Cryoprotection of tissues

Abstract

This disclosure relates to a novel procedure for cryoprotection of tissues for imaging procedures such as correlative light and electron microscopy (CLEM) and immunogold electron microscopy (iEM) and fluorescence imaging, in which harvested organs or tissues are perfusion-fixed with dimethyl sulfoxide (DMSO) and freeze-substituted with organic solvents to reliably freeze larger tissue blocks while preserving their ultrastructure for imaging analysis.

Description

[Technical Field]

[0001] This disclosure relates to a novel procedure for cryoprotection of tissues for imaging procedures such as correlative light and electron microscopy (CLEM) and immunogold electron microscopy (iEM) and fluorescence imaging, in which harvested organs or tissues are perfusion-fixed with dimethyl sulfoxide (DMSO) and freeze-substituted with organic solvents to reliably freeze larger tissue blocks while preserving their ultrastructure for imaging analysis. [Background technology]

[0002] Correlated light-electron microscopy (CLEM) and immunogold electron microscopy (iEM) are important methods in the realm of experimental strategies applied in the fields of drug discovery and safety assessment of potential therapeutic molecules. The precise localization of drug targets and drug candidates in cultured cells, organoids, and mammalian tissues plays a major role in elucidating the mechanism of action of drug candidates and better understanding safety concerns and pathologies associated with potential therapeutic molecules.

[0003] Cryofixation by plunge freezing or high-pressure freezing followed by freeze substitution and cryotreatment of samples into Lowicryl® resin has been used to prepare specimens for CLEM and iEM and may better preserve ultrastructural details, membrane contrast, epitopes, and fluorescence emission compared to other conventional methods. However, this cryofixation method is only effective for very small and thin samples (less than 400 µm) and is not suitable for most mammalian tissues and organoids without first performing vibratomy sectioning, which can be difficult when the region of interest and histopathology are unclear or very small and focal. For example, when imaging such thin samples to understand biological processes, such as drug localization within tissues, the information derived from the images may be relatively limited. Furthermore, the collection and sectioning of organs and tissues from mammals (mice, rats, monkeys) and tissues by vibratomy generally requires chemical prefixation of the tissue by perfusion fixation or drop fixation to avoid artifacts from tissue collapse, and the use of strong fixatives may further limit the benefits of this method.

[0004] Therefore, there is a need for more versatile methods of preparing tissue for CLEM and iEM and other imaging methods. Summary of the Invention

[0005] The present disclosure provides novel methods for preparing samples for, among other things, CLEM and iEM and related imaging applications, including fluorescence imaging. In some embodiments, the methods avoid the use of vibratory ablation and / or high-pressure fixation, allowing larger, thicker samples to be prepared and analyzed. In some aspects, the methods herein enable high-quality imaging of the localization of molecules, such as drugs, within cells, organelles, or tissues, and also allow imaging of a broader and greater selection of surrounding tissue, thereby providing more complete information about the interaction of the imaged molecules with cells, for example, in culture. In some embodiments, the methods herein are also cheaper and easier to perform because they do not require either high-pressure equipment to fix cells and / or vibrating structure equipment, which can be expensive.

[0006] The methods herein include, for example, a method of cryoprotecting a tissue sample, the method comprising: (a) exposing a tissue sample having an average thickness of 2 mm or less to a dimethyl sulfoxide (DMSO) solution; (b) freezing the sample of (a) in liquid nitrogen; (c) freeze-substituting the sample of (b); and (d) adding an embedding resin compatible with temperatures of -60°C to -80°C to the sample of (c) and polymerizing the resin. In some cases, the tissue sample of (a) has an average thickness of 1.5 mm or less. In some cases, the tissue sample of (a) has an average thickness of 1.0 mm or less. In some cases, the tissue sample of (a) has an average thickness of 0.4 mm to 1.5 mm. In some cases, the tissue sample of (a) has an average thickness of at least 0.5 mm (e.g., 0.5 to 2 mm, 0.5 to 1.5 mm, or 0.5 to 1 mm). In some cases, the tissue sample of (a) is not prepared by vibrotomy, and / or the sample is manually prepared or sliced. In some cases, the tissue sample is perfused with an optical fixative or is obtained from a tissue or organ perfused with an optical fixative, and / or the tissue sample is not high-pressure frozen. In some cases, the optical fixative contains 1-4% paraformaldehyde. In some cases, the sample (b) is freeze-substituted in an acetone solution, such as 100% acetone, or a mixture of 100% acetone and at least one heavy metal stain, such as uranyl acetate and / or osmium tetroxide. In some cases, the sample (b) is freeze-substituted in a mixture of 100% acetone and 0.01-0.2% uranyl acetate, and optionally osmium tetroxide, such as 0.001%-0.002% osmium tetroxide. In some cases, the embedding resin is a non-polar resin. In some cases, the embedding resin is a polar resin. In some cases, the embedding resin is an acrylate or methacrylate resin, and / or the resin is polymerized by ultraviolet light at a wavelength, such as 360 nm. Optionally, the method further includes staining the sample of (d), e.g., with one or more fluorescent stains and / or heavy metal stains, e.g., osmium, lead, or gold stains. Optionally, the method is carried out at atmospheric pressure.In some cases, the sample is a tissue block from a solid organ. In some cases, the method further includes preparing a tissue sample of an average thickness of 2 mm or less by obtaining a tissue sample or organ perfused with an optical fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less. In some cases, the method further includes, prior to step (a), perfusing the tissue sample or organ with an optical fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less. In some cases, the DMSO solution contains 40-60% DMSO, such as 40%, 45%, 50%, 55%, or 60% DMSO.

