Method for rapidly transparentizing biological tissue

Abstract

The invention relates to a method for rapidly transparentizing biological tissues, which comprises the following steps: carrying out degreasing treatment on a fixed biological tissue sample by using a degreasing solution which is an aqueous solution containing tetrahydrofuran and tetrahydroxypropyl ethylenediamine (EDTP); the degreased biological tissue sample is subjected to refractive index matching with a refractive index matching solution, transparency treatment is completed, and the refractive index matching solution is an aqueous solution containing urea, iohexol and EDTA. According to the method for transparentizing the biological tissue, visceral organs can be transparentized quickly, tissue shrinkage is not caused, and the tissue form is reserved. In addition, the method for transparentizing the biological tissue according to the invention has the advantages of fluorescence protection, morphology retention and high biological safety.

Description

Technical Field

[0001] This invention relates to the field of biological tissue transparency processing technology, and specifically to a method for making biological tissue transparent. Background Technology

[0002] Tissue transparency technology is an effective means of obtaining the three-dimensional structure of biological tissues and studying gene expression, cell morphology, and other related issues. Traditional methods for high-resolution three-dimensional imaging of biological tissues require slicing, two-dimensional imaging, and three-dimensional reconstruction, which are inefficient and involve complex sample pretreatment. Tissue transparency technology overcomes these obstacles, enabling cutting-edge three-dimensional fluorescence microscopy imaging techniques to efficiently acquire structural information of various biological tissues.

[0003] Biological tissue clearing techniques can be categorized into hydrophobic, hydrophilic, and hydrogel-based methods. Different methods have varying impacts on the three-dimensional imaging results of samples. Hydrophobic methods, represented by DISCO, utilize organic solvents for dehydration, defatting, and refractive index matching, exhibiting strong transparency but damaging endogenous fluorescent proteins and limiting high-resolution imaging [1-4]. Hydrogel-based methods, represented by CLARITY, fix most amino-containing proteins through cross-linking and treat with detergents to bring the sample's refractive index within the range of most high numerical aperture objectives, but the procedure is complex and prone to damaging the sample and causing loss of endogenous fluorescent proteins [5-8]. Hydrophilic methods, represented by the CUBIC series, use chemical reagents for defatting and refractive index matching, offering advantages in preserving endogenous fluorescent proteins and being suitable for clearing treatment with labeling staining. The final refractive index is approximately 1.49, facilitating high-resolution imaging, but the speed is relatively slow [7,9-11].

[0004] Therefore, there is an urgent need to develop a rapid tissue transparency method that can preserve endogenous fluorescent proteins and is compatible with immunofluorescence labeling, in order to meet the requirements of high-resolution three-dimensional imaging of intact organs.

[0005] References:

[0006] 1.Pan, C., et al., Shrinkage-mediated imaging of entire organs andorganisms using uDISCO.Nat Methods, 2016.13(10):p.859-67.

[0007] 2.Belle,M.,et al.,A simple method for 3D analysis of immunolabeledaxonal tracts in a transparent nervous system.Cell Rep,2014.9(4):p.1191-201.

[0008] 3.Renier,N.,et al.,Mapping of Brain Activity by Automated VolumeAnalysis of Immediate Early Genes.Cell,2016.165(7):p.1789-1802.

[0009] 4.Hahn,C.,et al.,High-resolution imaging of fluorescent whole mousebrains using stabilised organic media(sDISCO).J Biophotonics,2019.12(8):p.e201800368.

[0010] 5.Tomer,R.,et al.,Advanced CLARITY for rapid and high-resolutionimaging of intact tissues.Nat Protoc,2014.9(7):p.1682-97.

[0011] 6.Greenbaum,A.,et al.,Bone CLARITY:Clearing,imaging,and computationalanalysis of osteoprogenitors within intact bone marrow.Sci Transl Med,2017.9(387).

[0012] 7.Tainaka,K.,et al.,Chemical Landscape for Tissue Clearing Based onHydrophilic Reagents.Cell Rep,2018.24(8):p.2196-2210e9.

[0013] 8. Park, YG, et al., Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat Biotechnol, 2018.

[0014] 9. Susaki, EA, et al., Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell, 2014.157(3):p.726-39.

[0015] 10. Susaki, EA, et al., Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc, 2015.10(11):p.1709-27.

[0016] 11. Kubota, SI, et al., Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution. Cell Rep, 2017.20(1):p.236-250. Summary of the Invention

[0017] To overcome the aforementioned shortcomings of existing biological tissue transparency technologies, the inventors have developed a novel method for rapidly transparentizing biological tissues. This method, through a combination of a specific degreasing solution and a refractive index-matching solution, enables rapid transparency of organs without causing tissue shrinkage and preserving tissue morphology. Furthermore, the method can also incorporate fluorescent protective components to retain endogenous fluorescent proteins.

