Tissue transparentizing reagent capable of regulating and controlling mechanical properties and use method thereof

Abstract

The invention relates to the field of biomedical imaging, and discloses a tissue transparentizing reagent capable of regulating and controlling mechanical properties and a use method thereof. The tissue transparentizing reagent comprises monomers, a deep eutectic solvent, a cross-linking agent and an initiator, the monomers comprise an acrylamide monomer and N, N-dimethylacrylamide, and the deep eutectic solvent comprises antipyrine, erythritol and imidazole. The tissue transparentizing reagent provided by the invention has excellent optical clearing capability and tissue optical clearing effect, can realize dual effects of tissue transparentizing and embedding, can play a good role in shaping biological tissues, and is beneficial to fixation and imaging of the biological tissues. Meanwhile, different tissue-gel co-transparent complexes are sliced by regulating and controlling the mechanical performance of the gel and combining a conventional frozen section technology, and the tissue can be converted from three dimensions to two dimensions after three-dimensional imaging.

Description

Technical Field

[0001] The present invention relates to the field of biomedical imaging, and specifically to a tissue clearing agent with adjustable mechanical properties and a method for using the same. Background Art

[0002] Three-dimensional images of tissues can reveal the structural morphology of tissues, the anatomical relationship of adjacent tissues, and physiological function information at the cellular and molecular levels. The rapid development of tissue clearing technology can reveal the internal structural morphology of tissues. It reduces the uneven scattering and absorption of light in tissues through fixation, degreasing, decolorization, refractive index matching and other steps to make tissues transparent. Combined with advanced spinning disk confocal microscopes, two-photon microscopes and light sheet microscopes, it can achieve high-resolution three-dimensional imaging of the whole body or whole organ at the single-cell level.

[0003] Tissue clearing technologies are divided into three categories, including organic solvent type: BABB, uDISCO, iDISCO, vDISCO, PEGASOS, etc.; hydrophilic reagent type: SeeDB, FRUIT, TDE, ClearT, Scale, CUBIC; gel type: CLARITY, PACT, PARS, SWITCH, SHIELD, TESOS, ELAST, MPC, etc. These clearing technologies are widely used in the field of life sciences. Among them, the gel-type clearing method has been successfully applied in isolated animal organs and plant tissues, and it has broad prospects in basic life science research, innovative drug development and clinical tumor precision diagnosis. This type of method has the advantages of improving tissue hardness; maximally protecting the internal structure of the tissue and fixing biomacromolecules, retaining and retrieving key phenotypic information about the constituent cells and molecules (and their joint relationships); being compatible with multiple staining labels; and preventing it from floating in the imaging fluid. However, in general, the tissue-gel complex formed is relatively soft, which is not conducive to further sectioning observation after three-dimensional imaging. Therefore, it is of great significance to develop a method for transparent gel with adjustable mechanical properties for rapid shaping and slicing of biological tissues. It can make up for the defects of traditional tissue slicing technology such as discontinuity and difficulty in image reconstruction, and realize the reverse tracing of tissue three-dimensional imaging to two-dimensional imaging. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a tissue clearing agent capable of regulating mechanical properties and a method of using the same. The tissue clearing agent provided by the present invention can make biological tissue transparent, quickly shape and continuously slice biological tissue, and regulate its mechanical properties.

[0005] The invention provides a tissue clearing agent with adjustable mechanical properties.

[0006] Specifically, a tissue clearing agent with adjustable mechanical properties comprises the following components, measured by weight: 35 to 50 parts of monomers and 50 to 65 parts of low eutectic solvents; the tissue clearing agent also includes a cross-linking agent and an initiator, the mass of the cross-linking agent accounts for 0.5% to 3% of the mass of the monomer; the mass of the initiator accounts for 0.1% to 3.0% of the mass of the monomer; the monomer includes acrylamide monomer and N,N-dimethylacrylamide, the low eutectic solvent includes antipyrine, erythritol and imidazole, and the mass ratio of the antipyrine to the erythritol and the imidazole is (1.0 to 2.5): (1.0 to 4.0): (1.0 to 2.5).

[0007] It should be understood that the tissue clearing reagent generally has its components packaged separately before use and mixed when used.

[0008] Preferably, the mass ratio of the antipyrine to the erythritol and the imidazole is (1.0-2.0):(1.0-3.0):(1.0-2.0); further preferably, the mass ratio of the antipyrine to the erythritol and the imidazole is (1.0-1.5):(1.2-2.0):(1.5-2.0).

