A DAPI-based staining method and three-dimensional imaging method for biological tissue samples

Abstract

The present invention relates to the field of biomedical technologies, and particularly to a staining method and a three-dimensional imaging method for biological tissue samples based on DAPI. The staining method for biological tissue samples based on DAPI provided by the present invention includes: sequentially dehydrating, performing the first cell membrane permeabilization, degreasing, performing the second cell membrane permeabilization, enriching water, and performing the third cell membrane permeabilization on the tissue sample, and then performing DAPI staining. By utilizing the characteristic that the DAPI dye can exhibit strong fluorescence after binding to double-stranded DNA, this method performs overall staining on biological tissue organs, promotes the tissue penetration of the DAPI dye by introducing an optimized cell membrane permeabilization method, thereby enhancing the overall staining effect of biological tissue samples. In combination with the three-dimensional imaging method, it can clearly display the three-dimensional effect at the cellular level inside the organ, and has the characteristics of being fast, economical, and of high quality.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a method for staining a biological tissue sample based on DAPI and a three-dimensional imaging method. Background Art

[0002] In traditional biological tissue staining and imaging methods, tissue blocks are first embedded and then tissue sections are prepared. After the sections are stained, imaging of the sections is performed. However, it is usually difficult for tissue section staining and imaging techniques to stain and image all sections in a tissue block. Therefore, the image information obtained is difficult to reflect the state of the entire tissue. If all sections are stained, the amount of reagents consumed for staining is large, and the number of sections to be observed is large, greatly increasing the labor and material costs. Moreover, there may be differences in the staining and imaging conditions of different sections. In particular, when these sections are subjected to three-dimensional imaging, the registration between layers (upper and lower layer sections) is difficult and is manual registration, with a large error. The overall staining technique of biological tissues can better solve the above problems.

[0003] The molecular formula of the DAPI fluorescent dye (4',6-diamidino-2-phenylindole) is C 16 H 15 N5〃2HCl, with a molecular weight of 350.25, is one of the dyes commonly used for staining live cells and fixed cells, showing blue fluorescence, with a maximum excitation wavelength of 340 nm and a maximum emission wavelength of 488 nm. Under a fluorescence microscope, the DAPI dye is excited by light with an ultraviolet wavelength, and the emitted light is blue, with only a small overlap with the emission wavelengths of the green fluorescent protein (GFP) and the red fluorescent dye (Texas Red dye). Therefore, this property can be used for multi-fluorescence staining on a single sample. When DAPI binds to double-stranded DNA, the maximum absorption / maximum emission wavelength is 358 nm / 461 nm. When it binds to RNA, the fluorescence intensity generated is much lower than that when binding to DNA, and the maximum emission wavelength moves to about 400 nm. In addition, after DAPI penetrates the cell membrane and binds to DNA, it can produce fluorescence more than 20 times stronger than that of DAPI itself. However, although DAPI can penetrate the cell membrane to achieve rapid staining of cells or tissue sections, compared with cells or sections, DAPI has poor permeability to intact tissues or tissues with a certain thickness compared with Nissl dyes (such as thionin). Therefore, the current application of DAPI is still limited to cell and tissue section staining, and there is no report on its use for the overall staining of intact biological tissues. Developing a method for the overall staining of biological tissue samples using DAPI is of great significance for realizing multi-fluorescence staining and simultaneously three-dimensionally reconstructing biological tissues. Summary of the Invention

[0004] The present invention provides a DAPI-based staining method and three-dimensional imaging method for biological tissue samples.

[0005] Aiming at the defect that it is difficult to achieve overall staining of large biological tissue samples by DAPI staining, the present invention quickly penetrates DAPI dye into the tissue interior by means of membrane permeabilization staining, so as to achieve overall staining of large samples of visceral tissues. When developing a method for overall staining of biological tissue samples using DAPI, the present invention found that even after conventional membrane permeabilization treatment of biological tissue samples, the permeability of DAPI is still difficult to be significantly improved. After continuous attempts, the present invention found that treating biological tissue samples with specific treatment agents for cell membrane permeabilization respectively after dehydration, defatting and water enrichment can significantly improve the permeability of DAPI to biological tissue samples with a certain thickness, even to complete organ samples, and significantly improve the overall staining effect of biological tissue samples.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] The present invention provides a DAPI-based staining method for biological tissue samples, the method comprising: dehydrating the biological tissue sample, performing first cell membrane permeabilization, defatting, second cell membrane permeabilization, water enrichment, third cell membrane permeabilization in sequence, and then performing DAPI staining.

