Cryogenic treatment of cryoprotected tissue

Abstract

The present disclosure relates to novel procedures for cryoprotection of tissue for imaging procedures such as optoelectronic correlation microscopy (CLEM) and immunogold electron microscopy (iEM) as well as fluorescence imaging. The procedure combines perfusion immobilization with cryoprotection of organs or tissues harvested in dimethyl sulfoxide (DMSO) and cryoreplacement in an organic solvent to reliably freeze and treat larger tissue blocks while retaining ultrastructures for image analysis.

Description

Technical Field

[0001] The present disclosure relates to a novel procedure for cryoprotecting tissues for imaging procedures such as light-correlated electron microscopy (CLEM) and immunogold electron microscopy (iEM) and fluorescence imaging. The procedure combines perfusion fixation with cryoprotection of harvested organs or tissues in dimethyl sulfoxide (DMSO) and freeze substitution in organic solvents to reliably freeze and process larger tissue blocks while preserving ultrastructure for image analysis. Background Art

[0002] Correlative electron microscopy (CLEM) and immunogold electron microscopy (iEM) are important methods in the arsenal of experimental strategies applied in the field 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 an important role in elucidating the mechanism of action of drug candidates and in better understanding the safety issues and pathologies associated with potential therapeutic molecules.

[0003] The samples were frozen by sudden or high pressure freezing followed by freeze substitution and cryogenic treatment to Cryofixation in resin has been used to prepare samples for CLEM and iEM, and can better maintain ultrastructural details, membrane contrast, epitopes, and fluorescence emission than other previous methods. However, this cryofixation method is only reliable for very small and thin samples (less than 400 μm), and is not suitable for most mammalian tissues and organoids without first thin sectioning by vibration microtomy, which may be challenging when the target area and tissue pathology are unclear or very small and concentrated. For example, when such thin samples are imaged to, for example, understand biological processes (such as drug localization in tissues), the information derived from the images may be relatively limited. In addition, the harvesting and resection of organs and tissues from mammals (mice, rats, monkeys) and the vibration microtomy of tissues generally require chemical pre-fixation of the tissues via perfusion fixation or drop fixation to avoid artifacts caused by tissue decay, which may further limit the benefits of this method when strong fixatives are used.

[0004] Therefore, more versatile methods are needed to prepare tissues for CLEM and iEM as well as other imaging methods. Summary of the invention

[0005] The present disclosure provides, inter alia, a novel method for preparing samples for CLEM and iEM and related imaging applications, including fluorescence imaging. In some embodiments, the method avoids the use of vibration microtomy and / or high-pressure fixation, and allows for the preparation and analysis of larger, thicker samples. For example, in some embodiments, the methods herein facilitate high-quality images of the localization of molecules (such as drugs) in cells, organelles, or tissues, and also allow for imaging of a wider, larger range of surrounding tissues, thereby providing more comprehensive information about the interaction of the imaged molecules with cells in culture. In some embodiments, since no high-pressure equipment is required for fixing cells and / or no potentially expensive vibration microtomy equipment is required, the methods herein are also less expensive and simpler to implement.

[0006] The method herein includes, for example, a method for cryoprotecting a tissue sample, comprising: (a) exposing a tissue sample of 2 mm or less average thickness 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 a temperature 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 has an average thickness of 1.0 mm or less. In some cases, the tissue sample 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 (such as 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) has not been prepared via vibration microtomy, and / or wherein the sample has been manually prepared or sliced. In some cases, the tissue sample has been perfused with a mild fixative, or obtained from a tissue or organ perfused with a mild fixative, and / or the tissue sample has not been frozen under high pressure. In some cases, the mild fixative comprises 1% to 4% paraformaldehyde. In some cases, the sample of (b) is freeze-substituted with an acetone solution (such as a mixture of 100% acetone or 100% acetone and at least one heavy metal stain such as uranyl acetate and / or osmium tetroxide). In some cases, the sample of (b) is freeze-substituted in a mixture of 100% acetone with 0.01% to 0.2% uranyl acetate and optionally osmium tetroxide (such as 0.001% to 0.002% osmium tetroxide). In some cases, the embedding resin is a non-polar resin. In other cases, the embedding resin is a polar resin. In some cases, the embedding resin is an acrylate and methacrylate resin and / or wherein the resin is polymerized by ultraviolet light such as 360nm wavelength. In some cases, the method further includes, for example, dyeing the sample of (d) with a fluorescent stain and / or a heavy metal stain (such as one or more of osmium, lead or gold stain). In some cases, the method is performed under atmospheric pressure. In some cases, the sample is a tissue block from a parenchymal organ. In some cases, the method further includes preparing a tissue sample of 2mm or less average thickness by obtaining a tissue sample or organ perfused with a light fixative and slicing the tissue sample or organ to an average thickness of 2mm or less. In some cases, the method further includes perfusing a tissue sample or organ in a light fixative before step (a) and slicing the tissue sample or organ to an average thickness of 2mm or less. In some cases, the DMSO solution comprises 40% to 60% DMSO, such as 40%, 45%, 50%, 55% or 60% DMSO.

