WO2020172247A1 - Iterative direct expansion microscopy - Google Patents

Abstract

The present invention provides biological samples of interest that have been iteratively expanded in a method referred to herein as iterative direct expansion microscopy (id-ExM). In the id-ExM method, biological samples of interest are permeated with a swellable material that results in the sample becoming embedded in the swellable material, and then the sample can be expanded isotropically in three dimensions. The process of iteratively expanding the samples can be applied to expand samples one or more additional times such that, for example, a 5-fold expanded sample can be expanded again to achieve high expansion factors, for example, 20x to 100x or more linear expansion.

Description

ITERATIVE DIRECT EXPANSION MICROSCOPY

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 62/809,062, filed on February 22, 2019, the contents of which are incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with U.S. Government Support under Grant Number 2014509, awarded by US-Israel Binational Science Foundation Grant. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Understanding the nanoscale organizations of biomolecules in complex biological systems such as the brain, can not only provide fundamental biological insights but also help in the discovery of new targets and technologies for treating diseases. Optical microscopy provides a convenient way for imaging biological samples using readily available

dyes/antibodies. However, the spatial resolution of conventional optical microscopes is limited to 300 nm due to the diffraction of light waves. On the other hand, existing super resolution optical techniques, face challenges in scalability to thick tissues and require extremely expensive hardware, which limits their application.

Recently discovered expansion microscopy (ExM), which is based on physically expanding the sample (embedded in a swellable gel) by about 4.5x and thus, achieving an effective resolution of 70 nm, is scalable and compatible with conventional optical hardware. The resolution can be improved through iterative ExM (iExM). The iExM process requires transfer of biomolecules from one gel to another, with the cleaving of the first gel, which makes the process complex and potentially non-compatible for imaging of RNA or biomolecular retention for post-processing.

SUMMARY OF THE INVENTION

The present invention biological samples of interest that have been iteratively expanded more than once, in a method referred to herein as iterative direct expansion microscopy (id-ExM). In the id-ExM method, biological samples of interest are permeated with a swellable material that results in the sample becoming embedded in the swellable material, and then the sample can be expanded isotropically in three dimensions The process of iteratively expanding the samples can be applied to expand samples one or more additional times such that, for example, a 5-fold expanded sample can be expanded again to achieve high expansion factors, for example, 20x to lOOx or more linear expansion.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided to the Office upon request and payment of the necessary fee.

Fig. 1 A through Fig. ID- One schematic diagram of the id-ExM process (a)

Biomolecules shown in red are attached to the 1^st polymer network or gel (represented by blue lines) (b) After addition of water, the positive ions get washed away and the repulsion between negative ions causes the gel to expand (c) The 2^nd polymer network (represented by grey dashed lines) is integrated into the 1^st polymer (d) A force F that enables the 2^nd polymer to expand is applied. For example, the 2^nd polymer can be expanded due to electrostatic forces on application of water. As the 2^nd polymer expands, it exerts mechanical force on the 1^st polymer which expands along with the 2^nd polymer.

Fig. 2A through Fig. 2D - A workflow and concept of Iterative Direct Anchoring Expansion Microscopy (idExM). (A) Anchoring and gelation. Samples are contacted with biomolecule anchoring reagents to retain endogenous biomolecules. Subsequently, the sample is embedded in the swellable hydrogel, mechanically homogenized and expanded in water. (B) Re-embedding. The expanded gel is then re-embedded in a charge-neutral gel followed by the formation of a 2° swellable hydrogel within the re-embedded first gel. (C) The specimen is then expanded via the addition of water. (D) Post-expansion staining. Post expansion labels against proteins can be applied in the form of antibodies.

Fig. 3A through Fig. 3F- Nanoscale imaging of dense protein structures in synapse. Confocal images showing synaptic proteins Bassoon (green) and PSD95 (red) without (a), after 5x (b) and 20x (c) expansion in the mouse cortex (d), (e), and (f) are close-up views of images corresponding to (a), (b) and (c) respectively. Staining is done after expansion with off-the-shelf antibodies. All scale bars are post-expansion.

Fig. 4A through Fig. 4C- Nanoscale imaging of RNA in cultured cells. Wide-field image showing UbiquitinC RNA smFISH staining before (a) and after about lOx expansion (b). While the denser RNA domains are not resolved before expansion, they are clearly resolved after expansion as clear from the inset images (c) Wide-field image showing NEAT1 smFISH staining in the nuclei of cultured HeLa cells after ~10x expansion.

Maximum intensity projection (MIP) image, as well as representative images taken at different heights axially along the cluster, is shown. All scale bars are post-expansion.

Fig. 5A through Fig. 5E- Nanoscale RNA imaging in the mouse brain tissue (a) Wide-field image showing HCR-FISH staining performed against the 28s rRNA (red) in a Thyl-YFP mouse brain slice (cortex) after ~10x expansion. Representative images of 28s rRNA staining in neurons expressing YFP (green) is shown (b) Wide-field image showing the distribution of 28s rRNA in dendrites. Confocal images of HCR-FISH staining performed against the beta-actin mRNA with two color imaging shown in magenta (d) and red (e) in a neuron expressing GFP (c) in a Thyl-YFP mouse brain slice (cortex). All scale bars are post expansion.

Fig. 6A and Fig. 6B - Confocal image of mouse brain tissue after post-expansion staining with idExM.

Fig. 7A through Fig. 71 - Nanoscale-resolution imaging of synapses with idExM.

DETAILED DESCRIPTION OF THE INVENTION

As used herein and in the appended claims, the singular forms“a”,“an”, and“the” are defined to mean“one or more” and include the plural unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as“solely,”“only” and the like in connection with the recitation of claim elements or use of a“negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

The present invention provides compositions and methods that allow enlarging a sample of interest to be performed iteratively without the need for gel cleaving or transfer of biomolecules. This technology, referred to herein as iterative direct ExM or id-ExM, utilizes electrostatic and/or mechanical forces for enlarging a sample to achieve high expansion factors (e.g., 20x to lOOx) and enables super-resolution imaging without the need for any specialized or expensive hardware. By“enlarging a sample of interest” it is generally meant that the sample is physically expanded, or enlarged, relative to the sample prior to be exposed to the method(s) described herein. Enlarging the sample can be achieved by binding, e.g., anchoring, key biomolecules to a polymer network and swelling, or expanding, the polymer network, thereby moving the biomolecules apart as further described below. As the biomolecules are anchored to the polymer network isotropic expansion of the polymer network retains the spatial orientation of the biomolecules resulting in an expanded, or enlarged, sample.

