Rapid three-dimensional imaging method based on tissue light transparency

Abstract

The invention discloses a rapid three-dimensional imaging method based on tissue light transparency, and belongs to the technical field of biomedical optical imaging. The method comprises the following steps: placing a biological tissue sample in a staining solution, and carrying out oscillation treatment to obtain a stained tissue sample; sequentially putting the dyed tissue sample into a dehydration solution, a degreasing solution and a refractive index matching solution, and carrying out oscillation treatment to obtain a transparent tissue sample; performing three-dimensional imaging on the transparent tissue sample to obtain a three-dimensional fluorescence image of the tissue sample; and converting the three-dimensional fluorescence image of the tissue sample, and performing three-dimensional reconstruction on the obtained dyeing bright field image to obtain a three-dimensional dyeing image of the tissue sample. According to the method, after the tissue is subjected to dyeing, transparentizing, imaging and three-dimensional reconstruction and analysis, the complete three-dimensional information of the tissue can be obtained, the result is stable and reliable, and the whole method is rapid and simple, so that the method has a relatively good application prospect in tissue pathology detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical optical imaging, and in particular relates to a rapid three-dimensional imaging method based on tissue optical transparency. Background Art

[0002] Conventional tissue pathology testing includes fixation, dehydration, paraffin embedding, sectioning, staining, imaging and other steps. Samples obtained by puncture biopsy or surgical resection will be immersed in a tissue fixative such as formaldehyde to coagulate proteins and prevent nucleic acid degradation. Subsequently, the tissue will be completely dehydrated in gradient ethanol so that the paraffin can fully immerse and embed the sample. Paraffin embedding can provide sufficient rigidity for the sample, thereby avoiding damage or deformation of the sample during sectioning. Tissues cut into 4-5μm thickness will be stained with dyes (such as H&E, Masson, etc.) to distinguish different structures in the tissue. The processed samples will be subjected to histological examination under a microscope to diagnose the disease by observing the morphology or type of cells in the tissue.

[0003] However, the existing conventional histopathology detection methods also have certain problems. First, the processing time of conventional histopathology detection is long, and the entire sample processing process usually takes about a week. This may cause the diagnosis result to lag behind the actual condition and delay the treatment of the disease to a certain extent. At the same time, the complex and numerous operation steps also make histopathology detection a labor-intensive process, which requires a lot of manpower to prepare samples. In pathology detection, only dozens of 4-5μm thick thin sections are extracted from the tissue for diagnostic analysis. Compared with the original millimeter-thick tissue, less than 5% of the samples will be stained and visualized, resulting in a large amount of information loss. Some structures that are relatively sparse in the tissue, such as tumor progenitor cells and residual lesions after treatment, may be missed during the slicing process, which ultimately makes these important pathological indicators difficult to be effectively identified and quantified in conventional histopathology detection. At the same time, the conventional histopathology detection process includes destructive slicing of samples, which makes it impossible to reuse samples, making it difficult to conduct subsequent analyses such as molecular detection that require a large number of tissue samples.

[0004] The difficulty in obtaining the three-dimensional structure of pathological tissue is also an important problem in conventional pathological testing. Biological tissues contain complex three-dimensional reticular structures such as vascular networks and glandular networks, and the occurrence and development of diseases often cause these three-dimensional structures to be destroyed or deformed. Therefore, the effective reconstruction and analysis of the three-dimensional structure in pathological tissue samples plays a very critical role in the diagnosis of diseases. However, in conventional tissue pathology testing, only two-dimensional images of tissue sections can be obtained, and the three-dimensional information of the tissue is lacking. Analyzing the actual three-dimensional structure of the sample through these limited two-dimensional images may lead to wrong judgments. For example, a healthy glandular structure may be reflected as a dysplastic structure in a two-dimensional image due to the selection of the section. At the same time, it is difficult for two-dimensional images to reflect the spatial position relationship and interaction between cells and cells, and between cells and the microenvironment. These problems limit the application of tissue pathology testing in diagnosing diseases.

