an objective lens having high numerical aperture (NA) values. For example, the NA may be at least about 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or higher. Those skilled in the art will appreciate that NA, being dependent upon the index of refraction of the medium in which the lens is working, may be higher including, for example, up to 1.0 for air, 1.33 for pure water, or higher for other media such as oils. However, other embodiments may have lower NA values than the examples listed above. In embodiments, the objective lens includes a numerical aperture less than 1.0. In embodiments, the objective lens includes a numerical aperture of 0.1 to 1.65. In embodiments, the objective lens includes a numerical aperture of 0.1 to 0.95. In embodiments, the objective lens includes a numerical aperture of 1.0 to 1.65. In embodiments, the objective lens includes a numerical aperture of at least 0.2, 0.3, 0.4, or 0.5. In embodiments, the objective lens includes a numerical aperture is no greater than 0.8, 0.7, 0.6 or 0.5. In embodiments, the objective lens includes a numerical aperture is no greater than 1.4, 1.3, 1.2, 1.1, or 1.0. Image data obtained by the optical assembly may have a resolution that is between 0.1 and 50 microns or, more particularly, between 0.1 and 10 microns. In embodiments, the numerical aperture for the camera is at least 0.2. In embodiments, the numerical aperture for the camera is no greater than 0.8. In embodiments, the numerical aperture for the camera is no greater than 0.5. Image systems described herein may have a resolution that is sufficient to individually resolve the features or sites that are separated by a distance of less than 10 pm, 5 pm, 2 pm, 1.5 pm, 1.0 pm, 0.8 pm, 0.5 pm, or less. In embodiments, the image systems described herein may have a resolution that is sufficient to individually resolve the features or sites that are separated by a distance of 100 pm at most. In embodiments, the imaging system may generate image data, for example, at a resolution between 0.1 and 50 microns, which is then forwarded to a control/processing system within the bioanalytical instrument. The control/processing system may perform various operations, such as analog-to-digital conversion, scaling, filtering, and association of the data in multiple frames to appropriately and accurately image multiple sites at specific locations on a sample. The control/processing system may store the image data and may ultimately forward the image data to a post-processing system where the data is further analyzed. For example, further analysis may include determining nucleotide sequence information from the image data. In embodiments, the control/processing system may include hardware, firmware, and software designed to control operation of the bioanalytical instrument. The image data may be analyzed by the bioanalytical instrument itself, or may be stored for analysis by other systems and at different times subsequent to imaging. The data and files generated from the methods and analyses described herein may be a typical format, such as FASTQ files, FASTA files, binary alignment files (bam), .bcl, .vcf, and/or .csv files. The output files may be in file formats that are compatible with available sequence data viewing, modification, annotation, and/or additional manipulation software.

[0304] In embodiments, the device as described herein detects scattered light from the sample. In embodiments, the device as described herein detects diffracted light from the sample. In embodiments, the device as described herein detects reflected light from the sample. In embodiments, the device as described herein detects absorbed light from the sample. In embodiments, the device as described herein detects refracted light from the sample. In embodiments, the device as described herein detects transmitted light not absorbed by the sample. In embodiments, the device is configured to determine the cell morphology (e.g., the cell boundary, granularity, or cell shape). For example, to determining the cell boundary includes comparing the pixel values of an image to a single intensity threshold, which may be determined quickly using histogram -based approaches as described in Carpenter, A. et al Genome Biology 7, R100 (2006) and Arce, S., Sci Rep 3, 2266 (2013)).

[0305] The device described herein leverages nucleic acid sequencing to enable multiomics analysis of single cells and tissues as both a universal detection method and in situ sequencing. Advantageously, the device is designed to provide high throughput analysis of nucleic acids and proteins, while also generating high resolution images of cellular morphology to enable computer-vision based analysis of cellular phenotype. This design reflects an appreciation of the tremendous potential for machine learning based image analysis to serve as a rich source of biomarker information for cancer and autoimmune disease translational research. The device described herein eliminates the need for customers to employ multiple systems over several day workflows, which is required by existing commercial methods. This enables researchers to perform large scale experiments that may fundamentally advance our understanding of biology, and, in turn, advance human health.