In one embodiment, the invention provides methods for enlarging a sample of interest for microscopy, the method comprising the steps of:

the steps of:

a) contacting the sample with a biomolecule anchoring agent (BAA), wherein the BAA comprises a biomolecule binding moiety and an anchor;

b) embedding the sample in a swellable material;

c) subjecting the sample to a disruption of the endogenous physical structure of the sample;

d) swelling the swellable material resulting in an enlargement of the sample; e) re-embedding the enlarged sample in a non-swellable material to form an enlarged sample hybrid;

f) embedding the enlarged sample hybrid in second swellable material; and g) swelling the second swellable material to further enlarge the sample.

In one embodiment, steps (e) through (g) are repeated to form a further enlarged sample. This further enlarged sample may be further enlarged by the same process if desired.

As used herein, the term“sample of interest” generally refers to, but is not limited to, a biological, chemical or biochemical sample. In one embodiment, the sample of interest includes, but is not limited to, a tissue sample, a cell, or any components thereof.

A sample of interest is used herein in a broad sense and is intended to include sources that contain biomolecules and can be fixed. Exemplary tissue samples include, but are not limited to liver, spleen, kidney, lung, intestine, thymus, colon, tonsil, testis, skin, brain, heart, muscle and pancreas tissue. Other exemplary tissue samples include, but are not limited to, biopsies, bone marrow samples, organ samples, skin fragments and organisms. Materials obtained from clinical or forensic settings are also within the intended meaning of the term tissue sample. In one embodiment, the sample is derived from a human, animal or plant. In one embodiment, samples are human. The sample can be obtained, for example, from autopsy, biopsy or from surgery. It can be a solid tissue such as, for example, parenchyme, connective or fatty tissue, heart or skeletal muscle, smooth muscle, skin, brain, nerve, kidney, liver, spleen, breast, carcinoma (e.g. bowel, nasopharynx, breast, lung, stomach etc.), cartilage, lymphoma, meningioma, placenta, prostate, thymus, tonsil, umbilical cord or uterus. The tissue can be a tumor (benign or malignant), cancerous or precancerous tissue.

The sample can be obtained from an animal or human subject affected by disease or other pathology or suspected of same (normal or diseased), or considered normal or healthy. As used herein, the term“tissue sample” explicitly excludes cell-free samples, for example cell extracts, wherein cytoplasmic and/or nuclear components from cells are isolated.

Tissue samples suitable for use with the methods and systems described herein generally include any type of tissue samples collected from living or dead subjects, such as, e.g., biopsy specimens and autopsy specimens. Tissue samples may be collected and processed using the methods and systems described herein and subjected to microscopic analysis immediately following processing, or may be preserved and subjected to

microscopic analysis at a future time, e.g., after storage for an extended period of time. In some embodiments, the methods described herein may be used to preserve tissue samples in a stable, accessible and fully intact form for future analysis. For example, tissue samples, such as, e.g., human brain tissue samples, may be processed as described above and cleared to remove a plurality of cellular components, such as, e.g., lipids, and then stored for future analysis.

Tissues that have been preserved, or fixed, contain a variety of chemical

modifications that can reduce the detectability of proteins in biomedical procedures. In some embodiments, the methods and systems described herein may be used to analyze a previously-preserved or stored tissue sample. Previously preserved tissue samples include, for example, clinical samples used in pathology including formalin-fixed paraffin-embedded (FFPE), hematoxylin and eosin (H&E)-stained, and/or fresh frozen tissue samples. If the previously preserved sample has a coverslip, the coverslip should be removed. The sample is treated to remove the mounting medium. Such methods for removing the mounting medium are well known in the art. For example, treating the sample with xylene to remove paraffin or other hydrophobic mounting medium.

Alternatively, if the sample is mounted in a water-based mounting medium, the sample is treated with water. The sample is then then rehydrated and subjected to antigen- retrieval. The term“antigen retrieval” refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored such as, but not limited to, enzyme induced epitope retrieval, heat induced epitope retrieval (HIER), or proteolytic induced epitope retrieval (PIER). For example, the antigen retrieval treatment can be performed in a 10 mM sodium citrate buffer as well as the commercially available Target Retrieval Solution (DakoCytomation) or such.

In one embodiment, the cell or tissue sample can be labeled or tagged with a detectable label. Typically, the label or tag will bind chemically (e.g., covalently, hydrogen bonding or ionic bonding) to a biomolecule of the sample, or a component thereof. The detectable label can be selective for a specific target (e.g., a biomarker or class of molecule), as can be accomplished with an antibody or other target specific binder. The detectable label may comprise a visible component, as is typical of a dye or fluorescent molecule; however, any signaling means used by the label is also contemplated. A fluorescently labeled biological sample, for example, is a biological sample labeled through techniques such as, but not limited to, immunofluorescence, immunohistochemical or immunocytochemical staining to assist in microscopic analysis. In one embodiment, the detectable label is chemically attached to the biological sample, or a targeted component thereof. In one embodiment, the detectable label is an antibody and/or fluorescent dye wherein the antibody and/or fluorescent dye, further comprises a physical, biological, or chemical anchor or moiety that attaches or crosslinks the cell or tissue sample to the polymer. The labeled sample may furthermore include more than one label. For example, each label can have a particular or distinguishable fluorescent property, e.g., distinguishable excitation and emission wavelengths. Further, each label can have a different target specific binder that is selective for a specific and

distinguishable target in, or component of the sample.