[0005] Optical imaging technology has developed rapidly in recent decades, and a series of microscopic imaging technologies with optical tomography capabilities have emerged, including two-photon microscopy, confocal microscopy, and light sheet microscopy. These methods allow the excitation and imaging of signals at a specific depth of the sample, thereby obtaining three-dimensional information of the sample. However, due to the high scattering and high absorption of light by biological tissues, the imaging depth of optical imaging methods is significantly limited, and usually only tissues at a depth of tens to hundreds of microns can be imaged.

[0006] Therefore, it is necessary to provide a rapid three-dimensional imaging method based on tissue optical transparency to solve the shortcomings of existing three-dimensional imaging of tissue samples. Summary of the invention

[0007] The purpose of the present invention is to provide a rapid three-dimensional imaging method based on tissue optical transparency. In the present invention, after staining, transparency, imaging, three-dimensional reconstruction and analysis of tissue, complete three-dimensional information of the tissue can be obtained, the result is stable and reliable, and the whole method is rapid and simple, therefore, it has good application prospects in tissue pathology detection.

[0008] In a first aspect, the present invention provides a rapid three-dimensional imaging method based on tissue optical transparency, comprising the following steps: placing a biological tissue sample in a staining solution, and obtaining a stained tissue sample after oscillation treatment; placing the stained tissue sample in a dehydration solution, a defatting solution, and a refractive index matching solution in turn for oscillation treatment to obtain a transparent tissue sample; performing three-dimensional imaging on the transparent tissue sample to obtain a three-dimensional fluorescence image of the tissue sample; converting the three-dimensional fluorescence image of the tissue sample, and performing three-dimensional reconstruction on the obtained stained bright field image to obtain a three-dimensional staining image of the tissue sample.

[0009] In the present invention, the inventors have found that, in the step of staining biological tissue samples, by adding inorganic salts and alcohol substances to the staining solution, the staining depth and staining speed of the biological tissue samples can be effectively improved, and stable and uniform staining of the samples can be achieved; in the step of transparentizing the stained tissue samples, chemical dehydration is used instead of traditional physical gradient dehydration, which greatly improves the transparentization speed while reducing the complexity of the transparent treatment and significantly improving the transparent effect; further, by performing three-dimensional imaging, conversion and three-dimensional reconstruction on the transparent tissue samples, a three-dimensional staining map of the tissue sample is obtained, and the three-dimensional staining map can effectively and accurately reflect the three-dimensional structural information of the sample, the results are stable and reliable, and the entire method is fast and simple.

[0010] In some embodiments, the biological tissue sample includes a mammalian tissue organ.

[0011] In some more preferred embodiments, mammals include humans, macaques, mice, rats, rabbits, fish, and insects.

[0012] 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.

[0013] In some embodiments, during the preparation of the stained tissue sample, the staining solution includes 1-10 nM (e.g., 1 nM, 3 nM, 5 nM, 7 nM, 10 nM or other values ​​within the range) of a nuclear stain, 10-100 nM (e.g., 10 nM, 30 nM, 50 nM, 70 nM, 100 nM or other values ​​within the range) of a cytoplasmic stain, 10-100 mM (e.g., 10 mM, 30 mM, 50 mM, 70 mM, 100 mM or other values ​​within the range) of a cytoplasmic stain, % (for example, 10wt%, 30wt%, 50wt%, 70wt% or other values ​​within the range) of inorganic salts and 10-70wt% (for example, 10wt%, 30wt%, 50wt%, 70wt% or other values ​​within the range) of alcohols, and the rest is distilled water; the oscillation treatment step specifically includes: oscillating for 6-48h, for example, 6h, 10h, 20h, 30h, 40h, 48h or other values ​​within the range, at a temperature of 20-25°C (for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or other values ​​within the range).

[0014] In some embodiments, the nuclear stain is selected from at least one of TO-PRO-3, DAPI and PI; the cytoplasmic stain is selected from at least one of eosin, Cy3 and NHS ester; the inorganic salt is selected from at least one of sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate; the alcohol substance is selected from at least one of methanol, ethanol and glycerol.

