Tissue light transparentizing reagent and rapid tissue light transparentizing method

Abstract

The invention discloses a tissue optical transparency reagent and a rapid tissue optical transparency method, and belongs to the technical field of biomedical optical imaging. The tissue optical transparency reagent comprises a dehydration liquid, wherein the dehydration liquid comprises polyhydric alcohol, amino alcohol, a reducing agent and a dehydrating agent; the mass volume ratio of the polyhydric alcohol to the dehydrating agent is 5%-30%, the mass volume ratio of the amino alcohol to the dehydrating agent is 5%-30%, the mass volume ratio of the reducing agent to the dehydrating agent is 0.1%-1%, and the dehydrating agent is selected from at least one of boric acid ester substances, methoxane substances and oxysilane substances. The dehydration liquid in the tissue optical transparent reagent provided by the invention can be used for mildly and efficiently dehydrating biological tissues, and the problems that gradient dehydration needs to be carried out and the reagent is frequently replaced in the existing physical dehydration are solved. Therefore, the tissue optical transparency reagent has a good application prospect in the field of biological tissue optical transparency.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical optical imaging, and in particular relates to a tissue optical clearing agent and a rapid tissue optical clearing method. Background Art

[0002] Traditional histopathology testing includes a series of time-consuming and laborious steps such as fixation, dehydration, paraffin embedding, sectioning, and staining, resulting in a long overall processing time, which may take several days to weeks to obtain the final test results. This not only makes patients anxious during the waiting process, but also makes the diagnosis results have a certain lag relative to the condition. At the same time, most of the operations in histopathology testing require manual operation, and the operation steps are complicated and lengthy, making traditional histopathology testing a labor-intensive task, causing practitioners to spend a lot of time on repetitive work. In traditional histopathology, the final test results are based on microscopic imaging of thin sections (5-10μm). This makes the sampling rate of existing pathology detection methods seriously insufficient, usually only 3%-5% of the tissue is analyzed, which ultimately leads to a decrease in the accuracy of the diagnosis results. At the same time, biological tissues contain complex glandular and vascular structures, and it is difficult to effectively analyze these complex three-dimensional structures with limited two-dimensional information. Therefore, it has become an urgent need for pathology detection to quickly and easily obtain high-resolution three-dimensional information of pathological tissues.

[0003] In recent years, the development of optical imaging technology and tissue optical transparency technology has made three-dimensional high-resolution imaging of pathological tissues possible. Tissue optical transparency technology can reduce the scattering and absorption of light by biological tissues through treatments such as decolorization and refractive index matching, thereby increasing the imaging depth of optical imaging methods. Light sheet microscopy can stimulate light sheets from the side of the sample and selectively excite signals at a specific depth of the sample. And through optical sectioning of the sample, non-destructive three-dimensional imaging of large-volume samples can be achieved. The combination of light sheet microscopy and tissue optical transparency technology is introduced into tissue pathology detection, allowing three-dimensional visualization of the entire tissue without two-dimensional sectioning, thereby realizing three-dimensional pathological detection of the tissue. This overcomes the problems of insufficient sampling rate and missing three-dimensional structure caused by sectioning in conventional two-dimensional pathology detection.

[0004] At present, a variety of different tissue optical transparency technologies, including CUBIC, ECi, CLARITY, etc., have been applied to three-dimensional pathology detection, and clinical pathology samples such as prostate, breast, lymph nodes, etc. have been transparent and three-dimensionally imaged. However, transparent methods based on hydrogel embedding such as CLARITY need to be combined with additional electrophoresis devices, and the operation is complicated and the technical threshold is high, which increases the difficulty of implementing three-dimensional pathology detection. Some transparent methods based on water-soluble methods such as CUBIC take a long time, and three-dimensional detection of pathological tissue may take several days to weeks to complete. Transparent methods based on organic solvents such as ECi have a faster transparency rate and do not require the introduction of additional processing equipment. However, among the existing organic solvent-based optical transparency methods, transparent methods such as ECi that use physical osmosis for dehydration require gradient dehydration and frequent reagent replacement, which limits the further improvement of optical transparency efficiency.

