EP4599224A1 - Rapid gelling aqueous mounting media - Google Patents

Abstract

Aqueous formulations that rapidly gel, their use for mounting biological specimens (e.g., cells and tissues) to a substrate, and methods and kits for visualizing biological specimens embedded in such mounting formulations are described.

Description

RAPID GELLING AQUEOUS MOUNTING MEDIA

RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional patent application number 63/477,312, filed December 27, 2022, and U.S. provisional patent application number 63/495,945, filed April 13, 2023, which are hereby incorporated herein by reference in their entirety for all purposes.

TECHNICAL FIELD

[0002] Rapid-gelling, aqueous formulations, their use for mounting biological specimens (e.g., cells and tissues) to a substrate, and methods of visualizing biological specimens embedded in such mounting formulations are described.

BACKGROUND

[0003] Tire ability to observe targets within cultured cell and animal tissue specimens is an important tool applied to understand biological process. Biological specimens often are mounted on an optically clear substrate, such as a glass microscope slide, for examination by light microscopy. Typically, the mounting process involves suspending a biological specimen (e.g., a fixed cell or tissue) in amounting solution, also referred to as a “mounting medium” or “mountant,” and then depositing the specimen onto the surface of the substrate, thereby embedding the specimen in the mounting solution. The mounting solution and embedded specimen can be dried prior to interrogation, typically under a cover slide. The mounting medium protects the specimen from physical damage and allows for extended storage of the specimen.

[0004] The selection of a mounting medium is dictated by the type of sample and substrate. The mounting medium can be liquid or can harden into a permanent mountant. In addition, the mounting media ideally does not react with the specimen and does not crystallize or darken over time. If the specimen is stained with a dye, the mountant also should not cause the dye to fade or bleach. Another factor in the selection of an appropriate mountant is its optical clarity. Optical clarity is influenced not just by how clear a mountant is, but by how well the refractive index (RI) of the mountant, sample, and glass or other substrate is matched. For example, cells and tissue typically have a RI of 1.35-1.42. If the RI of a mountant falls below the RI of the glass used in microscope slides and coverslips (1.50-1.54) and/or does not match the RI of the biological specimens, the mountant does not provide optimal optical clarity. Thus, there is a need for improved formulations for mounting biological specimens that have a RI that more closely matches the RI of glass and/or the biological specimen under interrogation. Although mounting media with high refractive indices (RI) that cure quickly are available, these have several drawbacks when used with biological samples. For example, commonly available materials that quickly form solid resins (e.g.. epoxy and photoactivatable) utilize organic solvents, which are not compatible with many types of biological samples. Such organic solvent-based formulations also tend to be highly viscous and become opaque upon curing. Further, fast curing mounting formulations often involve non- reversible free radical polymerization that can damage fluorophores. For biological specimens, it can be preferable to utilize an aqueous mounting medium that can maintain the sample in hydrated form and docs not suffer from the disadvantages associated with organic-based mounting formulations. Tirus, there is a need for aqueous formulations that rapidly form a solid mountant at room temperature, allow imaging quickly after sample mounting, and can maintain a biological sample in a hydrated (“native”) form for extended periods of time.

SUMMARY

[0005] Disclosed herein are formulations and methods of preparing such fonnulations. methods for embedding biological samples and for retaining such samples on a solid substrate, as well as kits for implementing such methods.

[0006] In one aspect, a method of embedding a biological specimen in a crosslinked hydrogel is provided, including: combining a biological specimen and an aqueous solution, the aqueous solution including: i. a copolymer including a plurality of hydrophilic monomer residues and at least 3 monomer residues that each include a phenyl boronic acid substituent; and ii. a water-soluble polyol, wherein the pH of the aqueous solution is 6 or greater, wherein a crosslinked hydrogel forms at room temperature within 2 hours or less after combining the aqueous solution with the biological specimen, wherein the biological specimen is embedded in the crosslinked hydrogel in a hydrated form, and the hydrogel has a refractive index of 1 .40 or greater. [0007] In another aspect, a method of mounting a biological specimen on a substrate is provided, including: a) depositing a biological specimen in hydrated form on the substrate; b) contacting tire biological specimen with a first aqueous solution at room temperature, wherein the pH of the first aqueous solution is 6 or greater, including a copolymer, wherein the copolymer includes a plurality of hydrophilic monomer residues and at least 3 monomer residues that each include a phenyl boronic acid substituent, to provide a composition including the copolymer and a biological specimen; and c) contacting the composition with a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater and includes a water-soluble polyol, to provide a crosslinked hydrogel including the biological specimen in the hydrated form mounted to the substrate, wherein within 2 hours or less of contacting the composition with the second aqueous solution at room temperature the crosslinked hydrogel has a refractive index of 1.40 or greater.

[0008] In yet another aspect, a method of mounting a biological specimen on a substrate is provided, including: a) depositing a biological specimen in hydrated form on the substrate at room temperature; b) contacting the biological specimen with a first aqueous solution, wherein the pH of the second aqueous solution is 6 or greater and includes a water-soluble polyol, to provide a first composition; and c) contacting the first composition with a second aqueous solution, wherein the pH of tire second aqueous solution is 6 or greater, including a copolymer, wherein the copolymer includes a plurality of hydrophilic monomer residues and at least 3 monomer residues that each include a phenyl boronic acid substituent, to provide a crosslinked hydrogel including the biological specimen in the hydrated form mounted to the substrate, wherein within 2 hours or less of contacting the first composition with the second aqueous solution at room temperature the crosslinked hydrogel has a refractive index of 1.40 or greater.

[0009] In yet another aspect, a method of mounting a biological specimen on a substrate in a crosslinked hydrogel is provided, including: a) contacting a first substrate with a first aqueous solution including a copolymer, wherein the copolymer includes a plurality of hydrophilic monomer residues and at least 3 monomer residues that each include a phenyl boronic acid substituent, wherein the pH of the first aqueous solution is about 6 or greater; b) combining the first aqueous solution with a second aqueous solution, wherein the second aqueous solution includes a water-soluble polyol, wherein the pH of the second aqueous solution is about 6 or greater, to provide a first composition disposed on the first substrate; c) providing a biological sample in a hydrated fonn disposed on a second substrate; and d) contacting the first composition disposed on the first substrate and the biological sample disposed on the second substrate, such as to generate a crosslinked hydrogel including the biological specimen in the hydrated form disposed between the first substrate and the second substrate within 2 hours or less, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater.

[0010J In yet another aspect, a method of mounting a biological specimen on a substrate is provided, including: depositing an aqueous solution onto the substrate at room temperature, wherein the pH of the aqueous solution is about 6 or greater, wherein the aqueous solution includes: i. a copolymer including a plurality of hydrophilic monomer residues and at least 3 monomer residues that each include a phenyl boronic acid substituent; ii. a water- soluble polyol; and iii. a biological specimen, to fonn a crosslinked hydrogel mounted on the substrate within 2 hours or less that includes the biological specimen in a hydrated form and has a refractive index of 1.40 or greater. In any of the methods described herein, the crosslinked hydrogel can form at room temperature in about 2 hours or less; or in about 1 hour or less; or in about 30 minutes or less; or in about 10 minutes or less; or in about 1 minute or less; and/or further include visualizing the biological specimen on the substrate with a microscope.

[0011] In yet another aspect, a kit for mounting a biological specimen in a crosslinked hydrogel on a substrate is provided, including: a) a first aqueous solution, wherein the pH of the first aqueous solution is about 6 or greater, including a copolymer including a plurality of hydrophilic monomer residues and at least 3 monomer residues that each include a phenyl boronic acid substituent; b) a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater, including a water-soluble polyol; and c) instructions for combining the first and second aqueous solutions with a biological specimen in hydrated form to form a crosslinked hydrogel on the substrate at room temperature within 2 hours or less, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater and includes the biological specimen in the hydrated form.

[0012] In yet another aspect, a biological specimen embedded in hydrated fonn in a crosslinked hydrogel is provided, wherein the hydrogel includes a hydrophilic copolymer that is crosslinked to a water-soluble polyol through at least 3 boronate ester linkages, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater. In certain embodiments, the embedded biological specimen is disposed on a substrate. Tire copolymer can be or include a synthetic polymer. For example, the copolymer can include a plurality of hydrophilic monomer residues that can include an N-/? acrylamide or N-/i methacrylamide monomer residue, wherein R is methyl, ethyl, propyl, isopropyl or H (e.g., N,N-dimethylacrylamide, N,N-dimethylmethacrylamide, N,N- diethylacrylamide,: N-2 -hydroxypropyl methacrylamide, dimethlylaminopropyl methacrylamide, or N-3- aminoproyl methacrylamide): or a monomer residue selected from the group consisting of 4- vinylbenzenesulfonate, acrylic acid, methacrylic acid, acrylate, and methacrylate monomer residues; or a monomer residue selected from dimethlylaminoethyl methacrylate, aminoethyl methacrylate, and PEGylated acrylates or methacrylates (e.g.. pol(ethylene glycol) acrylate, pol(ethylene glycol) methacrylate, pol(ethylene glycol) methyl ether acrylates). The phenyl boronic acid monomer can be selected from the group consisting of 3- acr lamidophcnyl boronic acid, 2-acrylamidophenyl boronic acid, 3 -methacrylamido phenyl boronic acid, 2- methacrylamido phenyl boronic acid, and 4-vinyl phenyl boronic acid. Tire water-soluble polyol is compound can include two or more 1, 2-cis-diol groups or 1,3-cis-diol groups. The water-soluble polyol can be selected from iohexol, iodixanol, glycerol, thiodiethanol, thiodipropanol, polyvinyl alcohol (PVA), poly(glycerol monomethacrylate), poly(vinyl catechol) (e.g., 3. 4 dihydroxy styrene, 3-vinyl catechol), rosmarinic acid, erythorbic acid, erythorbic acid, fructose, sucrose, galactose, maltose, and a combination thereof. The hydrophilic copolymer and/or the water soluble polyol can have a weight average molecular weight of about 10 kDa to about 100 kDa. Hie weight ratio of the hydrophilic copolymer to the water-soluble polyol in the crosslinked hydrogel can be about 1:9 to about 1:30. Tire substrate can be a microscope slide, cuvette, well or dish. The biological specimen is a cell, tissue, 3D cell culture, a whole organism and can, optionally, be labeled with a fluorescent dye or fluorescent protein.