[0015] In some embodiments, during the preparation of the transparentized tissue sample, the dehydrating solution includes 5-30 wt % (e.g., 5 wt %, 10 wt %, 15 wt %, 20 wt %, 25 wt %, 30 wt % or other values ​​within the range) of a polyol, 5-30 wt % (e.g., 5 wt %, 10 wt %, 15 wt %, 20 wt %, 25 wt %, 30 wt % or other values ​​within the range) of an amino alcohol, and 0.1-1 wt % (e.g., 0.1 wt %, 0.3 wt %, 0.5 wt %, 0.7 wt %, 1 wt % or other values ​​within the range) of a hydroxyl alcohol. , and the rest are dehydrating agents; and the dehydrating agent is selected from at least one of tri(4-chlorophenyl) borate, triphenyl borate, trimethyl borate and tributyl borate; the step of placing the stained tissue sample in the dehydrating solution for oscillation treatment specifically comprises: oscillating for 20-180 minutes, for example, 20 minutes, 50 minutes, 80 minutes, 110 minutes, 140 minutes, 180 minutes 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).

[0016] 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.

[0017] In some embodiments, during the preparation of the transparentized tissue sample, the degreasing liquid includes 0.1-1wt% (for example, 0.1wt%, 0.3wt%, 0.5wt%, 0.7wt%, 1wt% or other values ​​within the range) of a reducing agent, and the rest is a degreasing agent; and the degreasing agent is selected from at least one of cyclohexane, ethanol, dichloromethane and benzyl benzoate; the step of placing the tissue sample treated with the dehydrating liquid in the degreasing liquid for oscillation treatment specifically includes: oscillating for 10-60min, for example, 10min, 20min, 30min, 40min, 50min, 60min 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).

[0018] In some embodiments, the reducing agent is selected from at least one of monothioglycerol, vitamin D, and vitamin C.

[0019] In some embodiments, during the preparation of the transparentized tissue sample, the refractive index matching liquid includes at least one of benzyl ether, benzyl benzoate, benzyl alcohol and ethyl cinnamate; the step of placing the tissue sample treated with the defatted liquid in the refractive index matching liquid for oscillation treatment specifically includes: oscillating for 5-60 minutes 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, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes or other values ​​within the range.

[0020] In some embodiments, during the three-dimensional fluorescence imaging of a tissue sample, the three-dimensional imaging step specifically includes: performing three-dimensional imaging of the transparentized tissue sample using a fluorescence microscope; wherein the fluorescence microscope includes a confocal microscope and / or a light sheet microscope.

[0021] In some preferred embodiments, the steps of performing three-dimensional imaging of transparent tissue samples using a confocal microscope specifically include: using a coverslip to clamp the sample and adding a refractive index matching liquid to fully infiltrate it; after using a 5X or 10X magnification microscope to obtain the overall distribution characteristics of the sample, continue to use a 20X magnification microscope to obtain the fine structure of the sample, and use the Alexa Fluor 555 channel and the Alexa Fluor 647 channel to respectively excite the cytoplasm and nucleus signals in the sample to perform three-dimensional imaging of the sample.

[0022] In some preferred embodiments, the steps of performing three-dimensional imaging of transparent tissue samples using a light sheet microscope specifically include: clamping the sample with a glass clamp and immersing it in an imaging tank filled with a refractive index matching liquid; using a 4X magnification lens to perform a rough scan of the sample, adjusting the positions of the scanning start point and the end point to ensure that the sample is completely scanned, and using the AlexaFluor 555 channel and the Alexa Fluor 647 channel to respectively excite the cytoplasm and nucleus signals in the sample; after the rough scan is completed, replacing the lens with a 25X magnification lens to perform three-dimensional imaging of the fine structure of the sample.

