A tissue optical clearing kit and method for maintaining sample morphology

Abstract

The present application belongs to the technical field of biomedical optical imaging, and more particularly relates to a tissue optical transparency kit capable of maintaining sample morphology and a method for optical transparency. The kit comprises a pretreatment liquid, a first buffer, a second buffer and a tissue dehydrating liquid. The pretreatment liquid, the first buffer, the second buffer and the tissue dehydrating liquid all contain a first alcohol substance and a surfactant, and the mass percentage of the alcohol substance in the pretreatment liquid, the first buffer, the second buffer and the tissue dehydrating liquid gradually increases. The first alcohol substance contains a polyhydric alcohol. The first alcohol substance, the surfactant and water are mixed to treat the tissue. Mixing the low-concentration first alcohol substance, the surfactant and water in a certain proportion can make the tissue sufficiently de-fat and swell. Mixing the high-concentration first alcohol substance and the surfactant in a certain proportion can moderately dehydrate the swollen tissue, so that the swollen tissue shrinks to the original size.

Description

Technical Field

[0001] This invention belongs to the field of biomedical optical imaging technology, and more specifically, relates to a tissue light transparency kit and a light transparency method that can maintain the morphology of a sample. Background Technology

[0002] Living organisms possess complex and diverse yet interconnected structures and functions. Obtaining the three-dimensional structure of biological tissues at the organ or even systemic level, and understanding the connections between central and peripheral nerves and blood vessels, is crucial for neuroscience and physiological and pathological research. Optical microscopy combined with fluorescence labeling technology can acquire the fine three-dimensional structure of biological tissues at the cellular and even subcellular resolution. However, the high scattering of turbid tissues limits the penetration depth of light within the tissue, often confining imaging to the superficial layers of biological tissues. The recently developed tissue light-transparency technique provides an important solution for three-dimensional structural imaging of tissues and organs. It employs various physical and chemical methods to homogenize the refractive index of biological tissues, thus making them "transparent" to light. Combining tissue light-transparency with various fluorescence labeling techniques and light-sheet illumination microscopy enables whole-body imaging of tissues and organs, providing a novel means for high-resolution acquisition of complete and specific neural and vascular network structures. It is now widely used in research across many biological and medical fields, including neurology, vascular medicine, and immunology.

[0003] Currently, optical clearing methods are mainly divided into two categories: those based on water-soluble reagents and those based on organic solvents. Water-soluble reagent-based methods offer better fluorescence compatibility, but their clearing effect is slightly inferior to organic solvent methods, and they suffer from swelling, complex processing, and longer processing times. In contrast, organic solvent-based methods achieve better clearing by dehydrating the tissue and applying a high-refractive-index organic solvent to the tissue sample for refractive index matching. These methods are also faster, and their fluorescence compatibility has improved significantly in recent years. Therefore, these methods are widely used.

[0004] However, current organic solvent-based methods require extensive dehydration of tissues, which causes severe tissue shrinkage. This inevitably reduces the resolution of the optical system on the measured object. More importantly, the shrinkage is not isotropic, especially for tissues with hollow structures, such as different ventricles, the heart, and the lungs. The deformation caused by shrinkage makes it impossible to accurately reconstruct their original morphological structure, which greatly hinders image registration and further analysis and processing. It makes it difficult to perform tasks such as cross-brain region neural tracking, registration of whole-brain vascular networks, and quantitative analysis. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a tissue light-transparency kit and method that maintains sample morphology. The method involves treating tissue by mixing alcohols with surfactants. A low-concentration mixture of alcohols, surfactants, and water in a specific ratio allows for sufficient defatting and swelling of the tissue. A high-concentration mixture of alcohols and surfactants in a specific ratio moderately dehydrates the swollen tissue, causing it to shrink back to its original size. This ensures adequate dehydration while restoring the original size of the biological sample tissue as much as possible. Tissue treated with this invention becomes highly transparent and largely retains its original morphology, making it suitable for overall three-dimensional fluorescence imaging. This enables high-resolution three-dimensional imaging and precise reconstruction of the whole-brain neural and vascular network structure. This invention solves the problems of existing organic solvent-based methods, which require extensive dehydration of the tissue, causing severe tissue shrinkage and inevitably reducing the optical system's resolution of the object being measured. This results in an inability to accurately reconstruct the original morphological structure, significantly hindering image registration and further analysis, and making it difficult to implement technical problems such as cross-brain region neural tracking, whole-brain vascular network registration, and quantitative analysis.

[0006] To achieve the above objectives, the present invention provides a kit for optically transparentizing one or more tissue samples, comprising a pretreatment solution, a first buffer solution, a second buffer solution, and a tissue dehydration solution. The pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution all contain a first alcohol and a surfactant, and the mass percentage of the first alcohol in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution gradually increases; the first alcohol contains a polyol.

[0007] Preferably, the mass percentage of the first alcohol in the pretreatment solution is less than or equal to 25%, and the mass percentage of the first alcohol in the tissue dehydration solution is greater than or equal to 70%.

[0008] Preferably, the mass percentage of surfactant in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution is less than or equal to 10%.