[0013] In any of the embodiments described herein, the refractive index of the crosslinked hydrogel matches the refractive index of soda-lime glass, borosilicate glass, glycerol, or immersion oil (e.g., RI of about 1.40 to about

1 .54), and/or the crosslinked hydrogel can have a specific storage modulus of 40Pa to 4000Pa (e.g., 40Pa to lOOOPa). Formulations provided herein can further include an anti-oxidant (e.g., 6-hydroxy-2,5,7,8- tetramethylchroman-2 -carboxylic acid, 3 -carboxy proxyl, hydroquinone, mequinol, sodium sulfite, sodium erythorbate, ascorbic acid, propyl gallate, caffeic acid, paraphenylenediamine, l,4-diazabicyclo[2.2.2]octane, or a combination thereof). For example, the crosslinked hydrogel further includes 1% by weight or less of the antioxidant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a schematic illustrating the formation of a crosslinked hydrogel after mixing a boronic acid copolymer (Component A) with a polyol (Component B).

[0015] FIG. 2 is an illustration of the crosslinked hydrogel formed after mixing Component A and Component B, as shown in FIG. 1.

[0016] FIG. 3 is a schematic illustrating a workflow using the components and methods disclosed herein.

[0017] FIG. 4 shows microscope images of Alexa Fluor 488 phalloidin-stained cells mounted to microscope slides in a Control Formulation, Formulation 1. and Formulation 8 after 1 hour and 72 hours from mounting start time.

[0018] FIG. 5 shows microscope images of Alexa Fluor 647 tubulin-stained cells mounted to microscope slides in a Control Fonnulation, Formulation 1, and Fonnulation 8 after 1 hour and 72 hours from mounting start time. [0019] FIG. 6 is a plot showing the increase in storage modulus over time for representative crosslinked hydrogel formulations.

[0020] FIG. 7 is a plot showing the increase in storage modulus over time for five different crosslinked hydrogel formulations.

DETAILED DESCRIPTION

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary’ skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents. applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0022] As used herein, "a" or "an" means "at least one" or "one or more."

[0023] As used herein, the tenn “about”, when used to describe a numerical value, encompasses a range up to ± 15% of that numerical value, unless the context clearly dictates otherwise.

[0024] While compositions and methods are described in terms of "comprising" various components or steps (interpreted as meaning "including, but not limited to"), the compositions and methods can also "consist essentially of’ or "consist of’ the various components and steps, such terminology should be interpreted as defining essentially closed-member groups.

[0025] “Refractive index” or “RI,” as used herein, is a measure of how fast light travels through a particular medium. When light travels between two media with different RI values, such as air and water, the path that it travels is bent, distorting the image. Refractive index also is a measure of how much the speed and the wavelength of radiation are reduced with respect to the wavelength of the light in a vacuum. Because RI is a ratio of two velocities, it is dimensionless.

[0026] "Water-soluble" is used herein to mean the compound can be soluble or dispersible in an aqueous-based solution, such as in water or water-based solutions or buffer solutions, including those used in biological or molecular detection systems as know n by those skilled in the art. A compound is considered water-soluble if it can be dissolved in an aqueous formulation at a concentration of 10 mg/mL or more. Compounds that can be dissolved in an aqueous system at a concentration of 100 mg/mL or greater are considered highly water-soluble. Compounds that can be dissolved in an aqueous system at a concentration of 1 mg/mL or less are considered to have poor water solubility.

[0027] “Biological specimen” or “biological sample.” as used herein encompasses hematological, cytological and histological specimens, such as cells. 3D cell cultures (e.g. spheroids and organoids), tissues, whole organisms (e.g. flies, wonns, zebrafish), cell-free extracts, or a fluid sample (e.g.. blood or sputum). A tissue specimen can be any type of nervous, epithelial, muscular, and connective tissue, including an organ tissue. Biological samples can be from a plant or animal (e.g., human, mouse, fly, worm, fish, frog, fungi, and the like). [0028] “Hydrogel” as used herein refers to a three-dimensional cross-linked polymeric network capable of absorbing a large amount of water, buffer, or biological fluid and maintains its three-dimensional structure without dissolving in water. The polymeric network can be formed from covalently crosslinking a synthetic and/or natural polymer(s).

[0029] In general, aqueous-based formulations are described herein that can rapidly gel to provide a solidified, hydrogel that exhibits a high refractive index (e.g., 1.40 or greater). The solidified hydrogel can be used as a media for retaining (i.e., mounting) a biological specimen onto the surface of a solid substrate. While aqueous mounting media with high RI properties are known in the art. aqueous-based compositions known in the art require prolonged curing times (e.g., 48 hours or greater) and/or elevated temperatures to solidify into a hardened matrix. The instant formulations can solidify (also referred to herein as “gel”, “cure”, or “harden”) more quickly than other types of aqueous mounting media known in the art. Aqueous-based fonnulations disclosed herein can solidify within one hour or less, which significantly decreases the time for processing a sample using existing mounting and imaging workflows.

[0030] Provided herein are formulations for use in mounting a specimen on a substrate are described herein. The described formulations are aqueous-based systems that provide a hardened mounting medium for protection and storage of the specimen. The formulations can be applied directly to cells and tissue samples, which optionally can be fluorescently labeled, on a substrate and can harden overtime. Formulations can harden without a coverslip. thus eliminating the requirement that a cover slip be used to protect the mounted specimen. The mounting formulations disclosed herein hold the specimen in place on the substrate, thereby stabilizing and preserving the biological specimen on the substrate for subsequent interrogation. For example, once mounted, the biological specimen can be imaged under a microscope.

[0031] The disclosed formulations can protect and extend the lifetime of a mounted biological sample, can slow irreversible photodegradation of the sample and fluorescent probes (if present) during imaging, thereby improving signal retention overtime. Hydrogel formulations disclosed herein can facilitate high-resolution imaging of various types of biological samples and are particularly advantageous for high volume 3D fluorescence imaging of cells and tissues.

[0032] The disclosed formulations have favorable optical characteristics that make these formulations ideal for use as a mounting medium for biological samples. In particular, the disclosed formulations exhibit a high refractive index. It is known that the RI of the specimen and mounting medium can significantly impact image quality. For example, the difference between the RI of the specimen and the surrounding mountant can influence how the specimen appears under a microscope. If the difference between the RI of the specimen and the surrounding medium is large, strong refraction of light at the specimen and mounting medium interface can occur. A large difference in RI can cause artifacts as light is refracted that can obscure details of the specimen. In contrast, small differences in RI between the specimen and the mounting medium can reduce light refraction, making many types of specimens appear brighter and/or more transparent under the microscope.

[0033] Provided herein arc formulations that can match the refractive index of a biological sample, as well as substrates, lens, coverslips, and other components commonly used in microscopy. Thus, the formulations described herein have a refractive index (RI) that matches the RI of the glass of the objective lens of the microscope, as well as the glass of the microscope coverslip or other components commonly used in microscopic imaging of samples such as immersion oil. By virtue of having high RI, the disclosed formulations minimize distortion of microscope image resulting from light refraction, making these formulations suitable for use under high magnification and/or with immersion oils, such as are typically used to minimize distortion of the microscope image. Hydrogel compositions disclosed herein have an RI that exceeds the RI of existing aqueousbased hard mount formulations, while reducing photobleaching of the sample and dyes, if present.

[0034] For biological specimens that are labeled with fluorescent dyes, selection of a suitable mountant often is governed by its ability to minimize photobleaching. Mountants disclosed herein can effectively reduce photobleaching of the fluorophores, while minimizing quenching of the initial fluorescence intensity. The disclosed compositions, minimize loss of fluorescence through irreversible photobleaching, which is known to lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration.

[0035] The formulations provided herein are optically clear, even once dried, are chemically compatible with the biological specimen and do not harm or degrade the biological specimen, even after prolonged storage. The described formulations also improve clarity of higher magnification and 3D reconstruction (Z-stack) images and can reduce spherical aberration or scattered light, thereby allowing for capture of clearer images at multiple depths. Due to the favorable physical and optical properties, the instant formulations can be implemented in three- dimensional imaging of various types of biological specimens (e.g., tissues and cells) using a range of microscope techniques such as those used for visualization of fluorophores at depth within a specimen (e.g., confocal fluorescence microscopy). Further, the disclosed formulations also exhibit low bubble formation, less shrinking than other commercially available hard set mountants, and do not crack upon drying and/or freezing. As a result, cell and tissue samples maintain their morphology and resist compression once embedded in a hydrogel matrix, as described herein. For example, compression or shrinkage of a cell is approximately 15 to 20% less in Formulation 2 (see Table 3) than for commercially available hard-set mountant media.

[0036] The hydrogel mountants do not discolor or shrink, making it possible to take high quality images weeks or even months after mounting the slides. The formulations disclosed herein also can minimize quenching of fluorescent dyes commonly used in cellular imaging applications. Formulations that include anti-fade reagents, for example, can resist quenching of dyes and particularly useful for imaging cells and tissues stained with fluorescent probes. The unique combination of attributes described above makes the described formulations particularly useful for long term storage of stained, biological samples.

[0037] Rapid fonnation of a transparent, crosslinked hydrogel in one hour or less is significantly faster than other types of aqueous mountants that require 48 to 96 hours to reach the desired RI. Because solidification of the hydrogel is a result of chemical crosslinking, rather than an evaporative process, the volume of the mountant does not significantly change. For example, other types of aqueous based formulations can lose about 60-70% of their volume before reaching an optimal RI. As a result, cell and tissue samples maintain their morphology and resist compression once embedded in a hydrogel matrix, as described herein. Thus, the instant formulations offer a unique combination of attributes not found in aqueous-based mounting media known to those in the art. In particular, the instant aqueous formulations can solidify rapidly and exhibit a RI comparable to other slower curing aqueous media. Further, aqueous-based formulations provided herein do not suffer from the disadvantages of media prepared with organic solvents.