[0018] This invention provides a method for producing transparent biological tissue, comprising the following steps:

[0019] (1) Degreasing: The fixed biological tissue sample is degreased with a degreasing solution, wherein the degreasing solution is an aqueous solution containing tetrahydrofuran (THF) and tetrahydroxypropylethylenediamine (EDTP);

[0020] (2) Refractive index matching: The defatted biological tissue sample is subjected to refractive index matching solution to complete the transparentization process. The refractive index matching solution is an aqueous solution containing urea, iohexol and EDTP.

[0021] In this invention, the biological tissue can be selected from biological tissues such as brain, heart, spinal cord, lung, kidney, liver, spleen, stomach, intestine, testis, glands, skin, and bones. The biological tissue sample can be the whole or a part of the aforementioned biological tissue. The organism can be selected from one or more biological research model animals. Examples of biological research model animals include nematodes, zebrafish, planarians, fruit flies, clawed frogs, salamanders, mice, rats, rabbits, pigs, and monkeys. Alternatively, the organism can be a vertebrate, including mammals, reptiles, and birds. Examples of mammals include humans, mice, dogs, cats, rabbits, pigs, monkeys, cattle, horses, and donkeys. Examples of reptiles include turtles, snakes, and lizards. Examples of birds include chickens, ducks, geese, and parrots.

[0022] In step (1) defatting (sometimes also called degreasing), there are no particular limitations on the fixation of the biological tissue sample, and any fixation method applicable in the art can be used. For example, freshly obtained biological tissue samples can be fixed using paraformaldehyde (PFA) (e.g., a 4% aqueous solution of PFA). In some embodiments, fixation can be performed under shaking conditions (e.g., on a shaker). After fixation, PFA is removed by washing the tissue with a PBS solution (e.g., a 0.01M PBS solution) to thoroughly remove residual PFA.

[0023] In step (1) degreasing, the degreasing solution comprises 40 to 60 ml (e.g., 45, 50, 55 ml) of THF, 4 to 6 g (e.g., 4.5, 5.0, 5.5 g) of EDTP, and 60 to 40 ml (e.g., 55, 50, 45 ml) of water. In some embodiments, the degreasing solution comprises 50 ml of THF, 5 g of EDTP, and 45 ml of water. Although the components of the degreasing solution are described here by volume or mass, those skilled in the art will understand that the above volume or mass indicates the proportion of these components in the degreasing solution, and the content of these components can be increased proportionally to prepare the desired degreasing solution. In some embodiments, the degreasing solution consists essentially of the above components, or is composed of the above components. Using the degreasing solution allows for rapid removal of lipids without causing tissue dehydration and deformation, thus preventing tissue shrinkage and preserving tissue morphology.

[0024] In some embodiments, the degreasing agent may be changed periodically or irregularly during the degreasing process. For example, the degreasing agent may be changed every 6 to 48 hours, such as every 12 hours, every 24 hours, or every 48 hours, but the invention is not limited thereto.

[0025] In the degreasing process, there is no particular limitation on the amount of degreasing reagent used in a single application, as long as it can submerge the biological tissue sample. In particular, the specific amount can be determined based on the volume of the biological tissue sample. For example, the amount of degreasing reagent used in a single application can be about 5 to 20 times the sample volume, preferably about 10 to 25 times, particularly 15 to 25 times, such as about 17, 18, 19, 20, 21, 22, 23, and 24 times.

[0026] The defatting process can take more than 5 minutes, more than 1 hour, or more than 1 day, but is not limited to these. The specific defatting time can be appropriately varied depending on the volume of the biological tissue sample, its age, and the amount of defatting reagent used. For example, for a mouse brain slice with a thickness of 200 micrometers, defatting can be achieved in about a few minutes; for the entire internal organs and nervous tissue (such as the whole brain and spinal cord) of a typical adult mouse, defatting can be completed in 24 hours; for tissues from older animals, the defatting time can be extended, and the overall defatting time can be 1 to 4 days.

[0027] Degreasing can be performed at 4°C, room temperature to 37°C, but the invention is not limited thereto. If the tissue contains intrinsic fluorescence, it is preferred to perform the treatment at 4°C.

[0028] After degreasing is complete, discard the degreasing solution and wash the tissue thoroughly with PBS solution (e.g., 0.01M PBS solution) to remove the degreasing solution.

[0029] In step (2) refractive index matching, the refractive index matching solution comprises 200 to 300 (e.g., 220, 240, 250, 260, 280) g / L urea, 600 to 800 (e.g., 650, 700, 750) g / L iohexol, and 15 to 25 (e.g., 17.5, 20, 22.5) g / L EDTP. In some embodiments, the refractive index matching solution comprises 250 g / L urea, 700 g / L iohexol, and 20 g / L EDTP. The refractive index solution is substantially composed of the above components, or is composed of the above components.