[0306] In recent years, systems have been developed for targeted gene sequencing in single cells, and for measuring levels of gene transcription in individual cells by sequencing readout. These tools have yielded new information that is not available from bulk sequencing measurements. However, current commercial methods have significant limitations. One limitation is that cells are broke open and tagged with DNA barcodes in droplets or emulsions, then pooled together into a sequencing run, thus losing information about cell morphology. Another limitation is the limited number of cells and samples that can be processed in an experiment. Finally, current methods struggle to achieve multiomics readout, with only limited ability to measure proteins along with DNA or RNA while maintaining cellular morphology.

[0307] For spatial analysis of tissue, the capabilities of current genomic technologies are even more limited. Most genomic analysis of tissue is done on a bulk basis, with no spatial resolution. Recently, several spatial analysis platforms have been developed and introduced commercially. However, these technologies currently have several limitations. First, most of these platforms currently have limited resolution and are unable to provide detailed information at the level of individual single cells. This limits information about subcellular localization and how the cells are organized in space within the tissue. Second, current commercial platforms are unable to provide high throughput. Experiments are limited to less than 20 samples per run, and in some cases just one sample per run, which limits the ability of users to conduct large scale experiments.

[0308] The device described herein is designed for multiomics detection. The device may identify specific RNA and proteins (e.g., through the use of oligo-conjugated antibodies) using nucleic acid sequencing either as a universal detection method or for in situ sequencing along with cellular morphology and tissue organization. This provides significantly more information than is available today with current commercial single cell technologies. The addition of the cellular morphology along with spatial organization of biomolecules within the tissue microenvironment provides a data rich solution across many research applications to better understand cell development, maturation, and pathogenesis. The combination of these useful datasets from individual cells provides a more complete cellular picture as it will combine both phenotypic data along with detailed molecular characterization.

[0309] The device described herein is designed for high throughput and large scale. The device is designed to be high throughput in order to enable researchers to perform large scale studies that are currently inaccessible but are needed for a more complete characterization and understanding of cells, and therefore biology. Current commercially available single cell technologies detect 10,000 to 100,000 cells in an experiment. The device described herein uses a microplate (e.g., multiwell-plate) approach (e.g., 4, 6, 12, 24, 48, 96, 384 or 1536 sample wells) designed to process 10,000 to 100,000 cells per well. For example, using a 96- multiwell-plate, the device is capable of a throughput of 1 million to 10 million cells. Current commercially available spatial analysis instruments can run an experiment involving only 4 to 20 tissue samples. With the devices described herein, it is possible to run up to 96 tissue samples per run. For tissue samples, the device is designed to retain the context of the cells within their cellular environment and to enable spatial analysis by returning both phenotypic data (cellular morphology, localization of different cell types and expression of different proteins within the context of the tissue) and molecular data at subcellular resolution and high throughput. The device can utilize fresh, fresh frozen, and FFPE samples.

[0310] The device includes a microplate (e.g., a multiwell plate), wherein within each well is one or more polynucleotides. The device is capable of performing a plurality of SBS sequencing cycles. Briefly, an SBS sequencing cycle includes extending a primer strand with modified nucleotides (e.g., labeled, terminated nucleotides). This is followed by a wash and an optional addition of unlabeled, terminated nucleotides. The microplate is imaged, thereby detecting the incorporated modified nucleotide. A cleaving agent is introduced into each well, cleaving the terminated nucleotide, followed by a wash. The number of sequencing cycles depends on the application. For example, for reading out a barcode the number of sequencing cycles is about 5-10 cycles. In embodiments, for variable region sequencing, the number of sequencing cycles includes about 30-80 cycles.

[0311] In embodiments, the device is configured to dispense one or more wash reagents into only a fraction of the entire microplate at a given time. In embodiments, the device is configured to dispense one or more wash reagents into 2-24 wells of a 96 well microplate a given time. In embodiments, the device is configured to dispense one or more wash reagents into 2-12 wells of a 48 well microplate a given time. In embodiments, the device is configured to dispense one or more wash reagents into only 25% of the entire microplate. In embodiments, the device is configured to dispense one or more wash reagents into only 50% of the entire microplate. In embodiments, the device is configured to dispense one or more wash reagents into only 75% of the entire microplate. In embodiments, the device is configured to dispense wash reagents in parallel to all or a fraction of the reaction chambers of the microplate.

