Embedding mold, preparation method of embedding sample and imaging method of biological tissue

Abstract

The invention provides an embedding mold, a preparation method of an embedding sample and an imaging method of biological tissue. Wherein the embedding mold comprises a bottom plate, a side surrounding plate, a side baffle and a supporting piece, the side surrounding plate is arranged on the bottom plate, and a containing cavity with a side opening is defined by the side surrounding plate; the side baffle is detachably combined with the side coaming at the side opening to form a shell structure with an embedding groove; the supporting piece is detachably mounted on the shell structure, can extend into the embedding groove from the outside of the shell structure, and is used for supporting the biological tissue to be embedded, so that the biological tissue to be embedded is suspended in the shell structure. The embedding sample obtained by virtue of the embedding mold can be stable in the whole slicing process, and the position of the biological tissue is relatively fixed, so that the imaging section integrity difference caused by the position difference of the biological tissue can be reduced, and the section imaging integrity after continuous slicing can be improved; and thus, complete and clear whole-tissue imaging can be obtained.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to an embedding mold, a method for preparing an embedded sample, and an imaging method for biological tissues. Background Art

[0002] Optical coherence tomography (OCT) is a high-resolution, non-contact three-dimensional imaging technology. By measuring the interference signal of the reference light and the sample light, it can achieve imaging at the micrometer scale in the three planes of XYZ and the millimeter-level penetration depth in the axial direction, thereby obtaining the structural information of the tissue, and is widely used in the biomedical field. At the same time, based on the birefringence characteristics of the tissue, the polarization OCT imaging technology (PSOCT) combined with polarization detection technology can further detect the polarization characteristics of the tissue, such as the birefringence characteristics of the myelin sheath in the brain tissue, thereby achieving micrometer-scale imaging of unlabeled tissue. For tissues whose size is much larger than the imaging depth, combining tissue slicing technology with continuous cross-sectional imaging technology has become an important tool for obtaining the full picture of the tissue. For this continuous cross-sectional imaging technology, vibration slicing technology is a suitable slicing method, which not only has low requirements for sample pre-processing, but also can retain the original physiological characteristics and tissue structure of the tissue to the greatest extent. For example, the equipment combining vibration slicing and OCT imaging, after completing the continuous slicing of the tissue, uses the OCT imaging equipment to obtain the cross-sectional imaging after slicing, until the slicing and cross-sectional imaging of the whole brain are completed, and then, by splicing the images of each cross section, the three-dimensional reconstruction of the complete biological tissue is achieved.

[0003] Before slicing, some supporting materials are usually used to embed the sample to ensure that the integrity of the tissue morphology and structure is maintained during the slicing process. However, the existing tissue embedding methods, such as using agarose with a mass concentration of 4%, or adding an embedding method that increases the surface adhesion of the sample (for example, sodium borohydride), do not penetrate into the sample, but only adhere to the sample on the surface of the sample, and the liquid agarose with a mass concentration of 4% has poor fluidity, and it is difficult to fill some concave and convex grooves and fine gaps on the surface of the tissue. Therefore, during the slicing process, it is easier for the sample surface and the agarose to separate, affecting the quality of the slice or section. After experiments, if the concentration is reduced to increase the fluidity of the agarose, the solidified agarose becomes soft and cannot provide sufficient hardness and support for the tissue. At the same time, sodium borohydride is a common material for hydrogen production, and there are safety hazards during the operation.

[0004] In order to obtain slices or sections of complete tissue, in the prior art, some embedding materials (e.g., agarose) are solidified before embedding as the base of the embedding block, and then the embedding material is poured again for secondary solidification. The incomplete filling and secondary solidification of the embedding material will cause the embedding material and the tissue surface to not fit tightly together. During multiple slicing processes, each time the blade moves forward, it will exert a certain pressure on the section to be cut of the embedded sample and is accompanied by lateral vibration. In the later slicing process of the tissue, the tissue sample is prone to partially loosen or even completely fall out of the embedding material, resulting in extremely uneven slices or sections, or even the inability to obtain slices or sections of the entire tissue, which in turn leads to abnormal imaging of uneven thickness slices or uneven sections and the inability to obtain the entire imaging of the complete tissue. Summary of the invention

[0005] The present application aims to at least solve the problem in the above-mentioned prior art or related art that the embedded samples are prone to partial or even complete detachment during the slicing process, resulting in uneven slicing or section surfaces, and further resulting in abnormal imaging of uneven thickness slices or uneven cross-sections and inability to obtain complete imaging of the intact tissue.

[0006] To achieve the above-mentioned purpose, the first aspect embodiment of the present application provides an embedding mold, wherein the embedding mold includes: a bottom plate, a side surrounding plate, a side baffle plate and a support member, wherein the bottom plate serves as a supporting part, the side surrounding plate is arranged on the bottom plate, and encloses to form a accommodating cavity with a side opening; the side baffle plate is detachably combined with the side surrounding plate at the side opening to form a shell structure with an embedding groove; the support member is detachably mounted on the shell structure, and can extend from the outside of the shell structure to the embedding groove, and is used to support the biological tissue to be embedded, so that the biological tissue to be embedded is suspended inside the shell structure.

[0007] In some embodiments, the embedding mold also includes a limiting member, which is detachably connected to the shell structure, can extend laterally into the embedding groove, and is located above the support member so that the biological tissue to be embedded can be limited between the support member and the limiting member.