[0009] Preferably, the polyol is one or more of ethylene glycol, glycerol, hexanediol, hexanetriol, and hexanetriol, and more preferably hexanediol or hexanetriol.

[0010] In some technical solutions, the first alcohol substance further contains one or more of methanol, tert-butanol and N-butyldiethanolamine, and the surfactant is a nonionic surfactant or anionic surfactant, more preferably one or more of Triton X-100, Tween-20 and sodium dodecyl sulfate.

[0011] Preferably, the kit further includes a refractive index matching solution, which comprises 50-70 parts by weight of an organic solvent, 20-40 parts by weight of a polymer and 0-10 parts by weight of a second alcohol.

[0012] Preferably, the organic solvent is one or more of dibenzyl ether, diphenyl ether, benzyl alcohol, and benzyl benzoate; the polymer is one or more of polyethylene glycol substances with a molecular weight of 200-1000; and the second alcohol is one or more of hexanediol, methanol, tert-butanol, and N-butyldiethanolamine.

[0013] According to another aspect of the present invention, a method for optically transparentizing tissues using the kit is provided, comprising the following steps:

[0014] (1) Immerse the fixed tissue in the pretreatment solution to allow the tissue to fully expand, thereby obtaining the expanded tissue;

[0015] (2) The expanded tissue is sequentially immersed in the first buffer solution and the second buffer solution to fix the tissue framework and stabilize the tissue morphology, thereby obtaining the buffer-treated tissue.

[0016] (3) The tissue treated with the buffer solution is placed in the tissue dehydration solution for dehydration treatment, so that the tissue shrinks to its original size and the overall texture of the tissue becomes hard and the shape is solidified, thus obtaining the dehydrated tissue;

[0017] (4) The dehydrated tissue is immersed in the refractive index matching solution to obtain an optically transparent tissue;

[0018] The pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution all contain a first alcohol and a surfactant, and the mass percentage of the first alcohol in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution gradually increases.

[0019] Preferably, in step (1), the fixed tissue is immersed in the pretreatment solution for 12-24 hours; in step (2), the swollen tissue is immersed in the first buffer solution and the second buffer solution for 6-12 hours respectively; in step (3), the tissue treated with the buffer solution is placed in the tissue dehydration solution for dehydration treatment for 24-36 hours.

[0020] Preferably, the refractive index matching solution in step (4) contains 50-70 parts by mass of an organic solvent, 20-40 parts by mass of a polymer and 0-10 parts by mass of a second alcohol; in step (4), the dehydrated tissue is immersed in the refractive index matching solution for 24-36 hours.

[0021] Preferably, the organic solvent is one or more of dibenzyl ether, diphenyl ether, benzyl alcohol, and benzyl benzoate; the polymer is one or more of polyethylene glycol substances with a molecular weight of 200-1000, more preferably one or more of PEG-200, PEG400, and PEGMMA500; the second alcohol is one or more of hexanediol, methanol, tert-butanol, and N-butyldiethanolamine.

[0022] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art:

[0023] (1) The present invention provides a tissue light-transparency kit that can maintain the morphology of samples, comprising a pretreatment solution, a first buffer solution, a second buffer solution, and a tissue dehydration solution. The pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution all contain a first alcohol and a surfactant, and the mass percentage of the first alcohol in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution gradually increases. The first alcohol containing polyol is mixed with a surfactant to treat the tissue. A low concentration of the first alcohol mixed with a surfactant and water in a certain proportion can fully degrease and swell the tissue, while a high concentration of the first alcohol mixed with a surfactant in a certain proportion can moderately dehydrate the swollen tissue, causing the swollen tissue to shrink back to its original size.

[0024] (2) This invention creatively mixes specific first alcohols with surfactants and water in different proportions. Although there seems to be a contradiction between sample degreasing, dehydration, transparency and morphology preservation, it is entirely feasible to maintain sample morphology and fluorescence signal while ensuring fast transparency and high transparency through clever reagent concentration design, compounding of reagents and setting of transparency steps. This concept is the first of its kind in the world.

[0025] (3) The tissue dehydration and transparency strategy proposed in this invention can maintain the sample morphology. The transparent sample has obvious advantages over the existing organic solvent-based methods in terms of transparency, sample morphology preservation and imaging quality.

[0026] (4) The tissue dehydration and transparency strategy proposed in this invention, which can maintain the morphology of the sample, not only provides a new means for high-resolution imaging and precise reconstruction of the neural, cellular and vascular structures of the whole brain and organs of mice, but also helps to accurately measure the physiological and pathological structures of tissues and organs and visualize them in three dimensions, which is of great importance to basic medical research. Attached Figure Description

[0027] Figure 1 This is a schematic flowchart of the method for making the whole brain of an adult mouse transparent according to an embodiment of the present invention.

[0028] Figure 2 These are intuitive images of the whole brain of a mouse in Example 1 before and after it has been transparent using the present invention.

[0029] Figure 3a These are intuitive images of the whole brain of a mouse in Example 2 before, after, and after being transparent using the present invention and the classic organic solvent transparent method.

[0030] Figure 3b This is a quantitative statistical analysis of the morphological change rate of the whole brain of mice before and after transparentization using the present invention and the typical organic solvent transparentization method uDISCO in Example 2.