[0038] The formulations provided herein exhibit a refractive index that matches the refractive index of substrates typically used in imaging applications (e.g., microscope slide, such as those made from soda-lime glass or borosilicate glass) or immersion oil. The refractive-index matching properties of the formulations described herein can improve image quality, making the described formulations ideal for use in imaging applications. Formulations provided herein exhibit an exceptionally high RI relative to standard mounting formulations known to those skilled in the art. Typically, the refractive index of the disclosed hydrogel once solidified exceeds 1.40. For biological samples, such as tissue, that exhibit a RI range from about 1.50 to 1.52, it can be desirable to utilize a formulation that can provide a solidified mountant having the same RI range. Hydrogel compositions are provided herein that can exhibit a RI of about 1.40 to about 1 .60. In some embodiments, the RI is about 1.40 to about 1 .50. In other embodiments, the RI is about 1.50 to about 1.55. In yet other embodiments, the RI of the water-soluble components is about 1.55 to about 1.70. The aqueous solutions including polymers and polyols, as disclosed herein, typically exhibit an RI that is slightly lower than that of the crosslinked hydrogel. Thus, also provided herein is an aqueous solution that includes components for generating a crosslinked hydrogel, wherein the solution has a refractive index of 1.33 or greater (e.g.. 1.333 to 1.530; or 1.330 to 1.420).

[0039] Compositions are provided herein for preparing crosslinked hydrogels useful for embedding biological specimens. In certain embodiments, the embedded biological specimens can be disposed on a solid support (e.g., a microscope slide). Tire crosslinked hydrogel can be prepared from an aqueous solution of a copolymer that includes hydrophilic monomer residues and a water-soluble polyol.

[0040] Aqueous formulations provided herein include polymer components that can chemically react to form a crosslinked hydrogel. The crosslinked hydrogel can be formed by combining a hydrophilic polymer that includes boronic acid side groups (e.g., side chains bearing boronic acid) with a water-soluble polyol in aqueous solution. A water-soluble polyol including diol (e.g., 1,2-diol or 1 ,3-diol) groups can rapidly react with boronic acid groups to form a hydrogel that is covalently crosslinked by boronate ester bonds (i.e., boronic esters (RB(0R)2)).

Gelation (i.e., solidification) of the solution can occur at an appropriate pH once the cross-linking reaction commences. FIG. 1 illustrates the linkage that is formed by reaction of an exemplary boronic acid-containing copolymer (e.g., poly(DMA-co-3APBA) and a 1 ,2-cis-diol (e.g., a polyvinyl alcohol) in an aqueous formulation at an appropriate pH. Formation of boronic ester linkages results in formation of a crosslinked hydrogel (see, FIG. 2). The pH at which the reaction occurs can be adjusted to optimize the time required from the solution to form into hydrogel and to adjust the stiffness of the formed matrix. Hie reaction between boronic acid and diol groups can occur at pH 6 or greater (e.g., pH ~ 6-10), where a basic pH may be desired to accelerate the reaction depending on the components of the system. At tire appropriate pH (e.g., about 6 or greater), the components in the aqueous solution can rapidly crosslink to form a hydrogel at room temperature. In certain embodiments, an optically clear, crosslinked hydrogel may advantageously be prepared within one hour or less from an aqueous formulation, as disclosed herein, at a basic pH (e.g., 8.5 or greater).

[0041] Also provided herein are methods for forming a hydrogel by reacting a copolymer that includes hydrophilic monomer residues with multiple boronic acid side groups and a water-soluble polyol (e.g., 1,2-diol or 1.3-diol) in aqueous solution at an appropriate pH. The resulting hydrogel forms a relatively stiff matrix. Stiffness refers to a material’s resistance to deformation and can be measured by applying a force to a sample and measuring the resulting defonnation. The type, molecular weight, concentration of polymer and/or polyol(s), as well as gelation time and other factors, can impact the stiffness of the hydrogel. One measure of hydrogel stiffness is the storage modulus. Certain hydrogels suitable for use as mounting media exhibit a storage modulus of at least 40Pa, when tested according to the procedure described herein. Depending on the desired stiffness of the matrix, hydrogels can be prepared to achieve a storage modulus that ranges from about 40 Pa to about 4000 Pa. In certain embodiments, the storage modulus of the hydrogel ranges from about 40 Pa to about 100 Pa; or about 100 Pa to about 1000 Pa; or about 1000 Pa to about 2000 Pa; or about 2000 Pa to about 3000 Pa; or about 3000 Pa to about

4000 Pa. [0042] The copolymer includes hydrophilic monomer residues, such as, for example, N-R acrylamide or N-/ methacrylamide monomer residues, wherein R is methyl, ethyl, propyl, isopropyl or H. Exemplary hydrophilic monomer residues include acrylamide, N-methyhnethacrylamide, N,N-dimethylacrylamide, N,N- dimcthylmcthacrylamidc. N.N-diethylacrylamidc.: N-2-hydroxypropyl methacrylamide, dimethlylaminopropyl methacrylamide. N-3-aminoproyl methacrylamide, 4-vinylbenzenesulfonate, acrylate and methacrylate residues. [0043] Other types of copolymers that can be used in the instant formulations include, without limitation, dimethlylaminoethyl methacrylate, aminoethyl methacrylate, acrylic acid, methacrylic acid, and PEGylated acrylates (e.g., pol(ethylene glycol) acrylate, pol(ethylene glycol) methacrylate, and pol(ethylene glycol) methyl ether acrylates).

[0044] A portion of monomer residues in the copolymer bear a phenyl boronic acid substituent. The monomer residue bearing a phenyl boronic acid group can be a hydrophilic or hydrophobic. In certain embodiments, the boronic acid bearing group is only hydrophilic at elevated pH. In certain embodiments, the copolymer can include three (3) or more monomer residues that each bear a phenyl boronic acid substituent. Suitable boronic acid groups include, without limitation, 3-acrylamidophcnyl boronic acid, 2-acrylamidophcnyl boronic acid, 3- methacrylamido phenyl boronic acid, or 2-mcthacrylamido phenyl boronic acid, and 4-vinyl phenyl boronic acid. [0045] Representative examples of copolymers for preparing a hydrogel, as disclosed herein, include polyfA' A— dimethylmethacrylamide-co-3-(acrylamido)phenylboronic acid) (poly(DMA-co-3APBA)), polyGV N— dimethylmethacrylamide-co-3-(methacrylamido)phenylboronic acid) (poly(DMA-co-3MAPBA)), and poly(A, N- methyl methacrylamide-co-3-(methacrylamido)phenylboronic acid) (poly(MMAm-co-3APBA)).

[0046] Formulations provided herein can include neutral or charged polymers. Certain formulations can be prepared by combining a neutral or charged polymer with the hydrophilic copolymer and/or the water-soluble polyol. A neutral (i.e., uncharged) polymer can be selected, such that it has minimal interaction with dyes and/or cellular proteins present in the sample. Exemplary neutral, water-soluble polymers include poly( acrylamide), poly(methacrylamide), poly(methyl vinyl ether), poly(vinyl pyrrolidone) (PVP), polyvinyl alcohol (PVA), poly(2- ethyl-2-oxazoline), and poly(2-methyl-2-oxazoline). Exemplary acrylamide and methacrylamide-based polymers can include residues of monomer units, such as, e.g., N-7? acrylamide or N-/ methacrylamide, wherein R is methyl, ethyl, propyl, isopropyl or H; N,N-dimethylacrylamide, N.N-dimcthylmethacrylamidc. N,N- diethylacrylamide, N, N diethylmethacrylamide, N-2 -hydroxypropyl methacrylamide), or a combination of these monomer units, hr certain embodiments, the uncharged, water-soluble polymer is poly(N -methyl methacrylamide) (PMMAm). Formulations including some neutral polymers, such as, e g., PMMAm, do not foam or form longterm air bubbles, as often occur with existing commercial hard mount formulations, and, therefore, can be pipetted onto a sample with very little risk of bubble formation. Alternatively, the water-soluble polymer can be a charged polymer (e g., a polyclcctrolytc). Charged polymers can be more soluble in water and more viscous than neutral polymers of equivalent MW. Charged polymers, therefore, can have certain advantages when used in mounting formulations where such properties are desired. Tire charged polymer can carry a positive or negative overall charge. Exemplar}’ charged, water-soluble polymers include polyacrylic acid, polymcthacr lic acid, poly(diallyldimethylammonium chloride), poly(sodium-4-styrenesulfonate), poly(ethyleneimine), poly(N,N- dimethylaminoethyl acrylate), poly(N,N-diethylethylamino acrylate), poly(allylamine), poly[bis(2- chloroethyl)ether-co-l,3-bis[3-(dimethylamino)propyl]urea], or poly (vinyl sulfonic acid, sodium salt).

[0047] The molecular weight of the hydrophilic polymer can be chosen based on the desired properties of the mountant and its intended use. For example, the molecular weight of the polymer affects the viscosity of the formulation. Generally, the molecular weight is chosen to achieve a formulation that can be easily handled and yet sufficiently viscous such that it stays in place on the substrate. Polymers for use in the disclosed formulations typically include a range of molecular weights. The lower end of the molecular weight (MW) range can be selected based on the desired solubility of the polymer in the formulation, and the upper end of the MW range can be selected based on its ability to fonn a hydrogel. For example, if the MW is too low, then the formulations may not form a rigid film even if complete crosslinking is achieved. In general, the water-soluble polymer can have a weight average molecular weight of about 1 kDa to about 100 kDa, e.g., 1 kDa to 15 kDa; or 15 kDa to 20 kDa; or 20 kDa to 40 kDa; or 40 kDa to 80 kDa; or 80 kDa to 100 kDa. In certain embodiments, the weight average molecular weight of the water-soluble polymer is about 40 kDa to about 80 kDa. Formulations that include a higher molecular weight polymer (e.g., greater than about 15 kDa) can readily solidify under mild conditions that maintain the integrity of the biological specimen. In some embodiments, the formulation can implement a lower molecular weight, polymer (e.g., less than 20 kDa) to provide a softer, more viscous mountant. A softer mountant can provide certain advantages depending on the application and for mounting certain types of samples. For example, a softer mountant can be used with a specimen with thickness >200 microns, when there is a need to minimize extended drying times, or when recovery of the sample from the formulation is desired.

