CN109406768B - Method for observing three-dimensional distribution of cells in micro tissue and counting cell number - Google Patents
Method for observing three-dimensional distribution of cells in micro tissue and counting cell number Download PDFInfo
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Abstract
The invention relates to a method for observing the three-dimensional distribution of cells in a tiny tissue and counting the number of the cells in the technical field of microscopic observation of biological tissues, which comprises the steps of cleaning the surface of an obtained tissue material to be observed, and then sequentially carrying out chemical fixation, dehydration treatment, immunofluorescence staining and transparentization treatment; and moving the tissue material and the transparent liquid to a culture dish special for a confocal microscope, carrying out layer-by-layer scanning photographing by using a laser confocal microscope to obtain cell distribution images of all layers of the whole tissue, observing the three-dimensional distribution of cells, processing cell distribution image data of all layers by Imaris software, reconstructing a three-dimensional model of the cell tissue, observing the three-dimensional distribution of cells marked by immunofluorescence in the three-dimensional model, and calculating the accurate number of the immunofluorescence marking cells by utilizing Imaris Bitplane Spot detection and analysis. The method of the invention keeps the integrity of the tissue to be observed, and has high definition and accuracy of cell distribution observation and more accurate cell number statistical result.
Description
Technical Field
The invention relates to the technical field of microscopic observation of biological tissues, in particular to a method for observing three-dimensional distribution of cells in a micro tissue and counting the number of the cells.
Background
In life science research and clinical pathology analysis, observation of microscopic distribution of cells in biological tissues and statistics of cell numbers have great significance in research on cell differentiation, tissue development, disease occurrence and the like. The conventional method for studying this process is to use immunohistochemical staining after serial sections of tissue, then to simulate and extrapolate the distribution of cells in the intact tissue by stitching and superimposing the image data between different sections, and then to calculate the number of specific cells. However, this conventional method has a limitation in that serial sections do not completely show true tissue overview, cell distribution, and specific cell numbers. The tissue section can only display the cell distribution and the number of a certain layer, moreover, when the section is actually made, the damage and the loss of the tissue are hard to avoid, the accuracy of the cell distribution and the number of a single section cannot be ensured, and simultaneously, the uniformity of different section thicknesses is hard to ensure, so that the superposed result is more inaccurate, even if the mathematical model is used for reconstruction and calculation, the final result is only predicted and estimated, and is not real image and data, so that the same biological tissue sample is caused, the variable coefficient of the final result is very large, and particularly, the reliability of the result of the cell number is not high.
How to observe the distribution and number of specific cells within a tissue while maintaining the integrity of the tissue is a major focus and focus of biological and medical research. Although the technologies of Computed Tomography (CT), positron emission tomography (PETCT), magnetic Resonance Imaging (MRI), etc. commonly used in clinical medicine are rapidly developed, the resolution thereof has not reached the cellular level or the sub-cellular level so far.
Disclosure of Invention
The invention aims to provide a method for observing the three-dimensional distribution of cells in a microtissue and counting the number of the cells while keeping the integrity of the tissue, and provides a method for observing the tissue and counting the number of the cells at a cellular level or a subcellular level for life science research and clinical pathology analysis.
In order to achieve the above object, the present invention provides a method for observing the three-dimensional distribution of cells in a microtissue, comprising the steps of: obtaining a tissue material to be observed and cleaning the surface of the tissue material; chemically fixing and dehydrating the obtained tissue material; performing immunofluorescence staining on the dehydrated tissue material; performing transparentization treatment on the tissue material subjected to immunofluorescence staining; and moving the tissue material subjected to transparentization treatment and the transparent liquid to a culture dish special for a confocal microscope, and scanning and photographing layer by using the confocal microscope to obtain cell distribution images of all layers of the whole tissue and observe the three-dimensional distribution of cells.
The method of the invention, through carrying on chemical fixation, dehydration treatment, immunofluorescence staining and transparentization treatment to the whole biological tissue, keep the integrality of the whole biological tissue obtained, identify and fluorescently mark the specific cell in the tissue to facilitate the observation, then scan and shoot layer by layer through the laser confocal microscope, obtain the cell distribution image of each layer of the whole tissue, carry on the observation of the three-dimensional distribution of the cell. In the method, the whole treatment and observation process of the biological tissue keeps the whole integrity of the tissue, does not need layered slice observation, and realizes high-resolution accurate observation of cell level or subcellular level.