[0030] In some embodiments, the refractive index matching solution can be replaced periodically or irregularly during refractive index matching. For example, the refractive index matching solution can be replaced every 6 to 48 hours, such as every 12 hours, every 24 hours, or every 48 hours, but the invention is not limited thereto. There is no particular limitation on the amount of refractive index matching solution used at one time, as long as it is sufficient to immerse the biological tissue sample. In particular, the specific amount can be determined based on the volume of the biological tissue sample, and the amount of refractive index matching solution used at one time can be 2 to 22 times the sample volume, preferably 7 to 17 times, particularly 9 to 15 times, for example 10, 11, 12, 13, or 14 times.

[0031] In some embodiments, the soaking time using the refractive index matching solution in the above-mentioned refractive index matching process can be more than 5 minutes, more than 1 hour, more than 1 day, etc., but is not limited to these. The specific processing time can be appropriately varied according to the volume of the biological tissue, its age, and the amount of refractive index matching solution used. For example, refractive index matching of adult mouse tissues and organs can generally be completed in 2 days.

[0032] In some embodiments, all steps in the method for the transparent biological tissue can be performed under oscillating conditions (e.g., on a shaker).

[0033] In some embodiments, in the method for making the transparent biological tissue, all steps are performed in the dark on a low-speed shaker at room temperature in order to protect the endogenous fluorescence signal.

[0034] In some embodiments, the method for transparent biological tissue according to the present invention may further include one or more steps selected from decolorization and decalcification prior to defatting. For example, when the biological tissue is a pigment-rich tissue, such as the heart, kidney, spleen, etc., decolorization may be performed first; when the biological tissue contains bone, such as the skull, decalcification may be performed.

[0035] Decolorization and decalcification can be performed using conventional methods in the art without particular limitation. For example, biological tissue can be placed in a decolorizing solution for decolorization, such as in a shaker at 37°C with slow shaking at 60 rpm for 24 hours. After decolorization, the next step can be performed directly without washing. The decolorizing solution can be a commonly used decolorizing solution, such as a hydrogen peroxide aqueous solution (10% H2O2 solution with a mass / volume concentration (g / ml)) or an EDTP aqueous solution (e.g., a 20% EDTP solution with a mass / volume concentration (g / ml)). Decalcification can be performed by placing biological tissue in a decalcification solution, such as in a shaker at room temperature, for a specific time until the bone softens. The decalcification solution can be a commonly used decalcification solution, such as an EDTA aqueous solution (e.g., a 10% EDTA aqueous solution with a mass / volume concentration (g / ml)). The decolorizing and decalcification solutions can be changed periodically or irregularly, such as daily.

[0036] In some embodiments, the method for clearing biological tissue according to the present invention may further include an immunostaining step of the biological tissue sample after defatting and before refractive index matching. Immunostaining can be performed using conventional methods in the art, without particular limitation. For example, the biological tissue sample can be blocked with a blocking solution and then incubated with a primary antibody, followed by incubation with a secondary antibody to achieve immunostaining, but the present invention is not limited thereto.

[0037] Figure 1 A schematic diagram illustrates a tissue transparency process according to an embodiment of the present invention, comprising four steps: decolorization, decalcification, degreasing, and refractive index matching, wherein decolorization and decalcification are optional steps. Figure 1 Figure B schematically illustrates a tissue transparency process according to another embodiment of the present invention, comprising three steps: decolorization, degreasing, and refractive index matching, wherein decolorization is an optional step. Most tissue types, such as the brain, lungs, and intestines, only require degreasing and refractive index matching steps to achieve tissue transparency. The brain of adult mice and most internal organs (heart, lungs, kidneys, intestines, etc.) can be transparentized in only 3-4 days. Figure 1 C shows the effect of using the transparent biological tissue method according to the present invention to treat a portion of biological tissue to make it transparent.

[0038] In another aspect, the present invention provides an imaging method for transparentizing biological tissues, the method comprising the step of transparentizing a biological tissue sample using the method for transparentizing biological tissues according to the present invention.

[0039] The imaging method may also include other steps required for imaging, such as obtaining and fixing biological tissue before clearing, and gel embedding, imaging, or staining, slide preparation and imaging after clearing, but the present invention is not limited thereto.

[0040] In one embodiment, the imaging method includes:

[0041] 1. Fix biological tissue samples;

[0042] 2. To perform transparentization treatment on the fixed biological tissue sample using the transparent biological tissue method according to the present invention;

[0043] 3. The transparentized biological tissue samples were then gel-embedded;

[0044] 4. Imaging of gel-embedded biological tissue samples.

[0045] In step 1 above, there are no particular limitations on the biological tissue samples used for fixation, and conventional methods in the art can be employed. For example, biological tissues isolated from organisms can be fixed by soaking them overnight in a paraformaldehyde solution (e.g., a 4% (g / mL) paraformaldehyde solution in 0.1M phosphate buffer at pH 7.2-7.4). However, the present invention is not limited thereto.

[0046] The description of the transparentization process in step 2 above is the same as that of the method for transparent biological tissue according to the present invention, and will not be repeated here.