[0007] The present disclosure also includes tissue samples prepared by the methods described above or as disclosed herein.

[0008] The present disclosure also includes kits for carrying out the methods described above or elsewhere herein. The kits include a freeze-substitution solution, such as a DMSO solution, 100% acetone, or a mixture of 100% acetone and at least one heavy metal stain (such as uranyl acetate and / or osmium tetroxide), and at least one embedding resin, such as a polar or non-polar acrylate or methacrylate resin, where the resin is compatible with temperatures between -60°C and -80°C, and optionally further include at least one of a plate or chip for immersing the tissue sample in liquid nitrogen, a fluorescent and / or electron microscope imaging slide, and instructions for use. The present disclosure also includes systems for carrying out the above methods or as elsewhere described herein. In some cases, the system automatically or semi-automatically performs the following method steps: A method comprising: (a) exposing a tissue sample having an average thickness of 2 mm or less to a dimethyl sulfoxide (DMSO) solution; (b) freezing the sample of (a) in liquid nitrogen; (c) freeze-substituting the sample of (b); and (d) adding to the sample of (c) an embedding resin compatible with temperatures of -60°C to -80°C and polymerizing the resin. Optionally, the system performs steps (a) through (d) on at least one plate or chip.

[0009] Further methods herein include, for example, a method for imaging a tissue sample having an average thickness of 2 mm or less prepared by a process comprising: (a) exposing the tissue sample to a dimethyl sulfoxide (DMSO) solution; (b) freezing the sample of (a) in liquid nitrogen; (c) freeze-substituting the sample of (b); and (d) adding and polymerizing an embedding resin compatible with temperatures of -60°C to -80°C to the sample of (c), the method comprising performing fluorescence microscopy, electron microscopy, or both fluorescence and electron microscopy on the tissue sample or a section of the tissue sample. In some cases, the tissue sample of (a) has an average thickness of 1.5 mm or less. In some cases, the tissue sample of (a) has an average thickness of 1.0 mm or less. In some cases, the tissue sample of (a) has an average thickness of 0.4 mm to 1.5 mm. In some cases, the tissue sample in (a) has an average thickness of at least 0.5 mm (e.g., 0.5-2 mm, 0.5-1.5 mm, or 0.5-1 mm). In some cases, the tissue sample in (a) is not prepared by vibrotomy, and / or the sample is manually prepared or sliced. In some cases, the tissue sample is perfused with an optical fixative or is obtained from a tissue or organ perfused with an optical fixative, and / or the tissue sample is not high-pressure frozen. In some cases, the optical fixative includes 1-4% paraformaldehyde. In some cases, the sample in (b) has been freeze-substituted with an acetone solution, such as 100% acetone, or a mixture of 100% acetone and at least one heavy metal stain, such as uranyl acetate and / or osmium tetroxide. In some cases, the sample in (b) is freeze-substituted in a mixture of 100% acetone, 0.01-0.2% uranyl acetate, and optionally osmium tetroxide, such as 0.001-0.002% osmium tetroxide. In some cases, the embedding resin is a non-polar resin. In some cases, the embedding resin is a polar resin. In some cases, the embedding resin is an acrylate or methacrylate resin, and / or the resin is polymerized by ultraviolet light, such as at a wavelength of 360 nm.Optionally, the method further comprises staining the sample of (d) with, for example, a fluorescent stain and / or a heavy metal stain, e.g., one or more of an osmium, lead, or gold stain. Optionally, the method is performed at atmospheric pressure. Optionally, the sample is a tissue block from a parenchymal organ. Optionally, the method further comprises preparing a tissue sample of an average thickness of 2 mm or less by obtaining a tissue sample or organ perfused with an optical fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less. Optionally, the method further comprises perfusing the tissue sample or organ with an optical fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less prior to step (a). Optionally, the DMSO solution comprises 40-60% DMSO, such as 40%, 45%, 50%, 55%, or 60% DMSO. In some cases, imaging involves electron microscopy, such as correlative light electron microscopy (CLEM), immunogold electron microscopy, or scanning electron microscopy (SEM), such as backscattered electron scanning electron microscopy (BSE-SEM). In some cases, the method can identify the location of drug molecules, such as lipid-coated drugs, antisense drugs, antibody drugs, polypeptide drugs, or small molecule drugs, within or adjacent to cells in the sample. In some cases, the method is performed on a section of the tissue sample having an average thickness of 300 nm to 1000 nm, e.g., 500 nm. In some cases, the method includes performing fluorescence microscopy on the sample or a section of the sample, as well as electron microscopy, such as CLEM, immunogold electron microscopy, SEM, or BSE-SEM.