[0005] Therefore, it is necessary to provide a tissue light-clearing reagent to solve the shortcomings of existing light-clearing reagents. Summary of the invention

[0006] The purpose of the present invention is to provide a tissue photoclearing reagent and a rapid tissue photoclearing method. The tissue photoclearing reagent of the present invention can gently and efficiently dehydrate biological tissues, can quickly make biological tissues transparent, and has a good transparent effect on biological tissues.

[0007] In a first aspect, the present invention provides a tissue phototransparency reagent, comprising a dehydrating liquid, the dehydrating liquid comprising a polyol, an amino alcohol, a reducing agent and a dehydrating agent; the mass volume ratio of the polyol to the dehydrating agent is 5%-30%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30% or other values ​​within this range; the mass volume ratio of the amino alcohol to the dehydrating agent is 5%-30%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30% or other values ​​within this range; the mass volume ratio of the reducing agent to the dehydrating agent is 0.1%-1%, for example, it can be 0.1%, 0.2%, 0.5%, 0.8%, 0.9%, 1% or other values ​​within this range; wherein the dehydrating agent is selected from at least one of borate substances, methoxyethane substances and oxysilane substances.

[0008] In the present invention, the inventors have found that when dehydrating agents such as borate substances, methoxyl substances and oxysilane substances are added to the dehydrating solution, biological tissues can be gently and efficiently dehydrated, solving the problem of the need for gradient dehydration and frequent reagent replacement in existing physical dehydration.

[0009] In some embodiments, the borate ester species includes at least one of triphenyl borate, trimethyl borate, and tributyl borate.

[0010] In the present invention, the inventors further studied and found that when triphenyl borate, trimethyl borate and tributyl borate are selected as dehydrating agents, the fluorescence signal of the sample can be maintained when the tissue sample is efficiently dehydrated.

[0011] In some embodiments, the methoxyl species include dimethoxypropane; and the oxysilane species include tetramethoxysilane.

[0012] In some embodiments, the polyol is selected from at least one of propylene glycol, glycerol, pentanediol and hexanediol; the amino alcohol is selected from at least one of triethanolamine, N-methyldiethanolamine and N-butyldiethanolamine; and the reducing agent is selected from at least one of thioglycerol, vitamin D and vitamin C.

[0013] In some embodiments, the tissue phototransparency reagent also includes a degreasing liquid, which includes a reducing agent and a degreasing agent; the mass volume ratio of the reducing agent to the degreasing agent is 0.1%-1%, for example, it can be 0.1%, 0.2%, 0.5%, 0.8%, 0.9%, 1% or other values ​​within this range; wherein the degreasing agent is selected from at least one of cyclohexane, ethanol, dichloromethane and benzyl benzoate.

[0014] In the present invention, the tissue light-transparent reagent also includes a degreasing liquid, which can remove lipid components in biological tissues, thereby further improving the transparency effect of tissue samples.

[0015] In some embodiments, the tissue light clearing agent further comprises a refractive index matching liquid comprising at least one of benzyl ether, benzyl benzoate, benzyl alcohol, and ethyl cinnamate.

[0016] In the present invention, the tissue optical transparency reagent also includes a refractive index matching liquid, which can maintain the fluorescence compatibility of the tissue sample and further improve the transparency speed and transparency effect of the sample tissue.

[0017] In some embodiments, the tissue photoclearing agent also includes a pretreatment liquid, which includes a polyol, an amino alcohol, a reducing agent and distilled water; the mass volume ratio of the polyol to the distilled water is 5%-30%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30% or other values ​​within this range; the mass volume ratio of the amino alcohol to the distilled water is 5%-30%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30% or other values ​​within this range; the mass volume ratio of the reducing agent to the distilled water is 0.1%-1%, for example, it can be 0.1%, 0.2%, 0.5%, 0.8%, 0.9%, 1% or other values ​​within this range.