[0007] The disclosure herein also includes tissue samples prepared by such methods as described above or otherwise described herein.

[0008] The present disclosure also includes a kit for performing the method as described above or otherwise described herein. The kit may include at least one of: a DMSO solution, a freeze substitution solution (such as a mixture of 100% acetone or 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 includes at least one of: a plate or chip for immersing a tissue sample in liquid nitrogen, a fluorescence and / or electron microscopy imaging slide, and instructions for use. The present disclosure also includes a system for performing the method as described above or otherwise described herein. In some cases, the system automatically or semi-automatically performs steps of the method, such as: (a) exposing a tissue sample of 2 mm or less average thickness 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 a temperature of -60°C to -80°C to the sample of (c), and polymerizing the resin. In some cases, the system performs steps (a) to (d) on at least one plate or chip.

[0009] Other methods herein include, for example, a method for imaging a tissue sample or a slice of a tissue sample, wherein the tissue sample has an average thickness of 2 mm or less and has been prepared by a process comprising: (a) exposing the sample to a dimethyl sulfoxide (DMSO) solution, (b) freezing the sample of (a) in liquid nitrogen, (c) freezing the sample of (b), and (d) adding an embedding resin compatible with a temperature of -60°C to -80°C to the sample of (c) and polymerizing the resin; the method comprising: performing fluorescence microscopy, electron microscopy, or a combination of fluorescence microscopy and electron microscopy on the tissue sample or the slice 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 has an average thickness of 1.0 mm or less. In some cases, the tissue sample 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 0.5mm (such as 0.5 to 2mm, 0.5 to 1.5mm or 0.5 to 1mm). In some cases, the tissue sample of (a) is not yet prepared via vibration microtome, and / or wherein the sample is manually prepared or sliced. In some cases, the tissue sample has been perfused with a slight fixative, or obtained from a tissue or organ perfused with a slight fixative, and / or wherein the tissue sample is not yet frozen under high pressure. In some cases, the slight fixative comprises 1% to 4% paraformaldehyde. In some cases, the sample of (b) is freeze-substituted with an acetone solution (such as a mixture of 100% acetone or 100% acetone and at least one heavy metal stain such as uranyl acetate and / or osmium tetroxide). In some cases, the sample of (b) is freeze-substituted in a mixture of 100% acetone and 0.01% to 0.2% uranyl acetate and optionally osmium tetroxide (such as 0.001% to 0.002% osmium tetroxide). In some cases, the embedding resin is a non-polar resin. In other cases, the embedding resin is a polar resin. In some cases, the embedding resin is an acrylate and methacrylate resin and / or wherein the resin is polymerized by ultraviolet light such as 360nm wavelength. In some cases, the method further includes, for example, dyeing the sample of (d) with a fluorescent dye and / or a heavy metal dye (such as one or more of osmium, lead or gold dye). In some cases, the method is performed at atmospheric pressure. In some cases, the sample is a tissue block from a parenchymal organ. In some cases, the method further includes preparing a tissue sample of 2mm or less average thickness by obtaining a tissue sample or organ perfused with a mild fixative and slicing the tissue sample or organ to an average thickness of 2mm or less. In some cases, the method further comprises perfusing the tissue sample or organ in a light fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less prior to step (a).In some cases, the DMSO solution comprises 40% to 60% DMSO, such as 40%, 45%, 50%, 55% or 60% DMSO. In some cases, imaging comprises electron microscopy, which is followed by photoelectric correlation microscopy (CLEM) or immunogold electron microscopy or scanning electron microscopy (SEM), such as backscattered electron scanning electron microscopy (BSE-SEM). In some cases, the method can distinguish the position of drug molecules in cells or adjacent cells in the sample, such as, for example, lipid-coated drugs, antisense drugs, antibody drugs, polypeptide drugs or small molecule drugs. In some cases, the method is to have 300nm to 1000nm, such as 500nm average thickness of the tissue sample slice is carried out. In some cases, the method includes fluorescence microscopy to the sample or to the slice of the sample, and electron microscopy, such as CLEM or immunogold electron microscopy or SEM or BSE-SEM.