[0048] The hydrogel -forming formulations provided herein can further include a water-soluble polyol. A ■‘polyol’’ refers to a compound that includes two or more hydroxyl groups. In certain embodiments, the polyol has 100 or more hydroxyl groups. In certain embodiments, the polyol has about 200 to about 2000 hydroxyl groups. In other embodiments, the polyol has about 500 to about 1000 hydroxyl groups.

[0049] Formulations provided herein further include a water-soluble polyol or a combination of two or more types of water-soluble polyols, where the polyol includes two or more 1, 2-cis-diol groups or 1.3-cis-diol groups that can react readily in aqueous solution at an appropriate pH with the phenylboronic acid groups on the copolymer to provide a crosslinked hydrogel. Water-soluble polyols that include 1,2- or 1,3-cis-diol groups include, for example, iohexol, iodixanol, polyvinyl alcohol (PVA), poly(glycerol monomethacrylate), and poly(vinyl catechol) (e.g., 3, 4 dihydroxy styrene, 3-vinyl catechol).

[0050] Hydrogel-forming formulations can include more than one type of water-soluble polyol. For example, a formulation can include a 1,2- or 1,3-cis-diol for reaction with boronic acid groups and a second type of water- soluble polyol to help plasticize the polymer in the formulation, such that when the mounting film solidifies, it does not become too brittle or start cracking. In some embodiments, the polyol is chosen to have a RI that closely matches that of the biological specimen and/or substrate or other components in the mounted system (e.g., 1.46 to 1.60). Further, a polyol that does not evaporate also can provide some permanent volume to the film, thereby preventing the film from becoming too thin. Ideally, the dried film maintains a volume similar to that of tire undried film. This can prevent defomration of a specimen due to shrinkage of dried material. Maintaining structure of the dried film is also important for sample archiving and storage at various temperatures (e.g. room temp, 4°C and -20 °C). [0051] For example, certain formulations provided herein can include a 1,2- or 1,3-cis-diol for reaction with boronic acid groups and a water-soluble polyol that serves to modify the rheological or other optical properties (e.g., RI) of tire hydrogel. Examples of water-soluble polyols that can be included in the formulations for such purposes include glycerol, diglycerol, polyglycerol, iohexol, rosmarinic acid, erythorbic acid, thiodiethanol, thiodipropanol, and ammonium thiocyante.

[0052] The amounts of copolymer including boronic acid groups and water-soluble polyol in the formulation can be independently varied to affect the rheological properties and/or the refractive index of the crosslinked hydrogel. In some embodiments, the concentration of copolymer relative to the total concentration of total water- soluble polyol(s) in the aqueous solution ranges from about 1 wt.% to about 15 wt. %. In other embodiments, the concentrations range from about 2 wt.% to about 10 wt.%. In other embodiments, the concentrations range from about 3 wt.% to about 7 wt.%. Typically, formulations intended for preparation of a hard mountant include a weight ratio of copolymer to total polyol of 0.06 or greater. In certain embodiments, the weight ratio of water- soluble polymer to total polyol ranges from 0.06: 1 to 4: 1. In certain embodiments, the weight ratio of water- soluble polymer to total polyol is; 0.06: 1 to 1: 1; 1: 1 to 2: 1; 2: 1 to 3: 1; or 3: 1 to 4: 1. In certain formulations, the w eight ratio of water-soluble polymer to total polyol is 1 : 1 to 3 : 1. In certain formulations, the weight ratio of water-soluble polymer to total polyol is about 0.5: 1.

[0053] Certain formulations can include two or more different polyols, where the two or more different polyols can affect the rheological and/or optical properties of the crosslinked hydrogel. For example, a formulation can include one type of polyol that serves as a crosslinker for the boronic acid copolymer and a second type of polyol (or a combination of two or more polyols) that modulates the optical properties of the crosslinked hydrogel. The amounts of copolymer including boronic acid groups and water-soluble polyol(s) in the fonnulation can be independently varied to affect the rheological properties and/or the refractive index of tire crosslinked hydrogel.

[0054] In an exemplary embodiment, tire fonnulation includes an amount of a first polyol (e.g., PVA) that is sufficient to crosslink the boronic acid containing copolymer (e.g., about 1% to about 10% by weight). The fonnulation can further include a different polyol (or a combination of two or more different polyols) that serves to modify the optical properties of the crosslinked hydrogel. For example, one or more polyols, which can be the same or different from the polyol used to crosslinked the copolymer, can be included in the formulation to modify the RI of the crosslinked hydrogel. Tire amount of additional polyol(s) in the formulation can range from 0% to about 70% by weight and typically ranges from about 6 times to about 20 times the amount of copolymer (by weight) in the formulation.

[0055] Formulations provided herein further include an aqueous component (e.g., water or a buffer). The aqueous component serves to dissolve and/or hydrate the biological specimen and components of the hydrogelforming formulation. Any biologically compatible buffer known to those skilled in the art can be used in the formulations described herein, such as, e.g., Tris, PBS, borate, and the like. Buffers that maintain the pH of the formulation above 7.4 are especially useful in certain formulations, such as, e.g., when fluorescent dyes are present.

[0056] Hie described formulations can solidify into a transparent matrix without the need for an additional clearing agent. However, a clearing agent optionally can be used for imaging tissue samples to further improve image quality. Thus, in certain embodiments, the formulations disclosed herein can be used in conjunction with a clearing agent. For samples including fluorescent materials, it can be preferable to implement aqueous clearing agents given their compatibility with many fluorophores and fluorescent proteins, as well as the instant formulations. Exemplary clearing agents include, e.g., detergent-based reagents, glycerol or thioglycerol solutions, mono- and polysaccharides (e.g.. fructose and sucrose), urea solutions, and commercially available reagents.

[0057] Formulations provided herein can resist formation of precipitates, in contrast to commonly used mountants that are known to form precipitates on samples over the course of as little as 2-3 days. Formulations described herein are compatible with components (e.g., anti-fade compounds) that prevent the sample and/or fluorescent labeling materials, if present, from photobleaching. Formulations suitable for mounting and imaging biological specimens stained with a fluorescent dye optionally can further include an anti-fade reagent to minimize degradation or photobleaching of the stained sample upon storage or interrogation. Thus, in certain embodiments, the formulations provided herein optionally include one or more antifade reagents and can be included in the formulation at about 1 wt.% or less. Antifade reagents are well known to those skilled in the art and include anti-oxidants such as, e.g., 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2 -carboxylic acid, 3-carboxy proxyl, hydroquinone, mequinol, sodium sulfite, sodium erythorbate, ascorbic acid, propyl gallate, caffeic acid, paraphenylenediamine, l ,4-diazabicyclo[2.2.2]octane], other nitroxides, or a combination thereof.

[0058] Formulations can further include additional components, such as, e.g., preservatives, to prevent degradation of the polymer and/or polyol during prolonged storage and/or to prevent or minimize bacterial growth. Suitable preservatives include those that do not absorb a significant amount of visible light or lower the RI of the formulation. Representative preservatives include, e.g., sodium benzoate, benzoic acid or sodium azide, and are typically present in the formulation of a concentration of less than 2% by weight. Additional components, such as dyes, also can be included in the formulations provided herein. In certain embodiments, the fonnulation can include a fluorescent dye for staining cells. In certain embodiments, the dye can stain the nucleus of a cell, such as, for example, Hoechst 33342 or DAPI.

[0059] Also provided herein are methods for mounting a sample on a substrate in crosslinked hydrogel. For example, a biological specimen can be embedded in a hydrogel fonnulation, as described herein, mounted on a substrate. A substrate can be any material having a surface suitable for supporting or containing the instant formulations. For example, the substrate can have a surface that is smooth or rough and can be flat or contain a cavity, channel or depression (e.g., a well for containing the formulation). In certain embodiments, the substrate has a relatively smooth and non-porous surface, such that the aqueous solution does not become absorbed into the substrate. Substrates can be optically transparent or non-transparent, and can be made from a variety of materials, depending on the ultimate end use. For microscopy applications, e.g., the substrate can be optically transparent. Substrates can be made from any appropriate material, including, e.g., glass or a polymer. Representative substrates include but are not limited to those commonly used in optical and imaging applications such as, e.g., a microscope slide, cuvette, imaging chamber, well-plate, coverslip or dish. In certain embodiments, the mounted sample can include a transparent substrate (e.g., a microscope slide), a biological specimen (e.g., tissue), and a solidified formulation surrounding the sample and adhering it to the substrate. Optionally, a coverslip can be disposed over the mounted sample prior to solidifying. The amount of solidified formulation on the mounted slide is sufficient to surround the biological specimen and adhere it to the substrate and/or coverslip. Mounted biological samples, as disclosed herein, can be stable for many months (e.g., 5 months or more) when stored at room temperature or less without degrading or fading the sample or fluorescent stains, if present.

[0060] The sample can be any biological material including, but not limited to, tissues, cells, 3D cultures, whole organisms (e.g., fruit fly, worm, zebra fish), blood, and the like. The biological sample can be stained with a fluorescent dye(s) or fluorescent protein prior to mounting on the substrate. Fluorescent proteins suitable for staining biological samples are well known in the art and include, e.g., without limitation, GFP, RFP. mCherry, and the like. Commonly used fluorescent dyes for biological specimens include, e.g., boron dipyrromethenes (4,4- difluoro-4-bora-3a,4a-diaza-s- indacenes), cyanines, xanthenes, sulfonated pyrenes, rhodamines, coumarins, and derivatives thereof. Exemplary organic dyes include BODIPY dyes, coumarins (e.g., PACIFIC BLUE, PACIFIC GREEN and PACIFIC ORANGE (available from Thermo Fisher Scientific; Waltham, MA)), rhodamines, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol; aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, silicon rhodamine, and thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanines (e.g.. cyanine 2, cyanine 3. cyanine 3.5, cyanine 5. cyanine 5.5, cyanine 7), oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyrene derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, aryhnethine derivatives, xanthene dyes, sulfonated xanthenes dyes, sulfonated pyrenes, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphyrin, phtalocyanine, bilirubin and bis-benzimides (Hoechst stains). In certain embodiments, the organic dye is a near-infrared dye, such as, e.g., CY5.5 (GE Healthcare Life Sciences; Pittsburgh, PA), IRDYE 800 (Li-Cor; Lincoln, NE), DYLIGHT 750 or DYLIGHT 800 (Thermo Fisher Scientific) or indocyanine green (ICG), or a cyanine dye, such as, e.g., cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7. In certain embodiments, the organic dye is a xanthene or sulfonated xanthenes dyes, such as those commercially available under the tradenames ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 680, and ALEXA FLUOR 700, ALEXA FLUOR 790 (Thermo Fisher Scientific). Additional examples of suitable commercially available dyes include ALEXA FLUOR 405, ALEXA FLUOR 488 and ALEXA FLUOR PLUS secondary antibodies (Thermo Fisher Scientific).