Preferably, the thickness of the tissue material is less than 500 μm, and the interval between the layer thicknesses of the layer-by-layer scanning pictures is 1-5 μm.
The chemical fixing step of the tissue material of the invention comprises: the step of chemically fixing the tissue material comprises: soaking the washed tissue material in 3.5-4.5% concentration water solution of paraformaldehyde at 2-5 deg.c for 2-2.5 hr; and then removing the paraformaldehyde aqueous solution, and respectively carrying out shaking elution on the tissue materials by using phosphate buffer at the temperature of 2-5 ℃ for 3-4 hours, wherein the phosphate buffer is replaced every 1 hour. Through the treatment of the step, the water solubility of various substances such as protein, peptide substances and the like in the tissues is reduced so as to facilitate the hardening and fixing of the tissue materials and ensure the integrity of the whole tissues.
In order to achieve thorough dehydration of cells within the tissue material, the dehydration treatment steps of the present invention include: removing phosphate buffer solution in the tissue material after dehydration treatment, and shaking and eluting the tissue material by using sucrose aqueous solution with the mass concentration of 9-11% and 19-21% for 3-4 hours at the temperature of 2-5 ℃; and then, continuously shaking and eluting the tissue material by using a sucrose aqueous solution with the mass concentration of 29-31% for 12-15 hours at the temperature of 2-5 ℃.
Further, the immunofluorescence staining step of the present invention comprises: removing the dehydrated sucrose solution, sequentially carrying out membrane permeation treatment and sealing treatment on the tissue material, sequentially incubating for 48-72 hours by using a primary antibody and a fluorescence-labeled secondary antibody respectively, and finally carrying out fluorescence dyeing by using a fluorescent dye.
Further, the permeable membrane treatment process comprises the following steps: and (2) oscillating and incubating the tissue material in a membrane permeation solution for 12-15 hours at the temperature of 20-25 ℃, wherein the membrane permeation solution is a polyethylene glycol octyl phenyl ether aqueous solution with the mass concentration of 0.4-0.6%. In the membrane penetration treatment process, the cell membrane permeability of the tissue material can be improved by controlling the membrane penetration time, and cells in the complete tissue can reach the same cell membrane permeability; so that the antibody (including primary antibody and secondary antibody) can smoothly enter the cells in the whole tissue in the subsequent treatment process.
Further, the sealing treatment process in the method of the invention is as follows: removing the membrane permeation liquid in the tissue material subjected to membrane permeation treatment, and oscillating and incubating the tissue material subjected to membrane permeation treatment in a confining liquid for 12-15 hours at the temperature of 4-5 ℃, wherein the confining liquid comprises the following water solutions of mass components: 0.4 to 0.6 percent of polyethylene glycol octyl phenyl ether, 0.10 to 0.20 percent of glycine, 9 to 11 percent of fetal bovine serum and 3 to 4 percent of bovine serum albumin. By the treatment, the nonspecific binding of the antibody in the subsequent treatment process can be reduced, and the occurrence of false positive signals can be eliminated.