[0008] In some embodiments, the side panel has a plurality of support member connecting holes formed on two opposite side surfaces respectively, and the support members are a plurality of support rods, and both ends of the plurality of support rods can be respectively inserted into the support member connecting holes corresponding to the two side surfaces of the side panel to form a support surface in the embedded groove.

[0009] In some embodiments, the side panel has a plurality of limiting holes formed on two opposite side surfaces, the limiting holes are arranged above the supporting member connecting hole, the limiting member is a plurality of limiting rods, and the two ends of the plurality of limiting rods can be respectively inserted into the limiting holes corresponding to the two side surfaces of the side panel to limit the biological tissue to be embedded above the biological tissue to be embedded.

[0010] In some embodiments, the embedding mold further includes a plurality of hole plugging plugs, which can cooperate with the support member connecting holes and the limiting holes to block unused support member connecting holes and the limiting holes.

[0011] In some embodiments, a card slot is provided on one of the side panel and the side baffle, and a card protrusion is provided on the other of the side panel and the side baffle, and the card protrusion can cooperate with the card slot so that both sides of the side baffle can be detachably connected to the side panel.

[0012] In some embodiments, the shell structure is made of silicone, and a mounting groove is provided on the bottom plate, and the lower end of the side baffle can be inserted into the mounting groove.

[0013] According to a second aspect of the present application, a method for preparing an embedded sample for optical coherence tomography is provided, wherein the preparation method utilizes the embedding mold described above, and the preparation method comprises: combining a side baffle and a side enclosure plate and enclosing them with a bottom plate to form a shell structure having an embedding groove; installing a support member on the shell structure; placing a biological tissue to be embedded on the support member so that the biological tissue to be embedded is suspended inside the shell structure; pouring a gelatin solution into the embedding groove to immerse the biological tissue to be embedded; and when the gelatin solution solidifies to a semi-solidified state, withdrawing the support member, and then, when it is completely solidified, removing the embedding mold, and performing a cross-linking treatment to obtain an embedded sample.

[0014] In some embodiments, the performing of the cross-linking treatment includes: after removing the embedding mold, placing the embedded sample in a cross-linking agent for cross-linking treatment, thereby obtaining an embedded sample in which the encapsulated gelatin is denatured and hardened into a solid.

[0015] In some embodiments, the embedding mold also includes a limiting member, and the supporting member and the limiting member are respectively detachably connected to the shell structure and extend laterally into the embedding groove. The preparation method also includes: after the biological tissue to be embedded is set on the supporting member, the limiting member is connected to the shell structure and is located above the biological tissue to be embedded, and the biological tissue to be embedded is limited above the biological tissue to be embedded.

[0016] In some embodiments, the preparation method further includes: when the gelatin solution solidifies and reaches a semi-solid state, removing the limiting component from the shell structure.

[0017] According to the third aspect of the present application, a method for cross-sectional imaging of biological tissue is provided, wherein the method comprises: embedding the biological tissue using the above-mentioned preparation method to obtain an embedded sample; performing continuous sectioning and cross-sectional imaging of the embedded sample; and reconstructing the image of the embedded sample based on the data of each cross-sectional imaging to obtain an overall image of the biological tissue.

[0018] According to the embedding mold, the method for preparing the embedded sample, and the method for imaging the biological tissue provided by the embodiments of the present application, a support is provided to fix the position of the biological tissue to be embedded before embedding, specifically to fix the position of the biological tissue to be embedded before filling the embedding material, so that the biological tissue to be embedded can be suspended in the shell structure and does not completely occupy the lateral space of the embedding groove, thereby reserving space for the smooth perfusion of the embedding material. In this way, in the corresponding method for preparing the embedded sample, the embedding material can be filled without filling the biological tissue to be embedded, and the embedding material can smoothly enter the embedding groove located at the bottom of the biological tissue to be embedded, so that the biological tissue to be embedded can be completely embedded through a one-time perfusion, thereby forming a compact and integrated embedded sample. The integrated embedded sample obtained with the aid of the embedding mold is more stable in structure, and the biological tissue and the embedding material are tightly combined. When the embedded sample is sliced ​​or continuously imaged after slicing, the embedded sample can be firmly fixed in the embedding material during the slicing process, thereby ensuring that the slice thickness or the section surface is uniform, and thus obtaining complete and clear tissue imaging, providing a more reliable and accurate basis for subsequent pathological diagnosis, scientific research, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will be described by taking the embedding of mouse brain and the use of PSOCT imaging technology to test the embedding effect as an embodiment or the accompanying drawings showing the embedding examples in the prior art as an example. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 It is a structural schematic diagram of the embedding mold provided by the present invention;

[0021] Figure 2 It is a schematic structural diagram of a side baffle in an embedding mold provided by the present invention;

[0022] Figure 3 It is a schematic structural diagram of the side panels and the bottom panel in the embedding mold provided by the present invention;

[0023] Figure 4 It is a schematic structural diagram of a support member in an embedding mold provided by the present invention;

[0024] Figure 5 It is a structural schematic diagram of a position-limiting member in an embedding mold provided by the present invention;

[0025] Figure 6 It is a schematic diagram of the structure of the hole plug in the embedding mold provided by the present invention;

[0026] Figure 7 It is a schematic structural diagram of the embedding mold provided by the present invention during demoulding;

[0027] Figure 8 It is a comparison diagram of PSOCT imaging of the embedded mouse brain obtained by the embedding mold and embedding method provided by the present invention and the embedded mouse brain slice obtained by the prior art, wherein A is the XY plane and YZ plane of PSOCT imaging of the embedded mouse brain section obtained by the prior art, and B is the XY plane and YZ plane of PSOCT imaging of the embedded mouse brain section obtained by the embedding mold and embedding method provided by the present invention;

[0028] Fig. 9 is a schematic diagram of mouse brain embedding after embedding using the embedding mold and embedding method provided by one embodiment of the present invention;

[0029] Fig.10 It is a comparison diagram of the whole-brain PSOCT imaging reconstruction effects of the mouse brain obtained using the embedding molds and embedding methods provided in the present invention and the prior art, wherein A is an imaging reconstruction effect diagram of the embedded mouse brain sample obtained using the prior art, and B is an imaging reconstruction effect diagram of the mouse brain sample embedded using the present invention.