[0009] Preferably, the mass ratio of the acrylamide monomer to the N,N-dimethylacrylamide is (1.0-4.0):(1.0-4.0); further preferably, the mass ratio of the acrylamide monomer to the N,N-dimethylacrylamide is (2.0-4.0):(1.25-3.0); more preferably, the mass ratio of the acrylamide monomer to the N,N-dimethylacrylamide is (2.0-3.0):(1.25-2.0).

[0010] Preferably, the tissue clearing agent comprises the following components, measured by weight: 40 to 48 parts of monomers and 55 to 65 parts of low eutectic solvents; the tissue clearing agent also comprises a cross-linking agent and an initiator, the mass of the cross-linking agent accounts for 0.5% to 2.0% of the mass of the monomer; the mass of the initiator accounts for 0.5% to 2.0% of the mass of the monomer.

[0011] Preferably, the initiator is a photoinitiator or a thermal initiator; further preferably, the initiator includes at least one of azobisisoheptanonitrile, azobisisobutyronitrile, and azobisisobutylimidazoline hydrochloride.

[0012] Preferably, the cross-linking agent includes at least one of tetraisocyanate, methacrylic acid, 1,4-butylene glycol diacrylate, diisopropylbenzene peroxide, ethylene glycol dimethacrylate, and isocyanate cross-linking agents.

[0013] The present invention also provides a method for using a tissue clearing agent capable of regulating mechanical properties.

[0014] Specifically, a method for using a tissue clearing agent with adjustable mechanical properties comprises the following steps: The monomer, the low eutectic solvent, the cross-linking agent and the initiator are mixed to obtain a tissue clearing agent; the fixed biological tissue is then placed in the tissue clearing agent, and then polymerized by a photo-initiated polymerization or a thermal-initiated polymerization process to obtain a hydrogel-embedded tissue-gel copolymer.

[0015] Preferably, when the polymerization is carried out by a heat-initiated process, the tissue clearing agent is heated for polymerization, the heating temperature is 37° C. to 80° C., and the heating time is 6 h to 48 h.

[0016] Preferably, the refractive index of the hydrogel is 1.470-1.550.

[0017] The tissue transparent reagent with adjustable mechanical properties provided by the present invention is mainly composed of monomers, low eutectic solvents, cross-linking agents and initiators. Antipyrine, erythritol and imidazole are used to form a ternary low eutectic solvent at a suitable ratio. The low eutectic solvent has significant effects on tissue clearance and refractive index matching. Antipyrine with aromatic amide groups can effectively solvate protein skeleton amides, which is helpful for refractive index matching homogenization and high clearance performance; in addition, even in concentrated solutions, antipyrine can avoid tissue shrinkage; the -NH2 group of imidazole can combine with lipids of tissues, causing lipids to seep out of tissues, thereby reducing the refractive index difference of various components in tissues; and erythritol can avoid the destruction of tissue structure and internal proteins, so that the tissue can maintain its original state. Antipyrine, erythritol and imidazole work together to make the tissue initially transparent. More importantly, the ternary low eutectic solvent formed by mixing antipyrine, erythritol and imidazole solution has many excellent physical properties, which can not only increase the solubility of polymer monomers, solve the problem that monomers have poor solubility and are easy to precipitate in a single solvent; and can reduce the melting point of the mixed solvent, so that the polymerization reaction is carried out at a lower temperature; it also has strong permeability characteristics, which can accelerate tissue penetration and shorten the time required for tissue transparency. In the present invention, acrylamide monomer and N, N-dimethylacrylamide are used as monomers, wherein the acrylamide copolymer formed by acrylamide monomer has a higher hardness, and the N, N-dimethylacrylamide polymer formed by N, N-dimethylacrylamide has higher toughness and viscosity. By controlling the content of the two, the mechanical properties of the copolymer gel can be adjusted. Further, the polymer formed by acrylamide monomer and N, N-dimethylacrylamide as monomers can form a strong and rich non-covalent cross-linking between its polymer chains, thereby forming a hard, tough and uniform network, and achieving the effect of shaping and slicing. The tissue is embedded in the hydrogel while being transparent, thereby forming a gel with strong mechanical properties. Especially for thin-walled soft tissues such as the small intestine and esophagus, the plasticity is high and the molding is stable, which is convenient for preparing tissue sections.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The tissue clearing reagent provided by the present invention has excellent optical clearing ability and is compatible with a variety of antibodies and dyes; the tissue clearing reagent has excellent tissue optical clearing effect, can achieve the dual effects of tissue clearing and embedding, and can play a good role in shaping biological tissues, which is beneficial to the fixation and imaging of biological tissues. At the same time, by changing the content of monomers to regulate the mechanical properties of the hydrogel, different tissue-gel co-transparent complexes can be sliced ​​in combination with conventional frozen sectioning technology, and the transformation of tissues from three-dimensional to two-dimensional can be achieved after three-dimensional imaging of the tissue. In addition, the tissue clearing reagent can preserve biological tissues for a long time and has good stability.