[0010] Additional objects and advantages will be set forth in part in the following description and will be understood in part from the description or may be learned through practice. These objects and advantages will be realized and obtained through the elements and combinations particularly indicated in the appended claims. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative and are not limitations of the claims. The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate certain embodiments and are used together with the specification to further explain certain principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] This U.S. provisional application includes at least one color drawing. If a non-provisional or PCT application is published in the future that claims priority to this U.S. provisional application and incorporates the content of this provisional application, the Office will provide copies of this provisional patent application (including color drawings) upon request and payment of the necessary fee.

[0012] Figures 1A to 1G The posterior colon ( Figure 1A , Figure 1B and Figure 1C ), colorectal cancer( Figure 1D and Figure 1E ),pancreas( Figure 1F ) and kidneys( Figure 1G ) samples. The samples were stained as follows: 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] FIG. 2A to FIG. 2C The fluorescence ( Figure 2A ) or BSE-SEM( Figure 2B ) or fluorescence and BSE-SEM ( Figure 2C ) imaging combination at 200x( Figure 2A ) or 250x( Figure 2B and Figure 2C ) magnification of islets of Langerhans in the mouse pancreas imaged in the presence of anti-insulin antibodies double stained with fluorescence and gold.

[0014] Figures 2D to 2F The fluorescence ( Figure 2D ) or BSE-SEM( Figure 2E ) or fluorescence and BSE-SEM ( Figure 2F ) imaging combination at 400x( Figure 2D ) or 1000x( Figure 2E and Figure 2F ) magnification of islets of Langerhans in the mouse pancreas imaged in the presence of anti-insulin antibodies double stained with fluorescence and gold.

[0015] FIG. 3A to FIG. 3B The fluorescence ( Figure 3A ) and BSE-SEM( Figure 3B ) at 1000x( Figure 3B ) and 20,000x( Figure 3A ) magnification of islets of Langerhans in the mouse pancreas imaged in the presence of anti-insulin antibodies double stained with fluorescence and gold.

[0016] Figure 4 Shown is the detection and localization of allele-specific oligonucleotide nanoparticles in ependymal cells of the choroid plexus chamber by BSE-SEM at 500x magnification following intracerebroventricular injection of the nanoparticles into the mouse brain. The samples were prepared by the methods described herein.

[0017] FIG. 5A to FIG. 5E Shown at 400x( Figure 5A )、5000x( Figure 5B and Figure 5C )、15,000x( Figure 5D ) and 20,000x( Figure 5E ) Detection and localization of allele-specific oligonucleotide nanoparticles in ependymal cells of the choroid plexus. A gold-labeled antibody specific for the nanoparticles was used and the nanoparticles are identified using arrows in the image. Figure 5B and Figure 5C The features circled in the upper part of Figure 5D and Figure 5E .

[0018] Implementation of Certain Embodiments

[0019] I. Definitions

[0020] Unless otherwise defined, scientific and technical terms related to the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0021] In this application, the use of "or" means "and / or" unless otherwise stated. In the context of multiple dependent claims, the use of "or" refers back to more than one preceding independent or dependent claim only in an alternative manner. In addition, unless specifically stated otherwise, terms such as "element" or "component" encompass elements and components comprising one unit as well as elements and components comprising more than one subunit.

[0022] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (such as one tenth and one hundredth of an integer) where appropriate.

[0023] Units, prefixes and symbols are expressed in the form accepted by its Système International de Unites (SI). Numerical ranges include the numbers defining the ranges. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0024] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0025] The term "cryoprotection" or "cryoprotecting" refers to the process of protecting a sample, such as a tissue sample, from damage (eg, to cell structure or cell integrity) that might occur by freezing the sample.

[0026] As used herein, "tissue sample" refers broadly to any sample from any organism containing cells or tissues. The sample can be derived from a solid organ (such as by sectioning the organ), or from a portion of an organ, such as a piece of tissue, or from a cell culture, etc.

[0027] As used herein, "freeze substitution" or "performing freeze substitution" refers to the process of using an organic solvent at very low temperatures to effectively dissolve frozen water in and / or surrounding a cryogenic tissue sample, thereby replacing water molecules with molecules of the organic solvent.