[0008] In the above method, the first cell membrane permeabilization, the second cell membrane permeabilization and the third cell membrane permeabilization respectively use a first treatment agent, a second treatment agent and a third treatment agent to treat the tissue sample;

[0009] Wherein, the first treatment agent and the second treatment agent are ethanol solutions containing DMSO, and the volume fraction of DMSO in the second treatment agent is 30-100% higher than that in the first treatment agent.

[0010] The present invention found that respectively using DMSO ethanol solutions with the above concentration gradients for the first cell membrane permeabilization and the second cell membrane permeabilization is more conducive to improving the permeability of DAPI to tissue samples, thereby improving the uniformity of the distribution of DAPI in the whole tissue sample, improving the uniformity of overall staining, and further accelerating the tissue penetration of DAPI, laying a foundation for the cell penetration of DAPI to combine with DNA to accelerate tissue staining.

[0011] Preferably, the volume fraction of DMSO in the second treatment agent is 50-100% higher than that in the first treatment agent.

[0012] More preferably, the first treatment agent is an ethanol solution containing 10-20% DMSO, and the second treatment agent is an ethanol solution containing 20-40% DMSO.

[0013] In the above method, the third treating agent is a PBS buffer solution containing a detergent, and the detergent is one or more selected from TritonX-100, SDS, SD (sodium deoxycholate), and CHAPS. Different from the first cell membrane permeabilization and the second cell membrane permeabilization, the present invention discovers that treating with a PBS buffer solution containing a specific detergent during the third cell membrane permeabilization can further improve the permeability of DAPI to the tissue sample and enhance the overall staining effect of the tissue sample.

[0014] Preferably, in the third treating agent, the concentration of the detergent is 0.1-1%.

[0015] In some embodiments of the present invention, the detergent is TritonX-100, and in the third treating agent, the concentration of the detergent is 0.2-0.5%.

[0016] The above three cell membrane permeabilization treatments can cooperate well. By first performing a preliminary membrane permeabilization treatment on the tissue with an ethanol solution containing a gradient concentration of DMSO, the combination of the detergent and the cell membrane can be accelerated, thereby better achieving the purpose of cell membrane rupture, achieving the result of rapid membrane rupture by the detergent in a short time, thereby accelerating the infiltration and staining of DAPI, and greatly shortening the experimental time.

[0017] In the above method, the treatment time of the first cell membrane permeabilization is 6-24h, and / or the treatment time of the second cell membrane permeabilization is 2-12h, and / or the treatment time of the third cell membrane permeabilization is 2-10d.

[0018] For the third cell membrane permeabilization, an appropriate time can be selected within the above treatment time range according to the tissue thickness. If the tissue is thicker, the time can be appropriately extended.

[0019] Preferably, the treatment time of the first cell membrane permeabilization is 6-12h, and / or the treatment time of the second cell membrane permeabilization is 2-5h, and / or the treatment time of the third cell membrane permeabilization is 2-5d.

[0020] In the above method, the staining is to treat the tissue sample with a DAPI solution having a volume fraction of 40-100% for 1-72h. The preferred staining time is 48-72h.

[0021] In the above method, the dehydration is to sequentially place the biological tissue sample in ethanol solutions with a volume fraction of 40-50%, 70-85%, and 95-100% for dehydration.

[0022] Preferably, the dehydration time in each ethanol solution is 1-4h (preferably 1-2h).

[0023] The defatting is to place the biological tissue sample in dichloromethane for defatting.

[0024] Preferably, the time for defatting is 1 - 4 h.

[0025] The water enrichment is to place the biological tissue sample in ethanol solutions with volume fractions of 95 - 100%, 70 - 85%, and 40 - 50% in sequence for water enrichment.