[0047] In step 3 above, gel embedding refers to embedding the refractive index-matched biological tissue sample in a gel. There are no particular limitations on the method for embedding the biological tissue sample in the gel; any known suitable method can be used. For example, a gel solution can be added to a mold, then the biological tissue sample can be placed inside, and then gelled to obtain the gel-embedded biological tissue sample.

[0048] The gel solution can be prepared by dissolving any suitable gelling agent in a refractive index-matching solution. In some embodiments, the gelling agent is agarose. In some embodiments, the gel solution is a solution of agarose in a refractive index-matching solution at a mass percentage concentration of 1.5% to 3%, preferably 1.8% to 2.5%, such as 1.85%, 1.90%, 1.95%, 2.0%, 2.05%, 2.10%, 2.15%, 2.20%, 2.25%, 2.30%, 2.35%, 2.40%, 2.45%, etc., particularly about 2% agarose in a refractive index-matching solution.

[0049] Gel formation can be achieved by dissolving the gelling agent in a refractive index-matching solution at high temperature and then cooling it; or by adding a crosslinking agent, but the present invention is not limited thereto.

[0050] In step 4 above, imaging can be performed using any imaging method known in the art. For example, the gel-embedded biological tissue sample can be fixed on the sample holder of an imaging microscope. During imaging, the biological tissue sample needs to be immersed in an imaging buffer.

[0051] Imaging buffer protects the sample from evaporation and dehydration during imaging, preventing it from shrinking and causing the excitation light to refract multiple times before entering the sample, thus affecting the image quality. Its refractive index is basically the same as that of the refractive index matching solution, and can be in the range of 99.8% to 100.06%, preferably 99.9% to 100.01%, of the refractive index of the refractive index matching solution.

[0052] In some embodiments, the imaging buffer is silicone oil AP 100 with a refractive index of 1.50.

[0053] In another aspect, the present invention provides a kit for clearing biological tissues, comprising:

[0054] (1) Degreasing solution, the composition of which is as described above;

[0055] (2) A refractive index matching solution, the composition of which is as described above;

[0056] Alternatively, the kit may contain:

[0057] (1) Degreasing agent, wherein the degreasing agent is THF and EDTP;

[0058] (2) Refractive index matching reagent, wherein the refractive index matching reagent is urea, iohexol and EDTP;

[0059] (3) Instructions on preparing the degreasing reagent and refractive index matching reagent into the degreasing solution and refractive index matching solution as described above.

[0060] The kit may also include one or more selected from phosphate buffer (PB), paraformaldehyde (PFA), gelation reagents, imaging buffers, staining agents, etc. Furthermore, the kit may include instructions for use, containing information about the reagents and teaching how to prepare them. These instructions may be printed on a suitable medium, such as paper, or stored in a suitable storage medium, such as film, disk, optical disc, USB flash drive, hard drive, etc. Alternatively, the kit may include media containing instructions on how to obtain the instructions, such as paper printed with a QR code or a link to the instructions, film containing a QR code or a link to the instructions, or a disk, optical disc, USB flash drive, hard drive, etc., storing a QR code image or link to the instructions. Scanning the QR code allows downloading the instructions or linking to a website where the instructions can be downloaded.

[0061] The phosphate buffer can be a phosphate buffer suitable for treating biological tissues, for example, a phosphate buffer composed of disodium hydrogen phosphate and sodium dihydrogen phosphate with a pH of 7.0 to 7.4, such as 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, etc., preferably about 7.2. Those skilled in the art can appropriately select the amount of disodium hydrogen phosphate and sodium dihydrogen phosphate according to the final desired pH. In the kit, the phosphate buffer can be a solid composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, prepared into a suitable solution for use, or it can be a phosphate buffer prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate. In one embodiment, a 0.01M phosphate buffer with a pH of 7.2, prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate, can be used. Here, the concentration of the phosphate buffer is calculated based on the concentration of phosphate ions.

[0062] The paraformaldehyde is used to prepare a fixative for treating biological tissues, which is then used to fix the biological tissues. In the kit, it can be a solid, prepared as a 4% (w / v) paraformaldehyde solution in phosphate buffer, or it can be a solution. In one embodiment, the paraformaldehyde is 4% paraformaldehyde (w / v / ml concentration) with a pH of 7.2-7.4, obtained by dissolving paraformaldehyde in the aforementioned phosphate buffer (e.g., 0.1M) and adjusting the pH.

[0063] The gelling agent can be agarose, used to prepare the gel solution by dissolving agarose in a refractive index-matching solution. In the kit, the gelling agent can be solid agarose, prepared into a suitable solution before use, or it can be a pre-prepared gel solution. In some embodiments, the gel solution is 1.5% to 3% by mass percentage, preferably 1.8% to 2.5%, for example 1.85%, 1.90%, 1.95%, 2.0%, 2.05%, 2.10%, 2.15%, 2.20%, 2.25%, 2.30%, 2.35%, 2.40%, 2.45%, etc., particularly about 2% agarose solution.

