CN106872252A - A kind of resin embedding method of smooth transparence biological tissue - Google Patents

Abstract

The invention discloses a resin embedding method for light-transparent biological tissue. By embedding the tissue with an embedding medium that matches the refractive index of the tissue itself, a resin-embedded tissue with a certain degree of transparency can be obtained. thing. The resin embedding method of light-transparent biological tissue of the present invention provides a certain degree of light transparency to the biological tissue on the basis of ensuring that the resin itself provides solid support, so that when the tissue is cut and imaged, the imaging depth of the sample is increased, The increased thickness of the z-axis ablation greatly reduces the time required for tissue imaging.

Description

A resin embedding method for phototransparent biological tissue

technical field

The invention belongs to the technical field of biomedical optical imaging, and specifically designs a resin embedding method for light-transparent biological tissue that can embed rat brain tissue and maintain a certain degree of transparency after monomer polymerization (embedding). .

Background technique

With the vigorous development of the subject of biomedical photonics, the analysis of the neural network structure in the brain and tissue by modern optical technology has entered a stage of rapid development. In brain imaging technology, extremely high resolution can be obtained through layer-by-layer cutting imaging, and people have obtained extremely fine 3D brain distribution maps of the whole brain of rats. However, due to the opacity of the brain tissue, the light penetration depth is insufficient, and the z-axis cutting thickness is small, resulting in the disadvantage of slow imaging speed. The ultimate goal of studying the structure of the brain neural network is to obtain a fine structure map of the brain nerves of primates, and then provide guidance for brain-related neurological diseases, brain activities and other fields. At present, a series of new technical difficulties have arisen in the process of transitioning the research topic from the mouse brain to the primate monkey brain. Among them, the change in intuitive volume from mouse brain (volume about 1 cm ³ ) to primate (volume about 10 ³ cm ³ ) leads to a sharp increase in the total amount of imaging and the corresponding amount of information. It takes more than a year to image a complete mouse brain, so if the imaging technology of the mouse brain is still used, the imaging time will become too long to be meaningful. Improving the imaging speed of layer-by-layer ablation technology has become extremely urgent.

Through a certain treatment of the tissue, a light-transparent biological sample can be obtained, which greatly enhances the light penetration ability, and thus greatly shortens the time required for imaging. However, these tissue light clearing techniques are almost exclusively concentrated in solution media and thus cannot be used in layer-by-layer ablation imaging systems. In the traditional tissue embedding technology, the embedding medium and the tissue are often opaque due to the mismatch of refractive index, which makes the z-axis imaging depth smaller, and the cutting thickness of a single imaging is too small, which makes the imaging time too long . With the help of tissue transparency technology, the light scattering of light in the tissue can be effectively reduced, the depth of z-axis imaging can be increased, and the time required for imaging can be shortened. If the tissue can still maintain a certain degree of transparency after embedding, then the depth and thickness of cutting imaging will be increased, and the time required for tissue imaging will be directly shortened, which will provide a better way for the imaging of larger primate monkey brains. play a key technical role. At the same time, most of the current tissue resin embedding techniques use temperatures above 50°C to effectively initiate monomer polymerization. However, fluorescent proteins tend to be denatured at higher temperatures, which weakens or even completely quenches the fluorescence, which is not conducive to imaging. Therefore, how to lower the polymerization temperature of the tissue during the embedding process will also better protect the fluorescent protein, thereby improving the quality of imaging is another issue that needs to be concerned about when tissue resin is embedded.

Contents of the invention

Aiming at the above defects or improvement needs of the prior art, the present invention provides a resin embedding method for light-transparent biological tissue, the purpose of which is to select and adopt an embedding medium that has a matching refractive index with the tissue itself to treat the tissue. Embedding, so as to obtain a resin-embedded tissue with a certain degree of transparency, thus solving the problem that the tissue transparency means of the prior art can only be carried out in a solution medium, and cannot be applied to cutting imaging; while the traditional tissue embedding technology can be applied to cutting imaging , but due to the opacity of the tissue after embedding, the Z-axis imaging depth is small during cutting imaging, and the imaging time is too long for technical problems.

In order to achieve the above object, according to one aspect of the present invention, a resin embedding method for light-transparent biological tissue is provided, using an embedding medium for resin embedding of biological tissue, and the refractive index of the biological tissue is related to the embedding method. The refractive index of the embedding medium is matched, so as to realize the resin embedding of the light-transparent biological tissue, and the embedding medium is a polymer formed by polymerization of a polymerization monomer initiated by an initiator.

Preferably, the polymerizable monomer is benzyl methacrylate, 2-acrylic acid-2-methyl-2-phenoxyethyl ester and/or 2-acrylic acid-2-hydroxyl-3-phenoxypropyl ester one or more of .