[0019] (2) The hydrogel-embedded tissue-gel copolymer prepared using the tissue clearing reagent provided by the present invention has good ductility and can be sliced ​​to observe pathological tissue and cell structure in a two-dimensional plane. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the nuclear magnetic hydrogen spectrum characterization diagram of the ternary deep eutectic solvent formed by antipyrine, erythritol and imidazole in Experiment 1; Figure 2 This is the NMR carbon spectrum characterization diagram of the ternary deep eutectic solvent formed by antipyrine, erythritol and imidazole in Experiment 1; Figure 3 This is the infrared characterization image of the ternary deep eutectic solvent formed by antipyrine, erythritol and imidazole in Experiment 1; Figure 4 This is a diagram showing the optical clearing effect of the tissue clearing agent in Example 2 on the heart, liver, spleen, lung and kidney of mice; Figure 5 This is a diagram showing the optical clearing effect of the tissue clearing agent on the mouse hemibrain, femur, small intestine, and esophagus in Example 2; Figure 6 This is a three-dimensional imaging effect diagram of multiple fluorescent labels of the tissue clearing reagent in Example 2; Figure 7 This is a two-dimensional imaging effect diagram of the tissue-gel co-transparent complex after slicing in Example 2. DETAILED DESCRIPTION

[0021] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0022] Unless otherwise specified, the raw materials, reagents or devices used in the following examples and comparative examples can be obtained from conventional commercial sources or by existing known methods.

[0023] Experiment 1: Study the effects of the content of antipyrine (ATP), erythritol (Ery), imidazole (IMi), and other substances on the tissue transparent organic solvent. Prepare directly according to the proportion, and then test the transparent effect of the solvent. See Table 1 for the refractive index.

[0024] Table 1 Group ATP(%) Ery(%) Imi(%) Other substances (%) transparency Refractive Index 1 0 30 35 Ethanol 35 Very low —— 2 35 0 25 Water 40 Very low —— 3 45 30 0 Ethylene glycol 25 Not completely soluble —— 4 0 50 50 —— Very low —— 5 20 55 25 —— high 1.5128 6 20 45 35 —— high 1.5024 7 15 45 40 —— high 1.5046 8 15 50 35 —— high 1.4953 As shown in Table 1, the contents of antipyrine (ATP), erythritol (Ery), and imidazole (IMi) are indispensable. If any one of the three substances is replaced by other substances, a uniform and transparent solution cannot be formed. The lower the content of antipyrine (ATP) and erythritol (Ery), the lower the refractive index of the solution. When the mass ratio of antipyrine to erythritol and imidazole is (1.0-2.5): (1.0-4): (1.0-2.5), a ternary low eutectic solvent can be formed, in which Figure 1 This is the nuclear magnetic hydrogen spectrum characterization diagram of the ternary deep eutectic solvent formed by antipyrine, erythritol and imidazole; Figure 2 This is the NMR carbon spectrum characterization diagram of the ternary deep eutectic solvent formed by antipyrine, erythritol and imidazole; Figure 3 This is an infrared characterization image of the ternary deep eutectic solvent formed by antipyrine, erythritol and imidazole. From the above characterization data, it can be seen that the ternary deep eutectic solvent of antipyrine, erythritol and imidazole was successfully constructed, and there is a rich non-covalent bond (hydrogen bond) network between the three.

[0025] Experiment 2: The effects of the contents of antipyrine (ATP), erythritol (Ery), imidazole (IMi), acrylamide (AAm) and N,N-dimethylacrylamide (N,N-AAm) on the properties of tissue clearing gel were studied. The raw material components with different mass ratios were mixed evenly and heated at 50°C for 6 hours to obtain tissue clearing gel. The transparency effect (measured by visible light transmittance), refractive index (measured by refractometer), hardness (measured by texture analyzer) and tensile ratio (measured by universal testing machine) of the obtained tissue clearing gel are shown in Table 2.