To facilitate the identification of a specific cell type, the primary and secondary antibody incubation processes of the invention comprise:
s1) removing sealing liquid of sealing treatment, transferring the tissue material of the sealing treatment into primary resistant solution at the temperature of 4-5 ℃, and incubating for 48-72 hours in a shaking table at the rotating speed of 50-60 rpm; the primary antibody is combined with the protein specifically expressed by a certain cell in the biological tissue through primary antibody incubation so as to achieve the purpose of identifying the certain cell in the tissue;
s2) removing the primary antibody solution, washing with 0.1% polyethylene glycol octyl phenyl ether aqueous solution at the temperature of 2-5 ℃, shaking and washing for 3-4 hours at the rotating speed of 50-60 rpm, and replacing the aqueous solution once every 1 hour; the primary antibody which is not subjected to specific binding can be washed away by the treatment;
s3) removing the cleaning aqueous solution in the previous step, transferring the tissue material into a second antibody solution, and incubating for 48-72 hours in a shaking table at a rotating speed of 50-60 rpm under the condition of keeping away from light at 2-5 ℃, wherein if the first antibody in the S1) is coupled with a fluorescent group, the step is omitted; the aim of the step is to carry out immunological recognition on the primary antibody in the S1) by using a fluorescence-labeled secondary antibody so as to be convenient for distinguishing specific cells by a fluorescence microscope in the subsequent step;
s4) removing the secondary antibody solution, transferring the tissue material into 0.1% polyethylene glycol octyl phenyl ether aqueous solution for cleaning at 42-5 ℃, washing for 3-4 hours by a shaking table at the rotating speed of 50-60 rpm, and replacing the aqueous solution once every 1 hour, wherein if S3) is omitted, the step is omitted;
s5) removing the cleaning solution in the previous step, transferring the tissue material into a DAPI diluent at the temperature of 2-5 ℃ in a dark condition, and incubating for 10-12 hours in a shaking table at the rotating speed of 50-60 rpm to identify and mark all cells in the tissue, wherein the DAPI diluent is a diluted aqueous solution of 4', 6-diamidino-2-phenylindole; by the process, the DAPI molecule can be combined with DNA of all cells in the tissue, so that the distribution of all cells in the tissue and the distribution of specific cells of the antibody recognition mark in the previous step in the tissue can be observed conveniently.
Further, the transparentizing treatment step includes: transferring the tissue material subjected to immunofluorescence staining into a transparent liquid, and incubating for 48-72 hours at a rotating speed of 50-60 rpm by a shaking table under the condition of keeping away from light at 20-25 ℃, wherein the transparent liquid is an aqueous solution containing the following components in parts by mass: 22 to 26 percent of urea, 8 to 12 percent of glycerol, 0.9 to 0.12 percent of polyethylene glycol octyl phenyl ether and 48 to 52 percent of sucrose. Through the treatment of this step, can make laser penetrate the tissue top layer, get into inside the tissue, and then make the microscope can discern the inside fluorescence signal of complete tissue.
According to the method, the biological tissue to be observed is subjected to the treatment, the integrity of the biological tissue to be observed is kept, meanwhile, a certain type of cells in the tissue are subjected to fluorescence labeling through a specific antibody, after the transparentization treatment, all fluorescence signals of the whole tissue are identified by using a laser confocal microscope, and the spatial distribution of the specifically labeled cells in the whole tissue is displayed in a real and three-dimensional manner. The invention solves the problems of fluorescent labeling and microscopic observation of cells in the complete tissue, and improves the observation of the complete tissue on the cell level and the three-dimensional level; in theory, it is possible to reach sub-cellular or molecular levels with the aid of high-resolution confocal microscopy. Particularly in the aspect of transparentization treatment, the invention carries out immunofluorescence staining before transparentization treatment, thus avoiding the damage of the transparentization treatment to the antigen and the loss or reduction of fluorescence signals caused by the damage; meanwhile, the optimized transparent liquid enables laser to penetrate through the complete tissue more easily, so that the fluorescent signal is clear and the background signal is weak.
The invention also aims to provide a method for counting the number of cells in the micro tissue, which comprises the steps of firstly obtaining cell distribution images of all layers of the micro tissue according to the method for observing the three-dimensional distribution of the cells in the micro tissue, processing the cell distribution image data of all layers through Imaris software, reconstructing a three-dimensional model of tissue cells, observing the three-dimensional distribution of cells marked by immunofluorescence in the three-dimensional model, and calculating the accurate number of the cells marked by the immunofluorescence by utilizing Imaris Bitplane Spot detection and analysis. According to the cell number statistical method, a three-dimensional model with high definition and high truth of cell distribution of the micro tissues is reproduced through Imaris three-dimensional software according to the cell distribution diagram of each layer of the micro tissues so as to accurately count the cell number of the whole biological tissues to be observed.
Drawings
Fig. 1 is a laser confocal whole tissue scanning layer diagram of isolated gonadal tissue of a 1-day-old mouse obtained by the method for observing the three-dimensional distribution of cells in the microtissue.