[0061] The mounted sample can be interrogated directly or can be stored for an extended period of time prior to interrogation. Advantageously, the refractive index of the mounted sample matches the RI of the substrate (e.g., microscope slide) and coverslip. Crosslinked hydrogels formed by reaction of a hydrophilic copolymer and water soluble polyol, as disclosed herein, have an RI of about 1.40 or greater. For some applications, it can be desirable to include additional components into the formulation to increase the RI of the resulting hydrogel so that it more closely matches the RI of sample and/or substrate. For example, a water-soluble polyol (e.g., glycerol or iohexol) that has a relatively high RI (e.g., greater than 1.45) can be added into the aqueous formulation to increase the RI of the crosslinked hydrogel. Tirus, formulations arc provided herein that include a combination of different water- soluble polyol(s) or other water-soluble compounds with high RI, such as ammonium thiocyanate. In some embodiments, incorporation of an additional polyol into the formulation can raise the RI to about 1.45 to 1.55.

[0062] The instant formulations can react rapidly to provide an optically clear, crosslinked hydrogel. Crosslinked hydrogels can be prepared by combining reactive components in aqueous solution with a biological sample at an appropriate pH and at ambient (e.g., room) temperature or at an elevated temperature. Hydrogel formation is generally conducted at a temperature below about 40°C to prevent damage to the biological specimen and/or degradation to the water-soluble components in the formulation. For example, a crosslinked hydrogel can be prepared from aqueous solutions disclosed herein within 2 hours or less, or within 1 hour or less; or in some cases 30 minutes or less; or even 15 minutes or less. Once mounted, tire sample can be interrogated directly (e.g.. imaged using a microscope). However, the sample is typically covered (e.g., with a coverslip) for protection during use and subsequent storage. The formulation containing the biological specimen then can be imaged or subsequently dried. [0063] A representative method of mounting a biological specimen on a substrate includes depositing the biological specimen on the substrate and then contacting the biological specimen with one or more components in aqueous solution, as disclosed herein, to provide a sample embedded in a crosslinked hydrogel, mounted to the substrate. FIG. 3 depicts an exemplary workflow using the components and methods disclosed herein. An aqueous solution at appropriate pH that includes a phenyl boronic acid substituted, hydrophilic copolymer, such as poly(DMA-co-3APBA) (Component A) is placed on a substrate. A water-soluble polyol, e.g., PVA (Component B) or an aqueous solution that includes the water-soluble polyol then is placed on top of Component A. A tissue or cell sample then is placed onto Component A and Component B. The sample optionally can be stained with a fluorescent dye. Within one hour or less, a transparent, crosslinked hydrogel forms that is ready for imaging. Although FIG. 3 depicts a specific order of steps in the workflow, the reactive components and sample can be combined in any order suitable for the desired application. For example, a biological specimen can be deposited on the substrate first. Then, the biological specimen is contacted with a first aqueous solution (pH 6 or greater) that includes a hydrophilic copolymer, as disclosed herein. A second aqueous solution including a water- soluble polyol then is deposited onto the substrate to provide a crosslinked hydrogel that includes the biological specimen in the hydrated form mounted to the substrate.

[0064] The mounting formulations provided herein can be used in various histochemistry, immunochemistry and cytochemistry applications, including, but not limited to mounting of hematological, histological, and cytological samples on microscope slides, including tissue, cultured cells and blood. The described formulations can be used to provide specimens mounted on substrates such as samples of blood, cells, tissue or other biological fluids or materials, including but not limited to, materials that have been stained to facilitate microscopic examination for research and/or diagnostic purposes. A mounted biological specimen prepared according to the methods disclosed herein can be visualized using imaging techniques that are well-known in the art (e g., 100 pm or less; or about 1- about 100 pm). Imaging can be performed, e.g., using an optical microscope. Tire high RI and optical clarity of the mounting formulations described herein also allow biological samples to be imaged to greater depths and to higher resolution than when using standard mountants with lower RI values. For example, fluorescently labeled targets within a sample can be detectable to a focal depth of about 100 pm or greater, while target resolution is maintained throughout the sample.

[0065] Samples can be prepared using standard ICC/IHC protocols. Any suitable biological sample can be evaluated using the methods disclosed herein, including, but not limited to solution- or suspension-based samples and tissue samples. In certain embodiments, the sample includes cells or a digested cells and fragments thereof. The cells can be dead (e.g., fixed) or live. Additional examples of biological materials that can be treated with the formulations disclosed herein include, a 3D cell culture (spheroid/organoid) or a whole organism (e.g., fruit fly, worm, zebra fish).

[0066] The formulations described herein can be used to identify features of different materials, including, without limitation, plant, microbial, animal, earth, blood and plasma samples, and other types of non-living organic and inorganic materials, including but not limited to soil particles and geological samples, without losing clarity, definition or resolution of the objective structures.

[0067] In order to identify different components in a cell, stains can be used to provide contrast between particular structures based on their chemical composition. The aqueous solutions disclosed herein do not interfere with the fluorescent dyes typically used for staining cells. Thus, in certain embodiments, the biological specimen can be labeled with a fluorescent dye including, but not limited to, those disclosed herein. For example, the sample can include cells that express surface antigens (e.g., cell surface receptors) that can be recognized by and bind to specific affinity molecules (e.g., antibodies). Cells can be treated with a conjugate that includes a fluorophore attached to an affinity molecule (e.g., an antibody) under conditions for binding the antigens on the surface of the cells to the affinity molecule to form a cell labeled with the conjugate. Alternatively, the fluorophore can be contained within the cells, e.g., in the cytoplasm or within an organelle or cellular membrane. Further, fonnulations provided herein can be used in imaging of stained cells and tissues and in fluorescent immunohistochemistry (IHC) applications.

[0068] A general method of preparing a specimen involves soaking the specimen applied to a substrate (e.g., a microscope slide, cuvette, or well), which can be optionally stained, in a sufficient quantity of the mounting solution to fully immerse the specimen within the solution. A cover slip can be applied over tire specimen prior to hydrogel formation. Mounted samples can be archived, or alternatively, the mounted sample can be interrogated, e.g., visualized under a microscope immediately after mounting. The mounting formulations are compatible with fluorescent microscopes and objectives, such as epi -fluorescent, wide-field, confocal, stimulated emission depletion (STED) and structured illumination microscopes (SIM). Due to the superior optical properties of the formulation, the specimen appears transparent, allowing visualization of cells and deeper layers of tissues without losing clarity.

[0069] The described formulations also are ideally suited for mounting tissue samples on a substrate. Whole tissue mounting and imaging has been challenging to date, because sample thickness, presence of extracellular materials, and reagent penetration can result in background fluorescent and poor image resolution, and decreased imaging depth. Hie described fonnulation can decrease the refraction of light as it passes through a coverslip and tissue sample allowing higher resolution and the ability to image to a greater depth. Because the disclosed formulations improve sample transparency, they are particularly advantageous for use with tissue samples.

[0070] A representative method is provided for mounting a tissue sample on a microscope slide using a formulation, as described herein. The tissue can be a sectioned or whole sample that can be fixed or unfixed. In general, the tissue sample is deposited onto the surface of a microscope slide and a formulation, as disclosed herein, is applied to the sample. The amount of the mounting medium used in the method can vary but is generally chosen to wet the surface of the tissue. Excess mounting medium can be removed using known methods, if desired. Once tire hydrogel forms, the tissue firmly adheres to the slide and the sample will be embedded within the mountant. If needed, an additional amount of the formulation can be added, and the process can be repeated.

[0071] Further provided herein are kits for mounting a biological sample to a substrate for subsequent imaging and/or storage of the sample. Thus, in yet another aspect, a kit for mounting a biological specimen on a substrate is provided that includes a hydrogel-forming composition, as disclosed herein; and instructions for mounting a biological specimen on the substrate. Additional components optionally can be included in the kit, including, e.g., glycerol.

[0072] The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor(s) to function well in tire practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the scope of the invention.

EXAMPLES

[0073] The examples provided herein utilize the following general methods unless indicated otherwise. Refractive indices of all formulations before and after curing/gelling were measured at 21 °C with a TORC5000 Thermo-oscillating refraction characterizer instrument (Anton Paar) equipped with a synthetic sapphire measuring prism with an operating LED light source at a wavelength of 589 nm. Molecular weights of poly(A N— dimethylmethacrylamide-co-3-(acrylamido)phenylboronic acid) (poly(DMA-co-3APBA)), poly(A N— dimethylmethacrylamide-co-3-(methacrylamido)phenylboronic acid) (poly(DMA-co-3MAPBA)), and poly(A, N- methyl methacrylamide-co-3-(methacrylamido)phenylboronic acid) (poly(MMAm-co-3APBA)) are listed as targeted number average molecular weight (M_n), and copolymer compositions were estimated from ¹ HNMR using a 400 MHz Bruker Instrument. Molecular weights of poly(A, N- methyl methacrylamide) (PMMAm) were measured by standard gel penneation chromatography (GPC) methods using 5 mM LiBr mcthanol/dimcthylformamidc (50/50 v/v%) as an eluent, TSkgcl® H _r organic size exclusion columns (TOSOH Bioscience LLC, King of Prussia, PA) with Wyatt Technology Corporation’s miniDAWN® TREOS® multiangle light scattering (MALS) and Optilab® T-rEX differential refractometer detectors (Santa Barbara, CA). EXAMPLE 1

Measuring Refractive Indices of Mountant Samples

[0074] Refractive index (RI) measurements on all samples were measured by carefully spreading 200 to 300 pL of cross-linked sample onto the refractometer prism. The cross-linked sample was immediately placed on the refractometer prism. Refractive indices of the samples were recorded after 1 hour on the prism.