[0018] In the present invention, the tissue light-transparent reagent also includes a pretreatment solution, which can remove the residual chromophores in the tissue sample and further improve the transparency effect of the sample.

[0019] In a second aspect, the present invention provides a method for rapid tissue optical clearing using any of the tissue optical clearing reagents described above, comprising the following steps: placing the biological tissue in a dehydrating liquid, and after oscillating treatment, obtaining a dehydrated tissue; placing the dehydrated tissue in a degreasing liquid, and after oscillating treatment, obtaining a defatted tissue; placing the defatted tissue in a refractive index matching liquid, and after oscillating treatment, obtaining a transparentized tissue.

[0020] In some embodiments, the biological tissue includes a mammalian tissue organ.

[0021] In some more preferred embodiments, mammals include humans, macaques, mice, rats, rabbits, fish, and insects.

[0022] It should be noted that the tissue optical clearing reagent of the present invention can quickly clear existing conventional biological tissues, including but not limited to humans, macaques, mice, rats, rabbits, fish and insects.

[0023] The rapid tissue optical clearing method provided by the present invention only requires a single-step dehydration to achieve rapid and efficient dehydration of tissue samples; and the method has the advantages of simple operation, fast clearing speed, high clearing effect, low reagent price and no reliance on expensive equipment.

[0024] In some embodiments, before the preparation of the dehydrated tissue, a step of pretreating the biological tissue is also included, and the pretreatment step specifically includes: placing the biological tissue in a pretreatment solution, and obtaining the pretreated tissue after oscillation treatment; wherein the oscillation treatment step specifically includes: oscillating for 6-24 hours at a temperature of 30-40°C (for example, 30°C, 32°C, 35°C, 38°C, 40°C or other values ​​within the range), for example, 6 hours, 10 hours, 14 hours, 18 hours, 22 hours, 24 hours or other values ​​within the range.

[0025] In some embodiments, during the preparation of dehydrated tissue, the oscillation treatment step specifically includes: oscillating for 6-24 hours, for example, 6 hours, 10 hours, 14 hours, 18 hours, 22 hours, 24 hours or other values ​​within the range, at a temperature of 30-40°C (for example, 30°C, 32°C, 35°C, 38°C, 40°C or other values ​​within the range); during the preparation of defatted tissue, the oscillation treatment step specifically includes: oscillating for 6-24 hours, for example, 6 hours, 10 hours, 14 hours, 18 hours, 22 hours, 24 hours or other values ​​within the range, at a temperature of 30-40°C (for example, 30°C, 32°C, 35°C, 38°C, 40°C or other values ​​within the range). The step of oscillating the transparent tissue comprises: oscillating at a temperature of 20-25°C (for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or other values ​​therein) for 1-6h, for example, 1h, 2h, 3h, 4h, 5h, 6h or other values ​​therein.

[0026] The beneficial effect of the present invention is that, different from the prior art, the tissue optical transparency reagent provided by the present invention includes a dehydrating liquid, and the dehydrating liquid includes dehydrating agents such as borate substances, methoxyl substances and oxysilane substances, which can gently and efficiently dehydrate biological tissues, solving the problem of the need for gradient dehydration and frequent reagent replacement in the existing physical dehydration. Therefore, the tissue optical transparency reagent has a good application prospect in the field of biological tissue optical transparency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The transparent effects of mouse brain tissue samples after dehydration by different dehydrating agents at different pH values ​​and the fluorescence preservation diagram of the samples in Example 1 of the present invention; Figure 2 This is a diagram showing the transparent effects of different borate esters on mouse hemi-brain samples after dehydration in Example 2 of the present invention; Figure 3 This is a diagram showing the transparency effect of dehydrating different organs of mice using the rapid tissue optical transparency method of the present invention in Example 3 of the present invention; Figure 4 This is a diagram showing the transparency effects of different tissues of the human body after dehydration using the rapid tissue optical transparency method of the present invention in Example 4 of the present invention; Figure 5 This is a graph showing the compatibility of a mouse brain tissue sample with a fluorescent dye after dehydration using the rapid tissue optical transparency method of the present invention in Example 5 of the present invention; Figure 6 This is a comparison chart of the transparency effects of dehydrating mouse brain tissue samples using the rapid tissue optical transparency method of the present invention and the traditional method in Example 6 of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0030] Example 1 This example studies the transparency effect of different dehydrating agents on mouse brain tissue samples after dehydration at different pH values ​​and the fluorescence preservation effect of the samples.