[0028] When applied to tissue samples, the term "average thickness" refers to the average overall thickness of a sample (e.g., a tissue sample arranged on a slide) across the length of the sample. The numerical value of the average thickness includes experimental and observational errors, which may be relatively large in this case because many samples may be sliced ​​manually rather than in a professional instrument.

[0029] The term "vibratome" refers to a specialized method for obtaining very thin tissue sections (eg, 0.4 mm thick or less), typically by using a specialized vibrometer instrument.

[0030] The term "high pressure fixation" or "high pressure freezing" refers to a method in which an organ or tissue sample is fixed by exposing it to both freezing temperatures and high pressures well above atmospheric pressure, with the goal of maintaining the organ or tissue without loss of ultrastructure.

[0031] The term "ultrastructure" refers to the cell or organelle structure within a tissue sample. The term "epitope" herein refers to a binding site for an antibody, polypeptide, oligonucleotide or other molecule that is intended to specifically target a particular molecule or structure in a cell. An epitope may include, for example, a specific binding site on a targeted molecule, or a specific binding site on a targeted structure of a cell such as an organelle.

[0032] The term "perfusion fixation" or "chemical fixation" refers 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 related structures of the organ or tissue sample. In some embodiments, the fixative chemical then forms crosslinks with the structural components of the organ or tissue, preserving the structure of these structural components.

[0033] As used herein, a "light fixative" is a fixation chemical (e.g., paraformaldehyde) that produces light crosslinks, such as single crosslinks from one fixative molecule to molecules in the fixed sample, as opposed to more robust crosslinks, such as double crosslinks (i.e., "strong fixatives," such as glutaraldehyde or osmium tetroxide). An example of a light fixative is 1% to 4% paraformaldehyde.

[0034] As described herein, "embedding resin" refers to a substance that is used to penetrate a tissue sample on a slide prior to imaging in order to fix the components of the tissue sample in place, such as by polymerization of the resin after it has penetrated the sample, and to allow the sample to be stored without degradation or movement of the ultrastructure and molecules to be imaged.

[0035] II. Exemplary Methods for Cryopreservation of Tissue Samples

[0036] The present disclosure encompasses methods such as preparing cryoprotected tissue samples and using such cryoprotected tissue samples to carry out fluorescence and / or electron microscopy. For example, such ultrastructural immunohistochemical applications can be ideally used to identify cell types and organelles, and to locate biological processes involving cells and organelles, such as via the location of specific molecules involved in biological processes. In some cases, such applications can ideally contribute to understanding and evaluating the mechanism of potential therapeutic agents. For example, a goal of this method is to be able to locate potential drug targets in a sample, or to locate therapeutic agents in a sample, to observe how they interact with their target molecules and other cell structures. Such imaging applications rely on high-quality tissue samples, which maintain both ultrastructure and binding epitopes, while allowing appropriate labeling density in fluorescence and electron microscopic imaging methods. This method solves the technical challenges of finding the best compromise between labeling density and ultrastructure maintenance in the sample and the fixation and processing conditions.

[0037] For example, a comparative method for preparing samples is to fix the samples by using high pressure and very low temperature, rather than by adding chemical fixatives (high pressure fixation). The advantage of the method may be that, since the initial tissue is not chemically fixed, they will not have the cross-linking that may cover the epitope or affect the ultrastructure, so that the antigen is well maintained in structure when performing fluorescence imaging, and the background is less. However, in the method, only small tissue samples can be frozen, such as samples with a thickness of less than 400 μm (such as 100 μm to less than 400 μm). Such small sample size may not be very useful for analysis. In addition, since the thickness of the sample must be so small, it is usually necessary to use vibration microtomy to prepare such samples. Both vibration microtomy and high pressure fixation are costly, and require professional instruments. In addition, due to the freezing in the tissue, there may be fixed artifacts.

[0038] In contrast, in the present disclosure, the inventors have found that cryoprotection of tissues that have been lightly chemically fixed (such as with 1%-4% paraformaldehyde) is simpler, less expensive, and allows for the preparation of larger samples of about 1 mm thickness. This allows for larger slices, which in some cases can be imaged by wide-field BSE-SEM imaging. The inventors have also found that when prepared according to the methods herein, such can be stored for reasonable periods of time and retain high-quality ultrastructure for imaging. Since the present method does not require either vibration microtomy or high-pressure fixation, the method is less expensive and simpler to perform. Although the use of chemical fixation may present challenges, such as the risk of artifacts produced by chemical fixation of the sample, and the possibility of loss of antigenicity, delocalization of antigens, increased background autofluorescence caused by the presence of aldehydes, and dimming of GFP fluorescence, the inventors have found that the methods herein produce relatively high-quality fluorescence and electron microscopy images.