[0023] In some embodiments, during reconstruction of a three-dimensional staining image of a tissue sample, the conversion step specifically includes: converting the three-dimensional fluorescence image of the tissue sample into Tif format, and obtaining a stained bright field image after conversion by color channel conversion software; the three-dimensional reconstruction step specifically includes: performing three-dimensional reconstruction using Imaris software.

[0024] In some preferred embodiments, the color channel conversion software includes falsecolor.

[0025] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention provides a rapid three-dimensional imaging method based on tissue optical transparency. Specifically, by sequentially performing the steps of staining, transparency, imaging, three-dimensional reconstruction and analysis on biological tissue samples, the complete three-dimensional structural information of the biological tissue samples can be obtained, the results are stable and reliable, and the entire method is rapid and simple. Therefore, the rapid three-dimensional imaging method has good application prospects in tissue pathology detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flow chart of the rapid three-dimensional imaging method based on tissue optical transparency in the present invention; Figure 2 This is a result diagram of rapid imaging of human uterine fibroid tissue using the rapid three-dimensional imaging method of the present invention in Example 1 of the present invention; Figure 3 This is a result diagram of rapid imaging of human colon cancer tissue using the rapid three-dimensional imaging method of the present invention in Example 2 of the present invention; Figure 4 This is a result diagram of rapid imaging of human breast cancer tissue using the rapid three-dimensional imaging method of the present invention in Example 3 of the present invention; Figure 5 This is a diagram showing the result of rapid imaging of normal breast tissue of a human body using the rapid three-dimensional imaging method of the present invention in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] See also Figure 1, which is a flow chart of the rapid three-dimensional imaging method based on tissue optical transparency in the present invention. Specifically, the method includes the following steps: placing a biological tissue sample in a staining solution, and obtaining a stained tissue sample after oscillation treatment; placing the stained tissue sample in a dehydration solution, a defatting solution and a refractive index matching solution in turn for oscillation treatment to obtain a transparent tissue sample; performing three-dimensional imaging on the transparent tissue sample to obtain a three-dimensional fluorescence image of the tissue sample; converting the three-dimensional fluorescence image of the tissue sample, and performing three-dimensional reconstruction on the obtained stained bright field image to obtain a three-dimensional staining image of the tissue sample.

[0030] Example 1 This embodiment uses the rapid three-dimensional imaging method of the present invention to perform rapid imaging of human uterine fibroid tissue.

[0031] Specifically, the steps include: 1) Tissue acquisition: The surgically removed uterine fibroid tissue was fixed in PFA overnight; 1) Sample staining: Soak human uterine fibroid tissue in staining solution (70wt% ethanol, 2nM TO-PRO-3, 10nM eosin, 10mM sodium chloride, the rest is distilled water, and the pH is adjusted to 4 with hydrochloric acid), incubate with shaking for 12h at 25°C, and then rinse with ethanol several times to obtain the stained tissue sample; 2) Sample transparentization: soak the stained sample obtained in step 1) in a dehydrating solution (10wt% N-butyldiethanolamine, 30wt% hexylene glycol, 0.5wt% thioglycerol, and the rest trimethyl borate), and incubate with shaking for 2h at 37°C; then soak the dehydrated sample in a defatting solution (0.5wt% thioglycerol, and the rest dichloromethane), and incubate with shaking for 30min at 37°C; finally soak the defatted sample in dibenzyl ether, and incubate with shaking for 30min at 37°C to obtain a transparent tissue sample; 3) Sample three-dimensional imaging: Use glass clamps to clamp the tissue sample obtained in step 2) and immerse it in an imaging tank filled with dibenzyl ether; Use a 4X magnification microscope to roughly scan the sample, adjust the positions of the scanning start and end points to ensure that the sample is completely scanned, and use the Alexa Fluor 555 channel and Alexa Fluor 647 channel to excite the cytoplasm and nuclear signals in the sample respectively. After the rough scan is completed, replace the lens with a 25X magnification microscope to perform three-dimensional imaging of the fine structure of the sample to obtain a three-dimensional fluorescence image; 4) Sample 3D reconstruction: Use FalseColor software to convert the fluorescence channel into HE style, and use Imaris to perform 3D reconstruction of the imaging data to obtain a 3D staining image of the tissue sample.