[0030] Reference numerals:

[0031] 10. Shell structure; 11. Bottom plate; 111. Mounting slot; 12. Side baffle; 121. Card slot; 13. Side panel; 131. Support member connecting hole; 132. Limiting hole; 133. Limiting line; 134. Card convex; 21. Support member; 22. Limiting member; 30. Hole plug; 100. Embedded sample. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are 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.

[0033] The terms "center", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "horizontal", "top", "bottom", "inside", "outside", etc. in the specification and claims of the present invention indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present invention, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as limiting the present invention.

[0034] In 3D imaging technology, the sliced ​​section is the basic unit for constructing a complete tissue image. Each sliced ​​section carries information about the tissue at a specific level, which is integrated in the subsequent image reconstruction process to form a comprehensive understanding of the tissue.

[0035] However, when the unevenness of the sliced ​​section reaches a value equal to or greater than the slice thickness, it means that a large amount of imaging information has been lost during the slicing process. Specifically, the unevenness will cause the sliced ​​section to fail to completely cover the tissue in some areas, resulting in information loss in the Z direction. This information loss cannot be compensated by simple interpolation or patching in subsequent image reconstruction, because the lost information is substantial and cannot be recovered from other sliced ​​section imaging.

[0036] For complete and lossless imaging of tissues, this information loss is undoubtedly fatal. It destroys the continuity and integrity of the image, making the reconstructed image unable to accurately reflect the true structure of the tissue. In addition, this information loss may also lead to misunderstanding or misjudgment of the tissue structure, thus having a negative impact on scientific research or clinical diagnosis.

[0037] In order to obtain images of intact biological tissues, the inventors of the present application have discovered that this problem can be solved by forming a more stable and better-bonded embedded sample.

[0038] According to the present application, an embedding mold is provided that can prepare an integrated and dense embedded sample. The biological tissue in the embedded sample is completely fitted with the embedding material, and the biological tissue can be firmly fixed in the embedding material during the slicing process, reducing the probability of the biological tissue falling off, so as to ensure that each time the sample is sliced ​​by the vibration slicing technology, the section surface is uniform and complete, providing guarantee for the imaging of all tissues.

[0039] According to a first aspect of the present application, there is provided an embedding mold, wherein: Figures 1 to 6As shown, the embedding mold includes a base plate 11, a side panel 13 and a side baffle 12. The side panel 13 is arranged on the base plate 11 and encloses a accommodating cavity with a side opening. The side baffle 12 is detachably combined with the side panel 13 at the side opening to seal the accommodating cavity to form a shell structure 10 with an embedding groove. The support member 21 is detachably mounted on the shell structure 10, and can extend from the outside of the shell structure 10 to the embedding groove and has a predetermined distance between the base plate 11. The support member 21 is used to support the biological tissue to be embedded before the gelatin is completely solidified, so that the biological tissue to be embedded is suspended inside the shell structure 10.

[0040] According to the embedding mold provided by the embodiment of the present application, by setting the support member 21, it is used to fix the position of the biological tissue to be embedded before embedding, specifically to fix the position of the biological tissue to be embedded before filling the embedding material, so that the biological tissue to be embedded can be suspended in the shell structure 10 and does not completely occupy the lateral space of the embedding groove, and space is reserved for the smooth perfusion of the embedding material. In this way, in the preparation method of the corresponding embedded sample 100, the embedding material can be injected without avoiding the biological tissue to be embedded, and the embedding material can smoothly enter the embedding groove located at the bottom of the biological tissue to be embedded, so that the biological tissue to be embedded can be completely embedded (covered) by a one-time perfusion, thereby forming a compact and integrated embedded sample 100. Compared with the method in which part of the embedding material needs to be solidified in advance as a base in the traditional method, the present application does not need to use part of the embedding material solidified as a base, thereby avoiding the problem of loose bonding between the base and the subsequent embedding material. The integrated embedded sample 100 obtained with the aid of the embedding mold is more stable in structure, and the biological tissue is tightly combined with the embedding material. When the embedded sample is sliced ​​or continuously imaged after slicing, the biological tissue can be firmly fixed in the embedding material during the slicing process, thereby ensuring that the slice thickness or the section is uniform, and thus obtaining complete and clear tissue imaging, providing a more reliable and accurate basis for subsequent pathological diagnosis, scientific research, etc.

[0041] In the embodiment of the present application, the biological tissue to be embedded refers to a biological tissue sample that has been fixed. Specifically, a fixative (such as 4% paraformaldehyde) can be used to soak fresh biological tissue for a certain period of time (3 hours to 12 hours) to maintain the morphology and structure of the biological tissue. Here, paraformaldehyde can be dissolved in lipid substances (such as the phospholipid bilayer of cells) and chemically cross-linked with amino groups in cells to form a cross-linked network, maintain the three-dimensional structure of cells and tissues, and prevent organelles from dissolving, autolyzing or deforming.