[0031] Specifically, after the mouse is perfused through the heart, the mouse brain is removed and fixed with PFA; then the mouse brain tissue sample is transparentized, specifically comprising the following steps: 1) The mouse brain tissue was placed in a dehydrating solution (30wt% hexylene glycol, 20wt% N-butyldiethanolamine, 0.5wt% thioglycerol, and the rest were dehydrating agents) containing different pH values ​​(3, 7, 9) and different dehydrating agents (trimethyl borate (TB), dimethoxypropane (DMP), and methyltrimethoxysilane (MTM)), and shaken at 37°C for 30 minutes to obtain the dehydrated tissue; 2) placing the dehydrated tissue obtained in step 1) in a defatting solution (0.5 wt% thioglycerol and the rest dichloromethane), shaking for 10 min at 37° C., to obtain a defatted tissue; 3) The defatted tissue obtained in step 2) is placed in dibenzyl ether and shaken at 25° C. for 5 minutes to obtain a transparent tissue.

[0032] The transparent tissue obtained in this example was photographed and fluorescently detected, and the results were as follows: Figure 1 shown.

[0033] from Figure 1It can be seen from the results that dimethoxypropane and methyltrimethoxysilane have good effects on dehydration and transparency of mouse brain tissue under acidic conditions, and average transparency effects under neutral and alkaline conditions; while trimethyl borate has good transparency effects on mouse brain tissue under acidic, neutral and alkaline conditions; in addition, under neutral and alkaline conditions, mouse brain tissue has good fluorescence signals. Therefore, in the present invention, trimethyl borate has a good transparency effect while maintaining a good fluorescence signal of the sample.

[0034] Example 2 This example studies the transparency effects of different borate esters on dehydrated mouse hemi-brain samples.

[0035] Specifically, after the mouse is perfused through the heart, the mouse brain is removed and fixed with PFA; then the mouse brain tissue sample is transparentized, specifically comprising the following steps: 1) The mouse brain tissue was placed in a dehydrating solution (30wt% hexylene glycol, 20wt% N-butyldiethanolamine, 0.5wt% thioglycerol, and the rest was a dehydrating agent) containing different dehydrating agents (trimethyl borate (TB), triethyl borate (TEB), tributyl borate (TBB), tripropyl borate (TPB)), and shaken for 10 hours at 37°C to obtain the dehydrated tissue; 2) placing the dehydrated tissue obtained in step 1) in a defatting solution (0.5wt% thioglycerol and the rest dichloromethane), shaking at 37°C for 3h to obtain defatted tissue; 3) The defatted tissue obtained in step 2) is placed in benzyl ether and shaken at 25° C. for 1 hour to obtain a transparent tissue.

[0036] The transparent tissue obtained in this example was photographed, and the results were as follows: Figure 2 shown.

[0037] from Figure 2 It can be seen that trimethyl borate, triethyl borate, tributyl borate and tripropyl borate all have good transparentizing effects on mouse brain tissue, and trimethyl borate has the best transparentizing effect.

[0038] Example 3 This example uses the rapid tissue optical clearing method of the present invention to clear different organs of mice.