[0026] Table 2 Transparency, refractive index, hardness and toughness of tissue clearing gel Group AAm / % N,N-AAm / % ATP / % Ery / % IMi / % transparency Refractive Index Hardness(N) Stretch ratio 1 15 25 20 30 10 Higher 1.5196 127.15 1.11 2 20 20 15 30 15 high 1.5185 134.52 1.09 3 25 15 20 25 15 high 1.5174 135.28 1.02 4 30 10 20 20 20 high 1.5179 135.64 1.00 5 10 10 25 35 20 high 1.5128 118.64 1.48 6 10 15 25 30 20 high 1.5024 115.29 1.55 7 15 10 30 25 20 high 1.5046 119.85 1.44 8 30 30 10 15 15 Low 1.4953 148.26 1.00 9 25 35 10 15 15 Low 1.4926 146.58 1.00 10 30 35 15 10 10 Low 1.4842 149.37 1.00 It can be seen from Table 2 that when the total monomer content is 20% to 30%, the gel is too soft, has high toughness and high tensile strength, which is not conducive to subsequent slicing operations. When the total monomer content is greater than 50%, the solution content is relatively low, the refractive index decreases, and the low transparency is not conducive to tissue transparency. With the increase of N,N-dimethylacrylamide content, the toughness of the hydrogel also increases; with the increase of acrylamide content, the hardness of the hydrogel also increases; with the increase of antipyrine content, the transparency of the hydrogel increases. Within the scope of protection of the present invention, by changing the content of the two polymerized monomers, the toughness and hardness of the gel can be regulated, thereby completing gel embedding and slicing processing for different tissues, such as bones, skin, small intestine, etc.

[0027] The following is a description of the specific embodiments. Example 1

[0028] A tissue clearing agent consists of the following components by mass fraction: 20% antipyrine, 20% erythritol, 17% imidazole, 21% acrylamide and 22% N,N-dimethylacrylamide; the initiator is azobisisobutylimidazoline hydrochloride, the mass of which is 1.5% of the mass of the mixed monomers; and the crosslinking agent is methacrylic acid, the mass of which is 0.8% of the mass of the mixed monomers.

[0029] The tissue clearing agent is prepared into a tissue clearing gel for performance testing, including the following steps: the above components are mixed evenly to obtain a tissue clearing agent; and then heated at 75°C for 4 hours to obtain a tissue clearing gel. The prepared tissue clearing gel has high transparency, a refractive index of 1.5138, and a hardness of 132.68N. Example 2

[0030] A tissue clearing agent consists of the following components by mass fraction: 24% antipyrine, 21% erythritol, 15% imidazole, 16% acrylamide and 24% N,N-dimethylacrylamide; the initiator is azobisisoheptanenitrile, the mass of which is 0.8% of the mass of the mixed monomers; and the crosslinking agent is methacrylic acid, the mass of which is 1.2% of the mass of the mixed monomers.

[0031] The tissue clearing agent is prepared into a tissue clearing gel for performance testing, including the following steps: the above components are mixed evenly to obtain a tissue clearing agent; and then heated at 50°C for 6 hours to obtain a tissue clearing gel. The prepared tissue clearing gel has high transparency, a refractive index of 1.5279, and a hardness of 136.68N.

[0032] The biological tissue was treated with the tissue clearing reagent of this embodiment, and the specific process was as follows: antipyrine, erythritol, imidazole, acrylamide, N,N-dimethylacrylamide, a cross-linking agent and an initiator were mixed evenly in proportion to prepare a tissue clearing reagent (i.e., a tissue clearing gel prepolymer solution), and fixed mouse tissues (heart, liver, spleen, lung, kidney, hemibrain, femur, esophagus, small intestine) were placed in the tissue clearing reagent, and then the mouse tissue and the tissue clearing reagent were placed in a mold together, and heated at 50° C. for 6 hours to perform thermal initiation polymerization to obtain a hydrogel-coated tissue-gel copolymer.