In one embodiment, the biomolecule anchoring agent (BAA) is a bi-functional linker wherein the bi-functional linker comprises a binding moiety and anchor, wherein the binding moiety binds to biomolecules in the sample. The anchor may be a physical, biological, or chemical moiety that attaches or crosslinks the sample to the swellable material. This may be accomplished by crosslinking the anchor with the swellable material, such as during or after the polymerization, i.e., in situ formation of the swellable material.

In some embodiments, the anchor may comprise a polymerizable moiety. The anchor may include, but is not limited to, vinyl or vinyl monomers such as styrene and its derivatives (e.g., divinyl benzene), acrylamide and its derivatives, butadiene, acrylonitrile, vinyl acetate, or acrylates and acrylic acid derivatives. The polymerizable moiety may be, for example, an acrylamide modified moiety that may be covalently fixed within a swellable material. In some embodiments, the biomolecule anchoring agent (BAA) comprises reactive groups to functional groups (e.g., primary amines or sulfhydryls) on biomolecules within the sample. The BAA may be used to chemically modify the amine group of biomolecules with a swellable material functional group, which enables antibodies and/or other endogenous biomolecules within the sample to be directly anchored to, or incorporate into, the swellable material.

In one embodiment, the biomolecule anchoring agent (BAA) is a hetero-bifunctional crosslinker. Hetero-bifunctional crosslinkers possess different reactive groups at either end of a spacer arm, i.e., atoms, spacers or linkers separating the reactive groups. These reagents not only allow for single-step conjugation of molecules that have the respective target functional group, but they also allow for sequential (two-steps) conjugations that minimize undesirable polymerization or self-conjugation. The biomolecule anchoring agent (BAA) may be a small molecule linker or a nucleic acid adaptor.

As used herein, a“nucleic acid adaptor” is a nucleic acid sequence having a binding moiety capable of attaching to a target nucleic acid and an anchor moiety capable of attaching to the swellable material. Attaching the nucleic acid adaptor to a target nucleic acid may be accomplished by hybridization or by ligation in situ. For example, DNA adaptors may be ligated to the 3’ ends of the RNAs in the sample with RNA ligases, such as T4 RNA ligase, or may be attached via a chemical linker such as a reactive amine group capable of reacting with target nucleic acid. Acrylamide modified oligonucleotide primers may be covalently fixed within a swellable material such as a polyacrylate gel. As used herein, the term

"acrylamide modified" in reference to an oligonucleotide means that the oligonucleotide has an acrylamide moiety attached to the 5' end of the molecule.

As used herein, a“small molecule linker” is a small molecule having a binding moiety capable of attaching to a target nucleic acid and an anchor moiety capable of attaching to the swellable material. Attaching the small molecule linker to the target nucleic acid may be accomplished by hybridization or by a chemical reactive group capable of covalently binding the target nucleic acid. For example, LABEL-IT^® Amine (MirusBio) is a small molecule with alkylating group that primarily reacts to the N7 of guanine, thereby allowing covalent binding of RNA and DNA. The small molecule linker may be, for example, acrylamide modified and therefore may be covalently fixed within a swellable material. As used herein, the term "acrylamide modified" in reference to a small molecule linker means that the small molecule linker has an acrylamide moiety. In one embodiment, the biomolecule anchoring agent (BAA) may comprise a protein- reactive chemical moiety and an anchor. The protein-reactive chemical group includes, but is not limited to, N-hydroxysuccinimide (NHS) ester, thiol, amine, maleimide, imidoester, pyridyldithiol, hydrazide, phthalimide, diazirine, aryl azide, isocyanate, or carboxylic acid, which, for example, can be reacted with amino or carboxylic acid groups on proteins or peptides. In one embodiment, the protein-reactive groups include, but are not limited to, N- succinimidyl ester, pentafluorophenyl ester, carboxylic acid, or thiol.

In one embodiment, the protein-reactive chemical group is a succinimidyl ester of 6- ((acryloyl)amino)hexanoic acid (acryloyl-X, SE; abbreviated“AcX”; Life Technologies). Treatment with AcX modifies amines on proteins with an acrylamide functional group. The acryi amide functional groups allows for proteins to be anchored to the swellable polymer as it is synthesized in situ.

In one embodiment, the proteins of the sample can be modified with the protein- reactive group and the anchor in separate steps using click chemistry. Click chemistry, also referred to as tagging, is a class of biocompatible reactions intended primarily to join substrates of choice with specific biomolecules. In this method, proteins of the sample of interest are treated with a protein-reactive group comprising a click group and then treated with an anchor comprising a complementary click group. Complementary groups include, but are not limited to, azide groups and terminal alkynes (see e.g., H. C. Kolb; M. G. Firm; K. B. Sharpless (2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie International Edition. 40(11): 2004 2021, which is incorporated herein by re ference).

As used herein, the term“attach” or“attached” refers to both covalent interactions and noncovalent interactions. In certain embodiments of the invention, covalent attachment may be used, but generally all that is required is that the bi-functional linker remain attached to the target nucleic acid under conditions for nucleic acid amplification and/or sequencing. Oligonucleotide adaptors may be attached such that a 3' end is available for enzymatic extension and at least a portion of the sequence is capable of hybridizing to a complementary sequence. Attachment can occur via hybridization to the target nucleic acid, in which case the attached oligonucleotide may be in the 3 '-5' orientation. Alternatively, attachment can occur by means other than base-pairing hybridization, such as the covalent attachment set forth above. The term“attach” may be used interchangeably herein with the terms, “anchor(ed)”, affix(ed), link(ed) and immobilize(d). As used herein, the terms“swellable material” and“swellable polymer” are used interchangeably and generally refers to a material that expands when contacted with a liquid, such as water or other solvent. Additionally, or alternatively, the swellable material can be expanded by any other means known to one of skill in the art. In some embodiments, the swellable material uniformly expands in three dimensions. Additionally, or alternatively, the material is transparent such that, upon expansion, light can pass through the sample. The first swellable material and the second swellable material may be the same or different swellable materials.