[0312] In embodiments, the device is configured to sequentially dispense and/or aspirate reagents into 25% of the entire plate (e.g., 24 wells of a 96 well microplate), followed by dispensing and/or aspirating reagents into another 25% of the entire plate (e.g., a different set of 24 wells of a 96 well microplate). In embodiments, the device is configured to sequentially dispense and/or aspirate reagents into another 25% of the entire plate (e.g., a different set from the first set of 24 wells and the second set of 24 wells of a 96 well microplate), followed by dispensing and/or aspirating reagents into a different 25% of the entire plate (e.g., a different set of 24 wells of a 96 well microplate).

[0313] In an additional example, patterns of gene expression are determined by analyzing a series of tissue sections, in a manner analogous to image reconstruction in CT scanning (e.g., reconstructing three dimensional sections). Such a method can be used to measure changes in gene expression in disease pathology, e.g., in cancerous tissue and/or a tissue upon injury, inflammation, or infection. With the devices and methods as described herein, more detailed information on gene expression and protein localization in complex tissues is obtained, leading to new insights into the function and regulation both in normal and diseased states, and provides new hypotheses that can be tested.

Example 3. Assembling a Microplate Assembly

[0314] Preparing a surface for tissue section mounting is a critical step in minimizing loss of tissue material during subsequent processing. For example, repeated exposure to immunohistochemistry reagents and solvents used during analysis may lead to loss of cellular or tissue section material if the tissue section is weakly bound to the surface. The conditions involved in in situ sequencing processes also involve elevated pH and incubation temperatures, in addition to the addition and removal of various fluids repeatedly. Lack of strong binding of the tissue section to the surface may therefore lead to detachment of the tissue section during in situ sequencing. Some common methods of preparing a surface for tissue section mounting, for example, a glass slide, include plasma treatment and functionalization with charged moi eties. To determine the optimal surface functionalization conditions, we performed a comparison of several surface functionalization reagents, including (3 -aminopropyl )triethoxysilane (APTES), (5,6-epoxyhexyl)triethoxysilane (EHTES), polyethyleneimine (PEI), or a combination thereof. Tissue sections were then transferred to the functionalized glass surfaces, and tissue integrity cycles in the presence of various buffers performed to assess for any tissue section detachment from the treated glass slides. [0315] Glass functionalization: Glass slides were washed three times in an acetone/ethanol bath while being sonicated. The glass slides were then oxygen plasma-treated (100 mTorr for vacuum, 1 Torr oxygen injection for 3 mins, plasma treatment at high power for 6 mins). Glass slides were then submerged in EtOH with either 1% APTES or 1% EHTES and incubated overnight. Following the overnight treatment, the slides were washed three times with EtOH and dried with an air gun. For PEI functionalization, the slides were incubated with 50 ug/mL in deionized water for 30 min followed by three washes with deionized water.

[0316] Plate assembly and rehydration: The glass slides were affixed to bottomless microplate inserts using a Kapton double-sided adhesive cut using a Silhouette Curio cutter. One side of the pressure sensitive adhesive tape (e.g., Kapton® tape) was removed and the adhesive portion was placed onto the microplate section. Firm pressure was applied. The other side of the pressure sensitive tape was peeled off and the the planar solid support (e.g., glass slide) was placed onto the adhesive. IOOUL of PEI was added to each well on the assembled plate and it was incubated for at least 5 minutes at room temperature. The PEI was removed and each well was washed out 3X with DI water. The plate was then covered with plastic cover or parafilm and stored at 4°C for further use.

[0317] Mouse intestine FFPE tissue sections were prepared and transferred to the bottom of different wells. 24 portions were cut and transferred to each glass slide. Following tissue section transfer, the slides were baked at 50°C for 15 mins. The slides were then baked at 60°C for 30 mins and placed in dark storage at room temperature overnight. The tissue sections were then deparaffinized using xylene followed by 100% EtOH incubation. The slides were dried at 37°C for 15 mins. Tissue sections were then fixed with 4% PFA in PBS for 30 min. Samples were then H&E stained using methods known in the art and imaged using a color camera.

NUMBERED EMBODIMENTS

[0318] Embodiment Pl . A microplate assembly, comprising: a microplate receiver frame defining a pocket; at least one microplate section; a planar support positioned on a bottom of the at least one microplate section, and wherein the at least one microplate section and the planar support have a similar coefficient of linear thermal expansion; wherein the pocket of the microplate receiver frame is sized and shaped to receive a plurality of microplate sections. [0319] Embodiment P2. The microplate assembly of Embodiment Pl, wherein a bottom region of the microplate section is glass.