Preferably, the initiator is azobisisoheptanonitrile.

According to another aspect of the present invention, a kind of described resin embedding method is provided, comprising the steps of:

(1) Degreasing the biological tissue fixed by a chemical method to obtain the degreased biological tissue;

(2) Soak the degreased biological tissue obtained in step (1) in polymerized monomers, then add an initiator, mix to fully dissolve the initiator, and then infiltrate at -20 to 0°C for 12 hours under dark conditions. ~24 hours to obtain the infiltrated biological tissue;

(3) Reacting the infiltrated biological tissue obtained in step (2) at 35-45° C. for 12-48 hours to obtain a light-transparent biological tissue resin.

Preferably, the chemical fixation in step (1) is fixation with formaldehyde solution.

Preferably, the specific step of the degreasing treatment described in step (1) is: performing degreasing treatment in different gradients of tetrahydrofuran-water mixed liquid.

Preferably, the dosage ratio of the polymerized monomer to the initiator in step (2) is 1 ml: 1-6 mg.

Preferably, the reaction temperature in step (3) is 40°C.

Generally speaking, compared with the prior art, the above technical solutions conceived by the present invention can achieve the following beneficial effects.

(1) The present invention provides a resin embedding method capable of optically translucent biological tissue, which provides a certain degree of optical transparency to biological tissue on the basis of ensuring that the resin itself provides solid support, so that the tissue can be cut During imaging, the depth of sample imaging increases, and the thickness of z-axis cutting increases, thereby greatly shortening the time required for tissue imaging.

(2) In the present invention, the polymerization monomer with a specific refractive index and the initiator with a specific polymerization initiation temperature are selected to be used together, and the selected initiator has a lower polymerization temperature to penetrate biological tissues at a low temperature, and then initiate polymerization at a lower temperature , the embedding medium after polymerization has a refractive index that matches the biological tissue, thereby realizing the transparent embedding of the biological tissue, and at the same time, due to the low polymerization temperature, the fluorescence of the fluorescent protein remains good, so the biological tissue of the present invention is transparent The chemical resin embedding method can not only realize the transparency of biological tissues, but also ensure that the fluorescence of fluorescent proteins in biological tissues remains good during the polymerization reaction without quenching, creating good conditions for fluorescence imaging.

(3) The transparent resin embedding method of the present invention finally obtains a set of overall technical solutions for biological tissue resin embedding and transparency through careful selection of polymerized monomers, initiators, and preferred embedding conditions. Finally, the transparent resin embedding of biological tissue is realized, and the fluorescence of biological tissue is kept good. The transparent biological tissue is applied to cutting imaging to increase the cutting thickness and shorten the time required for whole brain imaging.

Description of drawings

Fig. 1 is the comparison figure of rat brain before (a) and after treatment (b) in different gradients of tetrahydrofuran-water mixed solutions and dichloromethane;

Figure 2 is an effect diagram of ordinary rat brain placed in (a) PBS solution and (b) embedded (polymerized) with benzyl methacrylate monomer;

Fig. 3 is an effect diagram of ordinary rat brain placed (a) in PBS solution and (b) embedded (polymerized) with 2-acrylic acid-2-hydroxy-3-phenoxyacrylic acid monomer.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The tissue transparency methods of the prior art can only be carried out in solution media, which cannot be applied to cutting imaging; while the traditional tissue embedding technology can be applied to cutting imaging, but because the embedding tissue is opaque, the Z-axis imaging depth during cutting imaging The technical problems of small size and long imaging time, the present invention creatively provides a resin embedding method for light-transparent rat brain tissue, using embedding medium for resin embedding of biological tissue, and the refractive index of biological tissue is related to the embedding method. Resin embedding of biological tissue is achieved by matching the refractive index of the embedding medium to achieve light transparency.

The selection of polymerized monomers is the key point of the present invention, and the conditions that meet the requirements of polymerized monomers are: the polymerized monomers are triggered by an initiator, and in the system where biological tissues exist, the polymerized monomers formed after the polymerization reaction occurs. The refractive index of the object must match that of the biological tissue. After a large number of basic experiments and creative work, the applicant of the present invention has obtained benzyl methacrylate, 2-acrylic acid-2-methyl-2-phenoxyethyl ester and/or 2-acrylic acid-2-hydroxyl -3-Phenoxypropyl ester is a viable polymerizable monomer that meets the requirements of the present invention. The initiator is an initiator that can initiate polymerization at a relatively low temperature, such as azobisisoheptanonitrile.