[0031] Figure 4a The neural structural information of mouse brain tissue after treatment with the transparency method in Example 3 is labeled with EGFP.

[0032] Figure 4b This is information on the vascular structure of mouse brain tissue after treatment with the transparency method in Example 3, labeled with Rhodamine.

[0033] Figure 4c This is information on the nuclear architecture of mouse brain tissue after treatment with the transparency method in Example 3, labeled with To-Pro-3.

[0034] Figure 5 The imaging results of whole-brain neural structure information obtained by light-sheet illumination imaging of mouse whole-brain samples after transparency in Example 4 are presented at different imaging depths.

[0035] Figure 6 The imaging results of whole-brain vascular network structure information obtained by light sheet illumination imaging of mouse whole-brain samples after transparency in Example 5 are presented at different imaging depths.

[0036] Figure 7 The results of three-dimensional reconstruction of the neural, vascular and cellular structures of the whole brain of a mouse sample after transparency according to Example 6 of the present invention are presented.

[0037] Figure 8 These are intuitive images taken during the transparentization process of an isolated mouse whole brain sample as described in Example 7.

[0038] Figure 9 These are intuitive images taken during the process of transparentizing an isolated mouse whole brain sample as shown in Comparative Example 1.

[0039] Figure 10 These are intuitive images taken during the transparentization process of an isolated mouse whole brain sample as shown in Comparative Example 2. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] This invention provides a tissue light transparency method that can maintain sample morphology, comprising the following steps:

[0042] (1) Immerse the fixed tissue in the pretreatment solution to allow the tissue to fully expand and obtain the expanded tissue;

[0043] (2) The expanded tissue is sequentially immersed in the first buffer solution and the second buffer solution to fix the tissue framework and stabilize the tissue morphology, thereby obtaining the buffer-treated tissue.

[0044] (3) The tissue treated with the buffer solution is placed in a tissue dehydration solution for dehydration treatment, so that the tissue shrinks to its original size and the overall texture of the tissue becomes hard and the shape is solidified, thus obtaining the dehydrated tissue;

[0045] (4) The dehydrated tissue is immersed in a refractive index matching solution to obtain an optically transparent tissue;

[0046] The pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution all contain a first alcohol and a surfactant, and the mass percentage of the first alcohol in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution gradually increases. The first alcohol contains polyols.

[0047] In some embodiments, the mass percentage of the first alcohol in the pretreatment solution is less than or equal to 25%, and the mass percentage of the first alcohol in the tissue dehydration solution is greater than or equal to 70%.

[0048] In some embodiments, the mass percentage of surfactant in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution is less than or equal to 10%.

[0049] This invention has shown through experiments that when a polyol, such as one or more of ethylene glycol, glycerol, hexanediol, hexanetriol, or hexanetetraol, is introduced into a first alcohol compound, the pretreatment solution prepared by combining the first alcohol compound containing the polyol with a surfactant under low concentration conditions can degrease and swell the tissue. Under high concentration conditions, the pretreatment solution causes moderate dehydration and shrinkage, ultimately ensuring good transparency while maintaining the sample morphology. In a preferred embodiment, the polyol is hexanediol or hexanetriol.

[0050] In other embodiments, the first alcohol also contains one or more of methanol, tert-butanol, and N-butyldiethanolamine.

[0051] In some embodiments, the surfactant is a nonionic or anionic surfactant, including but not limited to Triton X-100, Tween-20, and / or sodium dodecyl sulfate. The surfactant primarily assists in degreasing, effectively breaking down the dense lipid structure in tissues, and works in conjunction with alcohols to achieve thorough degreasing of the tissue.

[0052] In some embodiments, step (1) involves immersing the fixed tissue in a pretreatment solution for 12-24 hours. The pretreatment solution in step (1) contains 10%-25% by mass of a first alcohol, less than or equal to 10% of a surfactant, and the remainder is water.

[0053] In some embodiments, in step (2), the first buffer solution contains 40%-50% by mass of a first alcohol, less than or equal to 5% by mass of a surfactant, and the remainder is water; the second buffer solution contains 60%-70% by mass of a first alcohol, less than or equal to 5% by mass of a surfactant, and the remainder is water. In step (2), the swollen tissue is sequentially immersed in the first buffer solution and the second buffer solution for 6-12 hours respectively.

[0054] The pretreatment solution, the first buffer solution, and the second buffer solution of this invention all contain a first alcohol, a surfactant, and water. The tissue dehydration solution contains a first alcohol and a surfactant, but no water. In some embodiments, the tissue dehydration solution in step (3) contains 70%-98% by mass of a first alcohol, with the remainder being a surfactant; the tissue treated with the buffer solution is placed in the tissue dehydration solution for dehydration treatment for 24-36 hours.

[0055] In some embodiments, the refractive index matching liquid in step (4) comprises 50-70 parts by mass of an organic solvent, 20-40 parts by mass of a polymer and 0-10 parts by mass of a second alcohol, preferably 5-10 parts by mass of an alcohol.