[0320] Embodiment P3. The microplate assembly of Embodiment Pl, wherein the pocket of the frame is sized and shaped to receive the plurality of the microplate sections arranged in a side-by-side configuration.

[0321] Embodiment P4. The microplate assembly of Embodiment Pl, wherein the microplate section includes a first retention element and the microplate receiver includes a second retention element, and wherein the first retention element mechanically interacts with the second retention element to retain the microplate section within the pocket.

[0322] Embodiment P5. The microplate assembly of Embodiment P4, wherein the first retention element is biased toward the second retention element when the microplate section is positioned in the pocket.

[0323] Embodiment P6. The microplate assembly of Embodiment P4, wherein first retention element is a prong and the second retention element is a slot.

[0324] Embodiment P7. The microplate assembly of Embodiment Pl, wherein the microplate assembly includes at least one well.

[0325] Embodiment P8. The microplate assembly of Embodiment Pl, wherein the microplate assembly does not include any wells.

[0326] Embodiment P9. The microplate assembly of Embodiment Pl, wherein the planar support is glass.

[0327] Embodiment P10. The microplate assembly of Embodiment Pl, wherein the microplate assembly includes 6, 8, 10, 12, 16, 18, 24, 30, 36, 40, 42, 46, 48, 96, 144, or 192 wells.

[0328] Embodiment P 11. The microplate assembly of Embodiment P 1 , wherein the planar support is attached via an adhesive to the microplate section.

[0329] Embodiment P12. The microplate assembly of Embodiment pl 1, wherein the adhesive is a black adhesive.

[0330] Embodiment P13. A method of imaging a cell, said method comprising: obtaining a sample from a subject, wherein said sample comprises a cell; contacting said microplate assembly of any one of Embodiments Pl to Pl 1 with the sample; and obtaining an image of the sample, thereby imaging the cell.

[0331] Embodiment 1. A microplate assembly, comprising: a microplate receiver frame defining a pocket; at least one microplate section; a planar support positioned on a bottom of the at least one microplate section, and wherein the at least one microplate section comprises a maximum coefficient of linear thermal expansion of about 2.0 xlO' ⁴ /K to about 1.0 X10' ⁵ /K; wherein the pocket of the microplate receiver frame is sized and shaped to receive a one or more microplate sections.

[0332] Embodiment 2. The microplate assembly of Embodiment 1, wherein the microplate section comprises a coefficient of linear thermal expansion of about 1.0 xl0' ⁵ /K, about 1.5 X10' ⁵ /K, about 2.0 xl0' ⁵ /K, about 2.5 xl0' ⁵ /K, about 3.0 xl0' ⁵ /K, about 3.5 xl0' ⁵ /K, about 4.0 X10' ⁵ /K, about 4.5 xl0' ⁵ /K, about 5.0 xl0' ⁵ /K, about 5.5 xl0' ⁵ /K, about 6.0 xl0' ⁵ /K, about 6.5 X10' ⁵ /K, about 7.0 xl0' ⁵ /K, about 7.5 xl0' ⁵ /K, about 8.0 xl0' ⁵ /K, about 8.5 xl0' ⁵ /K, about 9.0 X10' ⁵ /K, about 9.5 xl0' ⁵ /K.

[0333] Embodiment 3. The microplate assembly of Embodiment 1, wherein the microplate section comprises a coefficient of linear thermal expansion of about 1.0 xl0' ⁵ /K to about 3.0x10' ⁵ /K.

[0334] Embodiment 4. The microplate assembly of Embodiment 1, wherein the microplate section comprises a coefficient of linear thermal expansion of about 1.8 xl0' ⁵ /K, 1.9 X10' ⁵ /K, 2.0 X10' ⁵ /K, 2.1 xl0' ⁵ /K, or about 2.2 xl0' ⁵ /K.

[0335] Embodiment 5. The microplate assembly of any one of Embodiments 1 to 4, wherein the planar support comprises a coefficient of linear thermal expansion less than the coefficient of linear thermal expansion of said microplate section.