In one embodiment, the swellable material is formed in situ from precursors thereof. Embedding the sample in the swellable material comprises permeating the sample with a composition comprising one or more precursors thereof throughout the sample and polymerizing and/or crosslinking the monomers or precursors to form the swellable material. In this manner the sample of interest is embedded in the swellable material.

In one embodiment, the sample of interest and each iteratively enlarged sample is permeated with one or more monomers or precursors or a solution comprising one or more monomers or precursors which are then reacted (e.g., polymerized) to form a swellable or non-swellable material depending on what step of the method is being performed.

By“precursors of a swellable material” it is meant hydrophilic monomers, prepolymers, or polymers that can be crosslinked, or“polymerized”, to form a three- dimensional (3D) hydrogel network. Precursors can also comprise polymerization initiators and crosslinkers.

In one embodiment the swellable material is a polyelectrolyte. In one embodiment, the swellable material is polyacrylate or polyacrylamide and copolymers or crosslinked copolymers thereof.

In some embodiments, one or more polymerizable materials, monomers or oligomers can be used, such as monomers selected from the group consisting of water soluble groups containing a polymerizable ethylenically unsaturated group. Monomers or oligomers can comprise one or more substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinylalcohols, vinylamines, allylamines, allylalcohols, including divinylic crosslinkers thereof (e.g., N, N-alkylene bisacrylamides).

In one embodiment, the precursor of the swellable material comprises at least one polyelectrolyte monomer and a covalent crosslinker. In one embodiment, the swellable material is a hydrogel. In one embodiment, the hydrogel is a polyacrylate hydrogel. In one embodiment, the precursor of the swellable material comprises acrylate, acrylamide, and a crosslinker selected from N, N-m ethyl enebi sacry 1 a i de (BIS), N,N’-(1,2- Dihydroxythylene)bisacryalmide) (DHEBA); and N,N’-Bis(acryloyl)cystamine (BAC).

The precursors of the swellable polymer may be delivered to the biological specimen by any convenient method including, but not limited to, permeating, perfusing, infusing, soaking, adding or other intermixing the sample with the precursors of swellable material. In this manner, the biological specimen is saturated with precursors of the swellable material, which flow between and around biomolecules throughout the specimen.

Following permeating the specimen, the swellable polymer precursors are

polymerized, i.e., covalently or physically crosslinked, to form a polymer network. The polymer network is formed within and throughout the specimen. In this manner, the biological specimen is saturated with the swellable material, which flow between and around biomolecules throughout the specimen.

Polymerization may be by any method including, but not limited to, thermal crosslinking, chemical crosslinking, physical crosslinking, ionic crosslinking, photo crosslinking, irradiative crosslinking (e.g., x-ray, electron beam), and the like, and may be selected based on the type of hydrogel used and knowledge in the art. In one embodiment, the polymer is a hydrogel. Once polymerized, a polymer-embedded biological specimen is formed.

In one embodiment, the swellable polymer is polyacrylate and copolymers or crosslinked copolymers thereof. For example, if the biological sample is to be embedded in sodium polyacrylate, a solution comprising the monomers sodium acrylate and acrylamide, and a crosslinker selected from A,/V-methylenebisacrylamide (BIS), N,N’-(1,2- Dihydroxythylene)bisacrylamide), and (DHEBA) N,N’-Bis(acryloyl)cystamine (BAC), are perfused throughout the sample.

In one embodiment, the swellable material is a swellable polymer or hydrogel. The hydrogel may be a poly electrolyte hydrogel. The poly electrolyte may be a polyacrylate.

By embedding a specimen in a swellable polymer that physically supports the ultrastructure of the specimen this technology preserves the biomolecules (e.g., proteins, small peptides, small molecules, and nucleic acids in the specimen) in their three-dimensional distribution, secured by the polymer network. By bypassing destructive sectioning of the specimen, subcellular structures may be analyzed. In addition, the specimen can be iteratively stained, unstained, and restained with other reagents for comprehensive analysis.

In some embodiments, native proteins anchored to the swellable polymer perfused throughout the sample as described herein can retain epitope functionality and be labeled post-expansion if the nonspecific proteolysis of ExM is replaced with modified post-gelation homogenization treatments. Such approaches may overcome the limitations inherent to delivering antibodies in the crowded environment of native tissue.

In some embodiments, the composition can comprise a detectable label, tag or other feature of interest (for example, fluorescent dye molecules that have been delivered to the biological sample via antibody staining) which can be anchored (e.g., chemically) into the hydrogel before expansion. Following anchoring, the sample is subjected to a disruption of the underlying network of biological molecules, leaving the tags of interest (e.g., the fluorescent dye molecules) intact and anchored to the gel. In this way, the mechanical properties of the swellable material-sample complex are rendered more spatially uniform, allowing isotropic expansion with minimal artifacts.

In one embodiment, the sample is anchored to the swellable material before expansion. This can be accomplished by chemically crosslinking the polymerizable moiety of the biomolecule anchoring agent with the swellable material, such as during or after the polymerization or in situ formation of the swellable material.

“Re-embedding” the expanded sample in a non-swellable material (also referred to as a re-embedding gel) comprises permeating (such as, perfusing, infusing, soaking, adding or other intermixing) the sample with the non-swellable material, preferably by adding precursors thereof. Alternatively, or additionally, embedding the sample in a non-swellable material comprises permeating one or more monomers or other precursors throughout the sample and polymerizing and/or crosslinking the monomers or precursors to form the non- swellable material or polymer. In this manner the first enlarged sample, for example, is embedded in the non-swellable material. Embedding the expanded sample in a non-swellable material prevent conformational changes (e.g., shrinkage) during the following steps despite salt concentration variation. The non-swellable material can be charge-neutral hydrogels. For example, it can be polyacrylamide hydrogel, composed of acrylamide monomers, bisacrylamide crosslinker, ammonium persulfate (APS) initiator and

tetramethylethylenediamine (TEMED) accelerator.

