CN116202845B - A biological tissue sample staining method and three-dimensional imaging method based on DAPI - Google Patents
A biological tissue sample staining method and three-dimensional imaging method based on DAPIInfo
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- CN116202845B CN116202845B CN202310052633.6A CN202310052633A CN116202845B CN 116202845 B CN116202845 B CN 116202845B CN 202310052633 A CN202310052633 A CN 202310052633A CN 116202845 B CN116202845 B CN 116202845B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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Abstract
本发明涉及生物医学技术领域，具体涉及一种基于DAPI的生物组织样本染色方法和三维成像方法。本发明提供的基于DAPI的生物组织样本染色方法包括：将组织样本依次进行脱水、第一次细胞膜通透、脱脂、第二次细胞膜通透、富水、第三次细胞膜通透后，再进行DAPI染色。该方法利用DAPI染料能够与双链DNA结合后呈现强荧光的特性，对生物组织器官进行整体染色，通过引入优化的细胞膜通透方法促进DAPI染料的组织渗透，进而提升生物组织样本的整体染色效果，配合三维成像方法，可以清晰地显示器官内部细胞层面立体的效果，具有快速、经济、高质量的特点。
The present invention relates to the field of biomedical technology, and in particular to a biological tissue sample staining method and a three-dimensional imaging method based on DAPI. The biological tissue sample staining method based on DAPI provided by the present invention comprises: sequentially dehydrating the tissue sample, performing the first cell membrane permeabilization, defatting, performing the second cell membrane permeabilization, enriching water, and performing the third cell membrane permeabilization, and then performing DAPI staining. The method utilizes the property that the DAPI dye can present strong fluorescence after binding to double-stranded DNA, and performs overall staining of biological tissue organs. By introducing an optimized cell membrane permeabilization method, the tissue penetration of the DAPI dye is promoted, thereby improving the overall staining effect of the biological tissue sample. In combination with a three-dimensional imaging method, the three-dimensional effect of the cell level inside the organ can be clearly displayed, and the method has the characteristics of being fast, economical, and high-quality.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a biological tissue sample staining method and a three-dimensional imaging method based on DAPI.
Background
The traditional biological tissue staining and imaging method is to prepare tissue slices after embedding tissue blocks, and then to image the slices after staining the slices, but the tissue slice staining and imaging technology is generally difficult to stain and image all the slices in the tissue blocks, so that the obtained image information is difficult to reflect the state of the whole tissue, if all the slices are stained, the amount of reagents consumed by staining is large, the number of the slices to be observed is large, the manpower and material cost are greatly increased, and the staining and imaging conditions of different slices may be different, in particular, when the slices are imaged in three dimensions, the registration between layers (upper and lower slices) is difficult, and the error is large. The whole staining technique of biological tissues can better solve the problems.
The molecular formula of the DAPI fluorescent dye (4', 6-diamidino-2-phenylindole) is C 16H15N5 & lt 2 & gt HCl, the molecular weight is 350.25, the DAPI fluorescent dye is one of dyes commonly used for staining living cells and fixed cells, and the DAPI fluorescent dye presents blue fluorescence, has the maximum excitation wavelength of 340nm and the maximum emission wavelength of 488nm. Under a fluorescence microscope, the DAPI dye is excited with light of ultraviolet wavelength, and the emitted light is blue, which overlaps with the emitted wavelengths of Green Fluorescent Protein (GFP) and Red fluorescent dye (Texas Red dye) only in a small part, so that multiple fluorescent staining can be performed on a single sample using this property. DAPI has a maximum absorption/maximum emission wavelength of 358nm/461nm when bound to double-stranded DNA, and the fluorescence intensity generated when bound to RNA is far lower than that when bound to DNA, and the maximum emission wavelength is shifted to about 400 nm. In addition, DAPI can generate more than 20 times more fluorescence than DAPI itself after penetrating the cell membrane and DNA binding. However, while DAPI can penetrate cell membranes to achieve rapid staining of cells or tissue of a section, DAPI is less permeable to intact tissue or tissue having a certain thickness than cells or sections, compared to Nissl dyes (e.g., thiols), etc. Thus, the current use of DAPI is still limited to staining cells and tissue sections, and it has not been seen to use DAPI for whole body staining of intact biological tissue. The development of a method for performing overall staining on biological tissue samples by utilizing DAPI has important significance for realizing multiple fluorescent staining and three-dimensional reconstruction of biological tissues.