[0336] Embodiment 6. The microplate assembly of any one of Embodiments 1 to 4, wherein the planar support comprises a coefficient of linear thermal expansion of about 0.5X10' ⁵ /K to about 8.0xl0' ⁷ /K.

[0337] Embodiment 7. The microplate assembly of any one of Embodiments 1 to 6, wherein a bottom region of the microplate section is glass. [0338] Embodiment 8. The microplate assembly of Embodiment 7, wherein said glass is functionalized glass, comprising one or more topographical modifications, bioconjugate reactive moieties, or biomolecules.

[0339] Embodiment 9. The microplate assembly of any one of Embodiments 1 to 8, wherein the microplate section comprises a gasket between said microplate section and said planar support.

[0340] Embodiment 10. The microplate assembly of any one of Embodiments 1 to 9, wherein the pocket of the frame is sized and shaped to receive a plurality of the microplate sections arranged in a side-by-side configuration.

[0341] Embodiment 11. The microplate assembly of any one of Embodiments 1 to 10, wherein the microplate section includes a first retention element and the microplate receiver includes a second retention element, and wherein the first retention element mechanically interacts with the second retention element to retain the microplate section within the pocket.

[0342] Embodiment 12. The microplate assembly of Embodiment 11, wherein the first retention element is biased toward the second retention element when the microplate section is positioned in the pocket.

[0343] Embodiment 13. The microplate assembly of Embodiment 11, wherein first retention element is a prong and the second retention element is a slot.

[0344] Embodiment 14. The microplate assembly of any one of Embodiments 1 to 13, wherein the microplate section comprises at least one well.

[0345] Embodiment 15. The microplate assembly of any one of Embodiments 1 to 13, wherein the microplate section comprises a plurality of wells.

[0346] Embodiment 16. The microplate assembly of Embodiment 15, wherein the microplate section comprises 6, 8, 10, 12, 16, 18, 24, 30, 36, 40, 42, 46, 48, 96, 144, or 192 wells.

[0347] Embodiment 17. The microplate assembly of any one of Embodiments 1 to 16, comprising 1, 2, 3, or 4 microplate sections.

[0348] Embodiment 18. The microplate assembly of any one of Embodiments 1 to 16, comprising 2 microplate sections. [0349] Embodiment 19. The microplate assembly of Embodiment 18, wherein each microplate section comprises 6, 8, 10, 12, 16, 18, 24, 30, 36, 40, 42, 46, 48, 96, 144, or 192 well.

[0350] Embodiment 20. The microplate assembly of any one of Embodiments 1 to 16, comprising 4 microplate sections.

[0351] Embodiment 21. The microplate assembly of Embodiment 20, wherein each microplate section comprises 6, 8, 10, 12, 16, 18, 24, 30, 36, 40, 42, 46, 48, 96, 144, or 192 wells.

[0352] Embodiment 22. The microplate assembly of any one of Embodiments 1 to 16, wherein the microplate section does not include any wells.

[0353] Embodiment 23. The microplate assembly of any one of Embodiments 1 to 22, wherein the planar support is glass.

[0354] Embodiment 24. The microplate assembly of Embodiment 23, wherein said planar support is bonded to said microplate section.

[0355] Embodiment 25. The microplate assembly of Embodiment 23, wherein said planar support is attached via an adhesive to said microplate section.

[0356] Embodiment 26. The microplate assembly of Embodiment 25, wherein said adhesive is a black adhesive.

[0357] Embodiment 27. The microplate assembly of Embodiment 23, wherein said glass is functionalized glass, comprising one or more topographical modifications, bioconjugate reactive moieties, or biomolecules.

[0358] Embodiment 28. The microplate assembly of Embodiment 23, wherein the microplate section comprises a gasket between said microplate section and said planar support.

[0359] Embodiment 29. A microplate section, comprising a planar support attached to a solid support comprising a plurality of wells; wherein solid support comprises a coefficient of linear thermal expansion of about 2.0xl0' ⁴ /K to about 1.0xl0' ⁵ /K; and and the planar support comprises a coefficient of linear thermal expansion of about 0.5xl0' ⁵ /K to about 8.0xl0' ⁷ /K. [0360] Embodiment 30. The microplate section of Embodiment 29, wherein the microplate section comprises a gasket between said solid support and said planar support.