[0033] Figure 4 , 5 These are comparison images of the optical clearing effects of the above tissues taken before and after transparentization. Figure 4 From left to right, they are the heart, liver, spleen, lung, and kidney tissues of mice. Figure 5 From left to right, they are the hemibrain, femur, esophagus, and small intestine. Figure 4 and Figure 5In the figure, the upper row of images are images before tissue transparent treatment. Due to the turbidity of the tissue, the grid below the tissue is covered by the tissue and is not visible; the lower row is the image of the same tissue after transparent treatment. At this time, the tissue becomes highly transparent, and the grid below the tissue is clearly visible (the background grid is 2mm*2mm). From the comparison of the pictures taken by the stereoscope before and after transparentization, it can be obtained that the tissue transparentizing agent provided by the present invention can achieve a good transparent effect by simple immersion on the basis of defatting, and has excellent effect in clearing tubular and thin-walled tissues such as the small intestine and esophagus, with the shortest time and unchanged tissue morphology; it has a certain transparent ability for hollow organs with thick myocardial walls and complex results, and this method also has a good clearing ability for solid organs with thick tissues such as the liver, spleen, and lungs, and can also achieve transparentization of lipid-rich brain tissue. The tissue transparentization actually has excellent transparent effects on solid organs, organs with more pigments, and hard tissues. Experiments have shown that the tissue transparentizing agent provided in Example 1 can also achieve an optical clearing effect similar to that of Example 2.

[0034] Figure 6 (a) shows the effect of tissue clearing reagent on three-dimensional imaging of mouse brain slices (300 μm) with multiple fluorescent labels, including red-vascular LECTIN; green-astrocyte GFAP; blue-nucleus DAPI, Figure 6 (b) is a three-dimensional imaging image of the fluorescent labeling confocal microscope of the small intestinal section vascular immunostaining using tissue clearing reagent. Figure 6 It can be seen that the tissue clearing gel is compatible with the tested fluorescent dyes and antibodies and has a good tissue three-dimensional imaging effect.

[0035] Figure 7 This is a two-dimensional confocal microscope imaging effect diagram of the mouse small intestine tissue-gel co-transparent complex after slicing. Figure 7 (a) is a slice magnified 10 times. Figure 7 (b) is a slice magnified 20 times. Figure 7 The middle (c) is a 40-fold magnification of the slice. The red in the figure is the blood vessel signal marked by LECTIN; the green is the neuron signal marked by MAP2; and the blue is the cell nucleus signal marked by DAPI. The mouse small intestine tissue-gel co-transparent complex was imaged in three dimensions by LS 18 light sheet microscope, and then directly sliced ​​by conventional frozen section technology to obtain 20μm thin slices, and then imaged in two dimensions by super-resolution spinning disk confocal microscope to observe the fine structure inside the tissue. Figure 7 It can be seen that the tissue clearing gel combined with conventional frozen section technology can directly slice after three-dimensional imaging of the tissue-gel co-clearing complex to further observe the fine structure inside the tissue, realizing the reverse tracing of the tissue from three-dimensional to two-dimensional.

[0036] The tissue immunolabeling process in the above experiment is as follows: Membrane disruption: The tissue was immersed in the membrane disruption solution and placed in a 37°C constant temperature shaker overnight (50 rpm / h).

[0037] Blocking: QuickBlock™ immunostaining blocking buffer (Biyuntian), incubate overnight at 37°C in a constant temperature shaker (50 rpm / h).

[0038] Immunolabeling: Antibody selection: GFAP (SAB, dilution concentration 1:100) was selected to label astrocytes in brain tissue; LECTIN was used to label blood vessels; DAPI (sigma, dilution concentration 1:50) was used to label cell nuclei. DyLightTM488-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, dilution concentration 1:100) was selected as the secondary antibody.

[0039] Labeling steps: After the tissue was permeabilized and blocked, it was placed on a 25℃ shaker (50rpm / h) and incubated with the primary antibody for 48 hours. Unbound primary antibody was washed off with PBS (3 times, 1h / time), and then the secondary antibody was added and incubated on a 25℃ shaker (50rpm / h) in the dark for 24 hours. Unbound secondary antibody was washed off with PBS (3 times, 1h / time), and DAPI was added and incubated on a 25℃ shaker (50rpm / h) for 12 hours to label the cell nucleus. After labeling, unbound DAPI was washed off with PBS (3 times, 1h / time). Example 3

[0040] A tissue clearing agent consists of the following components by mass fraction: 16% antipyrine, 25% erythritol, 16% imidazole, 21% acrylamide and 22% N,N-dimethylacrylamide; the initiator is azobisisoheptanenitrile, the mass of which is 1.4% of the mass of the mixed monomers; and the crosslinking agent is 1,4-butylene glycol diacrylate, the mass of which is 1.5% of the mass of the mixed monomers.