EXAMPLE 2 Mounting Procedure [0075] Biological specimens (e.g., cultured monolayer cells) are mounted onto a substrate by applying enough of the first mountant component, typically 15 to 50 pL, to a specimen disposed on a substrate until fully covered or immersed and a sufficient amount of the second mountant component, typically 15 to 50 pL, to a microscope slide. Hie substrate containing the biological specimen was then placed on top of the microscope slide to allow both mountant components to mix and initiated the “curing” or gelation reaction. Alternatively, a sufficient amount of the first mountant component, typically 15 to 50 pL, can be placed on a microscope slide followed by the addition of the second mountant component, typically 15 to 50 pL, added to the first component. Hie two components are then briefly mixed with a pipette and then the biological specimen deposited on substrate is added on top of the microscope slide containing the premixed mountant components, 1 & 2. The biological specimen can be unstained or can be stained prior to mounting using procedures w ell known to those in the art. The specimen then is covered with an 18 mm x 18 mm coverslip and any excess mountant is wiped or pipetted away from the edges of the coverslip. The mounted specimen is allowed to dry for at least 1 hour at room temperature, although temperatures as high as 40 °C are acceptable. After the mounted specimen is completely solidified, the specimen can be imaged using fluorescent microscopy techniques that are well known to the person skilled in the art.

[0076] The following protocol can be used for mounting biological specimens that are 30-100 pm and thicker. Allow the mountant components to warm to room temperature for 1 hour before mounting coverslips. Remove excess liquid from the sample by gently tapping the edge of the coverslip or slide on a laboratory wipe. For slide- mounted specimens, apply 60-100 LIL of the first mountant component followed by 60-100 pL of the second mountant component directly to the specimen, then carefully lower a coverslip onto the mountant to avoid trapping any air bubbles. For specimens stained in well plates or culture dishes, carefully move sample to a microscope slide. The addition of 10 pL of mountant to the slide can assist with manipulating the sample into place. For three-dimensional (3-D) cultured cells or spheroids, move 3-D cultures or spheroids to a microscope slide using a ImL pipette with the end of the tip removed. Place the 3-D culture with buffer on a microscope slide prepared with an appropriate spacer to ensure integrity of the sample. The spacer should allow sufficient room for the sample while minimizing the volume of mountant required. Spacers allowing open edges decrease the curing time of the sample. If needed, gently tap the coverslip to remove air bubbles. Gently tap to remove air bubbles from around the sample. Failure to sufficiently cover the sample can lead to mountant contraction and reduce the imaging area. Place the mounted sample on a flat, dry surface, and allow it to cure at room temperature in the dark for 1 hour.

EXAMPLE 3

Formulation of Mounting Media Component 1 with poly(DMA-co-3APBA) 10 mol% Phenyl Boronic Acid Containing Copolymer

[0077] Formulation of a first mountant component was done by adding 40 to 100 mg of (poly(DMA-co- 3APBA), where 3APBA was 10 mol%. (Target Mn ranging from 20,000 to 100,000 g/mol), zero to 0.4 gram of glycerol (Fisher Scientific, Fair Lawn, NJ), 0.30 to 0.55 gram of iohexol (TCI America Portland, OR), and pH 8.6 50 mM Tris Buffer (TRIS base; Sigma-Aldrich, St. Louis, MO) prepared in deionized water to a 1.5 mL centrifuge tube or 4 mL scintillation vial. The vial was then sonicated at room temperature until complete dissolution was observed. Table 1 lists formulation details for Component 1.

Table 1. Component 1 Formulation Details

EXAMPLE 4

Formulation of Mounting Media Component 1 with poly(DMA-co-3APBA) 20 mol% Phenyl Boronic Acid Containing Copolymer

[0078] Formulation of a first mountant component was done by adding 50 mg of (poly(DMA-co-3APBA), where 3APBA was 20 mol%, (Target M_n was 94,000 g/mol), 0.40 gram of glycerol (Fisher Scientific, Fair Lawn, NJ), 0.35 gram (Component Ih) of iohexol. and 0.263 mL of pH 8.6 50mM Tris Buffer prepared in deionized water to a 1.5mL centrifuge tube or 4mL scintillation vial. The vial was then sonicated at room temperature until complete dissolution was observed.

EXAMPLE 5

Formulation of Mounting Media Component 1 with poly(MMAm-co-3APBA) Phenyl Boronic Acid Containing Copolymer

[0079] Formulation of the first mountant component was done by adding 50 mg of poly(MMAm-co-3APBA), where 3 APB A was 20 mol%, (Target M_n was 94,000 g/mol), 0.40 gram of glycerol (Fisher Scientific, Fair Lawn, NJ), 0.35 gram (Component li) of iohexol, and 0.263 mL of pH 8.6 50mM Tris Buffer prepared in deionized water to a 1.5mL centrifuge tube or 4mL scintillation vial. The vial was then sonicated at room temperature until complete dissolution was observed. EXAMPLE 6

Formulation of Mounting Media Component 2 with Polyvinyl Alcohol (PVA)

[0080] Formulation of the second mountant component was done by adding zero to 0.40 g of glycerol. 10 to 75 mg of PVA (M_w ~ 31,000 g/mol; Sekisui America Corporation, Secaucus, NJ, USA), 0.30 to 0.60 (Component 2b) gram of iohexol ( TCI America, Portland, OR), and pH 8.6 50mM Tris Buffer (TRIS base; Sigma-Aldrich, St.

Louis, MO) prepared in deionized water to a 1.5mL centrifuge tube or 4mL scintillation vial. The vial was then sonicated at room temperature until complete dissolution was observed. Table 2 lists formulation details for Component 2.

Table 2. Component 2 Formulation Details

EXAMPLE 7

Formulation of Mounting Media Component 1 with Thiodiethanol

[0081] Formulation of a first mountant component was done by adding 50 mg of (poly(DMA-co-3APBA), where 3APBA was 10 mol%, (Target M_n = 86,600 g/mol), 0.050 of iohexol. 0.25mL of thiodiethanol (Alfa Aesar, Ward Hill, MA) and 0.263 mL of pH 8.6 50mM Tris Buffer prepared in deionized water to a 1.5mL centrifuge tube or 4mL scintillation vial. The vial was then sonicated at room temperature until complete dissolution was observed

(Component Ij).

EXAMPLE 8

Formulation of Mounting Media Component 1 with Ammonium Thiocyanate

[0082] Formulation of the first mountant component was done by adding 50 mg of (poly(DMA-co-3APBA), where 3APBA molar composition was 10mol%, (Target Mn 86,600 g/mol), 0.30 gram of ammonium thiocyanate (TCI America, Portland, OR) and 0.650 mL of pH 8.6 50mM Tris Buffer (TRIS base: Sigma-Aldrich, St. Louis, MO) prepared in deionized water to a 1.5mL centrifuge tube or 4mL scintillation vial (Component Ik). The vial was then sonicated at room temperature until complete dissolution was observed.

EXAMPLE 9

Fonnulation of Mounting Media Component 1 with PMMAm

[0083] Formulation of the first mountant component was done by adding 50 mg of (poly(DMA-co-3APBA), where 3APBA molar composition was 10mol%, (Target Mn 86,600 g/mol), 50 mg of PMMAm (Mn = 1 1,000 g/mol), 0.25mL of thiodiethanol and 0.263 mL of pH 8.6 50mM Tris Buffer prepared in deionized water to a 1.5mL centrifuge tube or 4mL scintillation vial (Component 11). The vial was then sonicated at room temperature until complete dissolution was observed.

EXAMPLE 10

Formulation of Mounting Media Component 2 with Higher Glycerol Content

[0084] Formulation of the second mountant component was done by adding 0.945 g of glycerol, 25 mg of PVA (M_w ~ 31,000 g/mol) and 0.250 mL of lOOmM pH 9.0 N-cyclohexyl-2 -aminoethanesulfonic acid (CHES) Buffer (Sigma-Aldrich, St. Louis, MO) prepared in deionized water to a 1.5mL centrifuge tube or 4mL scintillation vial (Component 2m). The vial was then sonicated at room temperature until complete dissolution was observed. EXAMPLE 11

Preparing Cross-linked Hydrogel for Refractive Index Measurements

[0085] To prepare a cross-linked hydrogel for RI measurements, first 100 to 150 pL of Component 1 was added to a 1 .5 mL vial. Then an equivalent volume of component 2 was then added on top of Component 1 and the two liquids were then mixed by manual or mechanical agitation to induce cross-linking. The formed hydrogel was then placed on the refractometer prism within 1 hour of cross-linking for refractive index determination. Table 3 lists RI values Ih after combining Component 1 and Component 2.

Table 3. Refractive indices of various mountant formulations upon dry ing for Ih at room temperature EXAMPLE 12

Formulation of Mounting Media with Polymer and Antifade Reagent

[0086] Antifade reagents can also be added to either component 1 and/or component 2 formulations. For example, formulation of the second mountant component was done by adding 0.656 g of glycerol, 50 mg of PVA (M_w ~ 31,000 g/mol), 0.49 mg of benzoic acid (4 pmol), 0.63 mg of sodium sulfite (5 pmol) and 0.330 mL of pH 8.6 lOOmM Tris Buffer prepared in deionized water to a 1.5mL centrifuge tube (Component 2n). The tube was then sonicated at room temperature until complete dissolution of all materials was observed. Component 1 in this example is the same as that prepared in Example 3, (Component le), and a cross-linked hydrogel was prepared the same ways described in Example 11. An RI measurement after Ih at 21 °C was measured to be 1.4542 and is listed in Table 3.