[0039] Specifically, after the mouse is perfused through the heart, different organs of the mouse (brain, long bones, lungs, spleen, liver, testicles, heart, kidneys, spinal cord, uterus) are removed and fixed with PFA; then, different organ samples of the mouse are transparentized, specifically including the following steps: 1) The different organs of the mouse were placed in a pretreatment solution (30wt% hexanediol, 20wt% N-butyldiethanolamine, 0.5wt% thioglycerol, and the rest was distilled water), and shaken for 10 hours at a temperature of 37°C to obtain pretreated tissues; 2) placing the pretreated tissues obtained in step 1) in a dehydrating solution (30 wt% hexylene glycol, 20 wt% N-butyl diethanolamine, 0.5 wt% thioglycerol, and the rest trimethyl borate) at 37° C. and shaking for 10 h to obtain dehydrated tissues; 3) The dehydrated tissues obtained in step 2) were placed in a defatting solution (0.5wt% thioglycerol and the rest dichloromethane), and shaken for 3 hours at 37°C to obtain defatted tissues; 4) The defatted tissues obtained in step 3) were placed in benzyl ether and shaken at 25° C. for 1 h to obtain transparent tissues.

[0040] The transparent tissue obtained in this example was photographed, and the results were as follows: Figure 3 shown.

[0041] from Figure 3 It can be seen that after pretreatment, the mouse organs can remove the remaining blood red in the tissues and improve the transparency of the tissues.

[0042] Example 4 This embodiment uses the rapid tissue optical transparency method of the present invention to transparentize different tissues of the human body.

[0043] Specifically, the hernia sac, foreskin, fallopian tube and vaginal wall removed by surgery were placed in PFA for fixation overnight, and then the tissue was transparentized, which specifically included the following steps: 1) placing the human tissue in a pretreatment solution (30wt% hexanediol, 20wt% N-butyldiethanolamine, 0.5wt% thioglycerol, and the rest is distilled water), shaking at 37°C for 20 hours to obtain pretreated tissue; 2) placing the pretreated tissues obtained in step 1) in a dehydrating solution (30 wt% hexylene glycol, 20 wt% N-butyl diethanolamine, 0.5 wt% thioglycerol, and the rest trimethyl borate) at 37° C. and shaking for 20 h to obtain dehydrated tissues; 3) The dehydrated tissues obtained in step 2) were placed in a defatting solution (0.5wt% thioglycerol and the rest dichloromethane), and shaken for 6 hours at a temperature of 37°C to obtain defatted tissues; 4) The defatted tissues obtained in step 3) were placed in benzyl ether and shaken at 25° C. for 2 h to obtain transparent tissues.

[0044] The transparent tissue obtained in this example was photographed, and the results were as follows: Figure 4 shown.

[0045] from Figure 4 It can be seen that after pretreatment, the residual blood red in the human tissue can be removed and the transparency of the tissue can be improved.

[0046] Example 5 This embodiment studies the compatibility of the rapid tissue optical clearing method of the present invention with fluorescent dyes.

[0047] Specifically, after the mouse is perfused through the heart, the mouse brain is removed and fixed with PFA; then the mouse brain tissue sample is transparentized, specifically comprising the following steps: 1) The mouse brain tissue was placed in different staining solutions, shaken at 37°C for 24 hours, and the brain slices were stained; 2) The brain slices stained in step 1) were placed in a dehydrating solution (30wt% hexanediol, 20wt% N-butyldiethanolamine, 0.5wt% thioglycerol, and the rest trimethyl borate), shaken at 37°C for 20 hours to obtain dehydrated tissues; 3) The dehydrated tissues obtained in step 2) were placed in a defatting solution (0.5wt% thioglycerol and the rest dichloromethane), and shaken for 3 hours at 37°C to obtain defatted tissues; 4) The defatted tissues obtained in step 3) were placed in benzyl ether and shaken at 25° C. for 1 h to obtain transparent tissues.

[0048] The transparent tissue obtained in this example was photographed and fluorescently imaged, and the fluorescence intensity before and after transparentization was quantitatively analyzed. The results are as follows: Figure 5 shown.

[0049] from Figure 5 It can be seen that the transparentization method of the present invention is compatible with different fluorescent dyes, and the fluorescence intensity remains basically unchanged before and after transparentization.