[0042] In the embodiment of the present application, the biological tissue to be embedded is a biological tissue whose size is much larger than the imaging depth of the imaging. Due to the limited imaging depth, it is unrealistic to directly image large biological tissues (biological tissues whose size is much larger than the imaging depth). Therefore, it is necessary to use biological tissue slicing technology to continuously slice the biological tissue sample, image the continuous sections after slicing, and sequentially splice each section image, so as to gradually construct a three-dimensional structural image of the entire tissue sample.

[0043] In the embodiment of the present application, the embedding mold includes a bottom plate 11, a side panel 13 and a side baffle 12. The bottom plate 11, as the basic part of the embedding mold, can provide a stable support surface and ensure the stability of the entire mold during use. The side panel 13 is arranged on the edge of the bottom plate 11 and extends upward. The side panel 13 and the bottom plate 11 can enclose a receiving cavity with a side opening, and the receiving cavity can be blocked or opened to form an embedding groove for accommodating biological tissues and embedding agents to be embedded. Specifically, the side baffle 12 is used to be detachably combined with the side panel 13 at the side opening, so that the operator can easily open or close the embedding groove when needed, thereby improving the convenience and flexibility of operation. When the side baffle 12 is installed in place, it can block the side opening of the receiving cavity, thereby providing a closed environment for filling the fluid embedding material, ensuring the smooth perfusion of the fluid embedding material. When the side baffle 12 is removed, the side opening of the accommodating cavity can be opened, so that the embedded sample can be smoothly and conveniently separated from the shell structure, avoiding the situation where the embedded sample is difficult to remove due to its small volume.

[0044] In the embodiment of the present application, the shell structure 10 has an embedding groove. Here, the shell structure 10 is a structure composed of a bottom plate 11, a side panel 13 and a side baffle 12. The shell structure 10 can be formed into any shape. As an example, the shell structure 10 can be a cube or a cylinder. The present application does not make too many restrictions on this. The embedding groove refers to the accommodating space for filling the embedding material. The volume size of the embedding groove is not less than the volume of the biological tissue to be embedded, specifically not less than 1.5 times the volume of the biological tissue to be embedded. In this way, when the biological tissue to be embedded is suspended in the embedding groove, it can have a certain distance from the bottom wall, side wall and top opening of the shell structure, so as to ensure that the filled embedding material completely wraps the biological tissue to be embedded, so as to avoid problems such as incomplete embedding of the biological tissue and deformation of the biological tissue, and further improve the embedding effect. As a specific example, the volume of the biological tissue to be embedded is 15x7x11mm 3 , the size of the embedding tank is 20x20x25mm 3 .

[0045] In the embodiment of the present application, the housing structure 10 has a bottom wall and a side wall, wherein the bottom wall is formed by a bottom plate, and the side wall is composed of a side baffle plate 12 and a side enclosure plate 13, and the support member 21 is detachably mounted on the side wall of the housing structure 10, specifically, the support member 21 is detachably mounted on the side enclosure plate 13, and can extend laterally into the embedding tank and have a predetermined distance with the bottom plate 11, so that when the biological tissue to be embedded can be suspended in the housing structure 10 and does not completely occupy the lateral space of the embedding tank, space is reserved for the smooth perfusion of the embedding material. In this way, the embedding material can be smoothly perfused upward from the bottom wall, so that an integrated embedded sample can be formed.

[0046] In some embodiments, the support member 21 is detachably mounted on the housing structure 10. Before the embedding material is in a semi-solidified state, the support member 21 is used to support the biological tissue to be embedded, so as to ensure that the biological tissue maintains the correct position and shape in the embedding tank until the embedding material gradually enters a semi-solidified state. In the process from semi-solidification to complete solidification, since the biological tissue to be embedded can be held up by the embedding material in a semi-solidified state, as the embedding material transitions from a semi-solidified state to a fully solidified state, the support member 21 needs to be pulled out in time to prevent it from becoming a foreign body in the embedded sample 100 and affecting the subsequent slicing effect.

[0047] According to the present application, the size of the support 21 should be as small as possible, so as to minimize the impact on the quality of the embedded sample 100 during the subsequent extraction process.

[0048] In some embodiments, Figure 3 As shown, the support member 21 is detachably mounted on the side panel 13 of the shell structure 10. The side panel 13 is formed with a plurality of support member connection holes 131 on the opposite side surfaces, and specifically, the plurality of support member connection holes 131 are arranged at intervals in the horizontal direction. The support member 21 is a plurality of support rods, and the two ends of the plurality of support rods can be respectively inserted into the support member connection holes 131 corresponding to the two side surfaces of the side panel 13 to form a support surface in the embedding tank. The support surface has a preset distance from the bottom plate and the notch of the embedding tank, so that it can not only ensure that the biological tissue to be embedded can be stably suspended in the middle of the shell structure 10 during the embedding process, but also avoid problems such as uneven embedding or difficulty in slicing caused by the tissue in the obtained embedded sample 100 being too close to the bottom plate or the notch. In addition, the use of a plurality of support rods with a diameter of no more than 3 mm to form a support surface as the support member 21 can make the interference to the embedded sample 100 in the later extraction process minimal, and the structure is simple and easy to install and disassemble. As the embedding material gradually solidifies, the operator can easily pull out the support rods to prevent them from becoming foreign matter in the embedded sample 100, thereby ensuring the smooth progress of subsequent slicing and imaging steps and providing strong support for obtaining high-quality, integrated embedded samples 100.