[0010] Additional objects and advantages will be set forth in part in the following description, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate specific embodiments and, together with the description, serve to further explain certain principles described herein. [Brief explanation of the drawings]

[0011] This U.S. provisional application contains at least one drawing executed in color. If a nonprovisional or PCT application claiming priority to and incorporating the subject matter of this provisional application is later published, copies of this provisional application with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0012] [Figure 1] Figures 1A-1G show imaging of colon (Figure 1A, 1B, and 1C), colorectal cancer (Figure 1D and 1E), pancreas (Figure 1F), and kidney (Figure 1G) samples after sample preparation using the methods described herein. Samples were stained with osmium tetroxide (OsO4) to stain lipids and cell membranes, uranyl acetate (UA) to stain proteins and nucleic acids, and lead citrate (LC) to enhance the contrast between stained and unstained areas.

[0013] [Figure 2] Figures 2A-2C show imaging of islet cells in the mouse pancreas in the presence of fluorescent and gold double-stained anti-insulin antibody at 200x (Figure 2A) or 250x (Figures 2B and 2C) magnification by either fluorescent (Figure 2A) or BSE-SEM (Figure 2B), or combined fluorescent and BSE-SEM (Figure 2C) imaging.

[0014] Figures 2D–2F show imaging by fluorescence (Figure 2D) or BSE-SEM (Figure 2E), or a combination of fluorescence and BSE-SEM (Figure 2F), at 400x (Figure 2D) or 1000x (Figures 2E and 2F) magnification using an anti-insulin antibody double-stained with fluorescence and gold.

[0015] [Figure 3] Figure 3A-Figure 3B show the imaging of islet cells in the mouse pancreas by fluorescence (Figure 3A) and BSE-SEM (Figure 3B) in the presence of fluorescent and gold double-stained anti-insulin antibodies at 1000x (Figure 3B) and 20,000x (Figure 3A) magnification.

[0016] [Figure 4] Figure 4 shows the detection and localization of allele-specific oligonucleotide nanoparticles in chromatid-plexed ependymal cells by BSE-SEM at 500x magnification after intracerebroventricular nanoparticle injection into mice. Samples were prepared by the methods described herein.

[0017] [Figure 5] Figures 5A-5E show the detection and localization of allele-specific oligonucleotide nanoparticles in ependymal cells of the choroid plexus at magnifications of 400x (Figure 5A), 5000x (Figures 5B and 5C), 15,000x (Figure 5D), and 20,000x (Figure 5E). Nanoparticles are identified using gold-labeled antibodies specific for the nanoparticles, indicated by arrows in the images. Boxed features at the top of Figures 5B and 5C are shown in more detail in Figures 5D and 5E, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0018] I. Definition Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

[0019] In this application, the use of "or" means "and / or" unless stated otherwise. In the context of a multiple dependent claim, the use of "or" refers to only one or more of the preceding independent or dependent claims. Also, terms such as "element" or "component" encompass both elements and components that comprise a single unit and elements and components that comprise more than one subunit, unless otherwise specified.

[0020] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include every integer value within the recited range, and fractions thereof, where appropriate (such as tenths and hundredths of integers).

[0021] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the present disclosure that may be had by reference to the entire specification. Accordingly, the terms defined below are more fully defined by reference to the entire specification.

[0022] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0023] The term "cryoprotection" or "cryoprotecting" refers to the process of protecting a sample, such as a tissue sample, from damage, eg, to cellular structure or cellular integrity, that can occur due to freezing.

[0024] As used herein, "tissue sample" refers broadly to any sample from any organism that contains cells or tissue. The sample may be derived from a solid organ, for example, by sectioning the organ, or from a part of the organ, such as a tissue slice, or from a cell culture, etc.

[0025] As used herein, "freeze substitution" or "freeze substituting" refers to the process of using an organic solvent at low temperature to effectively dissolve the frozen water in and / or around a cryogenically frozen tissue sample, replacing the water molecules with molecules of the organic solvent.

[0026] The term "average thickness" when applied to a tissue sample refers to the average general thickness of the sample over the length of the sample, e.g., a tissue sample placed on a slide. The average thickness figure incorporates experimental and observational error, which can be relatively large in this example, in that many samples may be sliced ​​manually rather than with specialized equipment.

[0027] The term "vibroablation" refers to a special method of obtaining very thin sections of tissue, for example less than 0.4 mm thick, generally by using special vibrometric instruments.

[0028] The terms "high pressure fixation" or "high pressure freezing" refer to a method of fixing an organ or tissue sample by exposing it to both freezing temperatures and high pressures significantly above atmospheric pressure, with the aim of preserving the organ or tissue without damaging its ultrastructure.

[0029] The term "ultrastructure" refers to the cellular or organelle structure within a tissue sample. The term "epitope" herein refers to a binding site for an antibody, polypeptide, oligonucleotide, or other molecule intended to specifically target a particular molecule or structure within a cell. An epitope can include, for example, a specific binding site on a target molecule or a specific binding site on a target structure of a cell, e.g., an organelle.

[0030] The terms "perfusion fixation" or "chemical fixation" refer to a method of fixing an organ or tissue sample in which a fixative chemical, such as formaldehyde or paraformaldehyde, is perfused into the blood vessels or associated structures of the organ or tissue sample. In some embodiments, the fixative chemical then forms structural components and crosslinks in the organ or tissue, preserving their structure.