[0032] The results are as follows Figure 2 shown.

[0033] from Figure 2 It can be seen that the figure shows the distribution of proteins and nucleic acids in uterine fibroid tissue at different angles (including sagittal, coronal and horizontal planes); by further enlarging the coronal plane figure, the cell morphology in the uterine fibroid tissue can be observed, and it can be seen that the cell morphology in this area is relatively regular and there is no obvious self-fusion. The above results show that the method of the present invention is fast and simple, and can obtain complete three-dimensional structural information of biological tissue samples, and the results are stable and reliable.

[0034] Example 2 This embodiment uses the rapid three-dimensional imaging method of the present invention to perform rapid imaging of human colon cancer tissue.

[0035] Specifically, the steps include: 1) Tissue acquisition: Surgically resected colon cancer tissue was fixed in PFA overnight; 2) Sample staining: Human colon cancer tissue was immersed in a staining solution (70wt% ethanol, 2nM TO-PRO-3, 10nM eosin, 10mM sodium chloride, the rest was distilled water, and the pH was adjusted to 4 with hydrochloric acid), incubated with shaking for 12h at 25°C, and then rinsed with ethanol several times to obtain a stained tissue sample; 3) Sample transparentization: soak the stained sample obtained in step 1) in a dehydrating solution (10wt% N-butyldiethanolamine, 30wt% hexylene glycol, 0.5wt% thioglycerol, and the rest trimethyl borate), and incubate with shaking for 2h at 37°C; then soak the dehydrated sample in a defatting solution (0.5wt% thioglycerol, and the rest dichloromethane), and incubate with shaking for 30min at 37°C; finally soak the defatted sample in dibenzyl ether, and incubate with shaking for 30min at 37°C to obtain a transparent tissue sample; 4) Sample three-dimensional imaging: Use glass clamps to clamp the tissue sample obtained in step 2) and immerse it in an imaging tank filled with dibenzyl ether; Use a 4X magnification microscope to roughly scan the sample, adjust the positions of the scanning start and end points to ensure that the sample is completely scanned, and use the Alexa Fluor 555 channel and Alexa Fluor 647 channel to excite the cytoplasm and nuclear signals in the sample respectively. After the rough scan is completed, replace the lens with a 25X magnification microscope to perform three-dimensional imaging of the fine structure of the sample to obtain a three-dimensional fluorescence image; 5) Sample 3D reconstruction: Use FalseColor software to convert the fluorescence channel into HE style, and use Imaris to perform 3D reconstruction of the imaging data to obtain a 3D staining image of the tissue sample.

[0036] The results are as follows Figure 3 shown.

[0037] from Figure 3 It can be seen that the figure is an imaging effect diagram of tissue at different depths. Further observation of the cell structure in the local area shows that some tumor cells undergo self-fusion and the cell morphology has no obvious regularity. The above results show that the method of the present invention is fast and simple, and can obtain complete three-dimensional structural information of biological tissue samples, and the results are stable and reliable.

[0038] Example 3 This embodiment uses the rapid three-dimensional imaging method of the present invention to perform rapid imaging of human breast cancer tissue.