[0049] According to the present application, the support rod itself is relatively small in size, so in the later extraction process, the interference to the embedded sample 100 will be minimized, thereby ensuring the high-quality embedding of the tissue. As a specific example, the support rod is adapted to the support member connection hole 131, and the support rod is columnar, specifically a stainless steel column or titanium column with a diameter of 0.5mm-1.5mm. The stainless steel column or titanium column of such size is relatively small in size, so in the later extraction process, the interference to the embedding process will be minimized, thereby ensuring the high quality of the embedded sample 100. In addition, the stainless steel column or titanium column has good corrosion resistance and great strength, and can effectively support the biological tissue to be embedded to ensure its positional stability. In addition, the stainless steel column or titanium column has a smooth surface, which can facilitate the extraction of the support member without affecting the integrity of the embedded sample 100.

[0050] In histological research, the tissues to be analyzed are different. For tissues with a large volume and an average tissue density greater than 10% gelatin solution, only the support member 21 is needed to stably position it in the shell structure 10. The support area and shape of the support member 21 can be set based on the shape and size of the tissue to ensure that the tissue will not be displaced or deformed during the embedding process. For tissues with a small volume (generally a length, width and height of more than ten millimeters) and an average tissue density not greater than 10% gelatin solution, it is easy to move or float during the filling of the embedding material.

[0051] In order to ensure the position stability of these small tissues during the embedding process, in some embodiments, the embedding mold also includes a limiter 22, which is detachably connected to the side panel 13, can extend laterally into the embedding groove, and is located above the support member 21, so that the biological tissue to be embedded can be limited between the support member 21 and the limiter 22.

[0052] In these embodiments, by setting the stopper 22, more sizes of tissues can be adapted to achieve effective position limiting of tissues of different sizes, ensuring that the step of filling the embedding material will not affect the position of the tissue, thereby not only ensuring the stability of the tissue position in a single experiment, but also maintaining the relative consistency of the tissue position in the embedded sample 100 in multiple experiments, which can greatly improve the reliability and repeatability of the experimental results. Specifically, when comparing and analyzing different samples, the tissue position is relatively fixed, and it is easier to find corresponding structures and features between different samples, which helps to establish an accurate comparison relationship between different samples, thereby revealing the differences in tissue structure and function, and drawing the most repeatable conclusions, providing strong support for further research. In addition, since the stopper 22 ensures the position stability of the tissue in the embedded sample 100, it is easier to find and observe the target structure when imaging, which can improve the efficiency of the experiment.

[0053] According to the present application, the size of the stopper 22 should be as small as possible, so as to minimize the impact on the quality of the embedded sample 100 during the subsequent extraction process.

[0054] In some embodiments, Figure 3 As shown, the limiting member 22 is detachably mounted on the side panel 13 of the shell structure 10. The side panel 13 is formed with a plurality of limiting holes 132 on the opposite side surfaces. Specifically, the plurality of limiting holes 132 are arranged at intervals in the horizontal direction. The limiting holes 132 are arranged above the support member connection hole 131. The limiting member 22 is a plurality of limiting rods. The two ends of the plurality of limiting rods can be respectively inserted into the limiting holes 132 corresponding to the two side surfaces of the side panel 13 to limit the biological tissue to be embedded above the biological tissue to be embedded. When the limiting rod is inserted into place, it will limit the movement of the biological tissue to be embedded during the embedding process, so that the position of the tissue in the embedding groove is relatively fixed, avoiding problems such as difficulty in slicing or reduction in sample quality due to positional displacement of the tissue during the embedding process.

[0055] According to the present application, the size of the limiting rod itself is relatively small, so in the later extraction process, the interference to the embedded sample 100 will be minimized, thereby ensuring the high quality of the sample. As a specific example, a plurality of limiting rods with a diameter of no more than 3 mm are used to form a pressing surface as the limiting member 22, which can make the interference to the embedded sample 100 in the later extraction process minimal, and the structure is simple and easy to install and disassemble. In the process of the embedding material gradually solidifying, the operator can easily pull out the limiting rods to prevent them from becoming foreign matter in the embedded sample 100, thereby ensuring the smooth progress of the subsequent slicing and imaging steps, and providing strong support for obtaining high-quality, integrated embedded samples 100.

[0056] As a specific example, the limiting rod is adapted to the limiting hole 132, and preferably, the limiting rod is interference fit with the limiting hole 132. The limiting rod is columnar, specifically a stainless steel column or titanium column with a diameter of 1mm-3mm and a length of 20mm-50mm. The stainless steel column or titanium column of such size is relatively small in size, so in the later extraction process, the interference with the embedding process will be minimized, thereby ensuring the high quality of the embedded sample 100. In addition, the stainless steel column and the titanium column have good corrosion resistance and great strength, and can be used as the limiting member 22 for limiting the biological tissue to be embedded, so as to ensure its position stability. In addition, the surface of the stainless steel column and the titanium column is smooth, which can facilitate the extraction of the limiting member 22 without affecting the integrity of the embedded sample 100.

[0057] In some embodiments, the embedding mold further includes a plurality of plugging holes 30, which can cooperate with the support member connecting holes 131 and the limiting holes 132, and are used to plug the unused support member connecting holes 131 and the limiting holes 132 before injecting the embedding material (e.g., gelatin), that is, plug the holes into which the limiting members 22 and the support members 21 are not inserted.