Fig. 2 is a partial cross-sectional view of the partial scan slice of fig. 1, wherein the left vertical bar is a scan labeled specifically green (converted to white in the figure) with TRA98 for germ cells within intact ex vivo gonadal tissue, the middle vertical bar is a plot labeled blue (converted to gray in the figure) with DAPI for all cells in intact ex vivo gonadal tissue, and the right vertical bar is a plot of the distribution of all cells in the whole tissue after the left and middle bars have been combined.
Fig. 3 is a three-dimensional rotation map of the synthesized isolated gonadal tissue reproduced by Imaris software.
FIG. 4 is a three-dimensional schematic representation constructed by Imaris software based on the fluorescently labeled specific germ cells of FIGS. 1-3.
Detailed Description
This example illustrates the isolated gonads of a mouse of one day of birth, and the method of the present invention is described in detail in steps with reference to the accompanying drawings.
1. The isolated gonadal tissue of a day-old mouse obtained in this example having a thickness of less than 500 μm and a thickness of 228 μm measured under a microscope was washed with PBS (phosphate buffered saline) or physiological saline to wash off unnecessary tissue on the surface of the tissue material.
2. Chemical fixation and dehydration treatment of isolated tissue
1. Transferring the in vitro tissue treated in the previous step into a 1.5 ml centrifuge tube, adding 1ml of freshly prepared paraformaldehyde aqueous solution with the mass concentration of 4%, and carrying out fixed incubation for 2 hours at the temperature of 4 ℃;
2. carefully removing the fixed paraformaldehyde aqueous solution, washing the isolated tissues for 3 hours by using a phosphate buffer solution respectively in a shaking table at the rotating speed of 500 rpm, replacing the phosphate buffer solution every 1 hour, and washing away the residual paraformaldehyde solution in the isolated tissues after the washing;
3. carefully removing phosphate buffer solution in the isolated tissue, adding 1ml of sucrose aqueous solution with the mass concentration of 10% into the centrifugal tube of the isolated tissue, and washing for 3 hours at 4 ℃ by a shaking table at the rotating speed of 50 rpm;
4. carefully removing the 10% sucrose aqueous solution, adding 1ml of 20% sucrose aqueous solution into the centrifuge tube of the isolated tissue, and washing for 3 hours at 4 ℃ in a shaking table at a rotating speed of 50 rpm;
5. the 20% sucrose solution was carefully removed, and 30% sucrose solution was added to the centrifuge tube of the isolated tissue, followed by shaking washing at 50rpm at 4 ℃ for 12 hours.
After the three steps of sucrose aqueous solution washing, the complete dehydration of the cells in the whole tissue can be realized.
3. Immunofluorescent staining of isolated tissue
1. Performing membrane permeation treatment, transferring the fixed and dehydrated in-vitro tissue into another centrifugal tube with the capacity of 1.5 ml, adding a 0.5 Triton X-100 aqueous solution (polyethylene glycol octyl phenyl ether aqueous solution) with mass concentration, and performing shaking incubation for 12 hours at 25 ℃ by a shaking table at a rotating speed of 50 rpm; by the membrane permeation treatment, the cell membrane permeability of tissue cells can be improved, and the cells in the complete tissue can reach the same cell membrane permeability; so that the antibodies (including primary antibody and secondary antibody) in the subsequent treatment process can smoothly enter the cells in the whole tissue.
2. And (3) sealing, carefully removing the membrane permeation solution, adding 1ml of sealing solution, and performing shaking incubation for 12 hours at the temperature of 4 ℃ and at the rotating speed of 50rpm, wherein the sealing solution is an aqueous solution of the following components in mass percent: 0.5% Triton X-100 (i.e., polyethylene glycol octylphenyl ether), 0.15% glycine, 10% fetal bovine serum, 3% BSA (bovine serum albumin); by the blocking treatment, the nonspecific binding of the antibody in the subsequent treatment process can be reduced, and the occurrence of false positive signals can be eliminated.