[0056] In some embodiments, the organic solvent is one or more of dibenzyl ether, diphenyl ether, benzyl alcohol, and benzyl benzoate; the polymer is a polyethylene glycol of different molecular weights, preferably a polyethylene glycol of molecular weight 200-1000, including but not limited to one or more of PEG-200, PEG400, and PEGMMA500; the main function of the polymer is to protect fluorescence. The second alcohol is one or more of hexanediol, methanol, tert-butanol, and N-butyldiethanolamine.

[0057] In some embodiments, step (4) involves immersing the dehydrated tissue in a refractive index matching solution for 24-36 hours.

[0058] In some embodiments, the tissue types include, but are not limited to, the whole brain, heart, liver, kidney, lung, spleen, stomach, intestine, and embryo of mice and rats.

[0059] The experimental subject of the optical transparency method of this invention is fixed biological tissue. The tissue fixation method can adopt conventional tissue fixation methods in the prior art, such as using formaldehyde, glutaraldehyde, or paraformaldehyde to fix the tissue. In the embodiments of this invention, paraformaldehyde is used to fix the tissue, resulting in fixed tissue.

[0060] Traditional tissue light-transparency methods using organic solvents mostly employ single dehydrating agents, such as methanol, ethanol, and tetrahydrofuran, to perform gradient dehydration of the tissue, resulting in severe tissue shrinkage. In a preferred embodiment of this invention, a novel combination of a hexanediol-containing primary alcohol and a surfactant is used to treat the tissue. A low concentration of the primary alcohol, surfactant, and water, mixed in a specific ratio, allows for thorough defatting and swelling of the tissue. A high concentration of the primary alcohol, mixed with the surfactant, moderately dehydrates the swollen tissue, causing it to shrink back to its original size. This ensures the degree of dehydration while restoring the original size of the biological sample tissue as much as possible. Furthermore, the addition of a polymer to the refractive index matching solution ensures fluorescence compatibility; and the organic solvent-based refractive index matching solution guarantees both the speed of transparency and the sample's transparency.

[0061] The pretreatment solution, first buffer, second buffer, and tissue dehydration solution in the light-transparency kit of this invention use the same type of main reagents. The only difference is that the concentration of the first alcohol containing hexanediol gradually increases. The continuous change in the concentration of the first alcohol produces different effects on the tissue: the first alcohol containing hexanediol used in this invention has a significant degreasing effect compared to traditional dehydration reagents; combined with a certain concentration of surfactant, at low concentrations, it can fully degrease and swell the tissue, while at high concentrations, it can moderately dehydrate the tissue and cause it to shrink, thus offsetting the swelling during degreasing; therefore, the tissue dehydrated and transparent by this invention not only has high transparency but also has a sample morphology almost identical to that before transparentization, which cannot be achieved by transparentization methods using traditional dehydration reagents. Furthermore, some traditional methods, in order to reduce sample shrinkage, pre-treat the sample before dehydration to swell the tissue before dehydration. While this can be effective to some extent, it has significant limitations: firstly, the extra step prolongs the clearing time and reduces the clearing efficiency; secondly, the swelling reagents used for tissue pre-swelling, such as urea and guanidine hydrochloride, have a significant impact on tissue structure and fluorescence signal. The method provided by this invention allows for sufficient tissue swelling at low concentration gradients, eliminating the need for an additional tissue swelling step; and the first alcohol substances used are all relatively mild reagents that have no significant impact on the internal structure of the tissue or the fluorescence signal.

[0062] Tissues treated with the optical transparency method of this invention can be used in fluorescence microscopy imaging such as confocal imaging, two-photon imaging, and light sheet imaging to achieve high-resolution and accurate reconstruction of the neurovascular network of the whole sample.

[0063] Accordingly, the present invention also provides a kit for preparing one or more tissue samples, comprising a pretreatment solution, a first buffer, a second buffer, and a tissue dehydration solution as described above, wherein the pretreatment solution, the first buffer, the second buffer, and the tissue dehydration solution all contain a first alcohol and a surfactant, and the mass percentage of the first alcohol in the pretreatment solution, the first buffer, the second buffer, and the tissue dehydration solution gradually increases, wherein the first alcohol contains a polyol.

[0064] In some embodiments, the pretreatment solution contains less than or equal to 25% by mass of alcohols, and the tissue dehydration solution contains more than or equal to 70% by mass of the first alcohol. The pretreatment solution, the first buffer solution, and the second buffer solution contain water, while the tissue dehydration solution does not contain water.

[0065] In some embodiments, the polyol is one or more of ethylene glycol, glycerol, hexanediol, hexanetriol, hexanetetraol, etc., preferably hexanediol or hexanetriol.

[0066] In other embodiments, the first alcohol also contains one or more of methanol, tert-butanol, and N-butyldiethanolamine.

[0067] In some embodiments, the kit further includes a refractive index matching solution. The refractive index matching solution used in this invention contains, in addition to commonly used components of refractive index matching solutions in the prior art, a second alcohol substance for protecting fluorescence. For example, in some embodiments, the refractive index matching solution comprises 50-70 parts by weight of an organic solvent, 20-40 parts by weight of a polymer, and 0-10 parts by weight of a second alcohol substance, preferably 5-10 parts by weight of the second alcohol substance.