[0026] Preferably, the time for water enrichment in each ethanol solution is 1 - 4 h (preferably 1 - 2 h).

[0027] The above dehydration, defatting, and water enrichment operations can better cooperate with the cell membrane permeabilization step, which is beneficial to improving the overall staining effect of biological tissues.

[0028] In the staining method described above, each step cooperates with each other. Among them, through gradient dehydration with ethanol, the purpose of preliminary dehydration of tissues is achieved, which can accelerate the penetration of DAPI while ensuring its uniform distribution in tissues; using an ethanol solution containing DMSO for cell membrane permeabilization can accelerate the combination of the detergent and the cell membrane in the third cell membrane permeabilization treatment. In the third cell membrane permeabilization treatment, the detergent can better achieve the purpose of cell membrane rupture, achieving the result of rapid membrane rupture by the detergent in a short time, thereby accelerating the infiltration and staining of DAPI, greatly shortening the experimental time. The three - time cell membrane permeabilization treatment not only facilitates the uniform distribution of DAPI in tissues, but also further accelerates the tissue penetration of DAPI, laying a foundation for the cell penetration of DAPI and its binding to DNA and accelerating tissue staining. The previous dehydration, defatting, and cell membrane permeabilization treatments not only lay a foundation for the uniform distribution and rapid penetration staining of DAPI in tissues, but also after the staining is completed, ethanol is used for dehydration again while the resin is infiltrated and embedded. In this process, DAPI will dissolve in the ethanol solution and the resin solution. However, through the previous dehydration, defatting, and cell membrane permeabilization treatments, the time for dehydration and infiltration during the later resin embedding will be correspondingly shortened, which also reduces the loss of DAPI dye to a certain extent, realizes the rapid embedding of resin samples, and better ensures the accuracy of later data collection and analysis results. Through gradient water enrichment with ethanol, it not only provides conditions for the third cell membrane permeabilization treatment to avoid tissue deformation caused by direct treatment and affecting the experimental results, but also further increases the tissue permeability for the ethanol dehydration treatment during resin embedding, providing conditions for the rapid penetration of DAPI.

[0029] In the staining method described above, the biological tissue sample is an animal tissue or organ sample.

[0030] In some embodiments of the present invention, the biological tissue sample is a complete animal organ sample, preferably a complete brain tissue sample.

[0031] In some embodiments of the present invention, the sources of the animal tissue samples include, but are not limited to, mammals, such as mice, humans, monkeys, rabbits, pigs, etc.

[0032] Preferably, before dehydrating the tissue sample, the staining method further includes the step of sequentially perfusing a PBS buffer solution and a paraformaldehyde solution into the animal heart and then obtaining the tissue sample. Before taking the tissue sample, perfusing the PBS buffer solution into the heart can eliminate the influence of blood fluorescence.

[0033] Among them, the concentration of the PBS buffer solution used for heart perfusion is preferably 0.001 - 10 M (preferably 0.001 - 0.1 M). The mass fraction of the paraformaldehyde solution is 3 - 5%.

[0034] In some embodiments of the present invention, the staining method for the biological tissue sample includes the following steps:

[0035] 1) Perfusing a PBS buffer solution into the animal heart;

[0036] 2) Perfusing a paraformaldehyde solution into the animal heart and then removing the biological tissue sample to be stained;

[0037] 3) Sequentially placing the biological tissue sample in ethanol solutions with a volume fraction of 40 - 50%, 70 - 85%, and 95 - 100% for gradient dehydration;

[0038] 4) Placing the dehydrated biological tissue sample in a first treatment agent for the first cell membrane permeabilization;

[0039] 5) Degreasing the biological tissue sample in dichloromethane;

[0040] 6) Placing the biological tissue sample in a second treatment agent for the second cell membrane permeabilization;

[0041] 7) Sequentially placing the biological tissue sample in ethanol solutions with a volume fraction of 95 - 100%, 70 - 85%, and 40 - 50% for gradient rehydration;

[0042] 8) Placing the rehydrated biological tissue sample in a third treatment agent for the third cell membrane permeabilization;

[0043] 9) Staining the biological tissue sample in a DAPI solution.