[0031] As used herein, a "photofixative" is a fixative chemical that creates a mild crosslink, such as a single crosslink from one fixed molecule to a molecule in the sample being fixed (e.g., paraformaldehyde), as opposed to a stronger crosslink, such as a double crosslink (i.e., a "strong fixative" such as glutaraldehyde or osmium tetroxide). An example of a photofixative is 1-4% paraformaldehyde.

[0032] As used herein, "embedding resin" refers to a substance used to infiltrate a tissue sample on a slide prior to imaging, for example, to fix components of the tissue sample in place by polymerization of the resin after infiltration of the sample, allowing the sample to be preserved without degradation or migration of the ultrastructure and molecules to be imaged.

[0033] II. Exemplary Methods for Cryoprotecting Tissue Samples The present disclosure encompasses, for example, methods for preparing cryoprotected tissue samples and methods for using such cryoprotected tissue samples for fluorescence and / or electron microscopy. For example, such ultrastructural immunohistochemistry applications would ideally be used to identify cell types and organelles and localize biological processes involving cells and organelles, e.g., via localization of specific molecules involved in the biological process. In some cases, such applications would ideally be useful for understanding and evaluating the mechanisms of potential therapeutics. For example, one goal of the present method is to localize potential drug targets in a sample, or to localize therapeutics in a sample to see how they interact with not only the target molecule but also other cellular structures. Such imaging applications rely on high-quality tissue samples that preserve both ultrastructure and binding epitopes while allowing for appropriate labeling density for fluorescence and electron microscopy imaging. The present method addresses the technical challenges of finding fixation and processing conditions that allow for the best compromise between labeling density and ultrastructural preservation in a sample.

[0034] One comparative method of sample preparation involves fixing samples using high pressure and ultra-low temperatures rather than adding chemical fixatives (high-pressure fixation). This method may have the advantage that because the starting tissue is not chemically fixed, it is not exposed to crosslinking, which could mask epitopes or affect ultrastructure, resulting in well-preserved antigens with low background when performing fluorescence imaging. However, this method can only freeze small tissue samples, such as those less than 400 µm thick (e.g., 100 µm to 400 µm thick). Such small sample sizes may be less useful for analysis. Furthermore, because the sample thickness must be very small, such samples generally must be prepared using vibrotomy. Both vibrotomy and high-pressure fixation are expensive and require specialized equipment. Furthermore, fixation artifacts due to tissue freezing may exist.

[0035] In contrast, in this disclosure, the inventors have found that cryoprotection of mildly chemically fixed tissue, such as with 1-4% paraformaldehyde, is simpler and less expensive and allows for the preparation of larger samples, approximately 1 mm thick. This, in some cases, allows for larger sections that can be imaged by wide-field BSE-SEM imaging. The inventors have also found that when prepared according to the methods described herein, such tissues can be preserved for reasonable periods and retain good ultrastructure for imaging. The present method is less expensive and easier to perform because it does not require either vibrotomy or high-pressure fixation. While the use of chemical fixation can present challenges, such as the risk of artifacts from chemical fixation of the sample, as well as the potential for loss of antigenicity, antigen delocalization, increased background autofluorescence due to the presence of aldehydes, and the potential for dimming of GFP fluorescence, the inventors have found that the methods described herein produce relatively high-quality fluorescence and electron microscopy images.

[0036] In some embodiments, the methods described herein involve cryoprotecting a tissue sample fixed with a photofixative, such as 1-4% paraformaldehyde, to an average thickness of 2 mm or less by first exposing the sample to a DMSO solution, then freezing the sample in liquid nitrogen, freeze-substituting the sample frozen in liquid nitrogen, and then adding a cryo-stable resin to the sample. For example, some embodiments involve cryoprotecting the tissue sample by a method comprising: (a) exposing a tissue sample with an average thickness of 2 mm or less to a dimethyl sulfoxide (DMSO) solution; (b) freezing the sample of (a) in liquid nitrogen; (c) freeze-substituting the sample of (b); and (d) adding an embedding resin, such as an acrylate or methacrylate resin compatible with temperatures of -60°C to -80°C, to the sample of (c) and polymerizing the resin.

[0037] In some embodiments, the sample is collected from tissues or organs that have previously been perfused with a photofixative or that have been perfused with a photofixative. Therefore, while the sample is subjected to chemical fixation, the degree of crosslinking in the sample is maintained at a minimal level by using a photofixative such as paraformaldehyde, which is maintained at a relatively low concentration, such as 1-4% paraformaldehyde, compared to stronger fixatives such as glutaraldehyde. In some embodiments, the photofixative is 2-4% paraformaldehyde. In some embodiments, the photofixative is 2% paraformaldehyde. In some embodiments, the photofixative is 3% paraformaldehyde. In some embodiments, the photofixative is 4% paraformaldehyde. In some embodiments, the perfusion fixation does not include glutaraldehyde or osmium tetroxide. In some embodiments, the tissue sample is not high-pressure frozen.

[0038] In some embodiments, the tissue sample to be cryoprotected has an average thickness of 0.4 mm to 2 mm, e.g., 0.4 mm to 1.5 mm, 0.5 mm to 2 mm, 0.5 mm to 1.5 mm, 0.8 mm to 1.2 mm, 0.5 mm to 1.0 mm, 0.4 mm to 1.0 mm, 1.0 mm to 2 mm, 1.0 mm to 1.5 mm, or 1.5 mm to 2 mm. In some embodiments, the average thickness is 0.8 mm to 1.2 mm. In some embodiments, the average thickness is 1 mm. In some embodiments, the sample is not prepared by vibrotomy and / or is manually prepared or sliced ​​to an average thickness of, e.g., 2 mm or less. In some embodiments, the sample has been subjected to chemical fixation, such as perfusion fixation with a photofixative, and is prepared to the thicknesses listed above.