[0064] The imaging buffer is a mixture of silicone oil and mineral oil, and its refractive index is substantially the same as that of the refractive index matching solution, which can be 99.8% to 100.06% of the refractive index of the refractive index matching solution, preferably 99.9% to 100.01%. In some embodiments, the imaging buffer is silicone oil AP 100 with a refractive index of 1.50.

[0065] In some embodiments, the kit includes:

[0066] 1. Degreasing solution (S1), which contains 50 ml of THF, 5 g of EDTP, and 45 ml of water;

[0067] 2. Refractive index matching solution (S2), which is an aqueous solution containing 250 g / L urea, 700 g / L iohexol and 20 g / L EDTP;

[0068] 3. Gel solution (S3), which is a solution of 2% agarose in a refractive index-matched solution by mass percentage;

[0069] 4. Imaging buffer (S4), which is silicone oil AP 100 with a refractive index of 1.50.

[0070] The method for transparent biological tissue according to the present invention can rapidly make internal organs transparent without causing tissue shrinkage and preserving tissue morphology. Furthermore, the method for transparent biological tissue according to the present invention has the advantages of fluorescence protection, morphological preservation, and high biocompatibility.

[0071] The present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments.

[0072] In this document, unless otherwise stated, the solution is an aqueous solution.

[0073] In this document, all features or conditions defined in the form of numerical ranges or percentage ranges are for the sake of brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be regarded as covering and specifically disclosing all possible sub-ranges and individual values ​​within the ranges.

[0074] In this document, numerical values ​​should be understood to have a precision with significant digits, provided that the inventive objectives are achieved. For example, the number 40.0 should be understood to cover a range from 39.50 to 40.49. Except in the detailed description of the working embodiments provided at the end, all numerical values ​​of parameters (e.g., quantities or conditions) in this application (including the appended claims) should in all cases be understood to be modified by the term “about,” regardless of whether “about” actually appears before the numerical value. “About” indicates that the numerical value allows for slight inaccuracies (some close to precision on that value; approximately or reasonably close to that value; approximate). If the inaccuracy provided by “about” is not understood in this common sense in the art, then “about” as used herein at least indicates a variation that can be produced by common methods of measuring and using these parameters. For example, “about” can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%, and in some respects, less than or equal to 0.1%.

[0075] The above description is intended to be illustrative and not restrictive. For example, the above embodiments (or one or more features thereof) may be used in combination with each other. Other embodiments may be used by those skilled in the art upon reading the above description. Furthermore, in the above specific embodiments, various features may be grouped together to simplify the disclosure. This should not be construed as an intention that a feature of the disclosure that is not claimed is necessary for any claim. Rather, the subject matter of this disclosure may be less than all the features of a particular disclosed embodiment. Thus, the claims are incorporated herein by reference as examples or embodiments, wherein each claim is an independent, separate embodiment, and these embodiments are contemplated as being possible in various combinations or arrangements with each other. The scope of this disclosure should be determined by reference to the appended claims and the full scope of their equivalents. Attached Figure Description

[0076] Figure 1 This document describes the specific process of the method for making biological tissues transparent according to the present invention, and provides an image showing the effect of tissue and organ transparency.

[0077] Figure 2 The image shows the intestinal tract of ChAT-GFP mice after it has been transparent using the transparent biological tissue method according to the present invention.

[0078] Figure 3 The method for transparent biological tissue according to the present invention is used to show the compatible immunofluorescence staining of adult mouse skin. Detailed Implementation

[0079] The following preferred embodiments are provided to better understand the present invention. However, the following embodiments are provided only to facilitate a clearer understanding of the invention, and the scope of the invention is not limited thereto. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments. The methods, reagents, and conditions used in the embodiments, unless otherwise stated, are conventional methods, reagents, and conditions in the art.

[0080] Example

[0081] Materials and Methods:

[0082] 1. Medicines and reagents

[0083] Urea was purchased from Sangon Biotech (Shanghai) Co., Ltd. (product A600148-9001).

[0084] Triton X-100 was purchased from Sangon Biotech (Shanghai) Co., Ltd., product A110694;

[0085] N,N,N′,N′-Tetra(2-hydroxyethyl)ethylenediamine (EDTP, Ethylenediamine-N,N,N′,N′-tetra-2-propanol) was purchased from Tokyo Chemical Industry Co., Ltd. (T0781);

[0086] Tetrahydrofuran (THF) was purchased from Tokyo Chemical Industry Co., Ltd., T0104;

[0087] Iohexol was purchased from Nodi Sodium (66108-95-0).

[0088] Agarose S was purchased from Nacalai Tesque, 01163-76-500g;

[0089] The silicone oil AP 100 was purchased from Sigma-Aldrich as 10838.

[0090] The 0.01M PBS solution is prepared as follows:

[0091] Weigh out 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve them in 800ml distilled water, add an appropriate amount of concentrated HCl to adjust the pH of the solution to 7.4, and finally add distilled water to make up to 1L.