In certain embodiments, the sample of interest, or a labeled sample, can, optionally, be treated with a detergent prior to being contacted with the one or more swellable material precursors. The use of a detergent can improve the wettability of the sample or disrupt the sample to allow the one or more swellable monomer precursors to permeate throughout sample. After the sample has been anchored to the polymer, the sample may be subjected to a disruption of the endogenous biological molecules or the physical structure of the biological sample. The disruption of the endogenous physical structure of the sample or of the endogenous biomolecules of the sample generally refers to the mechanical, physical, chemical, biochemical or, enzymatic digestion, disruption or break up of the sample so that it will not resist expansion. In this way, the mechanical properties of the sample-swellable material complex are rendered more spatially uniform, allowing greater and more consistent isotropic expansion.

It is preferable that the disruption does not impact the structure of the swellable material but disrupts the structure of the sample. Thus, the sample disruption should be substantially inert to the swellable material. The degree of digestion can be sufficient to compromise the integrity of the mechanical structure of the sample or it can be complete to the extent that the sample-swellable material complex is rendered substantially free of the sample.

In one embodiment, the physical disruption of the sample is accomplished by a mild disruption treatment that minimizes damage to the individual proteins, allowing staining and other treatments on the proteins to be carried out after expansion. In some embodiments, such milder treatment is performed by using LyC. In some embodiments, such milder treatment is performed by heating the sample. In some embodiments, heating the sample is performed by autoclaving the sample.

The expandable cell or tissue sample can be expanded by contacting the sample- polymer complex with a solvent or liquid to cause the polymer to swell. By expanding, or swelling, the expandable sample it is generally meant that the sample is physically expanded, or enlarged, relative to the sample prior to be exposed to the method(s) described herein.

The swelling of the swellable material results in the sample itself expanding (e.g., becoming larger). This is because the swellable material is embedded throughout the sample, therefore, by binding, e.g., anchoring, biomolecules to the swellable material and swelling, or expanding, the swellable material, the biomolecules are thereby moved apart. In one embodiment, the swellable material expands (swells) isotropically. As the biomolecules are anchored to the polymer network isotropic expansion of the polymer network retains the spatial orientation of the biomolecules resulting in an expanded, or enlarged, sample.

The expanded sample can then be subjected to microscopic analysis. By

“microscopic analysis” it is meant the analysis of a sample using any technique that provides for the visualization of aspects of a sample that cannot be seen with the unaided eye, i.e., that are not within the resolution range of the normal eye.

The expanded sample-polymer complex can be imaged on any optical microscope, allowing effective imaging of features below the classical diffraction limit. Since the resultant expanded sample can be transparent, custom microscopes capable of large volume, wide field of view, 3D scanning may also be used in conjunction with the expanded sample.

Because biomolecules of the sample are anchored to a polymer that physically supports the ultrastructure of the sample, cellular components (e.g. lipids) that normally provide structural support but that hinder visualization of subcellular proteins and molecules may be removed while preserving the 3-dimensional architecture of the cells and tissue. This removal renders the interior of sample substantially permeable to light and/or

macromolecules, allowing the interior of the sample, e.g. cells and subcellular structures, to be microscopically visualized without time-consuming and disruptive sectioning.

Additionally, the sample can be iteratively stained, unstained, and re-stained with other reagents for comprehensive analysis.

By“biomolecules” it is generally meant, but not limited to, proteins, lipids, steroids, nucleic acids, and sub-cellular structures within a tissue or cell.

By“macromolecules” is meant proteins, nucleic acids, or small molecules that target biomolecules within the sample. These macromolecules are used to detect biomolecules within the sample and/or anchor the biolmolecules to the swellable polymer. For example, macromolecules may be provided that promote the visualization of particular cellular biomolecules, e.g., proteins, lipids, steroids, nucleic acids, etc. and sub-cellular structures. In some embodiments, the macromolecules are diagnostic. In some embodiments, the macromolecules are prognostic. In some embodiments, the macromolecules are predictive of responsiveness to a therapy. In some embodiments, the macromolecules are candidate agents in a screen, e.g., a screen for agents that will aid in the diagnosis and/or prognosis of disease, in the treatment of a disease, and the like.

As an example, the sample may be contacted with one or more polypeptide macromolecules, e.g. antibodies, labeled peptides, and the like, that are specific for and will bind to particular cellular biomolecules for either direct or indirect labeling by color or immunofluorescence. By immunofluorescence it is meant a technique that uses the highly specific binding of an antibody to its antigen or binding partner in order to label specific proteins or other molecules within the cell. A sample is treated with a primary antibody specific for the biomolecule of interest. A fluorophore can be directly conjugated to the primary antibody or peptide. Alternatively, a secondary antibody conjugated to a detection moiety or fluorophore, which binds specifically to the first antibody can be used. Peptides that are specific for a target cellular biomolecule and that are conjugated to a fluorophore or other detection moiety may also be employed.

Another example of a class of agents that may be provided as macromolecules is nucleic acids. For example, a sample may be contacted with an antisense RNA that is complementary to and specifically hybridizes to a transcript of a gene of interest, e.g., to study gene expression in cells of the sample. As another example, a sample may be contacted with a DNA that is complementary to and specifically hybridizes to genomic material of interest, e.g., to study genetic mutations, e.g., loss of heterozygosity, gene duplication, chromosomal inversions, and the like. The hybridizing RNA or DNA is conjugated to detection moieties, i.e., agents that may be either directly or indirectly visualized

microscopically. Examples of in situ hybridization techniques may be found at, for example, Harris and Wilkinson. In situ hybridization: Application to developmental biology and medicine, Cambridge University Press 1990; and Fluorescence In Situ Hybridization (FISH) Application Guide. Liehr, T, ed., Springer-Verlag, Berlin Heidelberg 1990.

In some embodiments, the fixed biological sample is subjected to passivation. As used herein the term“passivation” refers to the process for rendering the sample less reactive with the components contained within the fixative such as by functionalizing the fixative with chemical reagents to neutralize charges within. For example, the carboxylic groups of acrylate, which may be used in the swellable gel, can inhibit downstream enzymatic reactions. Treating the swellable gel composed of acrylate with l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) allows primary amines to covalently bind the carboxylic groups to form charge neutral amides and passivate the swellable gel.