[0039] The DMSO solution used for cryoprotection can be, for example, 40-60% DMSO, 40-50% DMSO, 50-60% DMSO, 40%, 45%, 50%, 55%, or 60% DMSO, or 35-70% DMSO. In some embodiments, the DMSO solution is 50% DMSO. In some embodiments, the DMSO solution is exposed to the tissue sample for at least 1 hour, e.g., 2, 4, 8, 10, 12, or 18 hours, or overnight. In some embodiments, exposure to the DMSO solution is performed at low temperatures, e.g., 2-8°C or 4°C. In some embodiments, samples subjected to chemical fixation, such as perfusion fixation with a photofixative, are prepared at the thicknesses described above and exposed to a DMSO solution having the DMSO concentration range described above.

[0040] After cryoprotection in DMSO, the sample can be frozen in liquid nitrogen or by a similar method of supercooling the sample. In some cases, for example, samples with an average thickness of 2 mm or less can be placed on a relatively flat surface suitable for immersion in liquid nitrogen, such as a plate, slide, chip, or tray that is stable in liquid nitrogen, and then immersed in liquid nitrogen. In some cases, the surface is a copper surface.

[0041] Once a sample is frozen in liquid nitrogen, freeze substitution can be performed to remove free water from the sample by effectively dissolving it in an organic solvent. In some embodiments, freeze substitution is performed by exposing the sample, while it is still frozen at -60 to -80°C, to a solvent such as acetone, isopropanol, or methanol. In some embodiments, freeze substitution is performed in acetone, i.e., 100% acetone. Thus, in some embodiments, freeze substitution is performed in a solution consisting essentially of acetone. In some cases, solvents used for freeze substitution, such as acetone, do not contain heavy metal stains such as uranyl acetate or osmium tetroxide. In other cases, 100% acetone is mixed with small amounts of other substances, such as uranyl acetate and / or osmium tetroxide, such as 0.01-2% uranyl acetate, 0.01-1% uranyl acetate, 0.01-0.1% uranyl acetate, 0.05-1% uranyl acetate, 0.05-0.1% uranyl acetate, 0.1-0.5% uranyl acetate, 0.1-0.2% uranyl acetate, 0.1% uranyl acetate, or 0.2% uranyl acetate. In some cases, small amounts of osmium tetroxide, such as 0.001%-0.01% or 0.001-0.002%, are added. In some cases, freeze substitution is performed at -60 to -80°C for at least 1 hour and up to 1 week, e.g., at least 12 to 96 hours or 48 to 96 hours. The freeze-substitution and cryogenic treatments used herein allow for ultrastructural preservation, eliminate the need for harsh fixatives such as glutaraldehyde (GA) and osmium tetroxide (OsO), preserve GFP fluorescence, preserve antigens, and reduce protein, lipid, and DNA / RNA extraction compared to other methods. Without being bound to any particular mechanism, freeze-substitution is based on the principle that frozen water contained in biological samples can be dissolved and replaced with organic solvents such as acetone, methanol, or isopropanol.Without being bound by any particular mechanism, it is believed that at very low temperatures, such as -80 °C, organic solvents that remain liquid tend to preferentially dissolve "free, unbound" water, leaving the more tightly bound hydration shell ("molecularly bound water") of biopolymers intact, thus allowing the biopolymers to remain properly folded. The hydration shell is believed to be essential for the proper folding and assembly of biopolymers. Therefore, preservation of the hydration shell during low-temperature dehydration aids in preserving the molecular structure, ultrastructure, and antigenicity of the sample.

[0042] After freeze substitution, the sample is then exposed to an embedding resin, such as an acrylate or methacrylate resin compatible with temperatures between -60 and -80°C, used for preservation, such as the nonpolar resin Lowacryl® HM23 or the polar resin Lowacryl® K11M. For example, in some embodiments, the resin material can be allowed to infiltrate the sample while it is maintained at low temperature. The embedding resin can then be polymerized according to the manufacturer's instructions. For example, in some embodiments, the resin can be polymerized by exposure to UV light, such as 360 nm light.

[0043] Such resin-embedded tissues, as used herein, have several advantages, such as forming stable, solid tissue blocks, being storable for months or years, being easily sectioned and capable of serial sectioning, being stable under potentially harsh antigen retrieval conditions, being easy to handle for complex applications such as CLEM, being capable of quantitative labeling on a nm-flat surface, being compatible with on-section fluorescence and / or immunogold labeling, and having an appearance similar to standard morphological transmission electron micrograph (TEM) images.