[0068] In some embodiments, the organic solvent is one or more of dibenzyl ether, diphenyl ether, benzyl alcohol, and benzyl benzoate; the polymer is one or more of PEG-200, PEG400, and PEGMMA500; and the second alcohol is one or more of hexanediol, methanol, tert-butanol, and N-butyldiethanolamine.

[0069] This invention provides a tissue dehydration and transparency strategy that maintains sample morphology. Various tissues treated with this method not only become highly transparent in a short time, but also maintain virtually no change in morphology or size compared to before transparency. The treated tissues can be used in whole-body fluorescence microscopy to achieve high-resolution three-dimensional fluorescence imaging and precise reconstruction of the neurovascular network structure of the entire brain and organs.

[0070] To make the objectives, features, and advantages of this invention more apparent and understandable, several embodiments are provided below to illustrate a tissue dehydration and transparency strategy for maintaining sample morphology and its application.

[0071] Example 1

[0072] In this embodiment, the biological tissue was obtained from the brain tissue of C57 mice. The fixed whole brain sample of the mice was processed using the transparency treatment method described in Example 1. Figure 1 As shown, the specific steps include the following:

[0073] (1) The fixed tissue is immersed in the pretreatment solution for 24 hours. The pretreatment solution will make the tissue fully swell. In this embodiment, the pretreatment solution consists of 15 wt% hexanediol, 5 wt% Triton-X 100, and the remainder is water.

[0074] (2) The tissues that have been fully soaked in the pretreatment solution are placed in buffer 1 and buffer 2 in sequence for 6 hours each to fix the tissue framework and stabilize the tissue morphology. In this embodiment, the components of buffer 1 include: 35 wt% hexanediol, 20 wt% methanol, 3 wt% Triton-X 100, and the remainder is water. The components of buffer 2 include: 50 wt% hexanediol, 20 wt% tert-butanol, 3 wt% Triton-X 100, and the remainder is water.

[0075] (3) The tissue treated in the buffer solution was placed in the tissue dehydration solution for dehydration treatment for 24 hours, so that the tissue shrank to its original size and the overall texture of the tissue became hard and the shape solidified. The tissue dehydration solution in this embodiment includes: 75wt% hexanediol, 20wt% tert-butanol, and 5wt% Triton-X 100.

[0076] (4) The dehydrated tissue was soaked in the refractive index matching solution for 24 hours, and finally a highly transparent tissue with the same shape and size as before the transparency was obtained. The refractive index matching solution in this embodiment includes: 20wt% benzyl alcohol, 40wt% benzyl benzoate, 30wt% PEGMMA500, and 10wt% butyl diethanolamine.

[0077] Figure 2 Visual images of isolated mouse whole brain samples before and after the transparency treatment described in this embodiment are presented. Figure 2 Content a is an image of the mouse brain before it becomes transparent; Figure 2 Content b is an image of the sample after optical transparency processing using the method of this embodiment. It can be seen that the tissue after transparency processing becomes transparent, and its morphology is basically the same as before transparency.

[0078] Example 2

[0079] In this embodiment, the biological tissue was obtained from the brain tissue of C57 mice. The fixed whole brain sample of the mice was processed using the transparency processing method of this embodiment, specifically including the following steps:

[0080] (1) The fixed tissue is immersed in the pretreatment solution for 24 hours. The pretreatment solution will make the tissue fully swell. In this embodiment, the pretreatment solution consists of 20 wt% hexanediol, 5 wt% Triton-X 100, and the remainder is water.

[0081] (2) The tissues that have been fully soaked in the pretreatment solution are placed in buffer 1 and buffer 2 in sequence for 6 hours to fix the tissue framework and stabilize the tissue morphology. In this embodiment, the components of buffer 1 include: 40 wt% hexanediol, 10 wt% methanol, 2 wt% Triton-X 100, and the remainder is water. The components of buffer 2 include: 60 wt% hexanediol, 10 wt% tert-butanol, 2 wt% Triton-X 100, and the remainder is water.

[0082] (3) The tissue that has been treated in the buffer solution is placed in the tissue dehydration solution for dehydration treatment for 24 hours, so that the tissue shrinks to its original size and the overall texture of the tissue becomes hard and the shape is solidified. The tissue dehydration solution in this embodiment includes: 70wt% hexanediol, 20wt% methanol, and 10wt% Triton-X 100.

[0083] (4) The dehydrated tissue was soaked in the refractive index matching solution for 24 hours, and finally a highly transparent tissue with the same shape and size as before the transparency was obtained. The refractive index matching solution in this embodiment includes: 20wt% benzyl alcohol, 40wt% benzyl benzoate, 30wt% PEGMMA500, and 10wt% butyl diethanolamine.

[0084] In addition, the fixed mouse whole brain sample was transparent using the existing mainstream transparency method and compared with the sample that was transparent in this embodiment.