[0041] The tissue clearing agent is prepared into a tissue clearing gel for performance testing, comprising the following steps: mixing the above components evenly to obtain a tissue clearing agent; and then heating at 55°C for 6 hours to obtain a tissue clearing gel. The prepared tissue clearing gel has high transparency, a refractive index of 1.5386, and a hardness of 146.68N. At the same time, the experiment proves that the tissue clearing agent provided in Example 3 can also achieve an optical clearing effect similar to that of Example 2. Example 4

[0042] A tissue clearing agent is composed of the following components by mass fraction: 20% antipyrine, 20% erythritol, 18% imidazole, 20% acrylamide and 22% N,N-dimethylacrylamide; the initiator is azobisisoheptanenitrile, the mass of which is 1.2% of the mass of the mixed monomers; and the crosslinking agent is 1,4-butylene glycol diacrylate, the mass of which is 1.2% of the mass of the mixed monomers.

[0043] The tissue clearing agent is prepared into a tissue clearing gel for performance testing, comprising the following steps: mixing the above components evenly to obtain a tissue clearing agent; and then heating at 70°C for 3 hours to obtain a tissue clearing gel. The prepared tissue clearing gel has high transparency, a refractive index of 1.5241, and a hardness of 138.41N. At the same time, the experiment proves that the tissue clearing agent provided in Example 4 can also achieve an optical clearing effect similar to that of Example 2.

[0044] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A tissue clearing reagent, characterized in that: The invention comprises the following components by weight: 35 to 50 parts of monomers and 50 to 65 parts of low eutectic solvents; the tissue clearing agent also comprises a crosslinking agent and an initiator, the mass of the crosslinking agent accounts for 0.5% to 3% of the mass of the monomers; the mass of the initiator accounts for 0.1% to 3.0% of the mass of the monomers; the monomers comprise acrylamide monomers and N,N-dimethylacrylamide, the low eutectic solvent comprises antipyrine, erythritol and imidazole, and the mass ratio of the antipyrine to the erythritol and the imidazole is (1.0 to 2.5): (1.0 to 4.0): (1.0 to 2.5).

2. The tissue clearing reagent according to claim 1, characterized in that The mass ratio of the antipyrine to the erythritol and the imidazole is (1.0-2.0): (1.0-3.0): (1.0-2.0).

3. The tissue clearing reagent according to claim 1, characterized in that The mass ratio of the acrylamide monomer to the N,N-dimethylacrylamide is (1.0-4.0): (1.0-4.0).

4. The tissue clearing agent according to any one of claims 1 to 3, characterized in that The invention comprises the following components: 40 to 48 parts of monomers and 55 to 65 parts of low eutectic solvents; the tissue clearing agent also comprises a crosslinking agent and an initiator, wherein the mass of the crosslinking agent accounts for 0.5% to 2.0% of the mass of the monomers; and the mass of the initiator accounts for 0.5% to 2.0% of the mass of the monomers.

5. The tissue clearing reagent according to claim 4, characterized in that: The initiator includes at least one of azobisisoheptanonitrile, azobisisobutyronitrile and azobisisobutylimidazoline hydrochloride.

6. The tissue clearing reagent according to claim 4, characterized in that The crosslinking agent includes at least one of tetraisocyanate, methacrylic acid, 1,4-butylene glycol diacrylate, diisopropylbenzene peroxide, ethylene glycol dimethacrylate and isocyanate crosslinking agents.

7. The method for using the tissue clearing agent according to any one of claims 1 to 6, characterized in that: The following steps are involved: The monomer, the low eutectic solvent, the cross-linking agent and the initiator are mixed to obtain a tissue clearing agent; the fixed biological tissue is then placed in the tissue clearing agent, and then polymerized by a photo-initiated polymerization or a thermal-initiated polymerization process to obtain a hydrogel-embedded tissue-gel copolymer.

8. The method of use according to claim 7, characterized in that: When the polymerization is carried out by a heat-initiated process, the tissue clearing agent is heated for polymerization, and the heating temperature is 37° C. to 80° C.

9. The method of use according to claim 7 or 8, characterized in that: The refractive index of the hydrogel is 1.470-1.550.

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