3. Primary antibody incubation, carefully removing the blocking solution after blocking treatment, adding 0.5ml of TRA98 primary antibody solution with the concentration of 2 μ g/ml to the isolated tissue, and incubating in a shaker at 50rpm for 48 hours at 4 ℃ for binding with the Germ cell specific antigen in the isolated tissue, wherein the primary antibody TRA98 in this example is Anti-Germ cell-specific antigen antibody purchased from abcam;
4. carefully removing the primary antibody solution, washing with 0.1% Triton X-100 aqueous solution (i.e., polyglykoctylphenyl ether aqueous solution), shaking at 50rpm for 3 hours at 4 deg.C, and replacing the aqueous solution every 1 hour;
5. removing the cleaning solution in the previous step, transferring the tissue material into a second antibody solution, and incubating for 48 hours in a dark condition at 4 ℃ in a shaking table at a rotating speed of 50rpm, wherein the second antibody in the step is goat anti-rat IgG H & L (Alexa Fluor 488) purchased from abcam company and used for being combined with primary anti-TRA 98, and if the primary antibody in 3 is coupled with a fluorescent group, the step is omitted; the purpose of the step is to identify and mark primary antibody without fluorescent group so as to facilitate the identification of specific cells by a laser confocal microscope;
6. removing the secondary antibody solution, transferring the tissue material into 0.1% Triton X-100 aqueous solution (namely polyethylene glycol octyl phenyl ether aqueous solution) at 4 ℃, cleaning, washing for 3 hours by a shaking table at the rotating speed of 50rpm, and replacing the aqueous solution every 1 hour, wherein if the 5 th step is omitted, the step is omitted;
7. removing the cleaning solution, transferring the tissue material into DAPI (fluorescent staining agent with molecular formula of 4', 6-diamidino-2-phenylindole) solution with concentration of 1 μ g/ml at 4 deg.C in dark, and incubating with a shaker at 50rpm for 12 hr; by the process, the DAPI molecule can be combined with all cells in the isolated tissue so as to observe the distribution of all cells in the tissue and the distribution of the specific cells of the previous identification marker in the tissue.
4. Carrying out transparentization treatment on the isolated tissue: the immunofluorescent-stained excised tissue was transferred to another new 1.5 ml centrifuge tube, 1ml of the clear solution was added, and the tube was incubated with a shaker at 50rpm for 48 hours at 25 ℃ in the dark. Wherein the transparent liquid is an aqueous solution with the following mass ratio: contains 24% of urea, 10% of glycerol, 0.1% of Triton X-100 (polyethylene glycol octyl phenyl ether), and 50% of sucrose. Through the step of transparentization treatment, the laser can penetrate through the tissue surface layer and enter the tissue, and then the laser confocal microscope can identify the fluorescent signal inside the complete tissue. Different from the prior transparentizing treatment, the transparentizing treatment is carried out after immunofluorescence staining, so that the damage of the transparentizing treatment to the antigen and the loss or reduction of fluorescence signals caused by the damage are avoided; meanwhile, the optimized transparent liquid enables the laser to penetrate through the complete tissue more easily, so that the fluorescent signal is clear and the background signal is weak.
Moving the transparent isolated tissue and the transparent liquid into a culture dish special for a confocal microscope, wherein the thickness of the culture dish is 0.1mm, scanning and photographing layer by using a laser confocal microscope, scanning and photographing layer by layer according to a scanning interval of 2 micrometers along a Z axis during scanning, and obtaining a whole cell distribution image of each layer of the whole tissue, as shown in figure 1, the thickness of the isolated tissue along the Z axis of the embodiment is 228 micrometers, 114 layers of scanning and photographing are performed in total, arabic number numbers in the figure represent the number of the scanned layers, and the white part in figure 1 shows that the cells are germ cells marked by fluorescence guns. Fig. 2 is a scanning picture of the 5 th, 10 th, 30 th, 64 th and 90 th layers in fig. 1, wherein the white cells in the left vertical row in the figure are the laser scanning pictures of the corresponding layer for carrying out the fluorescent marking on the germ cells in the whole in-vitro tissue by using the TRA98 antibody, the middle vertical row is the laser scanning picture of the corresponding layer for carrying out the fluorescent staining on all the cells in the whole in-vitro tissue by using DAPI, and the right vertical row is the picture of the distribution of all the cells in the whole tissue of the corresponding layer after the left vertical row and the middle vertical row are superposed. The distribution of specific germ cells in the whole tissue can be observed in an all-round way through the series of graphs, and from the graphs of each row on the left side, a single germ cell marked as green by TRA98 is clearly visible, clear in outline and clear in boundary, the single germ cell can be seen on the periphery and the inner part of the gonadal tissue, most of the germ cells are distributed on the periphery of the gonadal tissue, and the inner part (medulla) of the gonadal tissue is less in distribution. Through the treatment mode of the mouse in vitro gonad tissue, the real cell distribution in the in vitro tissue can be comprehensively observed and known.