EXAMPLE 13

Post-polymerization Synthesis of Water-Soluble Phenyl Boronic Acid Containing Copolymers

[0087] Copolymers with amine reactive side chains can be used to introduce a phenyl boronic acid moiety to tire copolymer side chain using standard activated ester amine chemistry. These copolymers can then be used in Component 1 formulations for mounting. For example, a copolymer including 10 mol% N-(3- aminopropyl)methacrylamide hydrochloride (APMA; Polysciences, Warrington, PA) and 90mol% JV-methyl methacrylamide (MMAm, TCI America, Portland, OR) with a target molecular weight of 34,500 g/mol was prepared by free radical polymerization. Poly(MMAm-co-APMA) was then reacted with 4-carbxoyphenyl boronic acid (Oakwood Chemical, Estill, SC) using carbodiimide chemistry'. Briefly. 0.5 g of poly(MMAm-co- APMA) and 144mg of 4-carbxoyphenyl boronic acid were weighed in a round bottomed flask equipped with a stir bar and dissolved in 43mL of deionized water to give an amine concentration of 20mM. pH can be adjusted to 9.0 to help dissolve all the 4-carbxoyphenyl boronic acid. Once dissolved the pH of the solution was adjusted to be between 7 and 8. 329mg of A-ethyl, A’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC; Sigma- Aldrich, St. Louis, MO) was then added to the solution and allowed to react overnight at room temperature. The phenylboronic acid modified copolymer was then isolated by first perfonning rotary evaporation to remove ~

30mL to concentrate the solution followed by precipitation into cold acetone and dry ing in vacuo overnight.

EXAMPLE 14

Imaging of Fluorescently-Labeled Cells in Mounting Media

[0088] This example shows results from imaging of fluorescently-labeled cells embedded in mounting media. FIG. 4 shows fluorescence microscope images of Alexa Fluor 488 phalloidin-stained cells mounted to microscope slides in a glycerol-based liquid mountant (Control Fonnulation). Formulation 1 (Component la and 2a). and Formulation 8 (Component le and 2b) after 1 hour and 72 hours from mounting start time. FIG. 5 shows fluorescence microscope images of cells immunostained for tubulin and detected with Alexa Fluor 647 antibody conjugate mounted to microscope slides in the Control Formulation, Fonnulation 1, and Fonnulation 8 after 1 hour and 72 hours from mounting start time. Images were collected using tire same acquisition settings for a given filter set. When comparing images taken at 1 hour and 72 hour time points, the data show that fluorescence was retained over sample storage time. Because Formulation 1 did not include antifade compounds, the 1 hour time point for this formulation in FIG. 5 appears dimmer as a result of photobleaching. The image panels also indicated that the base formulations were compatible with multiple fluorophore scaffold chemistries, and cytoskeletal morphologies appeared the same in the control and test formulations.

EXAMPLE 15

Measurement of Storage Modulus

[0089] Stiffness of various crosslinked hydrogel materials prepared as described in Example 3 & 6 were evaluated by measuring the increase in storage (i.e., the solid “like” component of the hydrogel) modulus (Pa) overtime using a HAAKE™ MARS™ 60 Rheometer (Thermo Fisher Scientific; Waltham, MA). As shown in FIG. 6, and FIG. 7 (comparing Formulations 2, 9, 10, 11 and 15), after combining the reactive components of the formulation, the storage modulus increases rapidly over the first 10 minutes and begins to level off over a 1 hour period. This measurement was performed by first adding 125 pL of component 1 to the bottom plate of the rheometer followed by adding 125 pL of component 2 on top of component 1. Hie two components were then mixed with a pipette tip 5 times and the rheometer experiment was immediately started using a 35mm cone/plate geometry (cone has 1° angle), with a frequency of 1Hz and a strain of 0.02. The experiment was run at 21 °C for 1 hour. The time course in FIG. 6 and FIG. 7 suggests hydrogel fonnation occurs in 1 hour or less which is a vast improvement 24-96 hours required for evaporative drying of standard mounting media.

[0090] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary⁷ to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[0091] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following clauses and claims, the terms used should not be construed to limit the clauses and claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such clauses and claims are entitled. Accordingly, the clauses and claims are not limited by the disclosure. Embodiments may be in accordance with the following numbered clauses:

1. A method of embedding a biological specimen in a crosslinked hydrogel, comprising: combining a biological specimen and an aqueous solution, the aqueous solution comprising: a. a copolymer comprising a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent; and b. a water-soluble polyol, wherein the pH of the aqueous solution is 6 or greater, wherein a crosslinked hydrogel forms at room temperature within 2 hours or less after combining the aqueous solution with the biological specimen, wherein the biological specimen is embedded in the crosslinked hydrogel in a hydrated form, and the hydrogel has a refractive index of 1.40 or greater.

2. A method of mounting a biological specimen on a substrate, comprising: a) depositing a biological specimen in hydrated form on the substrate; b) contacting the biological specimen with a first aqueous solution at room temperature, wherein the pH of the first aqueous solution is 6 or greater, comprising a copolymer, wherein the copolymer comprises a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent, to provide a composition comprising the copolymer and a biological specimen; and c) contacting the composition with a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater and comprises a water-soluble polyol, to provide a crosslinked hydrogel comprising the biological specimen in the hydrated form mounted to the substrate, wherein within 2 hours or less of contacting the composition with the second aqueous solution at room temperature the crosslinked hydrogel has a refractive index of 1.40 or greater. A method of mounting a biological specimen on a substrate, comprising: a) depositing a biological specimen in hydrated form on the substrate at room temperature; b) contacting the biological specimen with a first aqueous solution, wherein the pH of the second aqueous solution is 6 or greater and comprises a water-soluble polyol, to provide a first composition; and c) contacting the first composition with a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater, comprising a copolymer, wherein the copolymer comprises a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent, to provide a crosslinked hydrogel comprising the biological specimen in the hydrated form mounted to the substrate, wherein within 2 hours or less of contacting the first composition with the second aqueous solution at room temperature the crosslinked hydrogel has a refractive index of 1.40 or greater. A method of mounting a biological specimen on a substrate in a crosslinked hydrogel, comprising: a) contacting a first substrate with a first aqueous solution comprising a copolymer, wherein the copolymer comprises a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent, wherein the pH of the first aqueous solution is about 6 or greater; b) combining the first aqueous solution with a second aqueous solution, wherein the second aqueous solution comprises a water-soluble polyol, wherein tire pH of the second aqueous solution is about 6 or greater, to provide a first composition disposed on tire first substrate; c) providing a biological sample in a hydrated form disposed on a second substrate; and d) contacting the first composition disposed on the first substrate and the biological sample disposed on the second substrate, such as to generate a crosslinked hydrogel comprising the biological specimen in the hydrated fonn disposed between the first substrate and the second substrate within 2 hours or less, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater. A method of mounting a biological specimen on a substrate, comprising: depositing an aqueous solution onto the substrate at room temperature, wherein the pH of the aqueous solution is about 6 or greater, wherein the aqueous solution comprises: a. a copolymer comprising a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent; b. a water-soluble polyol: and c. a biological specimen, to form a crosslinked hydrogel mounted on the substrate within 2 hours or less that comprises the biological specimen in a hydrated form and has a refractive index of 1.40 or greater. Tire method of any one of the preceding clauses, wherein the crosslinked hydrogel forms at room temperature in about 1 hour or less: 30 minutes or less: or in about 10 minutes or less; or in about 1 minute or less. Tire method of any one of the preceding clauses, further comprising visualizing tire biological specimen on the substrate with a microscope. A kit for mounting a biological specimen in a crosslinked hydrogel on a substrate, comprising: a) a first aqueous solution, wherein the pH of the first aqueous solution is about 6 or greater, comprising a copolymer comprising a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent; b) a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater, comprising a water-soluble polyol; and c) instructions for combining the first and second aqueous solutions with a biological specimen in hydrated form to fomi a crosslinked hydrogel on the substrate at room temperature within 2 hours or less, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater and comprises the biological specimen in the hydrated form. A biological specimen embedded in hydrated form in a crosslinked hydrogel, wherein the hydrogel comprises a hydrophilic copolymer that is crosslinked to a water-soluble polyol through at least 3 boronate ester linkages, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater. The biological specimen of clause 9, wherein the embedded biological specimen is disposed on a substrate. The method, biological specimen, or kit of any one of the preceding clauses, wherein the plurality of hydrophilic monomer residues, comprises an N-/? acrylamide or N-7? methacrylamide monomer residue, wherein R is methyl, ethyl, propyl, isopropyl or H (e.g., N,N-dimethylacrylamide, N,N- dimethylmethacrylamide, N,N-diethylacrylamide,; N-2 -hydroxypropyl methacrylamide, dimethlylaminopropyl methacrylamide, or N-3-aminoproyl methacrylamide). The method, biological specimen, or kit of any one of the preceding clauses, wherein the plurality of hydrophilic monomer residues comprises a monomer residue selected from the group consisting of 4- vinylbenzenesulfonate, acrylic acid, methacrylic acid, acrylate, and methacrylate monomer residues. The method, biological specimen, or kit of any one of the preceding clauses, wherein the hydrophilic monomer residue is selected from dimethlylaminoethyl methacrylate, aminoethyl methacrylate, and PEGylated acry lates or methacry lates (e.g., pol(ethylene glycol) acrylate, pol(ethylene glycol) methacrylate, pol(ethylene glycol) methyl ether acrylates). The method, biological specimen, or kit of any one of the preceding clauses, wherein the phenyl boronic acid monomer is selected from the group consisting of 3-acrylamidophenyl boronic acid, 2- acry lamidophcnyl boronic acid, 3-mcthacry lamido phenyl boronic acid, 2-mcthacry lamido phenyl boronic acid, or 4-vinyl phenyl boronic acid. The method, biological specimen, or kit of any one of the preceding clauses, wherein the water-soluble polyol is compound comprising two or more 1, 2-cis-diol groups or 1,3-cis-diol groups. The method, biological specimen, or kit of any one of the preceding clauses, wherein the water-soluble polyol is selected from iohexol, iodixanol, glycerol, thiodiethanol, thiodipropanol, polyvinyl alcohol (PVA), poly(glycerol monomethacrylate), poly(vinyl catechol) (e.g., 3, 4 dihydroxy styrene, 3-viny 1 catechol), rosmarinic acid, erythorbic acid, fructose, sucrose, galactose, maltose, and a combination thereof. The method, biological specimen, or kit of any one of the preceding clauses, wherein the hydrophilic copolymer and/or the water soluble polyol has a weight average molecular w eight of about 10 kDa to about 100 kDa. The method, biological specimen, or kit of any one of the preceding clauses, wherein the weight ratio of the hydrophilic copolymer to the water-soluble polyol in the crosslinked hydrogel is about 1:9 to about 1:30. The method, biological specimen, or kit of any one of the preceding clauses, wherein the substrate is a microscope slide, cuvette, well or dish. The method, biological specimen, or kit of any one of the preceding clauses, wherein the biological specimen is a cell, tissue, 3D cell culture, a whole organism. Tire method, biological specimen, or kit of any one of tire preceding clauses, wherein the biological specimen is labeled with a fluorescent dye or fluorescent protein. The method, biological specimen, or kit of any one of the preceding clauses, wherein the refractive index of the crosslinked hydrogel matches the refractive index of soda-lime glass, borosilicate glass, glycerol, or immersion oil. The method, biological specimen, or kit of any one of the preceding clauses, wherein the crosslinked hydrogel has a refractive index of about 1.40 to about 1.60 ; or about 1.40 to about 1.54. The method, mounted biological specimen, or kit of any one of the preceding clauses, wherein the crosslinked hydrogel has a specific storage modulus of 40Pa to 4000Pa. The method, mounted biological specimen, or kit of any one of the preceding clauses, wherein the crosslinked hydrogel has a specific storage modulus of 40Pa to lOOOPa. Hie method, mounted biological specimen, or kit of any one of the preceding clauses, wherein the crosslinked hydrogel further comprises an anti-oxidant (e.g., 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid, 3-carboxy proxyl, hydroquinone, mequinol, sodium sulfite, sodium erythorbate, ascorbic acid, propyl gallate, caffeic acid, paraphenylenediamine, l,4-diazabicyclo[2.2.2]octane, or a combination thereof). The method, mounted biological specimen, or kit of any one of the preceding clauses, wherein the crosslinked hydrogel further comprises 1% by weight or less of the anti-oxidant. Tire method, mounted biological specimen, or kit of any one of the preceding clauses, wherein the copolymer is or comprises a synthetic polymer.