[0039] In some embodiments, the methods herein include cryoprotecting a tissue sample, which in some embodiments has been fixed with a light fixative such as 1%-4% paraformaldehyde and has 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 include cryoprotecting a tissue sample by 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); (d) adding an embedding resin compatible with temperatures of -60°C to -80°C, such as an acrylate and methacrylate resin, to the sample of (c), and polymerizing the resin.

[0040] In some embodiments, the sample is previously perfused and fixed with a mild fixative, or is taken from a tissue or organ that has been perfused and fixed with a mild fixative. Therefore, while the sample is chemically fixed, the degree of cross-linking in the sample is maintained at a minimum level by using a mild fixative (such as paraformaldehyde) and at a relatively low concentration (such as 1%-4% paraformaldehyde) relative to a strong fixative such as glutaraldehyde. In some embodiments, the mild fixative is 2%-4% paraformaldehyde. In some embodiments, the mild fixative is 2% paraformaldehyde. In some embodiments, the mild fixative is 3% paraformaldehyde. In some embodiments, the mild fixative is 4% paraformaldehyde. In some embodiments, perfusion fixation does not include glutaraldehyde or osmium tetroxide. In some embodiments, the tissue sample is not high-pressure frozen.

[0041] In some embodiments, the tissue sample to be cryoprotected has an average thickness of 0.4 mm to 2 mm, such as 0.4 to 1.5 mm, 0.5 to 2 mm, 0.5 to 1.5 mm, 0.8 to 1.2 mm, 0.5 to 1.0 mm, 0.4 mm to 1.0 mm, 1.0 to 2 mm, 1.0 to 1.5 mm, or 1.5 to 2 mm. In some embodiments, the average thickness is 0.8 to 1.2 mm. In some embodiments, the average thickness is 1 mm. In some embodiments, the sample is not prepared by vibration microtomy, and / or has been manually prepared or sliced, for example, to an average thickness of 2 mm or less. In some embodiments, the sample has been chemically fixed, such as fixed by perfusion with a mild fixative, and has also been prepared to the above-mentioned thickness.

[0042] The DMSO solution used for cryoprotection can be, for example, 35%-70% DMSO, such as 40%-60% DMSO, 40%-50% DMSO, 50%-60% DMSO, 40%, 45%, 50%, 55% or 60% 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, such as 2, 4, 8, 10, 12 or 18 hours, or overnight. In some embodiments, exposure to the DMSO solution and low temperature, such as at 2°C-8°C or at 4°C. In some embodiments, samples that have been chemically fixed (such as perfusion fixation with a mild fixative) have been prepared to the above-mentioned thickness and have been exposed to a DMSO solution having the above-mentioned DMSO concentration range.

[0043] After cryoprotection in DMSO solution, the sample can be frozen in liquid nitrogen or frozen with an equivalent method of supercooling the sample. In some cases, for example, a sample with an average thickness of 2 mm or less can be placed on a surface suitable for immersion in liquid nitrogen (such as a relatively flat surface, 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.

[0044] Once the sample is frozen in liquid nitrogen, freeze substitution can be performed to remove the free water in the sample by effectively dissolving the free water with an organic solvent. In certain embodiments, freeze substitution is performed by exposing the sample kept frozen at -60°C to -80°C to a solvent such as acetone, isopropanol or methanol. In certain embodiments, freeze substitution is performed in acetone, i.e. 100% acetone. Therefore, in certain embodiments, freeze substitution is performed in a solution consisting essentially of acetone. In some cases, the solvent used for freeze substitution, such as acetone, does 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, a small amount of osmium tetroxide is added, such as 0.001% to 0.01%, or 0.001%-0.002%. In some cases, freeze substitution is performed at -60°C to -80°C for at least 1 hour to up to a week, for example, for at least 12 hours to 96 hours, or 48 to 96 hours. As used herein, freeze substitution and cryogenic treatment allow to maintain ultrastructure, omit the need for strong fixatives such as glutaraldehyde (GA) and osmium tetroxide (OsO4), allow to retain GFP fluorescence, maintain antigens, and reduce protein, lipid and DNA / RNA extraction compared with other methods. Freeze substitution is not bound by any specific mechanism, and relies on the following principle: the frozen water found in biological samples can be dissolved and replaced by organic solvents (such as acetone, methanol or isopropanol, etc.). Freeze substitution is not bound by any specific mechanism, and it can be considered that at extremely low temperatures, such as -80 ° C, organic solvents that keep liquid preferentially dissolve "free unbound" water and tend to keep the more tightly bound hydration layer ("molecularly bound water") of biopolymers intact, thereby ensuring that biopolymers remain correctly folded. The hydration layer is considered to be crucial for the correct folding and assembly of biopolymers. Therefore, the maintenance of the hydration layer during low temperature dehydration helps to maintain the molecular structure, ultrastructure and antigenicity of the sample.