[0092] 2. Solution preparation

[0093] 1) 80% (m / m) EDTP

[0094] Weigh 80g of EDTP, add 20g of dd H2O, stir well, and store away from light.

[0095] 2) Degreasing solution (S1).

[0096] Add 50ml THF, 6.25ml 80% (m / m) EDTP and 43.75ml dd H2O to a beaker, stir well and store away from light.

[0097] 3) Refractive index matching solution (S2).

[0098] Weigh 25g urea, 70g iohexol, and 2.5ml 80% EDTP, add about 50ml deionized water, heat to dissolve, and bring the volume to 100ml. After mixing thoroughly, filter using a 0.45μm microporous membrane to remove impurities. Finally, adjust the refractive index to 1.50 and set aside.

[0099] 4) Gel solution (S3).

[0100] Weigh 2 grams of agarose S and 98 grams of S2, mix them thoroughly, and microwave until the agarose S dissolves. Store the prepared gel solution S3 at 37°C. The prepared gel solution is a pale yellow, clear liquid. The gel solution can be stored at 37°C for 1-2 weeks.

[0101] 5) Imaging buffer (S4).

[0102] S4 is silicone oil AP 100 with a refractive index of 1.50. S4 can be reused. Shake well and remove air bubbles before use.

[0103] Example 1: Transparency of intact organs in adult mice

[0104] 1) Material collection and post-fixation (1 day)

[0105] Adult mice (2 months old) were deeply anesthetized by intraperitoneal injection of 2% sodium pentobarbital. Cardiac perfusion was performed using a peristaltic pump. First, room temperature 0.01M PBS was perfused at a rate of ~10 ml / min until complete blood clearance (the liver turned completely white as the standard). Then, approximately 50 ml of pre-cooled 4% PFA was perfused until the liver hardened. During PFA perfusion, tail lifting and muscle twitching were observed. After perfusion, the target tissues and organs (brain, heart, kidney, testis) were dissected and placed in 50 ml centrifuge tubes. 4% PFA was added, and the tubes were slowly shaken at 4°C for post-fixation. The volume of 4% PFA was not less than 20 times the sample volume. After fixation, the PFA was removed, and the tissues were washed three times with 0.01M PBS for 2 hours each time, with the washing process performed on a shaker to completely remove any remaining PFA.

[0106] 2) Decolorization (~1 day)

[0107] Organs such as the heart and kidneys are rich in pigments and require decolorization first. Place the tissue in 10 times its volume of decolorizing solution (20% EDTP aqueous solution, g / ml) and gently shake at 60 rpm for 24 hours at 37°C. No washing is required; proceed directly to the next step.

[0108] 3) Degreasing (~1 day)

[0109] Place the tissue in a 30ml centrifuge tube and add 20ml of defatting solution S1. Place the centrifuge tube in a 37℃ shaker and gently shake to defatt the tissue. Defatting of visceral and nerve tissues in adult mice is generally completed within 24 hours.

[0110] After degreasing, discard solution S1 and wash the tissue with 0.01M PBS to thoroughly remove the degreasing solution. Wash for 2 hours each of the first three times, and the last wash can be overnight to completely remove the degreasing solution. The washing process is performed on a shaker.

[0111] 4) Refractive index matching (2 days)

[0112] After defatting, the tissue was placed in 15 ml of solution S2 and subjected to refractive index matching on a shaker at 25°C at a speed of less than 60 rpm until the sample was completely transparent. Refractive index matching of adult mouse tissues and organs can generally be completed within 2 days.

[0113] The standard for successful matching is as follows: Place the petri dish containing the sample on the graduation line (or any pattern) of the bottom stage (Nuohai, Cat#: NH210901), ensuring the sample is submerged in solution S2. If the black graduation line is clear and undistorted when observed through the sample, the matching process is complete. Figure 1 As shown in C.

[0114] 5) Gel embedding (approximately 5 hours)

[0115] The transparent sample was embedded using the sandwich method. First, a 2mm depth of 37°C gel solution S3 was added to the appropriately sized mold cavity, and the mold was cooled on ice for approximately 30 minutes to bring the gel solution to a semi-solid state. Then, the refractive index-matched sample was placed into the mold, and gel solution S3 was added until flush with the mold surface. A coverslip was placed on top, and the sample was further cured on ice for approximately 4 hours to accelerate the process. At this point, the gel block was not transparent; it returned to transparency after standing at room temperature. The embedded sample can be stored in the accompanying imaging solution at room temperature, protected from light. Imaging is recommended as soon as possible.

[0116] Figure 1 C represents representative images of adult mouse tissues and organs before and after being transparent using the transparent biological tissue method of this invention. Each organ was degreased at 37°C for 1 day and then matched at room temperature for 2 days.

[0117] Example 2: The method of the present invention is compatible with pre-sampling fluorescent labeling methods for endogenous fluorescent proteins, fluorescent dyes, etc.