Claims

CLAIMS What is claimed is:

1 . A method of embedding a biological specimen in a crosslinked hydrogel, comprising: combining a biological specimen and an aqueous solution, the aqueous solution comprising: a. a copolymer comprising a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent; and b. a water-soluble polyol, wherein the pH of the aqueous solution is 6 or greater, wherein a crosslinked hydrogel forms at room temperature within 2 hours or less after combining the aqueous solution with the biological specimen, wherein the biological specimen is embedded in the crosslinked hydrogel in a hydrated form, and the hydrogel has a refractive index of 1.40 or greater.

2. A method of mounting a biological specimen on a substrate, comprising: a) depositing a biological specimen in hydrated form on the substrate; b) contacting the biological specimen with a first aqueous solution at room temperature, wherein the pH of the first aqueous solution is 6 or greater, comprising a copolymer, wherein the copolymer comprises a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent, to provide a composition comprising the copolymer and a biological specimen; and c) contacting the composition with a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater and comprises a water-soluble polyol, to provide a crosslinked hydrogel comprising the biological specimen in the hydrated form mounted to the substrate, wherein within 2 hours or less of contacting the composition with the second aqueous solution at room temperature the crosslinked hydrogel has a refractive index of 1.40 or greater.

3. A method of mounting a biological specimen on a substrate, comprising: a) depositing a biological specimen in hydrated form on the substrate at room temperature; b) contacting the biological specimen with a first aqueous solution, wherein the pH of the second aqueous solution is 6 or greater and comprises a water-soluble polyol, to provide a first composition; and c) contacting the first composition with a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater, comprising a copolymer, wherein the copolymer comprises a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent, to provide a crosslinked hydrogel comprising the biological specimen in the hydrated form mounted to the substrate, wherein within 2 hours or less of contacting the first composition with the second aqueous solution at room temperature the crosslinked hydrogel has a refractive index of 1.40 or greater.

4. A method of mounting a biological specimen on a substrate in a crosslinked hydrogel, comprising: a) contacting a first substrate with a first aqueous solution comprising a copolymer, wherein the copolymer comprises a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent, wherein the pH of the first aqueous solution is about 6 or greater; b) combining the first aqueous solution with a second aqueous solution, wherein the second aqueous solution comprises a water-soluble polyol, wherein the pH of the second aqueous solution is about 6 or greater, to provide a first composition disposed on the first substrate; c) providing a biological sample in a hydrated form disposed on a second substrate; and d) contacting the first composition disposed on the first substrate and the biological sample disposed on the second substrate, such as to generate a crosslinked hydrogel comprising the biological specimen in the hydrated form disposed between the first substrate and the second substrate within 2 hours or less, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater.

5. A method of mounting a biological specimen on a substrate, comprising: depositing an aqueous solution onto the substrate at room temperature, wherein the pH of the aqueous solution is about 6 or greater, wherein the aqueous solution comprises: a. a copolymer comprising a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent; b. a water-soluble polyol; and c. a biological specimen, to form a crosslinked hydrogel mounted on the substrate within 2 hours or less that comprises the biological specimen in a hydrated form and has a refractive index of 1.40 or greater.

6. The method of claim 1. wherein the crosslinked hydrogel fonns at room temperature in about 1 hour or less or about 30 minutes or less.

7. The method of claim 1, further comprising visualizing the biological specimen on the substrate with a microscope.

8. A kit for mounting a biological specimen in a crosslinked hydrogel on a substrate, comprising: d) a first aqueous solution, wherein the pH of the first aqueous solution is about 6 or greater, comprising a copolymer comprising a plurality of hydrophilic monomer residues and at least 3 monomer residues that each comprise a phenyl boronic acid substituent; e) a second aqueous solution, wherein the pH of the second aqueous solution is 6 or greater, comprising a water-soluble polyol; and f) instructions for combining the first and second aqueous solutions with a biological specimen in hydrated form to form a crosslinked hydrogel on the substrate at room temperature within 2 hours or less, wherein the crosslinked hydrogel has a refractive index of 1 .40 or greater and comprises the biological specimen in the hydrated form.

9. A biological specimen embedded in hydrated form in a crosslinked hydrogel, wherein the hydrogel comprises a hydrophilic copolymer that is crosslinked to a water-soluble polyol through at least 3 boronate ester linkages, wherein the crosslinked hydrogel has a refractive index of 1.40 or greater.

10. The biological specimen of claim 9, wherein the embedded biological specimen is disposed on a substrate.

11. The method of claim 1. wherein the plurality of hydrophilic monomer residues, comprises an N-/ acrylamide or N-/? methacrylamide monomer residue, wherein R is methyl, ethyl, propyl, isopropyl or H (e.g., N,N-dimethylacrylamide, N.N-dimethylmcthacrylamidc. N,N-diethylacrylamide,; N-2- hydroxypropyl methacrylamide, dimethlylaminopropyl methacrylamide. or N-3-aminoproyl methacrylamide) .

12. The method of claim 1. wherein the plurality of hydrophilic monomer residues comprises a monomer residue selected from the group consisting of 4-vinylbenzenesulfonate, acrylic acid, methacrylic acid, acrylate, and methacrylate monomer residues.

13. Tire method of claim 1. wherein the hydrophilic monomer residue is selected from dimethlylaminoethyl methacrylate, aminoethyl methacrylate, and PEGylated acrylates or methacrylates (e.g., pol(ethylene glycol) acrylate, pol(ethylene glycol) methacrylate, pol(ethylene glycol) methyl ether acrylates).

14. The method of claim 1, wherein the phenyl boronic acid monomer is selected from the group consisting of 3-acrylamidophenyl boronic acid, 2-acrylamidophenyl boronic acid, 3 -methacrylamido phenyl boronic acid, 2-methacrylamido phenyl boronic acid, or 4-vinyl phenyl boronic acid.

15. The method of claim 1. wherein the water-soluble polyol is compound comprising two or more 1, 2-cis- diol groups or 1,3 -cis-diol groups.

16. Tire method of claim 1. wherein tire water-soluble polyol is selected from iohexol, iodixanol, glycerol, thiodiethanol, thiodipropanol, polyvinyl alcohol (PVA), poly(glycerol monomethacrylate). poly( vinyl catechol) (e.g., 3, 4 dihydroxy styrene, 3-vinyl catechol), rosmarinic acid, erythorbic acid, fructose, sucrose, galactose, maltose, and a combination thereof.

17. The method of claim 1. wherein the hydrophilic copolymer and/or the water soluble polyol has a weight average molecular weight of about 10 kDa to about 100 kDa.

18. Tire method of claim 1, wherein the weight ratio of the hydrophilic copolymer to the water-soluble polyol in the crosslinked hydrogel is about 1:9 to about 1:30.

19. The method of claim 1, wherein the substrate is a microscope slide, cuvette, well or dish.

20. Tire method of claim 1. wherein tire biological specimen is a cell, tissue, 3D cell culture, a whole organism.

21. The method of claim 1, wherein the biological specimen is labeled with a fluorescent dye or fluorescent protein.

22. The method of claim 1, wherein the refractive index of the crosslinked hydrogel matches the refractive index of soda-lime glass, borosilicate glass, glycerol, or immersion oil.

23. The method of claim 1, wherein the crosslinked hydrogel has a refractive index of about 1 .40 to about 1.60; or about 1.40 to about 1.54.

24. The method of claim 1. wherein the crosslinked hydrogel has a specific storage modulus of 40Pato 4000Pa.

25. Tire method of claim 1. wherein tire crosslinked hydrogel further comprises an anti-oxidant (e.g., 6- hydroxy-2,5, 7, 8-tetramethylchroman-2 -carboxylic acid, 3-carboxy proxyl, hydroquinone, mequinol, sodium sulfite, sodium erythorbate, ascorbic acid, propyl gallate, caffeic acid, paraphenylenediamine, 1,4- diazabicyclo[2.2.2]octane, or a combination thereof).

26. The method of claim 1, wherein the crosslinked hydrogel further comprises 1% by weight or less of the anti-oxidant.

27. The method of claim 1 , wherein the copolymer is or comprises a synthetic polymer.