[0050] Example 6 This example compares the transparency effects of the rapid tissue optical clearing method of the present invention and the traditional method.

[0051] Specifically, after the mouse was perfused through the heart, the mouse brain was removed and fixed with PFA; then the mouse brain tissue sample was transparentized using different methods, as follows: 1) The method of the present invention: The mouse brain was placed in a dehydrating solution (30wt% hexylene glycol, 20wt% N-butyldiethanolamine, 0.5wt% thioglycerol, and the rest trimethyl borate) and shaken for 20 hours at 37°C. It was then placed in a defatting solution (0.5wt% thioglycerol, and the rest dichloromethane) and shaken for 4 hours at 37°C. Finally, it was placed in dibenzyl ether and shaken for 2 hours at 25°C.

[0052] 2) SHANEL method: The mouse brain was placed in a defatting solution (10wt% CHAPS, 25wt% N-methyldiethanolamine, and the rest water) and shaken for 3 days at 25°C. It was then placed in a gradient tetrahydrofuran aqueous solution for dehydration, with a concentration gradient of 50%, 70%, 80%, 100%, 100%, and 100% (V / V) at 25°C for 12 hours per step. Finally, it was placed in dibenzyl ether and shaken for 6 hours at 25°C.

[0053] 3) iDISCO method: The mouse brain was placed in a gradient of 20%, 40%, 60%, 80%, 100% (V / V) methanol-water solution for dehydration at room temperature, and then the sample was placed in a 66% ethanol solution for degreasing at room temperature, each step for 12 hours. After degreasing, the sample was rinsed twice in 100% methanol, 1 hour each time. Then it was destained with 5% hydrogen peroxide solution and treated at 4°C for 12 hours. The destained sample was rehydrated in a gradient of 80%, 60%, 40%, 20% methanol solution and PBS, and treated at room temperature for 12 hours each step. The rehydrated sample was dehydrated again in a gradient of 20%, 40%, 60%, 80%, 100%, 100%, 100% (V / V) methanol-water solution, and each step was 12 hours at room temperature. The dehydrated sample was degreased in 100% DCM and refractive index matched in DBE, and each step was 12 hours at room temperature.

[0054] 4) ECI method: The mouse brain was placed in a gradient of 50%, 70%, 80%, 100%, 100%, 100% (V / V) ethanol-water solution for dehydration at room temperature, with each step lasting 24 hours. The dehydrated samples were then immersed in ethyl cinnamate for refractive index matching at room temperature for 12 hours.

[0055] 5) CLARITY method: The mouse brain was placed in A4P4 solution (4% (W / V) acrylamide, 4% (W / V) paraformaldehyde, 0.25% (W / V) VA-044 dissolved in PBS) and fixed at 4°C for 12 hours. The fixed samples were then placed in a vacuum chamber at 37°C for 3 hours for gel embedding. The embedded samples were placed in an electrophoresis machine and degreased using a 4% SDS solution at 37°C for 24 hours. The degreased samples were rinsed several times in PBST and then placed in RIMS (75% (W / V) sorbitol dissolved in water) for refractive index matching at room temperature for 24 hours.

[0056] 6) CUBIC method: The mouse brain was placed in CUBIC-L solution (10% (W / V) Triton X-100, 10% (V / V) N-butyldiethanolamine, dissolved in water) and degreased at 37°C for 4 days. The sample was then rinsed several times in PBS and then transferred to CUBIC-R (45% (W / V) antipyrine, 30% (W / V) nicotinamide, dissolved in water) and refractive index matched at room temperature for 3 days.

[0057] 7) EZ Clear method: The mouse brain was placed in a 50% (N / V) tetrahydrofuran aqueous solution and defatted at room temperature for 16 h. The defatted samples were then rinsed several times with distilled water and the samples were refractive index matched using EZ View solution (80% iohexol, 7 M urea, 0.05% sodium azide, dissolved in PBS) and treated at room temperature for 24 h.