[0039] Specifically, the steps include: 1) Tissue acquisition: Surgically removed breast cancer tissue was fixed in PFA overnight; 2) Sample staining: Soak human breast cancer tissue in staining solution (70wt% ethanol, 2nM TO-PRO-3, 10nM eosin, 10mM sodium chloride, the rest is distilled water, and the pH is adjusted to 4 with hydrochloric acid), incubate with shaking for 12h at 25°C, and then rinse with ethanol several times to obtain the stained tissue sample; 3) Sample transparentization: soak the stained sample obtained in step 1) in a dehydrating solution (10wt% N-butyldiethanolamine, 30% hexylene glycol, 0.5wt% thioglycerol, and the rest trimethyl borate), and incubate with shaking for 2h at 37°C; then soak the dehydrated sample in a defatting solution (0.5wt% thioglycerol, and the rest dichloromethane), and incubate with shaking for 30min at 37°C; finally soak the defatted sample in dibenzyl ether, and incubate with shaking for 30min at 37°C to obtain a transparent tissue sample; 4) Sample three-dimensional imaging: Use glass clamps to clamp the tissue sample obtained in step 2) and immerse it in an imaging tank filled with dibenzyl ether; Use a 4X magnification microscope to roughly scan the sample, adjust the positions of the scanning start and end points to ensure that the sample is completely scanned, and use the Alexa Fluor 555 channel and Alexa Fluor 647 channel to excite the cytoplasm and nuclear signals in the sample respectively. After the rough scan is completed, replace the lens with a 25X magnification microscope to perform three-dimensional imaging of the fine structure of the sample to obtain a three-dimensional fluorescence image; 5) Sample 3D reconstruction: Use FalseColor software to convert the fluorescence channel into HE style, and use Imaris to perform 3D reconstruction of the imaging data to obtain a 3D staining image of the tissue sample.

[0040] The results are as follows Figure 4 shown.

[0041] from Figure 4 It can be seen that the figure is a 3D fluorescence image and a 3D staining image during the imaging process of breast cancer tissue. Although both images can show the cell morphology of the tissue, the 3D staining image is closer to the existing histopathology detection habits. After further local magnification of the 3D staining image, it can be found that the fat tissue is distorted and presents an irregular shape. The above results show that the method of the present invention is fast and simple, and can obtain complete 3D structural information of biological tissue samples, and the results are stable and reliable.

[0042] Example 4 This embodiment uses the rapid three-dimensional imaging method of the present invention to rapidly image normal breast tissue of a human body.

[0043] Specifically, the steps include: 1) Tissue acquisition: Surgically removed breast tissue was fixed in PFA overnight; 2) Sample staining: normal human breast tissue was immersed in a staining solution (70wt% ethanol, 2nM TO-PRO-3, 10nM eosin, 10mM sodium chloride, the rest was distilled water, and the pH was adjusted to 4 with hydrochloric acid), incubated with shaking for 12h at 25°C, and then rinsed with ethanol several times to obtain a stained tissue sample; 3) Sample transparentization: soak the stained sample obtained in step 1) in a dehydrating solution (10wt% N-butyldiethanolamine, 30wt% hexylene glycol, 0.5wt% thioglycerol, and the rest trimethyl borate), and incubate with shaking for 2h at 37°C; then soak the dehydrated sample in a defatting solution (0.5wt% thioglycerol, and the rest dichloromethane), and incubate with shaking for 30min at 37°C; finally soak the defatted sample in dibenzyl ether, and incubate with shaking for 30min at 37°C to obtain a transparent tissue sample; 4) Sample three-dimensional imaging: Use glass clamps to clamp the tissue sample obtained in step 2) and immerse it in an imaging tank filled with dibenzyl ether; Use a 4X magnification microscope to roughly scan the sample, adjust the positions of the scanning start and end points to ensure that the sample is completely scanned, and use the Alexa Fluor 555 channel and Alexa Fluor 647 channel to excite the cytoplasm and nuclear signals in the sample respectively. After the rough scan is completed, replace the lens with a 25X magnification microscope to perform three-dimensional imaging of the fine structure of the sample to obtain a three-dimensional fluorescence image; 5) Sample 3D reconstruction: Use FalseColor software to convert the fluorescence channel into HE style, and use Imaris to perform 3D reconstruction of the imaging data to obtain a 3D staining image of the tissue sample.

[0044] The results are as follows Figure 5 shown.

[0045] from Figure 5 It can be seen that the figure is a three-dimensional fluorescence image and a three-dimensional staining image during the imaging process of normal breast tissue. Although both images can show the cell morphology of the tissue, the three-dimensional staining image is closer to the existing histopathological detection habits. After further local amplification of the three-dimensional staining image, it can be found that the breast sample is rich in connective tissue and adipose tissue; at the same time, the mammary ducts are enlarged and the mammary lobules are difficult to distinguish, which is consistent with the morphology of breast tissue in postmenopausal women. The above results show that the method in the present invention is fast and simple, and can obtain complete three-dimensional structural information of biological tissue samples, and the results are stable and reliable.