[0045] After freeze substitution, the sample is exposed to an embedding resin compatible with the temperature of -60°C to -80°C used to store the sample, such as acrylate and methacrylate resins such as HM23 (which is a non-polar resin) or K11M (which is a polar resin). For example, in some embodiments, the resin material can penetrate the sample while the sample is maintained at such a 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 360nm light).

[0046] As used herein, such resin-embedded tissues have a variety of advantages, including: forming stable and solid tissue blocks; being able to be stored for months or years; being easily sectioned and potentially serially sectioned; being stable under potentially harsh antigen retrieval conditions; being more easily handled for complex applications such as CLEM; being quantitatively labeled on a nano-flat surface; being compatible with fluorescent and / or immunogold labeling on sections; and having an appearance similar to standard morphological transmission electron microscopy (TEM) images.

[0047] In some embodiments, after the polymerization of the resin, the sample can be further prepared for imaging. For example, in some embodiments, a suitable dye (such as a fluorescent dye or label, or a heavy metal dye for electron microscopy, such as osmium tetroxide, uranyl acetate and lead citrate, etc.) can be added for any one or both of fluorescence or electron microscopy imaging. In some cases, the sample can be further sliced ​​for imaging, such as cut or sliced ​​into slices with an average thickness of less than 1mm (such as 0.5mm) or less (such as 100-1000nm, 300-1000nm, 400-600nm, 400nm, 500nm, or 600nm). In some cases, the sample or the slice of the sample can then be placed on an imaging plate or a slide for imaging by fluorescence and / or electron microscopy.

[0048] Therefore, in further embodiments of the present disclosure, methods include methods of performing fluorescence and / or electron microscopic imaging of samples prepared as described herein. Examples of electron microscopy include, for example, CLEM, BSE-SEM, and immunogold EM. In some cases, samples prepared as described herein can distinguish the position of macromolecules (e.g., 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 tissue samples. In other cases, the methods herein can distinguish the position of certain small molecules in tissue samples herein.

[0049] III. Kits, Systems, and Tissue Samples Prepared by the Methods

[0050] The present disclosure also includes tissue samples prepared according to the methods herein. Examples include tissue samples maintained in polymerized embedding resins and sections of such samples, which may or may not be placed on slides or plates for storage and imaging.

[0051] The present disclosure also includes a kit for performing the method of tissue sample preparation described herein. In some embodiments, the kit includes one or more of the reagents used in the methods described herein, such as DMSO solution (such as 40%-60% DMSO, such as 40%, 45%, 50%, 55% or 60% DMSO), a freeze substitution solution (such as a mixture of 100% acetone or 100% acetone and at least one heavy metal stain (such as uranyl acetate and / or osmium tetroxide)), and an embedding resin (such as polar or non-polar acrylate and methacrylate resins), wherein the resin is compatible with a temperature of -60°C to -80°C. In some embodiments, the kit also includes a surface for immersing the tissue sample in liquid nitrogen for cooling, such as a plate, a chip or a slide (i.e., one or more such surfaces). In some embodiments, the kit includes fluorescence and / or electron microscopy imaging slides (i.e., one or more such slides). In some embodiments, the kit includes instructions for use. In some cases, the kit includes all of the above components.

[0052] The disclosure also contemplates a system for performing the methods described herein. In some embodiments, the system may include a surface, such as a plate, a chip or a slide (i.e., one or more such surfaces), which is used to immerse the tissue sample in liquid nitrogen for cooling, and for performing other reactions herein, such as exposure to a DMSO solution, freeze substitution of the sample, and / or adding an embedding resin and polymerizing it. For example, in some embodiments, all method steps may be performed in the system so that at least one of the methods is automatically performed, or all method steps are automatically performed. In some cases, the system also includes a test kit as described above, to provide, for example, a reagent for the steps of the method, a suitable slide or surface on which the sample can be placed during the reaction, and / or instructions for use.