The innovation enables physical expansion of common clinical tissue sample based on the unique physical and chemical properties of clinical tissue samples. Clinical tissue samples are usually highly fixed, tightly attached on the superfrost glass slides, and embedded in the paraffin (or stained and mounted in a mounting medium) for long-term storage. Some clinical tissue samples are stained with dyes, such as hematoxylin and eosin (H&E), which are incompatible with fluorescence imaging. To apply ExM to clinical samples, de-paraffmization, antigen retrieval and aggressive protease digestion are integrated in a comprehensive workflow to handle various kinds of common clinical samples. De- paraffmization and antigen retrieval address the recovery of archived clinical samples, while aggressive protease digestion is critical for the success of sample expansion, as most of the human tissues contain abundant hard-to-digest structural proteins, such as collagen and fibronectin, which prevent homogeneous expansion of the sample. Taken together, the present invention allows for the application of ExM to the enormous amount of archived clinical samples and enable super-resolution optical interrogations of mechanisms of a broad range of diseases by conventional optical microscopy.

This invention provides a comprehensive workflow to facilitate expansion of common types of clinical samples for super-resolution molecular imaging. The methods described herein will result in optimal outcomes, such as proper immunostaining, sufficient digestion of tissue, high quality of polymer synthesis, and maintenance of proteins of interest during expansion.

The invention also describes the reutilization of classic H&E stained slides for further biomolecular interrogation in nanoscale level. In general, H&E stained slides are not considered suitable for further downstream processing due to the difficulty in removing the stain and mounting medium. Thus, the invention describes a unique and cost-effective approach to overcome this barrier and enable the extraction of more information from the used H&E slides. In one embodiment, the method of expanding H&E stained slides for further utilization combines xylene-ethanol-water sequential washing, protein anchoring and in situ polymer synthesis.

The subject methods find many uses. For example, the subject methods may be applied to preparing specimens for the study of the connectivity of the central nervous system.“Connectivity” as used herein generally means the connections between neurons, and includes connections at the single cell level, e.g., synapses, axon termini, dendritic spines, etc., as well as connections between groups of neurons and regions of the CNS as major axon tracts, e.g., corpus callosum (CC), anterior commissure (AC), hippocampal commissure (HC), pyramidal decussation, pyramidal tracts, external capsule, internal capsule (IC), cerebral peduncle (CP), etc. A whole brain and/or spinal cord specimen or region thereof (e.g. cerebrum (i.e., cerebral cortex), cerebellum (i.e., cerebellar cortex), ventral region of the forebrain (e.g., striatum, caudate, putamen, globus pallidus, nucleus accumbens; septal nuclei, subthalamic nucleus); regions and nuclei of the thalamus and hypothalamus; regions and nuclei of the deep cerebellum (e.g., dentate nucleus, globose nucleus, emboliform nucleus, fastigial nucleus) and brainstem (e.g., substantia nigra , red nucleus, pons, olivary nuclei, cranial nerve nuclei); and regions of the spine (e.g., anterior horn, lateral horn, posterior horn)) may be prepared post-mortem by the subject methods and the connectivity of the neurons therein microscopically analyzed, e.g., obtained, stored, rendered, used, and actuated, e.g., to provide the full connectivity of a brain, e.g., a human brain, after death. Such studies will contribute greatly to the understanding of how the brain develops and functions in health and during disease, and of the underpinnings of cognition and personality.

As another example, the subject methods may be employed to evaluate, diagnose or monitor a disease.“Diagnosis” as used herein generally includes a prediction of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identification of cancerous states, stages of cancer, likelihood that a patient will die from the cancer), prediction of a subject's responsiveness to treatment for a disease or disorder (e.g., a positive response, a negative response, no response at all to, e.g., allogeneic hematopoietic stem cell transplantation, chemotherapy, radiation therapy, antibody therapy, small molecule compound therapy) and use of therametrics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy). For example, a biopsy may be prepared from a cancerous tissue and microscopically analyzed to determine the type of cancer, the extent to which the cancer has developed, whether the cancer will be responsive to therapeutic intervention, etc.

Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term "detecting" may also optionally encompass any of the above.

As another example, a biopsy may be prepared from a diseased tissue, e.g. kidney, pancreas, stomach, etc., to determine the condition of the tissue, the extent to which the disease has developed, the likelihood that a treatment will be successful, etc. The terms “treatment”,“treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. The terms“individual,”“subject,” “host,” and“patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Examples of diseases that are suitable to evaluation, analysis, diagnosis, prognosis, and/or treatment using the subject methods and systems include, but are not limited to, cancer, immune system disorders, neuropsychiatric disease, endocrine/reproductive disease,

cardiovascular/pulmonary disease, musculoskeletal disease, gastrointestinal disease, and the like.

The subject methods may also be used to evaluate normal tissues, organs and cells, for example to evaluate the relationships between cells and tissues of a normal tissue sample, e.g., a tissue sample taken from a subject not known to suffer from a specific disease or condition. The subject methods may be used to investigate, e.g., relationships between cells and tissues during fetal development, such as, e.g., during development and maturation of the nervous system, as well as to investigate the relationships between cells and tissues after development has been completed, e.g., the relationships between cells and tissues of the nervous systems of a fully developed adult sample.

The subject methods also provide a useful system for screening candidate therapeutic agents for their effect on a tissue or a disease. For example, a subject, e.g. a mouse, rat, dog, primate, human, etc. may be contacted with a candidate agent, an organ or a biopsy thereof may be prepared by the subject methods, and the prepared sample microscopically analyzed for one or more cellular or tissue parameters. Parameters are quantifiable components of cells or tissues, particularly components that can be accurately measured, desirably in a high throughput system. The subject methods may also be used to visualize the distribution of genetically encoded markers in whole tissue at subcellular resolution, for example, chromosomal abnormalities (inversions, duplications, translocations, etc.), loss of genetic heterozygosity, the presence of gene alleles indicative of a predisposition towards disease or good health, likelihood of responsiveness to therapy, ancestry, and the like. Such detection may be used in, for example, diagnosing and monitoring disease as, e.g., described above, in personalized medicine, and in studying paternity.