Disclosure of Invention
The invention provides a biological tissue sample staining method and a three-dimensional imaging method based on DAPI.
Aiming at the defect that the overall staining of a large biological tissue sample is difficult to realize by DAPI staining, the DAPI dye is quickly permeated into the tissue by means of membrane permeation staining, so that the overall staining of the large organ tissue sample is realized. The present invention has been developed in the course of a method for bulk staining of biological tissue samples using DAPI, and has found that DAPI permeability is difficult to be significantly improved even after conventional membrane permeation treatment of biological tissue samples. Through continuous attempts, the invention discovers that the cell membrane permeation treatment of the biological tissue sample by adopting the specific treating agent after dehydration, degreasing and water enrichment can obviously improve the permeability of DAPI to the biological tissue sample with a certain thickness, even to the complete organ sample, so that the overall staining effect of the biological tissue sample is obviously improved.
Specifically, the invention provides the following technical scheme:
The invention provides a biological tissue sample staining method based on DAPI, which comprises the steps of sequentially dehydrating a biological tissue sample, performing first cell membrane permeation, degreasing, performing second cell membrane permeation, enriching water, performing third cell membrane permeation, and then performing DAPI staining.
In the method, the first cell membrane permeation, the second cell membrane permeation and the third cell membrane permeation are respectively used for treating the tissue sample by using a first treating agent, a second treating agent and a third treating agent;
the first treating agent and the second treating agent are ethanol solutions containing DMSO, and the volume fraction of DMSO in the second treating agent is increased by 30-100% compared with the volume fraction of DMSO in the first treating agent.
The invention discovers that the first cell membrane permeation and the second cell membrane permeation respectively adopt the DMSO ethanol solution with the concentration gradient, which is more beneficial to improving the permeability of DAPI to tissue samples, further improving the uniformity of the distribution of DAPI in the whole tissue samples, improving the uniformity of integral staining, further accelerating the tissue permeation of DAPI, and laying a foundation for accelerating the tissue staining by combining the cell permeation of DAPI with DNA.
Preferably, the volume fraction of DMSO in the second treatment agent is increased by 50-100% over the volume fraction of DMSO in the first treatment agent.
Further 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.
In the above method, the third treating agent is PBS buffer solution containing a detergent, and the detergent is one or more selected from Triton X-100, SDS, SD (sodium deoxycholate) and CHAPS. Unlike the first cell membrane permeation and the second cell membrane permeation, the invention discovers that the treatment of the third cell membrane permeation by using PBS buffer solution containing a specific detergent can further improve the permeability of DAPI to the tissue sample and improve the overall staining effect of the tissue sample.
Preferably, in the third treatment agent, the concentration of the detergent is 0.1 to 1%.
In some embodiments of the invention, the detergent is triton x-100 and the concentration of the detergent in the third treatment agent is 0.2-0.5%.
The three times of membrane permeation treatment can well cooperate, and the combination of the detergent and the cell membrane can be quickened by adopting the ethanol solution containing the gradient concentration DMSO to carry out the preliminary membrane permeation treatment on the tissue, so that the purpose of cell membrane rupture is better realized, the result of rapid membrane rupture of the detergent is achieved in a short time, the infiltration and dyeing of DAPI are quickened, and the experimental time is greatly shortened.
In the above method, the treatment time for the first cell membrane permeation is 6-24 hours, and/or the treatment time for the second cell membrane permeation is 2-12 hours, and/or the treatment time for the third cell membrane permeation is 2-10 days.
The third cell membrane permeation may be performed for a suitable time within the above treatment time range according to the tissue thickness, and if the tissue is thicker, the time may be prolonged appropriately.
Preferably, the treatment time for the first cell membrane permeation is 6-12h, and/or the treatment time for the second cell membrane permeation is 2-5h, and/or the treatment time for the third cell membrane permeation is 2-5d.
In the above method, the staining is to treat the tissue sample with a 40-100% DAPI solution by volume fraction for 1-72 hours. The preferred dyeing time is 48-72 hours.
In the method, the dehydration is to sequentially dehydrate the biological tissue sample in ethanol solution with the volume fraction of 40-50%, 70-85% and 95-100%.
Preferably, the time for dehydration in each ethanol solution is 1 to 4 hours (preferably 1 to 2 hours).
The degreasing is to put the biological tissue sample into methylene dichloride for degreasing.
Preferably, the degreasing time is 1-4 hours.