[0053] Examples

[0054] The following examples illustrate further embodiments of the methods herein.

[0055] Example 1. Preparation of Colon, Colorectal Cancer, Pancreas, and Kidney Samples for Imaging

[0056] To prepare tissue samples for imaging of colon, colorectal cancer (CRC) cells, pancreas, and kidney tissue, samples in the form of approximately 1 mm slices were obtained from larger samples that had been perfused with 4% paraformaldehyde (FPA). The 1 mm sliced ​​samples were infiltrated with 50% DMSO at 4°C for at least one hour to overnight, placed on a copper plate and frozen in liquid nitrogen. Freeze substitution was performed at -80°C in 100% acetone (without uranyl acetate (UA), osmium tetroxide (Os), or GA (glutaraldehyde)). The freeze-substituted samples were then embedded in HM23 resin and kept until imaged. Prior to imaging, the samples were stained with osmium tetroxide, uranyl acetate, and lead citrate to visualize the ultrastructure. The results are shown in Figures 1A to 1E , and compared with samples that were similarly treated but freeze-substituted in 100% acetone containing 0.1% uranyl acetate and without GA or osmium tetroxide, e.g. Figure 1F and Figure 1G shown.

[0057] Example 2. Preparation of samples for imaging of anti-insulin antibodies in islet cells of Langerhans

[0058] Pancreatic slices (islets of Langerhans in mouse pancreas) were prepared similarly to Example 1 and embedded on carbon-coated glass slides. In HM23. After blocking, anti-insulin monoclonal antibody ab6995 was added, followed by anti-mouse biotinylated secondary antibody (Jackson Immuno) and fluorescently labeled avidin (avidin A488) and 10 nm gold conjugate (Invitrogen) to stain the anti-insulin antibody with both fluorescence and gold. Hoechst staining was applied to the sample to provide fluorescence contrasting with the anti-insulin antibody dye. Osmium tetroxide, uranyl acetate and lead citrate were added to the sample at the same time to make the cell structure visible under electron microscopy. Figure 2A A fluorescence image of the sample at 200x magnification is shown, where the fluorescently stained antibodies are shown as light-colored markers. A 250x BSE-SEM image of the sample (taken by Zeiss Gemini 300) 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 to 2F and FIG. 3A to FIG. 3B Medium, shot at a higher magnification level.

[0059] Example 3. Preparation of samples for imaging of oligonucleotide nanoparticles in cells

[0060] Allele-specific oligonucleotide nanoparticles (ASO nanoparticles) have been explored as potential therapeutic agents. Figure 4 and FIG. 5A to FIG. 5E Images from intracerebroventricular (ICV) injection of ASO nanoparticles into the mouse brain are shown to discuss which tissues, cells, and organelles the ASO nanoparticles accumulate in and whether the encapsulation of ASOs into lipid nanoparticles changes their uptake and localization. Brain tissue samples were processed for imaging according to the methods herein. Specifically, brain tissue was fixed in 4% PFA and 0.1% GA and prepared into 1 mm thick slices, which were then trimmed to a size of 2×2×1 mm. The trimmed samples were cryoprotected in 50% DMSO at 4°C for at least one hour to overnight, placed on a copper plate and frozen in liquid nitrogen, then freeze-substituted at -80°C in acetone containing 0.5% GA, 0.01% UA, and 0.001% OsO4 for 96 hours, washed with ethanol at -80°C, and treated to -70°C. HM23 resin. BSE-SEM imaging was then performed on 500 nm thick sections stained with heavy metals on carbon-coated glass slides. The results are shown in Figure 4 .

[0061] Figures 5A to 5E Detection of ASO nanoparticles in ependymal cells of the choroid plexus is shown. Samples for imaging were divided into 500 nm slices and pre-blocked. Anti-ASO rabbit antibodies specific to the ASO analyzed were added, followed by anti-rabbit biotinylated secondary antibodies (Jackson Immuno). For fluorescence microscopy, avidin Ax568 was added to fluorescently stain the ASO particles, while DAPI dye (Invitrogen) was added to stain the nuclei. Figure 5A The arrows in the figure show the location of the ASO nanoparticles in the cells. For immunogold electron microscopy examination, avidin and 20 nm gold conjugate (Abcam) were added, and OsO4, UA, and LC were further added to stain the cell structure. BSE-SEM was performed using a Zeiss Gemini 300 instrument, BSD1 detector 3 kV. The results are shown in FIG. 5B to FIG. 5E The circles show the localization of ASO nanoparticles at 5000x magnification ( Figure 5B and 5C ), while arrows depict the localization of ASO nanoparticles at 15,000x or 20,000x magnification ( Figure 5D and 5E ).