The present invention will be better understood in connection with the following Examples. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention. Various changes and modifications will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims.

Examples

Example-1. id-ExM

The mechanism of id-ExM and concept of electrostatic and mechanical expansion is illustrated through the diagram as shown in Fig. 1. When the 1st gel is cast, the positive and negative ions are in a state of equilibrium (Fig. la). When water is added to the gel, the positive sodium ions get washed away and the negative ions in the polymer network repel each other (Fig. lb), causing the gel to expand. At this stage the gel is in equilibrium again and will not expand further. The 1 st polymer network has still room to stretch out and expand but since electrostatic equilibrium is reached, there is no net force available to stretch the polymer further. Integrating a 2nd polymer network into the 1st polymer network (Fig. lc), such that the 2nd polymer is expandable then, as the 2nd polymer expands, it will exert mechanical force on the 1st polymer, which will thus expand along with the 2nd polymer (Fig. Id). Throughout this process the biomolecules remain attached to the 1st polymer network and there is no need to transfer the biomolecules to the second polymer.

Example 2 - Iterative-Direct Expansion (idExM) of Tissue Sections

Figure 2 provides an example workflow of Iterative Direct Anchoring Expansion Microscopy (idExM). In this embodiment, the process of idExM begins with a fixation step that equips proteins with a biomolecule anchoring agent (e.g., a gel-anchorable chemical groups). Samples are contacted with biomolecule anchoring reagents to retain endogenous biomolecules, with acrylamide added during fixation to retain proteins. Subsequently, the sample is embedded in the swellable hydrogel, mechanically homogenized and expanded in water. (B) Re-embedding. The expanded gel is then re-embedded in a charge-neutral gel followed by the formation of a 2^nd swellable hydrogel within the re-embedded first gel. (C) The specimen is then expanded via the addition of water. (D) Post-expansion staining. Post expansion labels against proteins can be applied in the form of antibodies.

Briefly, to prepare a sample, in this example a mouse brain section, mice are transcardially perfused with a fixative consisting of 4% Paraformaldehyde and 30%

Acrylamide in lx PBS, after which the brain is removed and sectioned on a vibratome at a thickness of 50-100 pm. Alternatively, fresh-frozen sections are fixed with the same fixative. After fixation, tissue sections are incubated with the idExM 1^st gel solution (8.625% (w/v) sodium acrylate, 2.5% (w/v) acrylamide, 0.075% (w/v) N,N'-methylenebisacrylamide, 0.2% (w/v) ammonium persulfate (APS) initiator, 0.2% (w/v) tetramethylethylenediamine

(TEMED) accelerator, 0.2% (w/v), 0.01% Hydroxy-TEMPO) at 4 °C for 30 minutes. Tissue sections are then embedded in the 1^st idExM gel by incubating them in an enclosed chamber surrounded by excess 1^st gel solution at 37 °C for two hours. After the formation of the 1^st gel, the tissue-gel hybrid is expanded by incubating it in a denaturation buffer (200 mM SDS, 200 mM NaCl, and 50 mM Tris pH 9) at 95 °C for one hour, and then fully expanded via repeated washes with deionized water. The expanded gel is then re-embedded by incubating for two hours at room temperature in the idExM re-embedding solution (13.75% (w/v) acrylamide, 0.038% (w/v) N,N'-methylenebisacrylamide, 0.025% (w/v) ammonium persulfate (APS) initiator, 0.025% (w/v) tetramethylethylenediamine (TEMED) accelerator) while shaking, followed by incubation in a chamber with excess re-embedding solution for two hours at 45 °C. The re-embedded gel is then washed several times with lx PBS.

Subsequently, the re-embedded gel is incubated for two hours in the idExM 3^rd gel solution (8.625% (w/v) sodium acrylate, 2.5% (w/v) acrylamide, 0.038% (w/v) N,N'- methylenebisacrylamide, 0.025% (w/v) ammonium persulfate (APS) initiator, 0.025% (w/v) tetramethylethylenediamine (TEMED) accelerator, lx PBS). The 3^rd idExM gel is then formed by placing the re-embedded gel in an enclosed chamber at 60 °C for one hour. After this step, the gel can be fully expanded in water and trimmed axially as needed to reduce thickness to facilitate subsequent immunostaining and imaging. Immunostaining is then performed as needed with the appropriate antibodies and blocking solution. Both primary and secondary antibodies are incubated at 4 °C overnight to allow sufficient antibody penetration into the gel. Example-3. Nanoscale imaging of dense protein structures in synapse

Apart from the general proteomic architecture, biological systems frequently consist of extremely-dense and complex protein networks such as those in synapses. Understanding in detail, the proteomic organization of synapse is of great interest as it regulates synaptic transmission and slight variations in the protein organization has been implicated in neural diseases. idExM was employed prepared mouse brain slices to decipher the nanoscale structure of synapses. The sample was stained with primary and secondary antibodies after the expansion process. Fig. 2 shows the imaging of two synaptic proteins bassoon (pre- synaptic) and PSD-95 (post-synaptic) in the mouse brain cortex. While before expansion or with 4.5x expansion, the synaptic organization of these two proteins is not clearly observable, after 20x expansion, the two proteins are distinctively resolved.