[0044] Based on the above staining method, the present invention further provides a three-dimensional imaging method for a biological tissue sample. The three-dimensional imaging method includes: staining the biological tissue sample using the above-described staining method for the biological tissue sample, embedding the stained tissue sample, and then performing three-dimensional imaging.

[0045] Preferably, a three-dimensional imaging is performed using a TDI-fMOST (Tomographic fluorescence microscopy optical tomography) camera. The TDI-fMOST camera can perform continuous section tomography on tissue samples.

[0046] Preferably, a resin-embedded and stained biological tissue sample is used.

[0047] The present invention has the following beneficial effects: The DAPI-based staining method for biological tissue samples provided by the present invention utilizes the characteristic that DAPI dye can exhibit strong fluorescence after binding to double-stranded DNA to perform overall staining on biological tissue organs. By introducing an optimized cell membrane permeabilization method to promote the penetration of DAPI dye in tissues that are not permeable or semi-permeable to the membrane, the DAPI dye can quickly penetrate into the tissue interior, improving the uniformity of the distribution of DAPI in the tissue sample, thereby enhancing the overall staining effect of the biological tissue sample. Combined with the three-dimensional imaging method, it can clearly display the three-dimensional effect at the cellular level inside the organ, and has the characteristics of being fast, economical, and of high quality.

[0048] The three-dimensional imaging method of the present invention is based on a three-dimensional optical imaging device. For the overall stained tissue sample, imaging is completed while slicing. The upper and lower layers of the slice are naturally registered, without artificial errors and with a high matching degree. Therefore, the accuracy of the reconstructed three-dimensional tissue result is relatively high. Description of the Drawings

[0049] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0050] Figure 1 It is a fine section diagram of the mouse cerebral blood vessels in Example 1 of the present invention (collected by a TDI-fMOST camera).

[0051] Figure 2 It is a continuous section diagram of the mouse brain in Example 1 of the present invention (collected by a TDI-fMOST camera), with a layer thickness of 2 μm. The numbers 1, 100, 200, 300, 400, 500, 600, 700, 800 below the picture respectively represent the slice numbers of the resin-embedded mouse brain sample. For example, 1 is the first layer and 100 is the 100th layer.

[0052] Figure 3This is the microscopic observation result diagram of the mouse brain tissue after staining in Example 2 of the present invention. Among them, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 below the picture respectively represent the mouse brain sample section layers. For example, 1 is the first layer and 55 is the 55th layer.

[0053] Figure 4 This is the cross-sectional view of the mouse brain tissue after staining in Comparative Example 1 of the present invention.

[0054] Figure 5 This is the cross-sectional view of the mouse brain tissue after staining in Comparative Example 2 of the present invention.

[0055] Figure 6 This is the cross-sectional view of the mouse brain tissue after staining in Comparative Example 3 of the present invention. The upper picture is the directly observed and photographed picture; the lower picture is the observed and photographed picture using a Mshot Mingmei microscope (model: MF43, company name: Guangzhou Mingmei Optoelectronic Technology Co., Ltd.). Detailed implementation manners

[0056] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0057] The DAPI (liquid dye) used in the following examples was purchased from Beyotime.

[0058] Example 1

[0059] This example provides a method for staining biological tissue samples based on DAPI, including the following steps:

[0060] 1) Perfuse a 0.001M PBS buffer solution into the mouse heart;

[0061] 2) Perfuse a 3% paraformaldehyde solution with a mass fraction into the mouse heart and then remove the mouse brain;

[0062] 3) Gradient dehydrate the mouse brain tissue in ethanol solutions with volume fractions of 50%, 75%, and 100% in sequence, and the dehydration time in each ethanol solution is 1 h;

[0063] 4) Place the dehydrated mouse brain tissue in an ethanol solution containing 10% DMSO for the first cell membrane permeabilization, and the treatment time is 6 h;

[0064] 5) Degrease the mouse brain tissue in dichloromethane, and the treatment time is 1 h;

[0065] 6) Place the dehydrated and defatted mouse brain tissue in an ethanol solution containing 20% DMSO for the second cell membrane permeabilization, and the treatment time is 2 h;

[0066] 7) Place the mouse brain tissue successively in ethanol solutions of 100%, 75%, and 50% for gradient water enrichment, and the water enrichment time in each ethanol solution is 1 h;

[0067] 8) Place the water-enriched mouse brain tissue in a PBS buffer solution containing 0.2% TritonX-100 for the third cell membrane permeabilization, and the treatment time is 2 d;

[0068] 9) Then place the mouse brain tissue in a 40% DAPI solution for staining, and the treatment time is 3 d.