[0118] To test the compatibility of this method with fluorescent signals such as fluorescent proteins and dyes that require labeling before sampling, adult ChAT-GFP transgenic mice were used. After injecting the heart with Lectin-647 fluorescent dye to label the blood vessels throughout the body, perfusion sampling (small intestine) was performed according to step 1) of Example 1. Subsequently, the small intestine was subjected to permeation treatment at room temperature using steps 3) and 4) of Example 1, including defatting for 1 day and matching for 2 days. Then, after gel embedding using step 5) of Example 1, 3D imaging analysis of the small intestine was performed. The results are as follows: Figure 2 As shown.

[0119] Figure 2 The image shows the imaging results of the ChAT-GFP mouse intestine after it has been transparent using the method of this invention. A is a three-dimensional rendering of the small intestine after transparency, where ChAT-GFP is cyan and Lectin-647 is red. B is a cross-sectional view of the yellow area shown in A. Scale bar: 200 μm.

[0120] Figure 2 The results showed that the neural signals labeled with ChAT-GFP endogenous fluorescent protein and the vascular signals labeled with Lectin-647 were both compatible with the method of the present invention, and indicated that the method of the present invention is applicable to transgenic fluorescent labeling and fluorescent dye labeling before sampling.

[0121] Example 3: The method of the present invention is compatible with post-sampling immunofluorescence labeling.

[0122] Besides labeling biological tissues with transgenes or other methods before sampling, post-sampling immunofluorescence staining is considered a crucial labeling method for biological samples, especially human samples. To evaluate the compatibility between the method of this invention and immunofluorescence staining, an immunolabeling mechanism was used to immunize adult mouse skin.

[0123] 1) Material collection and post-fixation (1 day)

[0124] Adult mice (2 months old) were deeply anesthetized by intraperitoneal injection of 2% sodium pentobarbital. Hair was removed from the skin surface by depilatory cream. After cardiac perfusion fixation according to step 1) of Example 1, the required skin (approximately 1 cm) was harvested. 2 The tissue was then fixed overnight. The next day, PFA was removed by washing the skin tissue three times with 0.01M PBS for 2 hours each time, with the washing process performed on a shaker to completely remove any remaining PFA.

[0125] 2) Degreasing

[0126] After cleaning, the skin sample was placed in a 30ml centrifuge tube and 20ml of degreasing solution S1 was added. The centrifuge tube was placed in a shaker at 37°C and the sample was slowly shaken to remove the degreasing solution. After 48 hours, solution S1 was discarded, and the skin sample was washed with 0.01M PBS to thoroughly remove the degreasing solution. The first three washes were each 2 hours long, and the last wash could be overnight to completely remove the degreasing solution. The washing process was carried out on a shaker.

[0127] 3) Immunostaining

[0128] After washing, skin samples were blocked with blocking buffer (0.01M PBS containing 0.1% Triton X-100, 2% bovine serum albumin (BSA), and 0.05% sodium azide, g / ml) for 1.5 hours. Then, the samples were completely immersed in primary antibody dilution buffer (primary antibodies diluted with blocking buffer, with anti-Vimentin diluted 1:200, anti-CD34 diluted 1:50, and anti-Cytokeratin diluted 1:200, volume ratio) and incubated at 4°C for 4 days. After primary antibody incubation, the skin samples were washed three times with 0.01M PBS for 2 hours each time. The samples were then completely immersed in secondary antibody dilution buffer (the secondary antibodies were diluted with blocking buffer, with goat-anti-rat AF488 diluted 1:500, goat-anti-mouse AF561 diluted 1:500, and goat-anti-rabbit AF647 diluted 1:500, volume ratio), and incubated at 4°C for 4 days. Afterwards, the skin samples were washed with 10% (v / v) Triton X-100 prepared in 0.01M PBS at room temperature for 15 minutes to remove non-specifically binding antibodies from the sample surface, and then washed three times with 0.01M PBS at room temperature for 2 hours each time, taking care to avoid light.

[0129] 4) Refractive index matching (2 days)

[0130] After washing, the skin sample was placed in 15 ml of S2 solution and subjected to refractive index matching on a shaker at 25°C at a speed of less than 60 rpm until the sample was completely transparent. Refractive index matching of adult mouse skin can be completed in about 1 day.

[0131] After sample preparation, multi-channel high-resolution three-dimensional imaging (25×, 0.6NA objective) was performed using a Nuohai LS18 flat sheet microscope.

[0132] Figure 3The image shows the three-dimensional imaging results of adult mouse skin after transparency and immunostaining using the method of this invention. A is a three-dimensional rendering of the skin after transparency, where DAPI is blue, anti-Vimentin is green, anti-CD34 is yellow, and anti-Cytokeratin is purple; B is a cross-sectional view of the yellow area in A, with a scale bar of 10 μm; C is a magnified view of the area shown in the white box in B, with a scale bar of 30 μm.