The resin embedding method of light-transparent mouse brain tissue provided by the present invention specifically comprises the following steps:

(1) Degreasing the biological tissue fixed by chemical method to obtain the degreased biological tissue; chemical fixation can be fixed by formaldehyde solution; degreasing treatment can be carried out in different gradients of tetrahydrofuran-water mixture .

(2) Soak the degreased biological tissue obtained in step (1) in the polymerized monomer, then add the initiator, mix to fully dissolve the initiator, and then infiltrate at -20～0°C under the condition of avoiding light 12-24 hours to obtain the infiltrated biological tissue;

Wherein, the polymerization monomer is preferably benzyl methacrylate, 2-acrylic acid-2-methyl-2-phenoxyethyl ester and/or 2-acrylic acid-2-hydroxyl-3-phenoxypropyl ester One or more; The initiator is preferably azobisisoheptanonitrile; The amount ratio of the polymerized monomer to the initiator is 1 milliliter: 1 to 6 milligrams, that is, 1 to 6 milligrams of the polymerized monomer per milliliter. the initiator.

(3) Polymerize the infiltrated biological tissue obtained in step (2) at 35-45°C for 12-48 hours, so that the polymerized monomers are polymerized under the initiation of the initiator to form a polymer, wherein the initiator initiates the polymerization The refractive index of the polymer after monomer polymerization is matched with that of the biological tissue, so that light-transparent biological tissue with different hardness can be obtained.

In general, the higher the polymerization reaction temperature, the faster the polymerization rate and the higher the degree of polymerization. However, in practical applications, it is necessary to consider the irreversible damage to fluorescent proteins in tissues caused by excessively high temperatures. In the prior art, the resin embedding of biological tissues generally selects the polymerization temperature at 50-70°C, which is relatively high, and it is easy to cause irreversible damage to the fluorescent protein and reduce or quench the fluorescence, which is not conducive to fluorescence imaging. Aiming at this problem, the present invention selects azobisisoheptanonitrile as the initiator, and the initiator can effectively initiate the polymerization of the monomer at a relatively low temperature (35-45° C.). Therefore, the resin embedding method of the present invention can not only realize the transparency of biological tissues, but also ensure that the fluorescent properties of the fluorescent proteins in the biological tissues remain good during the polymerization reaction without quenching or reducing quenching.

The key technology of the present invention is that the embedding medium matches the refractive index of the tissue itself. If the refractive index of the resin is too high or too low, the embedded biological tissue will be opaque, and the embedding medium is a polymer formed by polymerization of monomers initiated by an initiator. In the prior art, the light-transparency treatment of biological tissue is usually performed in a solution medium, and it is only necessary to find a chemical reagent that matches the refractive index of the biological tissue to realize the transparentization of the biological tissue. However, solution media cannot be used in cutting fluorescence imaging. However, the present invention is not to transparentize biological tissue in a solution medium, but to find a resin embedding method for optically transparent biological tissue for the purpose of fluorescence cutting and imaging, in order to realize the biological tissue after resin embedding. If the reagent matching the refractive index of biological tissue is selected as the polymerized monomer, the refractive index of the polymerized monomer will change after polymerization under the trigger of the initiator. It is not feasible to use monomers and initiators. The polymerized monomers and initiators selected in the present invention can ensure that the embedding medium formed after polymerization matches the refractive index of the biological tissue itself, so the transparent embedding process of the biological tissue is finally guaranteed , applied to cutting fluorescence imaging, the cutting thickness increases, and the imaging time is greatly increased compared with the prior art.

The following are examples:

Example 1

A resin embedding method for phototransparent mouse brain tissue, comprising the steps of:

(1) Place the rat brain fixed with formaldehyde solution in a tetrahydrofuran-water mixture (with a tetrahydrofuran content of 50%) for 12 hours; then replace the solution with a mixed solution with a tetrahydrofuran ratio of 80% for 1 hour; continue to replace until 1 h in pure THF, twice. Then place the treated rat brain in methylene chloride for half an hour (as shown in Figure 1);

(2) Add the monomer benzyl methacrylate dropwise on the basic alumina (200-300 mesh) column chromatography column prepared in advance, flow out under the pressure of the air pump, and remove the polymerization inhibitor therein;

(3) In a serum bottle with a volume of 10ml, soak the treated rat brain in benzyl methacrylate monomer (2ml) (polymerization monomer), then add the initiator azobisisoheptanonitrile (4mg ) to fully dissolve it. Then wrap the bottle with tinfoil for shading, store it at -10°C for 24 hours for permeation treatment, transfer it to a 40°C oven for heating and polymerization for 1 day, you can see that the monomers are polymerized and become solid, tissue and embedding medium When the refractive index of the tissue matches, the tissue becomes transparent.