[0085] Figure 3a Visual images of the isolated mouse brain before, after the transparency treatment described in this embodiment, and after transparency using other methods (uDISCO) are presented. The leftmost image is of the mouse brain before transparency; due to the turbidity of brain tissue, the grid lines beneath the tissue are completely obscured. The remaining images are of the samples after light transparency treatment. It can be seen that the tissue treated in this embodiment becomes highly transparent, the grid lines beneath the tissue are clearly visible, and the morphology is basically the same as before transparency, which has a significant advantage compared to samples transparent using other methods. Figure 3b Quantitative statistics are presented on the tissue size change rates of mouse whole brain samples before and after clearing using this embodiment and the typical organic solvent clearing method uDISCO. It can be seen that the morphology of the mouse whole brain tissue treated with this embodiment is basically consistent with that before clearing, while the mainstream organic solvent method uDISCO shows severe shrinkage.

[0086] Example 3

[0087] In this embodiment, the biological tissues were obtained from brain slices of Thy1-GFP-M transgenic mice and C57 mice. The neural, vascular, and nuclear structural information of the brain slices was fluorescently labeled. The fluorescently labeled brain slices were then processed using the clearing method described in Embodiment 3, specifically including the following steps:

[0088] (1) The fixed tissue is immersed in the pretreatment solution for 24 hours. The pretreatment solution will make the tissue fully swell. In this embodiment, the pretreatment solution consists of 10 wt% hexanediol, 3 wt% Triton-X 100, and the remainder is water.

[0089] (2) The tissues that have been fully soaked in the pretreatment solution are placed in buffer 1 and buffer 2 in sequence for 6 hours each to fix the tissue framework and stabilize the tissue morphology. In this embodiment, the components of buffer 1 include: 45 wt% hexanediol, 5 wt% methanol, 2 wt% Triton-X 100, and the remainder is water. The components of buffer 2 include: 55 wt% hexanediol, 15 wt% tert-butanol, 2 wt% Triton-X 100, and the remainder is water.

[0090] (3) The tissue that has been treated in the buffer solution is placed in the tissue dehydration solution for dehydration treatment for 24 hours, so that the tissue shrinks to its original size and the overall texture of the tissue becomes hard and the shape is solidified; the tissue dehydration solution in this embodiment includes: 70wt% hexanediol, 20wt% methanol, and 10wt% Triton-X 100.

[0091] (4) The dehydrated tissue was soaked in the refractive index matching solution for 24 hours, and finally a highly transparent tissue with the same shape and size as before the transparency was obtained. The refractive index matching solution in this embodiment includes: 20wt% benzyl alcohol, 40wt% benzyl benzoate, 35wt% PEGMMA500, and 5wt% butyl diethanolamine.

[0092] (5) The transparent tissue treated by the dehydration and transparency strategy was used in confocal imaging to obtain high-resolution structural information of nerves, blood vessels and cell nuclei on mouse brain slices.

[0093] Figure 4a , Figure 4b and Figure 4c Fluorescence images of fluorescently labeled brain slices, processed using the transparency method of Example 3 of this invention, and captured by laser confocal microscopy, are presented. Figure 4a Neural structural information labeled with EGFP, Figure 4b Information on vascular structures labeled with Rhodamine. Figure 4cInformation on the nuclear structure of cells labeled with To-Pro-3. It can be seen that the transparency method in this embodiment is compatible with many mainstream fluorescent labeling methods.

[0094] Example 4

[0095] In this embodiment, the biological tissue was obtained from the whole brain of Thy1-GFP-M mice. The whole brain of the mice was processed using the clearing method described in Embodiment 4 of this embodiment, specifically including the following steps:

[0096] (1) The fixed tissue is immersed in the pretreatment solution for 24 hours. The pretreatment solution will make the tissue fully swell. In this embodiment, the pretreatment solution consists of 10 wt% hexanediol, 3 wt% Triton-X 100, and the remainder is water.

[0097] (2) The tissues that have been fully soaked in the pretreatment solution are placed in buffer 1 and buffer 2 in sequence for 6 hours each to fix the tissue framework and stabilize the tissue morphology. In this embodiment, the components of buffer 1 include: 45 wt% hexanediol, 5 wt% methanol, 2 wt% Triton-X 100, and the remainder is water. The components of buffer 2 include: 55 wt% hexanediol, 15 wt% tert-butanol, 2 wt% Triton-X 100, and the remainder is water.

[0098] (3) The tissues treated in the buffer solution were placed in the tissue dehydration solution for dehydration treatment for 36 hours, so that the tissues shrank to their original size and the overall texture of the tissues became hard and the shape solidified. The tissue dehydration solution in this embodiment includes: 70wt% hexanediol, 20wt% methanol, and 10wt% Triton-X 100.

[0099] (4) The dehydrated tissue was soaked in the refractive index matching solution for 36 hours, and finally a highly transparent tissue with the same shape and size as before the transparency was obtained. The refractive index matching solution in this embodiment includes: 20wt% benzyl alcohol, 40wt% benzyl benzoate, 35wt% PEGMMA500, and 5wt% butyl diethanolamine.

[0100] (5) The transparent tissue treated by the dehydration and transparency strategy was used in confocal imaging to obtain high-resolution structural information of nerves, blood vessels and cell nuclei in mouse brain tissue;

[0101] Figure 5 The paper presents three-dimensional neural structure images of the mouse whole brain sample after transparency in this embodiment, acquired using a light-slide illumination microscope at different imaging depths. It can be seen that the neural structure information of the mouse whole brain is clearly visible at different imaging depths.