[0058] As a specific example, the support member connection hole 131 and the limiting hole 132 are circular through holes. Figure 6 As shown, the hole plug 30 has a stepped shape, including a first column section and a second column section. The first column section can be embedded in the circular through hole, closely fitting the hole wall of the circular through hole to form an effective plug. The second column section can cover the outside of the circular through hole to plug the circular through hole, avoiding the problem of difficulty in removal caused by excessive embedding, and improving the reliability and durability of the hole plug 30. In addition, when in use, the second column section can also be used as a gripping portion for user operation.

[0059] Specifically, the first column segment and the second column segment are cylindrical, the diameter of the first column segment is smaller than the diameter of the second column segment, the diameter of the first column segment is 0.5mm-1.5mm, and the diameter of the second column segment can be 3mm-4mm. As a specific example, Figure 6 As shown, the size of the first column section is φ1mm and the length is 2mm, the size of the second column section is φ3mm and the length is 3mm, the first column section is inserted into the support member connecting hole 131 and the limiting hole 132 from the outside of the side panel 13, and the second column section covers the outside of the support member connecting hole 131 and the limiting hole 132 to seal the support member connecting hole 131 and the limiting hole 132.

[0060] In some embodiments, a card slot 121 is provided on one of the side panel 13 and the side baffle 12, and a card protrusion 134 is provided on the other of the side panel 13 and the side baffle 12. The card protrusion 134 can cooperate with the card slot 121, so that both sides of the side baffle 12 can be detachably connected to the side panel 13.

[0061] Refer to 1 to Figure 3 The side baffle 12 is provided with a slot 121, and the side panel 13 is provided with a protrusion 134, which can cooperate with the slot 121, so that the two sides of the side baffle 12 can be detachably connected to the side panel 13. The side panel 13 is formed into a U-shaped frame, which is composed of three vertical panels and is designed as an integral whole. It is formed as a whole on the upper surface of the bottom plate and together with the bottom plate, it forms an enclosure structure with a accommodating cavity with a side opening, and the protrusions 134 are provided on the edges of the two side panels.

[0062] In these embodiments, when the embedding mold needs to be assembled, the operator only needs to align the clamping protrusion 134 on the side baffle 12 with the clamping groove 121 on the side enclosure 13 and gently push it in, so that the side enclosure 13 and the side baffle 12 can be connected in the first direction. This connection method is not only simple and easy to operate, but also ensures the close fit between the side baffle 12 and the side enclosure 13, and effectively prevents the embedding material leakage problem that may occur during the embedding process. As an example, the clamping protrusion 134 is formed on the two side walls of the side baffle 12, specifically, on the left and right walls of the side baffle 12, and the side baffle 12 is installed on the side enclosure 13 along the horizontal direction.

[0063] In some embodiments, the shell structure 10 is made of silicone. The shell structure 10 formed of silicone has good flexibility and durability. Peeling the shell structure 10 outward facilitates demolding of the embedded block. At the same time, it can ensure that the embedded block can be demolded without loss after solidification, thereby maintaining the integrity of the embedded block.

[0064] In order to further improve the sealing and stability of the detachable connection between the side panel 13 and the side baffle 12, in some embodiments, a mounting groove 111 is provided on the bottom plate 11, and the lower end of the side baffle 12 can be inserted into the mounting groove 111. On the one hand, the sealing of the detachable connection between the side panel 13 and the side baffle 12 can be ensured, and on the other hand, the side panel 13 and the side baffle 12 can be limited from a second direction different from the first direction, thereby ensuring the stability and reliability of the detachable connection between the side panel 13 and the side baffle 12.

[0065] In some embodiments, the embedding mold further includes a limit line 133 extending in the horizontal direction. Figure 3 As shown, the limit line 133 is set on the side panel 13 and is located above the limit hole 132 of the installation limit member 22. In this way, when the embedding material is poured, the limit line 133 can easily remind the operator that the pouring operation is completed to ensure that the embedding material can completely cover the biological tissue to be embedded and ensure the integrity of the embedded sample 100.

[0066] According to a second aspect of the present application, a method for preparing an embedded sample 100 is provided, wherein the preparation method uses the embedding mold described above, and the preparation method comprises:

[0067] Step S101 , combining the side baffle plate 12 and the side enclosure plate 13 and enclosing them with the bottom plate 11 to form a shell structure 10 with an embedded groove.

[0068] Step S102 : installing the support member 21 on the shell structure 10 .

[0069] Step S103 , placing the biological tissue to be embedded on the support 21 , so that the biological tissue to be embedded is suspended in the shell structure 10 .

[0070] Step S104, a gelatin solution is poured into the embedding tank once to immerse the biological tissue to be embedded. Specifically, the gelatin solution is poured until the limit line 133 of the shell structure.

[0071] Step S105 , when the gelatin solution solidifies to a semi-solidified state, the support member 21 is pulled out, and then, when it is completely solidified, the embedding mold is removed, and a cross-linking treatment is performed to obtain the embedded sample 100 .

[0072] According to the preparation method of the embedded sample 100 provided in the embodiment of the present application, it can be obtained by a one-time perfusion of a flowing embedding material (gelatin) followed by solidification. This operation method simplifies the embedding step, and at the same time helps to form an integrated solidification of the tissue and the sample, thereby improving the embedding effect. After the gelatin is perfused, it is placed in a paraformaldehyde solution for cross-linking, and the resulting solidified embedded sample 100 can meet the requirements for subsequent slicing and post-slicing cross-sectional PSOCT imaging.