[0361] Embodiment 31. The microplate section of Embodiments 29 or 30, comprising one or more bores, slots, seats, retention mechanisms, clips, or other structures sized and shaped to receive, align, and secure a microplate section within a microplate receiver.

[0362] Embodiment 32. The microplate section of any one of Embodiments 29 to 31, wherein the planar support comprises a functionalized glass surface or a functionalized plastic surface.

[0363] Embodiment 33. The microplate section of any one of Embodiments 29 to 32, wherein the planar support is borosilicate glass.

[0364] Embodiment 34. The microplate section of any one of Embodiments 29 to 33, wherein the planar support comprises a cell-permissive coating capable of adhering live cells.

[0365] Embodiment 35. The microplate section of Embodiment 34, wherein the functionalized glass surface includes (3 -aminopropyl )triethoxysilane (APTES), (3- Aminopropyl)trimethoxysilane (APTMS), y-Aminopropylsilatrane (APS), N-(6- aminohexyl)aminomethyltriethoxysilane (AHAMTES), polyethylenimine (PEI), 5,6- epoxyhexyltriethoxysilane, or triethoxysilylbutyraldehyde, or a combination thereof.

[0366] Embodiment 36. The microplate section of any one of Embodiments 29 to 35, wherein the planar support comprises a nanoimprint resist.

[0367] Embodiment 37. The microplate section of any one of Embodiments 29 to 35, wherein the planar support comprises a polymer layer, wherein said polymer layer comprises polymerized units of alkoxysilyl methacrylate, alkoxysilyl acrylate, alkoxysilyl methylacrylamide, alkoxysilyl methylacrylamide, or a copolymer thereof.

[0368] Embodiment 38. The microplate section of any one of Embodiments 29 to 35, wherein the planar support comprises a hydrogel.

[0369] Embodiment 39. The microplate section of Embodiment 38, wherein said hydrogel comprises polymerized monomers of acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, polyethylene glycol) and derivatives thereof (e.g., PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, or combinations thereof.

[0370] Embodiment 40. The microplate section of any one of Embodiments 29 to 39, wherein the solid support comprises a thermoplastic.

[0371] Embodiment 41. The microplate section of any one of Embodiments 29 to 39, wherein the thermoplastic comprises polyetherimide (PEI).

[0372] Embodiment 42. The microplate section of any one of Embodiments 29 to 39, wherein the solid support comprises a liquid crystal polymer.

[0373] Embodiment 43. The microplate section of any one of Embodiments 29 to 42, wherein the planar support is attached via an adhesive to the solid support.

[0374] Embodiment 44. The microplate section of Embodiment 43, wherein said adhesive is a pressure sensitive adhesive.

[0375] Embodiment 45. A method of imaging a cell, said method comprising: obtaining a sample from a subject, wherein said sample comprises a cell; contacting said microplate assembly of any one of Embodiments 1 to 28 or a microplate section of any one of Embodiments 29 to 44 with the sample; and obtaining an image of the sample, thereby imaging the cell.

[0376] Embodiment 46. A method of detecting a biomolecule in a tissue section, the method comprising: immobilizing the tissue section onto the microplate section of any one of Embodiments 29 to 44, optionally permeabilizing the immobilized tissue section; and contacting the biomolecule in the tissue section with a detection agent thereby detecting the biomolecule in the tissue section.

[0377] Embodiment 47. The method of Embodiment 41, wherein detecting comprises expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy, confocal microscopy, visual identification, and combinations thereof. [0378] Embodiment 48. A cell comprising a polymerase complex, wherein the polymerase complex comprises a polymerase bound to a double stranded nucleic acid molecule, wherein one strand of said double-stranded nucleic acid molecule comprises a modified nucleotide, wherein said cell is within the well of any one of Embodiments 14 to 28.

[0379] Embodiment 49. The cell of Embodiment 48, wherein said modified nucleotide comprises a label.

[0380] Embodiment 50. The cell of Embodiment 48 or 49, wherein said modified nucleotide comprises a reversible terminator.

[0381] Embodiment 51. A plurality of cells, wherein one or more cells comprise a polymerase complex, wherein the polymerase complex comprises a polymerase bound to a double stranded nucleic acid molecule, wherein one strand of said double-stranded nucleic acid molecule comprises a modified nucleotide, wherein said one or more cells are attached within one or more wells of the microplate section of any one of Embodiments 29 to 44.