[0102] Example 5

[0103] In this embodiment, the biological tissue was obtained from the whole brain of Thy1-GFP-M mice. The whole brain sample of the mice was processed using the clearing method described in Embodiment 5 of this embodiment, specifically including the following steps:

[0104] (1) The fixed tissue is immersed in the pretreatment solution for 24 hours. The pretreatment solution will make the tissue fully swell. In this embodiment, the pretreatment solution consists of 20 wt% hexanediol, 5 wt% Triton-X 100, and the remainder is water.

[0105] (2) The tissues that have been fully soaked in the pretreatment solution are placed in buffer 1 and buffer 2 in sequence for 6 hours each to fix the tissue framework and stabilize the tissue morphology. In this embodiment, the components of buffer 1 include: 45 wt% hexanediol, 5 wt% methanol, 2 wt% Triton-X 100, and the remainder is water. The components of buffer 2 include: 55 wt% hexanediol, 15 wt% tert-butanol, 2 wt% Triton-X 100, and the remainder is water.

[0106] (3) The tissue that has been treated in the buffer solution is placed in the tissue dehydration solution for dehydration treatment for 36 hours, so that the tissue shrinks to its original size and the overall texture of the tissue becomes hard and the shape is solidified; the tissue dehydration solution in this embodiment includes: 75wt% hexanediol, 20wt% methanol, and 5wt% Triton-X 100.

[0107] (4) The dehydrated tissue was soaked in the refractive index matching solution for 36 hours, and finally a highly transparent tissue with the same shape and size as before the transparency was obtained. The refractive index matching solution in this embodiment includes: 20wt% benzyl alcohol, 40wt% benzyl benzoate, 35wt% PEGMMA500, and 5wt% butyl diethanolamine.

[0108] (5) The transparent tissue treated by the dehydration and transparency strategy was used in confocal imaging to obtain high-resolution structural information of nerves, blood vessels and cell nuclei in mouse brain tissue;

[0109] Figure 6 Images of the three-dimensional vascular network structure of the mouse whole brain, obtained by light-sheet illumination microscopy after being transparentized according to this invention, are presented at different imaging depths. It can be seen that the vascular network structure information of the mouse whole brain is clearly visible at different imaging depths.

[0110] Example 6

[0111] In this embodiment, the biological tissue was obtained from the whole brain of Thy1-GFP-M mice, and the blood vessels and cell nuclei of the whole brain were stained. The whole brain sample of mice was processed by the clearing method in embodiment 5, which specifically includes the following steps:

[0112] (1) The fixed tissue is immersed in the pretreatment solution for 24 hours. The pretreatment solution will make the tissue fully swell. In this embodiment, the pretreatment solution consists of 20 wt% hexanediol, 5 wt% Triton-X 100, and the remainder is water.

[0113] (2) The tissues that have been fully soaked in the pretreatment solution are placed in buffer 1 and buffer 2 in sequence for 6 hours each to fix the tissue framework and stabilize the tissue morphology. In this embodiment, the components of buffer 1 include: 45 wt% hexanediol, 5 wt% methanol, 2 wt% Triton-X 100, and the remainder is water. The components of buffer 2 include: 55 wt% hexanediol, 15 wt% tert-butanol, 2 wt% Triton-X 100, and the remainder is water.

[0114] (3) The tissue that has been treated in the buffer solution is placed in the tissue dehydration solution for dehydration treatment for 36 hours, so that the tissue shrinks to its original size and the overall texture of the tissue becomes hard and the shape is solidified; the tissue dehydration solution in this embodiment includes: 75wt% hexanediol, 20wt% methanol, and 5wt% Triton-X 100.

[0115] (4) The dehydrated tissue was soaked in the refractive index matching solution for 36 hours, and finally a highly transparent tissue with the same shape and size as before the transparency was obtained. The refractive index matching solution in this embodiment includes: 20wt% benzyl alcohol, 40wt% benzyl benzoate, 35wt% PEGMMA500, and 5wt% butyl diethanolamine.

[0116] (5) The transparent tissue treated by the dehydration and transparency strategy was used in confocal imaging to obtain high-resolution structural information of nerves, blood vessels and cell nuclei in mouse brain tissue;

[0117] Figure 7 The paper presents the three-dimensional reconstruction results of whole-brain neural, vascular, and cellular structures of a mouse brain sample after transparency according to this invention, acquired using a light-slide illumination microscope. It can be seen that this invention can effectively obtain high-resolution structural information of the neural, vascular, and cellular nuclei structures of the entire mouse brain.

[0118] Example 7

[0119] In this embodiment, the biological tissue was obtained from the brain tissue of C57 mice. The fixed whole brain sample of the mice was processed using the transparency treatment method described in Example 1. Figure 1 As shown, the specific steps include the following:

[0120] (1) The fixed tissue is immersed in the pretreatment solution for 24 hours. The pretreatment solution will make the tissue fully swell. In this embodiment, the pretreatment solution consists of 15 wt% hexanediol, 5 wt% Triton-X 100, and the remainder is water.