[0069] This embodiment also provides a three-dimensional imaging method for biological tissue samples, and the method is as follows: Embed the mouse brain tissue stained by the above steps 1)-9) with resin, and collect it using a TDI-fMOST camera.

[0070] The imaging results of the stained mouse brain tissue are as Figure 1 and Figure 2 shown. As can be seen from Figure 1 and Figure 2 , the overall staining of the mouse brain sample is uniform, the cell bodies are clearly visible in each layer of sections, which is beneficial to observing and analyzing the morphology and quantity of the cell bodies. The fluorescence (white dots (cell bodies) in the figure) effect is good, and strong fluorescence can be maintained under the irradiation of laser, realizing the overall staining of biological tissue in a short time. By using the method of collecting sections of resin-embedded tissue samples with TDI-fMOST, imaging of the overall DAPI-stained samples is achieved, thereby obtaining a three-dimensional structure diagram of the whole mouse brain with a layer thickness of 1-10 μm.

[0071] Example 2

[0072] This embodiment provides a DAPI-based staining method for biological tissue samples, including the following steps:

[0073] 1) Perfuse the mouse heart with 0.1M PBS buffer;

[0074] 2) Perfuse the mouse heart with a 5% paraformaldehyde solution by mass fraction and then remove the mouse brain;

[0075] 3) Place the mouse brain tissue successively in ethanol solutions with volume fractions of 40%, 70%, and 95% for gradient dehydration, and the dehydration time is 2 h;

[0076] 4) Place the dehydrated mouse brain tissue in an ethanol solution containing 20% DMSO for the first cell membrane permeabilization, and the treatment time is 12 h;

[0077] 5) Place the mouse brain tissue in dichloromethane for defatting, with a treatment time of 4 h;

[0078] 6) Place the dehydrated and defatted mouse brain tissue in an ethanol solution containing 30% DMSO for the second cell membrane permeabilization, with a treatment time of 3 h;

[0079] 7) Place the mouse brain tissue successively in ethanol solutions of 95%, 70%, and 40% for gradient water enrichment, with a water enrichment time of 2 h;

[0080] 8) Place the water-enriched mouse brain tissue in a PBS buffer containing 0.5% TritonX-100 for the third cell membrane permeabilization, with a treatment time of 3 d;

[0081] 9) Then place the mouse brain tissue in an 80% DAPI solution for staining, with a treatment time of 2 d.

[0082] This embodiment also provides a three-dimensional imaging method for a biological tissue sample, and the method is as follows: Embed the mouse brain tissue stained by the above steps 1)-9) with resin, and use a TDI-fMOST camera for acquisition.

[0083] The microscopic observation results of the stained mouse brain tissue are as Figure 3 shown (150 μm per layer). It can be Figure 3 seen that the overall staining of the mouse brain sample is uniform, the cell bodies are clearly visible in each layer of section, which is beneficial to observing and analyzing the morphology and quantity of the cell bodies. The blue fluorescence (blue dots in the figure) has a good effect and can maintain strong fluorescence under the irradiation of the laser, achieving the overall staining of biological tissues in a short time. Further, by using the method of slicing and collecting the resin-embedded tissue sample with TDI-fMOST, the imaging of the overall DAPI-stained sample is realized, so as to obtain a three-dimensional structure diagram of the whole mouse brain with a layer thickness of 1-10 μm.

[0084] Comparative Example 1

[0085] This comparative example provides a DAPI-based staining method for biological tissue samples, and the difference from the method of Example 1 is only that: all cell membrane permeabilization steps are removed.