The water enrichment is to sequentially place the biological tissue sample in ethanol solution with volume fraction of 95-100%, 70-85% and 40-50% for water enrichment.
Preferably, the time for enriching the water in each ethanol solution is 1-4 hours (preferably 1-2 hours).
The dehydration, degreasing and water-rich operation can better cooperate with the cell membrane permeation step, and is beneficial to improving the overall dyeing effect of biological tissues.
The method comprises the steps of carrying out gradient dehydration on ethanol to achieve the aim of preliminary dehydration on tissues, accelerating DAPI permeation and guaranteeing uniformity of distribution in the tissues, adopting ethanol solution containing DMSO to permeate cell membranes, accelerating combination of a detergent and the cell membranes in the third cell membrane permeation treatment, enabling the detergent to achieve the aim of cell membrane rupture better in the third cell membrane permeation treatment, and achieving the effect of rapid membrane rupture of the detergent in a short time, thereby accelerating DAPI permeation and dyeing, greatly shortening experimental time, facilitating DAPI tissue uniform distribution in the third cell membrane permeation treatment, further accelerating DAPI tissue permeation, laying a foundation for DAPI cell permeation and DNA combination and accelerating tissue dyeing. The early dehydration, degreasing and cell membrane permeation treatment not only lays a foundation for uniform tissue distribution and rapid permeation and dyeing of the DAPI, but also adopts ethanol to dehydrate and simultaneously carry out resin permeation and embedding after the dyeing is finished, in the process, the DAPI can be dissolved in ethanol solution and resin solution, and the time of dehydration and permeation during the later resin embedding can be correspondingly shortened after the early dehydration, degreasing and cell membrane permeation treatment, so that the loss of DAPI dye is reduced to a certain extent, the rapid embedding of a resin sample is realized, and the accuracy of the later data acquisition and analysis result is better ensured. The gradient water enrichment of the ethanol provides conditions for the third cell membrane permeation treatment, avoids tissue deformation caused by direct treatment, influences experimental results, and provides conditions for the rapid permeation of DAPI for further increasing the permeability of tissues by ethanol dehydration treatment during resin embedding.
In the above-described staining method, the biological tissue sample is an animal tissue organ sample.
In some embodiments of the invention, the biological tissue sample is a whole animal organ sample, preferably a whole brain tissue sample.
In some embodiments of the invention, the source of the animal tissue sample includes, but is not limited to, a mammal, such as a mouse, human, monkey, rabbit, pig, and the like.
Preferably, the staining method further comprises the step of obtaining the tissue sample after sequentially pouring the PBS buffer and the paraformaldehyde solution into the animal's heart before dehydrating the tissue sample. Perfusion of the heart with PBS buffer prior to taking the tissue sample can exclude the effects of blood fluorescence.
Among them, the concentration of PBS buffer used for cardiac perfusion is preferably 0.001-10M (preferably 0.001-0.1M). The mass fraction of the paraformaldehyde solution is 3-5%.
In some embodiments of the invention, the method of staining a biological tissue sample comprises the steps of:
1) Pouring PBS buffer solution into the heart of the animal;
2) Pouring paraformaldehyde solution into animal hearts, and then taking down a biological tissue sample to be dyed;
3) Sequentially placing the biological tissue sample into ethanol solution with volume fraction of 40-50%, 70-85% and 95-100% for gradient dehydration;
4) Placing the dehydrated biological tissue sample in a first treating agent for first cell membrane permeation;
5) Degreasing a biological tissue sample in dichloromethane;
6) Performing a second cell membrane permeabilization in a second treatment agent of the biological tissue sample;
7) Sequentially placing the biological tissue sample into ethanol solution with volume fraction of 95-100%, 70-85% and 40-50% for gradient water enrichment;
8) Placing the biological tissue sample rich in water into a third treating agent for third cell membrane permeation;
9) The biological tissue samples were stained in DAPI solution.
On the basis of the dyeing method, the invention also provides a three-dimensional imaging method of the biological tissue sample, which comprises the steps of dyeing the biological tissue sample by using the dyeing method of the biological tissue sample, embedding the dyed tissue sample, and then performing three-dimensional imaging.
Preferably, three-dimensional imaging is performed using a TDI-fMOST (fluorescence micro optical tomography) camera. The TDI-fMOST camera can perform continuous slice tomographic imaging of the tissue sample.
Preferably, the stained biological tissue sample is embedded with a resin.