Example-4 Nanoscale imaging of RNA in cultured cells

Nanoscale resolution imaging of RNA is critical for identifying cell types, distinguish between normal and pathological states of cell as well as understanding local RNA

processing. Apart from proteomic mapping, idExM was applied for nanoscale imaging of RNA. For high-resolution imaging of RNA, a bifunctional crosslinker (such as the mixing of Label-IT Amine and 6-((acryloyl)amino) hexanoic acid which provide an alkylating group that primarily reacts to the N7 of guanine and an acrylamide moiety that can get incorporated in the swellable material during or after polymerization) was used to anchor the nucleic acid to the swellable material network. Read out of the RNA was done using fluorescent in-situ hybridization (FISH) technique. Fig. 3 shows the wide-field image showing UbiquitinC RNA smFISH staining before (a) and after about lOx expansion (b). While the denser RNA domains are not resolved before expansion, they are clearly resolved after expansion as clear from the inset images. Fig. 3(c) shows the wide-field image of NEATl smFISH staining in the nuclei of cultured HeLa cells after ~10x expansion. Maximum intensity projection (MIP) image, as well as representative images taken at different heights axially along the cluster, is shown. These images illustrate the capability of idExM in nanoscale imaging of RNA.

Example-5. Nanoscale RNA imaging in the mouse brain tissue.

Apart from cultured cells, idExM was applied to image RNA in mouse brain tissue samples, which were then subjected to FISH staining. Fig. 4 (a) show wide-field image of HCR-FISH staining performed against the 28s rRNA (red) in a Thyl-YFP mouse brain slice (cortex) after ~10x expansion. Representative images of 28s rRNA staining in neurons expressing YFP (green) is shown (b) Wide-field image showing the distribution of 28s rRNA in dendrites. Confocal images of HCR-FISH staining performed against the beta-actin mRNA with two color imaging are shown in magenta (d) and red (e) in a neuron expressing GFP (c) in a Thy 1 -YFP mouse brain slice (cortex). These images illustrate that it is not only possible to use idExM for nanoscale imaging of RNA in cultured cells but also in mouse brain tissue.

Example 6

Mouse brain tissue was prepared by idExM according to Example 2.

Figure 6A and 6B depict confocal images of mouse brain tissue after post-expansion staining with idExM. (A) Low-magnification widefield image of a mouse brain slice stained with DAPI showing the somatosensory cortex region imaged after expansion in (B). (B) Confocal image of Layer 1 somatosensory cortex after post-expansion immunostaining with antibodies against P/Q-type calcium channel (red), PSD-95 (green) and RIMl/2 (blue) (i-iv) shows confocal images of highlighted four regions.

Figure 7A through Fig. 71 depict nanoscale-resolution imaging of synapses with idExM. (a-b) provide a low-magnification widefield image of a mouse brain slice with DAPI staining showing the somatosensory cortex region. Confocal images of the specimen after immunostaining with antibodies against calcium channel (c), RIMl/2 (d), PSD95 (e), Syngap (f), Homerl (g), Bassoon (h), and Shank3 (i). For pre-expansion staining, primary and secondary antibodies were applied before expansion, secondary antibodies were anchored to the gel by AcX treatment, and tertiary antibodies were applied after expansion to visualize the pre-expansion staining. For post-expansion staining, the same primary and secondary antibodies were applied after expansion. Antibodies against Shank3 (c, d, f, g) or Homerl (e, h, i) were used as a reference channel after expansion to help identify putative synapses. Confocal images of Cortex Layers 2/3 (top) showing merged pre-, post-expansion staining and reference channels. Below f-i, zoomed-in images of putative synapses (i-iii) show separate channels for pre-expansion staining (green), post-expansion staining (red), reference staining (cyan), and merge.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. It should also be understood that the preferred embodiments described herein are not mutually exclusive and that features from the various preferred embodiments may be combined in whole or in part in accordance with the invention.

Claims

CLAIMS What is claimed is:

1. A method for iteratively enlarging a sample of interest for microscopy, the method comprising the steps of:

a) contacting the sample with a biomolecule anchoring agent (BAA), wherein the BAA comprises a biomolecule binding moiety and an anchor;

b) embedding the sample in a first swellable material;

c) subjecting the sample to a disruption of the endogenous physical structure of the sample;

d) swelling the swellable material resulting in an enlargement of the sample; e) re-embedding the enlarged sample in a non-swellable material to form an enlarged sample hybrid;

f) embedding the enlarged sample hybrid in second swellable material; and g) swelling the second swellable material to further enlarge the sample.

2. The method according to claim 1, wherein embedding the sample in the first swellable material in step b) comprises permeating the sample with a composition comprising one or more water soluble monomer precursors; and polymerizing the composition within the sample to form the first swellable material, wherein said polymerizing results in anchoring of the biomolecules of the sample to the first swellable material to form a sample-swellable material complex.

3. The method according to claim 1, wherein embedding the sample in the second

swellable material in step f) comprises permeating the sample with a composition comprising one or more water soluble monomer precursors; and polymerizing the composition within the sample to form the second swellable material, wherein the biomolecules remain anchored to the first swellable material.

4. The method according to claim 2 or 3, wherein the composition comprises at least one polyelectrolyte monomer and a covalent crosslinker.

5. The method according to claim 2 or 3, wherein the swellable material is a hydrogel.

6. The method according to claim 5, wherein the hydrogel is a polyacrylate hydrogel.

7. The method according to claim 6, wherein the solution comprises acrylate, acrylamide, and a crosslinker selected from N, N-m ethyl enebi sacry 1 a i de (BIS), N,N’-(1,2- Dihydroxythylene)bisacryalmide)(DHEBA); and N,N’-Bis(acryloyl)cystamine (BAC).

8. The method of claim 1, where embedding the enlarged sample in a non-swellable

material comprises permeating the sample with a composition comprising precursors of a non-swellable material and polymerizing the composition within the sample to form a non-swellable material.

9. The method of claim 1, wherein the second swellable material is the same as the first.

10. The method of claim 1, wherein the second swellable material is different than the first.

11. The method of claim 1, wherein prior to step g) a slice of the sample is taken and

swelled.

12. The method according to claim 1, wherein swelling the swellable material comprises adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex, which results in an enlarged sample.

13. The method of claim 12, wherein the aqueous solvent or liquid is water.

14. The method of claim 1, further comprising the step of producing a high-resolution image of the sample by viewing the enlarged sample under a microscope.

15. The method of claim 1, further comprising the step of optically imaging the enlarged sample by viewing the sample under a microscope.