[0058] The transparent tissues obtained by different methods in this example were photographed and observed. Figure 6 shown.

[0059] from Figure 6 It can be seen that compared with the traditional method, the method of the present invention has a better transparent effect on biological tissues, and the transparent speed of the present invention is significantly faster than that of the traditional method. Therefore, the method of the present invention has a good application prospect in the field of rapid tissue transparentization.

[0060] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0061] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. 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 light clearing reagent, characterized in that: The dehydrating liquid comprises a polyol, an amino alcohol, a reducing agent and a dehydrating agent; the mass volume ratio of the polyol to the dehydrating agent is 5%-30%, the mass volume ratio of the amino alcohol to the dehydrating agent is 5%-30%, and the mass volume ratio of the reducing agent to the dehydrating agent is 0.1%-1%; Wherein, the dehydrating agent is selected from at least one of borate substances, methoxy substances and oxysilane substances.

2. The tissue light clearing reagent according to claim 1, characterized in that: The borate ester substance includes at least one of triphenyl borate, trimethyl borate and tributyl borate.

3. The tissue light clearing reagent according to claim 1, characterized in that: The methoxyl group substance includes dimethoxypropane; and the oxysilane group substance includes tetramethoxysilane.

4. The tissue light clearing reagent according to claim 1, characterized in that: The polyol is selected from at least one of propylene glycol, glycerol, pentanediol and hexanediol; the amino alcohol is selected from at least one of triethanolamine, N-methyldiethanolamine and N-butyldiethanolamine; and the reducing agent is selected from at least one of thioglycerol, vitamin D and vitamin C.

5. The tissue light clearing reagent according to claim 1, characterized in that: It also includes a degreasing liquid, which includes a reducing agent and a degreasing agent; the mass volume ratio of the reducing agent to the degreasing agent is 0.1%-1%; Wherein, the reducing agent is selected from at least one of thioglycerol, vitamin D and vitamin C; the degreasing agent is selected from at least one of cyclohexane, ethanol, dichloromethane and benzyl benzoate.

6. The tissue light clearing reagent according to claim 1, characterized in that: The invention also includes a refractive index matching liquid, wherein the refractive index matching liquid includes at least one of dibenzyl ether, benzyl benzoate, benzyl alcohol and ethyl cinnamate.

7. The tissue light clearing agent according to claim 1, characterized in that: It also includes a pretreatment liquid, which includes polyol, amino alcohol, reducing agent and distilled water; the mass volume ratio of polyol to distilled water is 5%-30%, the mass volume ratio of amino alcohol to distilled water is 5%-30%, and the mass volume ratio of reducing agent to distilled water is 0.1%-1%.

8. A method for rapid tissue photoclearing using the tissue photoclearing reagent according to any one of claims 1 to 7, characterized in that: The steps include: The biological tissue is placed in a dehydrating solution and subjected to shaking treatment to obtain a dehydrated tissue; placing the dehydrated tissue in a defatting solution and shaking the tissue to obtain a defatted tissue; The defatted tissue is placed in a refractive index matching liquid and subjected to an oscillation treatment to obtain a transparent tissue.

9. The method according to claim 8, characterized in that Before the dehydrated tissue is prepared, the method further includes a step of pre-treating the biological tissue, wherein the pre-treating step specifically includes: placing the biological tissue in a pre-treating solution and oscillating the solution to obtain the pre-treated tissue; The shaking treatment step specifically includes shaking for 6-24 hours at a temperature of 30-40°C.

10. The method according to claim 8, characterized in that In the preparation process of the dehydrated tissue, the shaking treatment step specifically includes: shaking for 6-24 hours at a temperature of 30-40°C; In the preparation process of the defatted tissue, the shaking treatment step specifically includes: shaking for 2-12 hours at a temperature of 30-40°C; In the preparation process of the transparent tissue, the shaking treatment step specifically includes: shaking for 1-6 hours at a temperature of 20-25°C.

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