The DAPI-based biological tissue sample staining method provided by the invention has the beneficial effects that the DAPI dye can be combined with double-stranded DNA to show strong fluorescence, the whole biological tissue organ is stained, the tissue permeation of the membrane-impermeable or semi-permeable DAPI dye is promoted by introducing an optimized cell membrane permeation method, so that the DAPI dye rapidly permeates into the tissue, the uniformity of the DAPI in the tissue sample is improved, the whole staining effect of the biological tissue sample is further improved, the three-dimensional effect of the cell layer in the organ can be clearly displayed by matching with a three-dimensional imaging method, and the DAPI-based biological tissue sample staining method has the characteristics of rapidness, economy and high quality.
The three-dimensional imaging method is based on three-dimensional optical imaging equipment, imaging is completed when a tissue sample is subjected to overall staining, natural registration is adopted between the upper layer and the lower layer of the slice, no artificial error exists, the matching degree is high, and therefore the accuracy of a reconstructed three-dimensional tissue result is high.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a fine sectional view of the cerebral vessels of the mice in example 1 of the present invention (TDI-fMOST camera acquisition).
FIG. 2 is a continuous sectional view of mouse brain (TDI-fMOST camera acquisition) of example 1 of the present invention, 2 μm in one layer, wherein 1, 100, 200, 300, 400, 500, 600, 700, 800 below the picture represent the number of slice layers of the mouse brain resin embedded sample, for example, 1 is layer 1 and 100 is layer 100.
Fig. 3 is a graph of microscopic observations of stained murine brain tissue in example 2 of the present invention, wherein 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 below the pictures represent the number of layers of the murine brain sample slice, for example, 1 is layer 1 and 55 is layer 55, respectively.
FIG. 4 is a sectional view of the stained murine brain tissue of comparative example 1 of the present invention.
FIG. 5 is a sectional view of the stained murine brain tissue of comparative example 2 according to the present invention.
FIG. 6 is a sectional view of the stained mouse brain tissue of comparative example 3 of the present invention, wherein the upper view is a direct view photograph, and the lower view is a view photograph using Mshot Ming's microscope (specification model: MF43, enterprise name: ming's photovoltaic technologies Co., ltd., guangzhou).
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
DAPI (liquid dye) used in the examples below was purchased from bi-cloudy days.
Example 1
The embodiment provides a biological tissue sample staining method based on DAPI, which comprises the following steps:
1) Perfusing the mouse heart with 0.001M PBS buffer;
2) Pouring paraformaldehyde solution with mass fraction of 3% into heart of mice, and taking down brain of mice;
3) Sequentially placing the rat brain tissue into ethanol solutions with volume fractions of 50%, 75% and 100% for gradient dehydration, wherein the dehydration time in each ethanol solution is 1h;
4) Placing the dehydrated mouse brain tissue into an ethanol solution containing 10% DMSO for the first cell membrane permeation, wherein the treatment time is 6 hours;
5) Degreasing the rat brain tissue in dichloromethane for 1h;
6) Placing the dehydrated and defatted mouse brain tissue in an ethanol solution containing 20% DMSO for the second cell membrane permeation, wherein the treatment time is 2 hours;
7) Sequentially placing the rat brain tissue in 100%, 75% and 50% ethanol solutions for gradient water enrichment, wherein the water enrichment time in each ethanol solution is 1h;
8) Placing the mouse brain tissue rich in water into PBS buffer solution containing 0.2% TritonX-100 for performing cell membrane permeation for the third time, wherein the treatment time is 2d;
9) The rat brain tissue was then stained in 40% DAPI solution for 3d.
The embodiment also provides a three-dimensional imaging method of the biological tissue sample, which comprises the steps of embedding the rat brain tissue dyed in the steps 1) to 9) by using resin and collecting by using a TDI-fMOST camera.
The imaging result of the stained mouse brain tissue is shown in fig. 1 and 2, and as can be seen from fig. 1 and 2, the whole staining of the mouse brain sample is uniform, the cell bodies in each slice are clearly visible, the shape and the number of the cell bodies can be observed and analyzed, the fluorescent (white spots (cell bodies) in the figure) effect is good, the strong fluorescent can be kept under the irradiation of laser, and the whole staining of the biological tissue in a short time is realized. Imaging of the whole DAPI staining sample is achieved by means of a method of carrying out section collection on a tissue sample embedded by the resin through TDI-fMOST, and therefore a whole rat brain three-dimensional structure diagram with a layer of 1-10 mu m is obtained.