[0044] In some embodiments, after polymerization of the resin, the sample may be further prepared for imaging. For example, in some embodiments, appropriate stains may be added for either or both fluorescence or electron microscopy imaging, such as fluorescent dyes or tags, or heavy metal stains for electron microscopy, such as osmium tetroxide, uranyl acetate, and lead citrate. In some cases, the sample may be further sectioned for imaging, e.g., cut or sliced ​​into sections having an average thickness of less than 1 mm, e.g., 0.5 mm, or less, e.g., 100-1000 nm, 300-1000 nm, 400-600 nm, 400 nm, 500 nm, or 600 nm. In some cases, the sample, or a portion of the sample, may then be placed on an imaging plate or slide for fluorescence and / or electron microscopy imaging.

[0045] Therefore, in a further embodiment of the present disclosure, the method includes performing fluorescence and / or electron microscopy imaging on the sample prepared as described herein.Examples of electron microscopy include, for example, CLEM, BSE-SEM, and immunogold EM.In some cases, the sample prepared as described herein can identify the location of macromolecules in tissue samples, such as labeled drug molecules, lipid-coated drug particles, antisense drugs or lipid-coated antisense drugs, antibodies, polypeptide drugs, or targets of antibodies, antisense drugs, or polypeptide drugs.In other cases, the method herein can identify the location of specific small molecules in the tissue samples herein.

[0046] III. Kits, Systems, and Tissue Samples Prepared by the Method The present disclosure also includes tissue samples prepared according to the methods herein, including, for example, tissue samples preserved in polymerized embedding resins and sections of such samples, which may or may not be placed on slides or plates for storage and imaging.

[0047] The present disclosure also includes kits for carrying out the methods of tissue sample preparation described herein. In some embodiments, the kits include one or more reagents used in the methods described herein, such as a DMSO solution, e.g., 40-60% DMSO, e.g., 40%, 45%, 50%, 55%, or 60% DMSO; a freeze-substitution solution, e.g., 100% acetone or a mixture of 100% acetone and at least one heavy metal stain, such as uranyl acetate and / or osmium tetroxide; and an embedding resin, such as a polar or non-polar acrylate or methacrylate resin, which is compatible at temperatures between -60°C and -80°C. In some embodiments, the kits also include a surface, e.g., a plate, chip, or slide (i.e., one or more such surfaces), for immersing the tissue sample in liquid nitrogen for cooling. In some aspects, the kits include fluorescent and / or electron microscope imaging slides (i.e., one or more such slides). In some embodiments, the kits include instructions for use. In some cases, the kits include all of the above components.

[0048] The present disclosure also relates to systems for carrying out the methods described herein. In some embodiments, the system includes a surface, e.g., a plate, chip, slide (i.e., one or more such surfaces), for immersing and cooling a tissue sample in liquid nitrogen, and may also include surfaces for carrying out other reactions described herein, such as exposure to a DMSO solution, freeze-substitution of the sample, and / or addition and polymerization of an embedding resin. For example, in some embodiments, all of the method steps may be carried out within the system, such that at least one of the methods is carried out automatically, or all of the method steps are carried out automatically. In some cases, the system also incorporates a kit, as described above, providing reagents for the method steps, suitable slides or surfaces on which the sample may remain during the reaction, and / or instructions for use. [Example]

[0049] The following examples illustrate further embodiments of the methods herein.

[0050] Example 1. Preparation of colon, colorectal cancer, pancreas, and kidney samples for imaging To prepare tissue samples for imaging of colon, colorectal cancer (CRC), pancreas, and kidney tissue, samples were obtained in approximately 1 mm slices from larger samples perfused with 4% paraformaldehyde (FPA). The 1 mm slices were infiltrated in 50% DMSO for at least 1 hour to overnight at 4°C, placed on copper plates, and frozen in liquid nitrogen. Freeze-substitution was performed in 100% acetone (without uranyl acetate (UA), osmium tetroxide (Os), or glutaraldehyde) at -80°C. The freeze-substituted samples were then embedded in Lowacryl® HM23 resin at -70°C and maintained until imaging. Samples were stained with osmium tetroxide, uranyl acetate, and lead citrate prior to imaging to visualize the ultrastructure. The results are shown in Figure 1A-1E and compared with samples treated similarly but freeze-substituted with 0.1% uranyl acetate in 100% acetone and without GA or osmium tetroxide, as shown in Figure 1F and 1G.

[0051] Example 2. Sample preparation for imaging of anti-insulin antibodies in islet cells Pancreatic sections (mouse pancreatic islets) were prepared as in Example 1 and embedded in Lowacry® HM23 on carbon-coated glass slides. After blocking, anti-insulin monoclonal antibody ab6995 was added, followed by anti-mouse biotinylated secondary antibody (Jackson Immuno) and fluorescently labeled streptavidin (streptavidin A488) and 10 nm gold conjugate (Invitrogen) to stain the anti-insulin antibody with both fluorescent and gold. Hoechst stain was applied to the sample to provide contrasting fluorescence to the anti-insulin antibody staining. Osmium tetroxide, uranyl acetate, and lead citrate were also added to the sample to visualize cellular structures under an electron microscope. Figure 2A shows a fluorescent image of the sample at 200x magnification, with fluorescently stained antibodies indicated as light-colored marks. A 250x BSE-SEM image (Zeiss Gemini 300) of the sample is shown in Figure 2B, and a composite image of the fluorescent and gold-stained images is shown in Figure 2C. Additional images are shown in Figures 2D-F, and images taken at higher magnification are shown in Figures 3A-B.