[0073] In addition, gelatin is used as an embedding material. Compared with other materials such as agarose, gelatin has better fluidity. During the embedding process, gelatin can easily fill the tiny gaps and concave and convex grooves on the tissue surface to better adapt to the concave and convex texture of the tissue surface, and achieve complete fit between the tissue sample and the embedding material, thereby improving the embedding quality. During the entire process of tissue sectioning, it can be firmly fixed in the embedding material, reducing the probability of tissue shedding, ensuring that the section is uniform and complete, and providing guarantee for the imaging of all tissues.

[0074] In some embodiments, performing the cross-linking treatment includes: placing the embedded sample 100 in a paraformaldehyde solution for reaction, and the gelatin molecules can be chemically cross-linked by the amine-aldehyde condensation reaction between the paraformaldehyde and gelatin. Specifically, the embedded sample is placed in a cross-linking agent for cross-linking treatment, so that the gelatin forms a fixed insoluble cross-linked product, and the cross-linking agent is selected from at least one of paraformaldehyde, formaldehyde and glutaraldehyde, and the concentration of the cross-linking agent is 3%-6%, for example, 4%, and the cross-linking treatment time is 24h-48h, so as to wrap the embedded sample in a solid state after the gelatin is denatured and hardened. Through the cross-linking treatment, the stability and hardness of the embedded sample can be enhanced, and the sample can be prevented from being deformed or broken during the subsequent sectioning process. In addition, by performing the cross-linking treatment with paraformaldehyde, etc., the initial embedded sample 100 can also be fixed for a second time, so that the embedded sample and the gelatin form a tighter and more stable combination, which is suitable for sectioning, thereby ensuring the flatness of the section, and then obtaining each complete section imaging.

[0075] In some embodiments, the embedding mold also includes a limiting member 22, and the support member 21 and the limiting member 22 are respectively detachably connected to the shell structure 10 and extend laterally into the embedding groove. The preparation method also includes: after the biological tissue to be embedded is set on the support member, the limiting member 22 is connected to the shell structure 10 and is located above the biological tissue to be embedded, and the biological tissue to be embedded is limited above the biological tissue to be embedded.

[0076] In some embodiments, the preparation method further includes: when the gelatin solution solidifies to a semi-solidified state, the stopper 22 is removed from the shell structure 10. That is, when the gelatin is strong enough to support the biological tissue to be embedded, but has not yet completely solidified, the stopper 22 is removed from the shell structure 10 to ensure that the removal of the stopper 22 will not cause damage to the tissue or gelatin, thereby ensuring that the removal of the stopper 22 will not affect the quality of the embedded sample.

[0077] According to the present application, waiting for the gelatin solution to solidify includes placing the embedded block containing the sample in a refrigerator at 3° C.-10° C. to solidify the gelatin solution until the gelatin is completely solidified.

[0078] According to the present application, the mass concentration of the gelatin solution is between 10% and 15%. The gelatin solution in this concentration range can gel and form a jelly. The jelly state can provide a stable supporting environment for biological samples and effectively prevent the movement or deformation of the samples during processing and imaging. Secondly, gelatin, as a natural polymer material, has good biocompatibility and degradability, and has little effect on the physiological activity of biological samples.

[0079] According to the third aspect of the present application, a method for imaging biological tissue is provided, wherein the method comprises: embedding the biological tissue using the above-mentioned preparation method to obtain an embedded sample; performing continuous section imaging or section imaging on the embedded sample; and reconstructing the image of the embedded sample based on the imaging data of each section to obtain an overall image of the biological tissue.

[0080] Taking continuous section imaging as an example, continuous section imaging of embedded samples includes using an imaging device to image the embedded sample with a first preset thickness, obtaining a first section imaging, and then performing a slicing operation to remove the surface layer of the embedded sample that is less than a second preset thickness. Then imaging the second preset thickness, obtaining a second section imaging, and then performing the next slicing operation to remove the surface layer of the embedded sample that is less than the first preset thickness. This is done until the required number of slices or the biological tissue sample is completely sliced, wherein the first preset thickness and the second preset thickness are not greater than the imaging depth of the imaging device. Since the thickness of each slice is less than the imaging thickness that has been imaged the current time, the first section imaging and the second section imaging have a certain overlapping area, which is conducive to image reconstruction and obtains the overall imaging of the biological tissue.

[0081] Specifically, the data of each cross-sectional imaging includes the first, second, ..., N groups of target cross-sectional imaging, where N is a positive integer. Based on the first, second, ..., N groups of target cross-sectional imaging, the image of the embedded sample is reconstructed using image processing software to obtain an overall imaging of the biological tissue.

[0082] Hereinafter, the beneficial effects of the inventive concept will be described in conjunction with specific examples.

[0083] In order to further demonstrate the significant effects of the embedding mold and embedding method provided by the present invention, mouse brain samples embedded in different ways were sliced ​​under the same slicing parameters, and PSOCT imaging was performed on the sliced ​​sections using the same imaging parameters to illustrate the beneficial effects of the embedding mold and embedding method provided by the present invention.

[0084] The method for preparing the embedded sample containing mouse brain in the Example group is carried out by the following steps:

[0085] Step 201) Install the side baffle 12 into the installation groove 111 of the bottom plate 11, and assemble the protrusion 134 on the side panel 13 into the groove 121 of the side baffle 12 to form a shell structure 10 with an embedded groove.