Fig. 1 is the comparison figure of mouse brain before (a) and after treatment (b) in the tetrahydrofuran-water mixed solution of different gradients and dichloromethane; After methyl chloride treatment, there were obvious changes in the brain volume of rats.

Figure 2 is a comparison of treated rat brain soaked in benzyl methacrylate monomer before and after polymerization. It can be seen from Figure 2 that the mouse brain cannot penetrate light before it is processed; after embedding, the mouse brain in the resin has an excellent transmittance to light. The transparent mouse brain prepared in this embodiment can greatly improve the depth of imaging, increase the thickness of cutting, and shorten the time required for whole brain imaging.

Example 2

Transparent embedding was performed using 2-acrylic acid-2-hydroxy-3-phenoxyacrylic acid as monomer.

(1) Place the rat brain fixed with formaldehyde solution in a tetrahydrofuran-water mixture (with a tetrahydrofuran content of 50%) for 12 hours; then replace the solution with a mixed solution with a tetrahydrofuran ratio of 80% for 1 hour; continue to replace until 1 h in pure THF, twice. Then place the treated rat brain in methylene chloride for half an hour (as shown in Figure 1);

(2) Add the monomer 2-acrylic acid-2-hydroxyl-3-phenoxyacrylic acid dropwise on the basic alumina (200-300 mesh) column chromatography column prepared in advance, and flow out under the pressure of the air pump, Remove the polymerization inhibitor therein;

(3) The rat brain after transparent treatment was placed in a serum bottle with a capacity of 10 ml. Soak the treated rat brain in 2-acrylic acid-2-hydroxyl-3-phenoxyacrylic acid monomer (2ml) (polymerizable monomer), then add initiator azobisisoheptanonitrile (4mg) to make fully dissolved. Then the bottle was wrapped with tinfoil for shading, and stored at -10°C for 24 hours for permeation treatment. Under the condition of water bath, the temperature was gradually increased from 20°C (the temperature increase range was 5°C), and heated to 25, 30, 35, and 40°C step by step, and the constant temperature was maintained for 6 hours in each stage, and then continued to be heated at 40°C for 6 hours. It can be seen from this that the monomers are polymerized and become solid, and when the refractive index of the tissue and the embedding medium match, the tissue becomes transparent, and at the same time, the mouse brain changes from milky white opaque before polymerization to transparent, as shown in Fig. Figure 3 shows.

In the transparent embedding technique of mouse brain, the key is to match the refractive index of the embedding medium with the tissue itself. Resin with a refractive index that is too high or too low will make the embedded mouse brain opaque. Matching refractive index refers to the same or close refractive index, the closer the two refractive indices are, the higher the degree of transparency. Therefore, any simple changes or modified designs of the design structure and idea of the present invention will fall within the scope of protection of the present invention.

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (8)

1. A resin embedding method for light-transparent biological tissue, characterized in that, adopting an embedding medium to carry out the resin embedding of biological tissue, the refractive index of the biological tissue matches the refractive index of the embedding medium, In this way, resin embedding of light-transparent biological tissues is realized, and the embedding medium is a polymer formed by polymerization of monomers initiated by an initiator. 2. resin embedding method as claimed in claim 1, is characterized in that, described polymerization monomer is benzyl methacrylate, 2-acrylic acid-2-methyl-2-phenoxyethyl ester and/or One or more of 2-acrylate-2-hydroxy-3-phenoxypropyl esters. 3. resin embedding method as claimed in claim 1, is characterized in that, described initiator is azobisisoheptanonitrile. 4. The resin embedding method according to any one of claims 1 to 3, characterized in that it comprises the steps of: (1) Degreasing the biological tissue fixed by a chemical method to obtain the degreased biological tissue; (2) Soak the degreased biological tissue obtained in step (1) in polymerized monomers, then add an initiator, mix to fully dissolve the initiator, and then infiltrate at -20 to 0°C for 12 hours under dark conditions. ~24 hours to obtain the infiltrated biological tissue; (3) Reacting the infiltrated biological tissue obtained in step (2) at 35-45° C. for 12-48 hours to obtain a resin-embedded light-transparent biological tissue. 5. The resin embedding method according to claim 4, characterized in that, the chemical fixation in step (1) is to use formaldehyde solution for fixation. 6 . The resin embedding method according to claim 4 , wherein the specific step of degreasing treatment in step (1) is: performing degreasing treatment in different gradients of tetrahydrofuran-water mixtures. 7 . 7. The resin embedding method according to claim 4, characterized in that, the dosage ratio of the polymerized monomer to the initiator in step (2) is 1 milliliter: 1 to 6 milligrams. 8. The resin embedding method according to claim 4, characterized in that the reaction temperature in step (3) is 40°C.