In order to facilitate accurate statistics of the number of germ cells in the in vitro tissue, image data of each layer of cells obtained in the process is processed through Imaris software, a three-dimensional model of the in vitro tissue is reconstructed, three-dimensional rotation observation is carried out, distribution of identified germ cells in the tissue is further known, and as shown in figure 3, the three-dimensional model rotates clockwise by 0 degree, 45 degrees, 90 degrees, 135 degrees and 180 degrees along the Y axis respectively.
The exact number of immunofluorescent-labeled cells was calculated by assay using Imaris Bitplane Spot detection. According to the cell number statistical method, a three-dimensional model with high degree of purity and high degree of reality of cell distribution of the micro tissue is reproduced through Imaris three-dimensional software according to a cell distribution diagram of each layer of the micro tissue, so that the cell number of the whole biological tissue to be observed is accurately counted, as shown in figure 4, one of screenshots of the three-dimensional model constructed for the reproductive cells is obtained, and through the three-dimensional model, multi-azimuth rotation observation statistical verification is carried out, wherein the accurate number of the reproductive cells is 2415.
The invention is not limited to the above-described embodiment of the mouse isolated gonadal tissue, and can also be used for observing and processing other micro tissue materials.
Claims (4)
1. A method for observing the three-dimensional distribution of cells in a micro tissue is characterized by comprising the following steps: obtaining a tissue material to be observed and cleaning the surface of the tissue material; chemically fixing and dehydrating the obtained tissue material; performing immunofluorescence staining on the dehydrated tissue material; performing transparentization treatment on the tissue material subjected to immunofluorescence staining; moving the tissue material subjected to transparentization treatment and transparent liquid to a culture dish special for a confocal microscope, and scanning and photographing layer by using the confocal microscope to obtain cell distribution images of all layers of the whole tissue and observe the three-dimensional distribution of cells;
the step of chemically fixing the tissue material comprises: soaking the washed tissue material in 3.5-4.5% concentration water solution of paraformaldehyde at 2-5 deg.c for 2-2.5 hr; then removing the paraformaldehyde aqueous solution, and respectively carrying out shaking elution on the tissue materials for 3-4 hours by using phosphate buffer at the temperature of 2-5 ℃, wherein the phosphate buffer is replaced every 1 hour;
the dehydration treatment step comprises: removing phosphate buffer solution in the tissue material after dehydration treatment, and respectively shaking and eluting the tissue material for 3-4 hours by using sucrose aqueous solutions with mass concentrations of 9-11% and 19-21% at the temperature of 2-5 ℃; then, continuously shaking and eluting the tissue material by using a sucrose aqueous solution with the mass concentration of 29-31% for 12-15 hours at the temperature of 2-5 ℃;
the immunofluorescent staining step comprises: removing the dehydrated sucrose solution, sequentially carrying out membrane permeation treatment and sealing treatment on the tissue material, sequentially incubating for 48-72 hours by using a primary antibody and a fluorescence-labeled secondary antibody respectively, and finally carrying out fluorescence dyeing by using a fluorescent dye;
the primary antibody and the secondary antibody incubation process comprises the following steps:
s1) removing sealing liquid of sealing treatment, transferring the tissue material of the sealing treatment into primary resistant solution at the temperature of 4-5 ℃, and incubating for 48-72 hours in a shaking table at the rotating speed of 50-60 rpm;
s2) removing the primary antibody solution, cleaning with 0.1% polyethylene glycol octyl phenyl ether aqueous solution at the temperature of 2-5 ℃, washing for 3-4 hours in a shaking table at the rotating speed of 50-60 rpm, and replacing the aqueous solution once every 1 hour;