[0121] (2) The tissues that have been fully soaked in the pretreatment solution are placed in buffer 1 and buffer 2 in sequence for soaking for 6 hours each to fix the tissue framework and stabilize the tissue morphology. In this embodiment, the components of buffer 1 include: 50 wt% hexanediol, 3 wt% Triton-X 100, and the remainder is water; the components of buffer 2 include: 70 wt% hexanediol, 3 wt% Triton-X 100, and the remainder is water.

[0122] (3) The tissues treated in the buffer solution were placed in the tissue dehydration solution for dehydration treatment for 24 hours, so that the tissues shrank to their original size and the overall texture of the tissues became hard and the shape solidified. The tissue dehydration solution in this embodiment includes: 95wt% hexanediol and 5wt% Triton-X 100.

[0123] (4) The dehydrated tissue was soaked in the refractive index matching solution for 24 hours, and finally a highly transparent tissue with the same shape and size as before the transparency was obtained. The refractive index matching solution in this embodiment includes: 20wt% benzyl alcohol, 40wt% benzyl benzoate, 30wt% PEGMMA500, and 10wt% butyl diethanolamine.

[0124] Figure 8 Visual images of isolated mouse whole brain samples taken during the clarification process described in this embodiment are presented. It can be seen that the tissue undergoing the clarification process first swells after being immersed in the pretreatment solution, then gradually shrinks, and finally becomes highly transparent in the refractive index matching solution, with its morphology essentially consistent with that before clarification.

[0125] Comparative Example 1

[0126] The other conditions are the same as in Example 7, except that hexanediol in steps (1) to (3) of Example 7 is replaced with methanol. Figure 9 Visual images of the isolated mouse whole brain samples from this comparative example, taken during the transparency process described in this embodiment, are presented. It can be seen that when hexanediol is replaced with methanol, the sample gradually shrinks during the transparency process, failing to effectively maintain its final morphology.

[0127] Comparative Example 2

[0128] The other conditions are the same as in Example 7, except that hexanediol in steps (1) to (3) of Example 7 is replaced with tert-butanol. Figure 10 Visual images of the isolated mouse whole brain samples from this comparative example, taken during the transparency process described in this embodiment, are presented. It can be seen that when hexanediol is replaced with tert-butanol, the sample gradually shrinks during the transparency process, failing to effectively maintain its final morphology.

[0129] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A kit for optically clearing one or more tissue samples, characterized in that, The kit comprises a pretreatment solution, a first buffer solution, a second buffer solution, and a tissue dehydration solution. All three solutions use the same type of main reagent, containing a first alcohol and a surfactant, with the mass percentage of the first alcohol gradually increasing in each. The first alcohol contains a polyol, specifically hexanediol. The surfactant is Triton X-100. The kit also includes a refractive index matching solution. The mass percentage of the first alcohol in the pretreatment solution is less than or equal to 25%, and the mass percentage of the first alcohol in the tissue dehydration solution is greater than or equal to 70%. The mass percentage of surfactant in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution is less than or equal to 10%.

2. The kit according to claim 1, characterized in that, The first alcohol also contains one or more of methanol, tert-butanol and N-butyldiethanolamine.

3. The kit according to claim 1, characterized in that, The refractive index matching liquid contains 50-70 parts by weight of an organic solvent, 20-40 parts by weight of a polymer and 0-10 parts by weight of a second alcohol.

4. The kit according to claim 3, characterized in that, The organic solvent is one or more of dibenzyl ether, diphenyl ether, benzyl alcohol, and benzyl benzoate; the polymer is one or more of polyethylene glycol substances with a molecular weight of 200-1000; the second alcohol is one or more of hexanediol, methanol, tert-butanol, and N-butyldiethanolamine.

5. A method for light-transparency treatment of tissues using the kit described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Immerse the fixed tissue in the pretreatment solution to allow the tissue to fully expand, thereby obtaining the expanded tissue; (2) The expanded tissue is sequentially immersed in the first buffer solution and the second buffer solution to fix the tissue framework and stabilize the tissue morphology, thereby obtaining the buffer-treated tissue. (3) The tissue treated with the buffer solution is placed in the tissue dehydration solution for dehydration treatment, so that the tissue shrinks to its original size and the overall texture of the tissue becomes hard and the shape is solidified, thus obtaining the dehydrated tissue; (4) The dehydrated tissue is immersed in the refractive index matching solution to obtain an optically transparent tissue; The pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution all contain a first alcohol and a surfactant, and the mass percentage of the alcohol in the pretreatment solution, the first buffer solution, the second buffer solution, and the tissue dehydration solution gradually increases; the first alcohol contains polyols.

6. The optical transparency method as described in claim 5, characterized in that, Step (1) Immerse the fixed tissue in the pretreatment solution for 12-24 hours; Step (2) Immerse the swollen tissue in the first buffer solution and the second buffer solution in sequence for 6-12 hours each; Step (3) Place the buffer-treated tissue in the tissue dehydration solution for dehydration treatment for 24-36 hours.

7. The optical transparency method as described in claim 5, characterized in that, The refractive index matching solution in step (4) contains 50-70 parts by mass of organic solvent, 20-40 parts by mass of polymer and 0-10 parts by mass of second alcohol; in step (4), the dehydrated tissue is immersed in the refractive index matching solution for 24-36 hours.

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