[0133] like Figure 3 As shown, after processing using the method of this invention, the epidermis, dermis, subcutaneous tissue, and hair follicles are clearly visible. Blue represents DAPI-labeled cell nuclei, green represents anti-Vimentin-labeled vimentin, yellow represents anti-CD34 positive cells, and purple represents anti-Cytokeratin-labeled keratin. This result demonstrates that the method of this invention is compatible with immunofluorescence multicolor labeling and can achieve high-resolution three-dimensional imaging.

[0134] This invention provides a novel method for rapidly transparentizing tissues and organs, allowing most soft tissues in adult mice to achieve complete organ transparency in just 3 to 4 days.

Claims

1. A method for using transparent biological tissue, comprising the following steps: (1) Degreasing: The fixed biological tissue sample is degreased with a degreasing solution, wherein the degreasing solution is an aqueous solution containing tetrahydrofuran (THF) and tetrahydroxypropylethylenediamine (EDTP); (2) Refractive index matching: The defatted biological tissue sample is subjected to refractive index matching solution to complete the transparentization process. The refractive index matching solution is an aqueous solution containing urea, iohexol and EDTP.

2. The method according to claim 1, wherein, The degreasing solution contains 40 to 60 ml of THF, 4 to 6 g of EDTP, and 60 to 40 ml of water; specifically, the degreasing solution contains 50 ml of THF, 5 g of EDTP, and 45 ml of water; and / or The amount of degreasing agent used in a single application is 5-20 times the sample volume, preferably 10-25 times, and particularly 15-25 times; and / or The refractive index matching solution comprises 200 to 300 g / L urea, 600 to 800 g / L iohexol, and 15 to 25 g / L EDTP; specifically, the refractive index matching solution comprises 250 g / L urea, 700 g / L iohexol, and 20 g / L EDTP; and / or The amount of refractive index matching solution used at one time is 2 to 22 times the sample volume, preferably 7 to 17 times, and particularly 9 to 15 times.

3. The method of claim 1, wherein, All steps were performed in the dark on a low-speed shaker at room temperature.

4. The method of claim 1, wherein, The method further includes: One or more steps selected from degreasing and decalcification prior to defatting; and / or The step of immunostaining biological tissue samples after defatting and before refractive index matching.

5. An imaging method for transparentizing biological tissue, the method comprising the step of transparentizing a biological tissue sample using the method according to any one of claims 1-4.

6. The imaging method of claim 5, wherein, The imaging method further includes: The steps of obtaining and fixing biological tissue prior to the transparentization process, and / or Gel embedding and imaging, or staining, slide preparation and imaging steps, are performed after the clearing process.

7. The imaging method of claim 5, wherein, The imaging method includes: (1) Fix biological tissue samples; (2) The fixed biological tissue sample is transparentized using the method according to any one of claims 1-4; (3) The transparent biological tissue samples were gel-embedded; (4) Imaging biological tissue samples embedded in gel.

8. A kit for clearing biological tissues, comprising: (1) A degreasing solution comprising 40 to 60 ml of THF, 4 to 6 g of EDTP, and 60 to 40 ml of water; particularly, the degreasing solution comprising 50 ml of THF, 5 g of EDTP, and 45 ml of water; (2) A refractive index matching solution comprising 200 to 300 g / L of urea, 600 to 800 g / L of iohexol, and 15 to 25 g / L of EDTP; in particular, the refractive index matching solution comprises 250 g / L of urea, 700 g / L of iohexol, and 20 g / L of EDTP. Alternatively, the kit may contain: (1) Degreasing agent, wherein the degreasing agent is THF and EDTP; (2) Refractive index matching reagent, wherein the refractive index matching reagent is urea, iohexol and EDTP; (3) instructions for formulating the delipidating reagent and the refractive index matching reagent as a delipidating solution and a refractive index matching solution, wherein, The degreasing solution contains 40 to 60 ml of THF, 4 to 6 g of EDTP, and 60 to 40 ml of water; specifically, the degreasing solution contains 50 ml of THF, 5 g of EDTP, and 45 ml of water; and the refractive index matching solution contains 200 to 300 g / L of urea, 600 to 800 g / L of iohexol, and 15 to 25 g / L of EDTP; specifically, the refractive index matching solution contains 250 g / L of urea, 700 g / L of iohexol, and 20 g / L of EDTP.

9. The kit of claim 8, wherein, The kit also includes one or more selected from phosphate buffer, paraformaldehyde, gel reagent, imaging buffer, and staining agent.

10. The kit of claim 8, wherein, The kit includes: (1) Degreasing solution (S1) contains 50 ml of THF, 5 g of EDTP and 45 ml of water; (2) Refractive index matching solution (S2), which is an aqueous solution containing 250 g / L urea, 700 g / L iohexol and 20 g / L EDTP; (3) Gel solution (S3), which is a solution of 2% agarose in a refractive index matched solution by mass percentage concentration; (4) Imaging buffer (S4), which is silicone oil AP 100 with a refractive index of 1.50.

Images