s3) removing the cleaning aqueous solution in the previous step, transferring the tissue material into a second antibody solution, and incubating for 48-72 hours in a shaking table at a rotating speed of 50-60 rpm under the condition of keeping away from light at 2-5 ℃, wherein if the first antibody in the S1) is coupled with a fluorescent group, the step is omitted;
s4) removing the secondary antibody solution, transferring the tissue material into 0.1% polyethylene glycol octyl phenyl ether aqueous solution for cleaning at the temperature of 2-5 ℃, washing for 3-4 hours by a shaking table at the rotating speed of 50-60 rpm, and replacing the aqueous solution once every 1 hour, wherein if S3) is omitted, the step is omitted;
s5) removing the cleaning solution in the previous step, transferring the tissue material into a DAPI diluent at the temperature of 2-5 ℃ in a dark condition, and incubating for 10-12 hours in a shaking table at the rotating speed of 50-60 rpm to identify and mark all cells in the tissue, wherein the DAPI diluent is a diluted aqueous solution of 4', 6-diamidino-2-phenylindole;
the transparentizing treatment step comprises the following steps: transferring the tissue material subjected to immunofluorescence staining into a transparent liquid, and carrying out shake-table incubation for 48-72 hours at a rotating speed of 50-60 rpm under the condition of keeping out of the sun at 20-25 ℃, wherein the transparent liquid is an aqueous solution containing the following components in parts by mass: 22 to 26 percent of urea, 8 to 12 percent of glycerol, 0.09 to 0.12 percent of polyethylene glycol octyl phenyl ether and 48 to 52 percent of sucrose;
a method for counting the number of cells in the microtissue based on the method for observing the three-dimensional distribution of the cells in the microtissue specifically comprises the following steps: obtaining cell distribution images of each layer of the micro tissue, processing cell distribution image data of each layer through Imaris software, reconstructing a three-dimensional model of the tissue, observing the three-dimensional distribution of cells marked by immunofluorescence in the three-dimensional model, and calculating the accurate number of the cells marked by the immunofluorescence by utilizing ImarisBitplaneSpot detection and analysis.
2. The method according to claim 1, wherein the thickness of the tissue material is less than 500 μm and the interval between the layer thicknesses of the layer-by-layer scan is 1-5 μm.
3. The method for observing the three-dimensional distribution of cells in a minute tissue according to claim 1, wherein the transmembrane processing comprises: and (2) oscillating and incubating the tissue material in a membrane permeation solution for 12-15 hours at the temperature of 20-25 ℃, wherein the membrane permeation solution is a polyethylene glycol octyl phenyl ether aqueous solution with the mass concentration of 0.4-0.6%.
4. The method for observing the three-dimensional distribution of cells in a microscopic tissue according to claim 1, wherein the sealing treatment process is: removing the membrane permeation liquid in the tissue material subjected to membrane permeation treatment, and oscillating and incubating the tissue material subjected to membrane permeation treatment in a confining liquid for 12-15 hours at the temperature of 4-5 ℃, wherein the confining liquid comprises the following water solutions of mass components: 0.4 to 0.6 percent of polyethylene glycol octyl phenyl ether, 0.10 to 0.20 percent of glycine, 9 to 11 percent of fetal bovine serum and 3 to 4 percent of bovine serum albumin.
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| CN112461733A (en) * | 2020-11-05 | 2021-03-09 | 闽江学院 | Method for observing three-dimensional structure of ciliate cell by multiple fluorescence markers |
| CN112435259B (en) * | 2021-01-27 | 2021-04-02 | 核工业四一六医院 | Cell distribution model construction and cell counting method based on single sample learning |
| CN113049804B (en) * | 2021-03-17 | 2022-09-09 | 上海交通大学 | Method for rapidly marking biological tissues |
| CN113138110A (en) * | 2021-04-27 | 2021-07-20 | 吉林大学 | Three-dimensional reconstruction method for pathological picture |
| CN113640520A (en) * | 2021-07-16 | 2021-11-12 | 南方医科大学珠江医院 | Application of tissue transparency method and histology method in combination for detecting bacteria in tumor |
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