[0046] 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.

[0047] 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 rapid three-dimensional imaging method based on tissue optical transparency, characterized in that: The steps include: The biological tissue sample is placed in a staining solution and subjected to shaking treatment to obtain a stained tissue sample; placing the stained tissue sample in a dehydrating liquid, a defatting liquid and a refractive index matching liquid in sequence for oscillation treatment to obtain a transparent tissue sample; Performing three-dimensional imaging on the transparent tissue sample to obtain a three-dimensional fluorescence image of the tissue sample; The three-dimensional fluorescence image of the tissue sample is converted, and the obtained stained bright field image is three-dimensionally reconstructed to obtain a three-dimensional staining image of the tissue sample.

2. The method according to claim 1, characterized in that: In the preparation process of the stained tissue sample, the staining solution includes 1-10 nM of a nuclear stain, 10-100 nM of a cytoplasmic stain, 10-100 mM of an inorganic salt, and 10-70 wt % of an alcohol substance, and the rest is distilled water; The shaking treatment step specifically includes: shaking for 6-48 hours at a temperature of 20-25°C.

3. The method according to claim 2, characterized in that The cell nucleus stain is selected from at least one of TO-PRO-3, DAPI and PI; the cytoplasm stain is selected from at least one of eosin, Cy3 and NHS ester; the inorganic salt is selected from at least one of sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate; the alcohol substance is selected from at least one of methanol, ethanol and glycerol.

4. The method according to claim 1, characterized in that: In the preparation process of the transparent tissue sample, the dehydrating liquid includes 5-30wt% of a polyol, 5-30wt% of an amino alcohol and 0.1-1wt% of a reducing agent, and the rest is a dehydrating agent; and the dehydrating agent is selected from at least one of tri(4-chlorophenyl) borate, triphenyl borate, trimethyl borate and tributyl borate; The step of placing the stained tissue sample in a dehydrating solution for shaking treatment specifically includes: shaking for 20-180 minutes at a temperature of 30-40°C.

5. The method according to claim 4, 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.

6. The method according to claim 1, characterized in that In the preparation process of the transparent tissue sample, the degreasing solution includes 0.1-1wt% of a reducing agent, and the rest is a degreasing agent; and the degreasing agent is selected from at least one of cyclohexane, ethanol, dichloromethane and benzyl benzoate; The step of placing the tissue sample treated with the dehydrating solution in the defatting solution for shaking treatment specifically includes: shaking for 10-60 minutes at a temperature of 30-40°C.

7. The method according to claim 6, characterized in that The reducing agent is selected from at least one of thioglycerol, vitamin D and vitamin C.

8. The method according to claim 1, characterized in that In the preparation process of the transparentized tissue sample, the refractive index matching liquid includes at least one of benzyl ether, benzyl benzoate, benzyl alcohol and ethyl cinnamate; The step of placing the tissue sample treated with the defatted liquid in the refractive index matching liquid for oscillation treatment specifically includes: oscillating for 5-60 minutes at a temperature of 30-40°C.

9. The method according to claim 1, characterized in that: In the three-dimensional fluorescence imaging process of the tissue sample, the three-dimensional imaging step specifically includes: performing three-dimensional imaging of the transparent tissue sample using a fluorescence microscope; Wherein, the fluorescence microscope includes a confocal microscope and / or a light sheet microscope.

10. The method according to claim 1, characterized in that In the reconstruction process of the three-dimensional staining image of the tissue sample, the conversion step specifically includes: converting the three-dimensional fluorescence image of the tissue sample into Tif format, and obtaining a staining bright field image after conversion by color channel conversion software; The three-dimensional reconstruction step specifically includes: performing three-dimensional reconstruction using Imaris software.

Images