[0052] Example 3. Sample preparation for imaging of oligonucleotide nanoparticles in cells Allele-specific oligonucleotide nanoparticles (ASO nanoparticles) have been investigated as potential therapeutic agents. Figure 4 and Figures 5A-5E show images of ASO nanoparticles administered intracerebroventricularly (ICV) to mouse brains, addressing the question of which tissues, cells, and organelles ASO nanoparticles accumulate and whether encapsulating ASOs in liposomes alters their uptake and localization. Brain tissue samples were processed for imaging according to the methods described herein. Specifically, brain tissue was fixed in 4% PFA and 0.1% GA, prepared into 1 mm-thick sections, and then trimmed to dimensions of 2 x 2 x 1 mm. Trimmed samples were cryoprotected in 50% DMSO at 4°C for at least 1 hour to overnight, placed on a copper plate, frozen in liquid nitrogen, and then freeze-substituted in acetone containing 0.5% GA, 0.01% UA, and 0.001% OsO at -80°C for 96 hours, washed in ethanol at -80°C, and processed in Lowacryl® HM23 resin at -70°C. BSE-SEM images of 500-nm-thick heavy-metal-stained sections on carbon-coated slides were then taken. The results are shown in Figure 4.

[0053] Figures 5A–5E show the detection of ASO nanoparticles in ependymal cells of the choroid plexus. For imaging, samples were cut into 500-nm sections and preblocked. An anti-ASO rabbit antibody specific for the analyzed ASO was added, followed by an anti-rabbit biotinylated secondary antibody (Jackson Immuno). For fluorescence microscopy, streptavidin Ax568 was added to fluorescently stain the ASO particles, and DAPI stain (Invitrogen) was also added to stain the nuclei. The arrows in Figure 5A indicate the location of the ASO nanoparticles within the cells. For immunogold electron microscopy, streptavidin-20 nm gold conjugate (Abcam) was added, followed by OsO4, UA, and LC to stain the cellular structures. BSE-SEM was performed using a Zeiss Gemini 300 instrument with a BSD1 detector at 3 kV. The results are shown in Figures 5B–5E. Circles indicate the localization of ASO nanoparticles at 5000x magnification (Figures 5B and 5C), and arrows indicate the localization of ASO particles at 15,000x or 20,000x magnification (Figures 5D and 5E).

[0054] The foregoing specification is deemed sufficient to enable one skilled in the art to practice the present invention. The foregoing description and examples detail certain embodiments and explain the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears, it will be understood that the present invention can be practiced in many ways and should be construed in accordance with the appended claims and their equivalents.

Claims

1. 1. A method of cryoprotecting a tissue sample, comprising: a) exposing a tissue sample with an average thickness of 2 mm or less to a dimethyl sulfoxide (DMSO) solution; b) freezing the sample of (a) in liquid nitrogen; c) freeze-substituting the sample of (b); and d) adding an embedding resin compatible with temperatures between −60° C. and −80° C. to the sample of (c) and polymerizing the resin; A method comprising:

2. 10. The method of claim 1, wherein the tissue sample of (a) has an average thickness of 1.5 mm or less.

3. 10. The method of claim 1, wherein the tissue sample of (a) has an average thickness of 1.0 mm or less.

4. 10. The method of claim 1, wherein the tissue sample of (a) has an average thickness of 0.4 mm to 1.5 mm.

5. 5. The method of any one of claims 1 to 4, wherein the tissue sample in (a) has an average thickness of at least 0.5 mm (e.g., 0.5 to 2 mm, 0.5 to 1.5 mm, or 0.5 to 1 mm).

6. The method of any one of claims 1 to 5, wherein the tissue sample in (a) is not prepared by a vibrating structure and / or the sample is manually prepared or sliced.

7. 7. The method of any one of claims 1 to 6, wherein the tissue sample is perfused with an optical fixative or is obtained from a tissue or organ that has been perfused with an optical fixative, and / or the tissue sample has not been high-pressure frozen.

8. The method of claim 7, wherein the photofixative comprises 1-4% paraformaldehyde.

9. 9. The method of any one of claims 1 to 8, wherein the sample of (b) is freeze-substituted with an acetone solution, such as 100% acetone, or a mixture of 100% acetone and at least one heavy metal stain, such as uranyl acetate and / or osmium tetroxide.

10. 10. The method of claim 9, wherein the sample of (b) is freeze-substituted in a mixture of 100% acetone and 0.01-0.2% uranyl acetate, and optionally osmium tetroxide, such as 0.001-0.002% osmium tetroxide.

11. The method according to any one of claims 1 to 10, wherein the embedding resin is a non-polar resin.

12. The method according to any one of claims 1 to 10, wherein the embedding resin is a polar resin.

13. The method according to any one of claims 1 to 12, wherein the embedding resin is an acrylate and methacrylate resin, and / or the resin is polymerized by ultraviolet light of a wavelength such as 360 nm.

14. 14. The method of any one of claims 1 to 13, further comprising staining the sample of (d) with, for example, a fluorescent stain and / or a heavy metal stain such as one or more of an osmium, lead, or gold stain.