[0086] Embed the mouse brain tissue stained by the method of this comparative example with resin, and use a TDI-fMOST camera for acquisition. The results are as Figure 4 shown. Figure 4 It can be clearly observed that the blue signal only stays in the cortex and does not penetrate into the white matter region. While using the staining method of Example 1, bright fluorescence signals (white dots) can be seen in each layer of section, the signals are uniform, and both the cortex and white matter in the section show fluorescence signals ( Figure 1 and Figure 2), indicating that the dye has completely penetrated into the middle region of the brain, achieving overall staining of biological tissues; thus, it can be seen that compared with the method of Example 1, the staining effect is significantly reduced when using the method of this comparative example for staining.

[0087] Comparative Example 2

[0088] This comparative example provides a method for staining biological tissue samples based on DAPI, which is only different from the method of Example 1 in that: the second and third cell membrane permeabilization steps are removed.

[0089] The mouse brain tissue stained by the method of this comparative example was embedded in resin and collected using a TDI-fMOST camera. The results are as Figure 5 shown, Figure 5 It can be seen that the mouse brain sections have obvious tissue fragmentation, and the blue signal only stays in the cortex and does not penetrate into the white matter area. While using the staining method of Example 1, the section surface of each layer is flat and no section fragmentation is observed, and bright fluorescence signals (white dots) can be observed. The signals are uniform and both the cortex and white matter in the sections show fluorescence signals ( Figure 1 and Figure 2 ), indicating that the dye has completely penetrated into the middle region of the brain, achieving overall staining of biological tissues; thus, it can be seen that compared with the method of Example 1, the staining effect is significantly reduced when using the method of this comparative example for staining.

[0090] Comparative Example 3

[0091] This comparative example provides a method for staining biological tissue samples based on DAPI, which is only different from the method of Example 1 in that: the time of the third cell membrane permeabilization treatment is 14 d.

[0092] The results show that obvious fragmentation appears in the tissue after staining ( Figure 6 ).

[0093] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for whole staining of biological tissue samples based on DAPI, characterized in that, The method includes: dehydrating a biological tissue sample, performing first cell membrane permeabilization, degreasing, second cell membrane permeabilization, water enrichment, and third cell membrane permeabilization in sequence, and then performing DAPI staining; The first cell membrane permeabilization, the second cell membrane permeabilization, and the third cell membrane permeabilization respectively use a first treatment agent, a second treatment agent, and a third treatment agent to treat the biological tissue sample; Among them, the first treatment agent is an ethanol solution containing 10-20% DMSO, and the second treatment agent is an ethanol solution containing 20-40% DMSO; the volume fraction of DMSO in the second treatment agent is 30-100% higher than the volume fraction of DMSO in the first treatment agent; The third treatment agent is a PBS buffer solution containing 0.2-0.5% TritonX-100; The treatment time of the first cell membrane permeabilization is 6-24 h, the treatment time of the second cell membrane permeabilization is 2-12 h, and the treatment time of the third cell membrane permeabilization is 2-10 d; The biological tissue sample is an animal tissue organ sample; The dehydration is to dehydrate the biological tissue sample by placing it in ethanol solutions with volume fractions of 40-50%, 70-85%, and 95-100% in sequence; The degreasing is to degrease the biological tissue sample by placing it in dichloromethane; The water enrichment is to enrich the water of the biological tissue sample by placing it in ethanol solutions with volume fractions of 95-100%, 70-85%, and 40-50% in sequence.

2. The whole tissue sample staining method according to claim 1, wherein The staining is to treat the biological tissue sample with a DAPI solution with a volume fraction of 40-100% for 1-72 h.

3. The whole tissue sample staining method according to claim 1, characterized in that, The dehydration time in each ethanol solution is 1-4 h.

4. The overall staining method for biological tissue samples according to claim 1, wherein The degreasing time is 1-4 h.

5. The whole staining method for biological tissue samples according to claim 1, characterized in that, The water enrichment time in each ethanol solution is 1-4 h.

6. A three-dimensional imaging method for a biological tissue sample, characterized in that, The method includes: staining a biological tissue sample by using the biological tissue sample overall staining method according to any one of claims 1 to 5, embedding the stained tissue sample, and then performing three-dimensional imaging.

7. The method according to claim 6, wherein Performing three-dimensional imaging by using a TDI-fMOST camera.

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