[0062] It is believed that the foregoing written description is sufficient to enable a person skilled in the art to practice the present embodiment. The foregoing description and examples detail certain embodiments and describe the best mode contemplated by the inventor. However, it should be understood that no matter how detailed the foregoing content is in the text, the embodiments can be practiced in many ways and should be interpreted according to the attached claims and any equivalent forms thereof.

Claims

1. A method for 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) performing freeze substitution on the sample of (b); as well as d) adding an embedding resin compatible with temperatures of -60°C to -80°C to the sample of (c) and polymerizing the resin.

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

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

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

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

6. The method according to any one of claims 1 to 5, wherein the tissue sample of (a) has not been prepared via vibratome microtome and / or wherein the sample has been manually prepared or sectioned.

7. The method according to any one of claims 1 to 6, wherein the tissue sample has been perfused with a light fixative, or has been obtained from a tissue or organ that has been perfused with a light fixative, and / or wherein the tissue sample has not been subjected to high pressure freezing.

8. The method of claim 7, wherein the mild fixative comprises 1% to 4% paraformaldehyde.

9. The method according to 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. A method according to claim 9, wherein the sample of (b) is freeze-substituted in a mixture of 100% acetone with 0.01% to 0.2% uranyl acetate and optionally with osmium tetroxide, such as 0.001% to 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. A method according to any one of claims 1 to 12, wherein the embedding resin is an acrylate or methacrylate resin and / or wherein the resin is polymerised by ultraviolet light, such as at a wavelength of 360 nm.

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

15. The process according to any one of claims 1 to 14, wherein the process is carried out at atmospheric pressure.

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

17. The method of any one of claims 1 to 6 or 8 to 16, wherein the method further comprises preparing the tissue sample having an average thickness of 2 mm or less by obtaining a tissue sample or organ that has been perfused with a light fixative and slicing the tissue sample or organ to an average thickness of 2 mm or less.

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

19. A method of 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 has been prepared by a process comprising: (a) exposing the sample to a dimethyl sulfoxide (DMSO) solution, (b) freezing the sample of (a) in liquid nitrogen, (c) subjecting the sample of (b) to freeze substitution, and (d) adding an embedding resin compatible with temperatures of -60°C to -80°C to the sample of (c) and polymerizing the resin; the method comprising: Electron microscopy is performed on the tissue sample or on the section of the tissue sample.

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

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

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

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

24. The method of any one of claims 19 to 23, wherein the tissue sample of (a) has not been prepared via vibratome microtome and / or wherein the sample has been manually prepared or sectioned.

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

26. The method of claim 25, wherein the mild fixative comprises 1% to 4% paraformaldehyde.

27. A method according to any one of claims 19 to 26, 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.

28. A method according to claim 27, wherein the sample of (b) is freeze-substituted in a mixture of 100% acetone with 0.01% to 0.2% uranyl acetate and optionally with osmium tetroxide, such as 0.001% to 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 of any one of claims 19 to 28, wherein the embedding resin is a polar resin.

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

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

33. The method according to any one of claims 19 to 32, wherein the method is 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 parenchymal organ.

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

36. The method of any one of claims 19 to 35, wherein the method is capable of discerning the location of drug molecules such as, for example, lipid-coated drugs, antisense drugs, antibody drugs, peptide drugs or small molecule drugs in or adjacent to cells in the sample.

37. The method according to any one of claims 19 to 36, wherein the method is performed on sections of the tissue sample having an average thickness of 300 nm to 1000 nm, such as 500 nm.

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

39. The method according to any one of claims 1 to 38, wherein the DMSO solution comprises 40% to 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. A kit for performing the method according to any one of claims 1 to 18 or 39, the kit 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 the kit optionally further comprising at least one of: a plate or chip for immersing tissue samples in liquid nitrogen; a fluorescence and / or electron microscopy imaging slide; and instructions for use.

42. A system for performing the method according to any one of claims 1 to 18 or 39, wherein the system automatically or semi-automatically performs the following steps: 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) performing freeze substitution on 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.

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

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