Example 2
The embodiment provides a biological tissue sample staining method based on DAPI, which comprises the following steps:
1) Perfusing the mouse heart with 0.1M PBS buffer;
2) Pouring paraformaldehyde solution with mass fraction of 5% into heart of mice, and taking down brain of mice;
3) Sequentially placing the rat brain tissue into ethanol solution with volume fractions of 40%, 70% and 95% for gradient dehydration, wherein the dehydration time is 2h;
4) Placing the dehydrated mouse brain tissue into an ethanol solution containing 20% DMSO for primary cell membrane permeation, wherein the treatment time is 12h;
5) Degreasing the rat brain tissue in dichloromethane for 4h;
6) Placing the dehydrated and defatted mouse brain tissue in an ethanol solution containing 30% DMSO for the second cell membrane permeation, wherein the treatment time is 3 hours;
7) Sequentially placing the rat brain tissue into 95%, 70% and 40% ethanol solution for gradient water enrichment for 2h;
8) Placing the mouse brain tissue rich in water into PBS buffer solution containing 0.5% TritonX-100 for performing cell membrane permeation for the third time, wherein the treatment time is 3d;
9) The rat brain tissue was then stained in 80% DAPI solution for 2d.
The embodiment also provides a three-dimensional imaging method of the biological tissue sample, which comprises the steps of embedding the rat brain tissue dyed in the steps 1) to 9) by using resin and collecting by using a TDI-fMOST camera.
The microscopic observation result of the stained mouse brain tissue is shown in fig. 3 (150 μm one layer), the whole staining of the mouse brain sample is uniform, the cell bodies in each layer of slice are clearly visible, the observation and analysis of the form and the quantity of the cell bodies are facilitated, the blue fluorescence (blue spots in the figure) effect is good, the strong fluorescence can be kept under the irradiation of laser, and the whole staining of the biological tissue in a short time is realized. Further, imaging of the whole DAPI staining sample is achieved by means of a method of carrying out section collection on the tissue sample embedded by the resin through TDI-fMOST, and therefore a whole rat brain three-dimensional structure diagram with a layer of 1-10 mu m is obtained.
Comparative example 1
This comparative example provides a method of staining a biological tissue sample based on DAPI which differs from the method of example 1 only in that all cell membrane permeabilization steps are removed.
Rat brain tissues stained by the method of this comparative example were embedded with resin and collected using a TDI-fMOST camera. As a result, as shown in fig. 4, it was clearly observed that the blue signal remained only in the cortex and did not penetrate into the white matter region, whereas with the staining method of example 1, each slice was able to see a bright fluorescent signal (white dot), the signal was uniform and the cortex or white matter in the slice exhibited fluorescent signals (fig. 1 and 2), indicating that the dye had completely penetrated into the middle region of the brain, and the overall staining of the biological tissue was achieved, whereby compared with the method of example 1, the staining effect was significantly reduced by the method of this comparative example.
Comparative example 2
This comparative example provides a method of staining a biological tissue sample based on DAPI which differs from the method of example 1 only in that the second and third cell membrane permeabilization steps are removed.
Rat brain tissues stained by the method of this comparative example were embedded with resin and collected using a TDI-fMOST camera. As a result, as shown in FIG. 5, it was seen in FIG. 5 that the rat brain sections had been significantly disintegrated in tissue and the blue signal remained only in the cortex and did not penetrate into the white matter region, whereas with the staining method of example 1, the section of each section was flat and no section disintegrated was observed, a bright fluorescent signal (white spots) was observed, the signals were uniform and the cortex or white matter in the section exhibited fluorescent signals (FIGS. 1 and 2), indicating that the dye had been completely penetrated into the middle region of the brain to effect overall staining of biological tissue, whereby the staining effect was significantly reduced by the method of this comparative example as compared with the method of example 1.
Comparative example 3
This comparative example provides a method for staining a biological tissue sample based on DAPI which differs from the method of example 1 only in that the third cell membrane permeabilization treatment time is 14d.
The results showed that after staining the tissue appeared to be significantly disintegrated (fig. 6).
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.
Claims (7)
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| CN106796165A (en) * | 2014-07-03 | 2017-05-31 | 理查德.托里斯 | Novel tissue processing and imaging methods |
| CN108088725A (en) * | 2017-12-28 | 2018-05-29 | 华中科技大学 | A kind of colouring method of biological tissue |
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