15. The method of any one of claims 1 to 14, carried out at atmospheric pressure.

16. The method of any one of claims 1 to 15, wherein the sample is a tissue block from a solid organ.

17. 17. The method of any one of claims 1-6 or 8-16, further comprising preparing the tissue sample of an average thickness of 2 mm or less by obtaining a tissue sample or organ perfused with optical fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less.

18. 18. The method of any one of claims 1 to 6 or 8 to 17, further comprising, prior to step (a), perfusing the tissue sample or organ with an optical fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less.

19. 1. A method for imaging a tissue sample or a section of a tissue sample, wherein the tissue sample has an average thickness of 2 mm or less and was prepared by a process comprising: (a) exposing the tissue sample to a dimethyl sulfoxide (DMSO) solution; (b) freezing the sample of (a) in liquid nitrogen; (c) freeze-substituting the sample of (b); and (d) adding to the sample of (c) an embedding resin compatible with temperatures between -60°C and -80°C and polymerizing the resin; and the method comprises performing electron microscopy on the tissue sample or the section of the tissue sample.

20. 20. The method of claim 19, wherein the tissue sample of (a) has an average thickness of 1.5 mm or less.

21. 20. The method of claim 19, wherein the tissue sample of (a) has an average thickness of 1.0 mm or less.

22. 20. The method of claim 19, wherein the tissue sample of (a) has an average thickness of 0.4 mm to 1.5 mm.

23. 23. The method of any one of claims 19 to 22, wherein the tissue sample in (a) has an average thickness of at least 0.5 mm (e.g., 0.5 to 2 mm, 0.5 to 1.5 mm, or 0.5 to 1 mm).

24. 24. The method of any one of claims 19 to 23, wherein the tissue sample in (a) is not prepared by a vibrating structure and / or the sample is manually prepared or sliced.

25. 25. The method of any one of claims 19 to 24, wherein the tissue sample is perfused with optical fixative or is obtained from a tissue or organ that has been perfused with optical fixative, and / or the tissue sample has not been high-pressure frozen.

26. 26. The method of claim 25, wherein the photofixative comprises 1-4% paraformaldehyde.

27. 27. The method of any one of claims 19 to 26, wherein the sample in (b) is freeze-substituted with an acetone solution, such as 100% acetone, or a mixture of 100% acetone and at least one heavy metal stain, such as uranyl acetate and / or osmium tetroxide.

28. 28. The method of claim 27, wherein the sample of (b) is freeze-substituted in a mixture of 100% acetone and 0.01-0.2% uranyl acetate, and optionally osmium tetroxide, such as 0.001-0.002% osmium tetroxide.

29. The method according to any one of claims 19 to 28, wherein the embedding resin is a non-polar resin.

30. The method according to any one of claims 19 to 28, wherein the embedding resin is a polar resin.

31. The method according to any one of claims 19 to 30, wherein the embedding resin is an acrylate and methacrylate resin, and / or the resin is polymerized by ultraviolet light of a wavelength such as 360 nm.

32. 32. The method of any one of claims 19 to 31, wherein the processes (a) to (d) further comprise staining the sample of (d) with, for example, a fluorescent stain and / or a heavy metal stain such as one or more of an osmium, lead, or gold stain.

33. A method according to any one of claims 19 to 32 carried out at atmospheric pressure.

34. The method of any one of claims 19 to 33, wherein the sample is a tissue block from a solid organ.

35. 35. The method of any one of claims 19 to 34, wherein the electron microscopy is correlative light electron microscopy (CLEM) or immunogold electron microscopy.

36. 36. The method of any one of claims 19 to 35, which is capable of identifying the location of drug molecules, such as lipid-coated drugs, antisense drugs, antibody drugs, polypeptide drugs or small molecule drugs, within or adjacent to cells in the sample.

37. 37. The method of any one of claims 19 to 36, performed on a section of the tissue sample having an average thickness of between 300 nm and 1000 nm, for example 500 nm.

38. 38. The method of any one of claims 19 to 37, further comprising performing fluorescence microscopy on the sample or a section of the sample.

39. 39. The method of any one of claims 1 to 38, wherein the DMSO solution comprises 40-60% DMSO, such as 40%, 45%, 50%, 55%, or 60% DMSO.

40. A tissue sample prepared by the method of any one of claims 1 to 18 or 39.

41. 40. A kit for performing the method of any one of claims 1 to 18 or 39, comprising at least one of a DMSO solution, a freeze substitution solution such as 100% acetone or a mixture of 100% acetone and at least one heavy metal stain such as uranyl acetate and / or osmium tetroxide, and an embedding resin such as a polar or non-polar acrylate and methacrylate resin, wherein the resin is compatible with temperatures of -60°C to -80°C, and optionally further comprising at least one of a plate or chip for immersion of the tissue sample in liquid nitrogen, a fluorescent and / or electron microscope imaging slide, and instructions for use.

42. A system for carrying out the method of any one of claims 1 to 18 or 39, comprising: a) exposing a tissue sample having an average thickness of 2 mm or less to a dimethyl sulfoxide (DMSO) solution; b) freezing the sample of (a) in liquid nitrogen; c) freeze-substituting the sample of (b); and d) adding to the sample of (c) an embedding resin compatible with temperatures between −60° C. and −80° C., and polymerizing the resin. A system that automatically or semi-automatically performs the above.

43. The system of claim 42, wherein steps (a) to (d) are carried out on at least one plate or chip.

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