[0086] Step 202) Use 1XPBS (1 times phosphate buffer) to prepare a gelatin flowing liquid with a mass concentration of 10%, then place it in a constant temperature hot water bath above 50-60 degrees Celsius for continuous shaking to ensure that the gelatin particles are evenly dissolved in PBS (phosphate buffer) and the number of bubbles is reduced.

[0087] Step 203) Insert multiple support rods into the corresponding support member connecting holes 131 on the two side panels of the side panel 13 in sequence, and put the biological tissue to be embedded into it, ensuring that the tissue is suspended in the shell structure 10 and does not contact the inner wall of the shell structure 10, and then insert multiple limiting rods into the corresponding limiting holes 132 on the two side panels of the side panel 13 in sequence to limit the biological tissue to be embedded above the biological tissue to be embedded; use plugging plugs to seal the unused support member connecting holes 131 and limiting holes 132.

[0088] Step 204) The prepared gelatin is slowly poured as an embedding material along a corner of the embedding mold until it reaches the limit line 133 on the side panel 13.

[0089] Step 205) Move the embedding mold and the sample to a 4° refrigerator to accelerate the solidification of the gelatin, and pull out the support rod and the limit rod when the gelatin reaches a semi-solidified state.

[0090] Step 206) Place the mold containing the embedded block of the sample in a 4° refrigerator again until the embedded block is completely solidified, remove the side baffle 12, and Figure 7 As shown, the two side panels of the side panel 13 are gently pried apart to separate the side panels from the surrounding solidified gelatin, and then taken out to obtain the initial embedded sample containing the mouse brain.

[0091] Step 207 ) The taken out initial embedded sample containing the mouse brain is immersed in a 4% PFA solution for cross-linking treatment at room temperature for 2 days to obtain an embedded sample 100 .

[0092] Step 208) Take out the embedded sample 100 in PFA, immerse it in a container filled with 1XPBS, and place the immersion container on a shaker for washing for one day.

[0093] Step 209) After being washed with 1XPBS, the embedded sample 100 is then immersed in new 1XPBS.

[0094] Step 210) The embedded sample 100 containing the mouse brain is glued to the slice buffer tank using 502 glue, and the slicing speed is 0.1 mm / s, the slice thickness is 200 μm, and the coronal section of the brain tissue is sliced ​​using Leica VT 1200s and a self-designed knife holder. The embedded sample is sliced ​​and the cross-section PSOCT imaging is performed after the section is cut. The PSOCT imaging of the tissue section at the same slicing position is selected as follows Figure 8 As shown in B.

[0095] The preparation method of the embedded sample containing mouse brain of Comparative Example 1 is carried out by the following steps:

[0096] Step 301) adopts the shell structure 10 with embedded grooves in the embodiment group, and uses plugging plugs to seal the support member connecting hole 131 and the limiting hole 132.

[0097] Step 302) solidifying agarose with a weight concentration of 4% to form a base.

[0098] Step 303) Agarose with a mass volume concentration of 4% is slowly poured along a corner of the embedding mold until it reaches the limit line 133 of the side panel 13.

[0099] Step 304) Move the embedding mold and the sample to a 4° refrigerator to accelerate the solidification of the agarose. When the embedding block is completely solidified, remove the side baffle 12. Figure 7 As shown, the two side panels of the side panel 13 are gently pried apart to separate the side panels from the solidified agarose on the periphery, and then taken out to obtain the initial embedded sample containing the mouse brain.

[0100] Step 305) The embedded sample 100 containing the mouse brain is glued to the slice buffer tank using 502 glue, and the slicing speed is 0.1 mm / s, the slice thickness is 200 μm, and the coronal section of the brain tissue is sliced ​​using Leica VT 1200s and a self-designed knife holder to obtain PSOCT imaging of the cross section of the mouse brain tissue after slicing. The imaging effect is as follows: Figure 8 As shown in A.

[0101] The preparation method of the embedded sample containing mouse brain of Comparative Example 2 is carried out by the following steps:

[0102] The embedding material in the example group was changed to agarose with a weight concentration of 4%, and the embedding sample of comparative example 2 was formed by the same manufacturing method as that of the example group.

[0103] The preparation method of the embedded sample containing mouse brain of Comparative Example 3 is carried out by the following steps:

[0104] The cross-linking step was not performed (ie, step 207 to step 209 were not performed), and the embedded sample of comparative example 3 was formed by the same manufacturing method as that of the embodiment group.

[0105] Conclusion: Through multiple verifications, the inventors found that the mold of the present application combined with gelatin as the embedding material, the embedded samples obtained in multiple embodiments are not prone to cracks and have few gaps under the same slicing parameters, and can obtain a flat cross section, thereby ensuring the integrity of the tissue. However, the embedded samples in Comparative Examples 1, 2 and 3 have cracks and gaps in the slices obtained under the same slicing parameters, and the integrity of the tissue is poor.

[0106] The imaging of the embedded samples obtained in one of the embodiments and the comparative example 1 after sectioning will be specifically described below.

[0107] like Figure 8 As shown in A and B, A is a cross-sectional image of the embedded mouse brain sample of Comparative Example 1, and B is a similar cross-sectional image of the embedded mouse brain sample of the embodiment of the present application. The figure shows the imaging in the XY and YZ directions, and the image display resolution is 10x10x10mm 3 .

[0108] from Figure 8It can be seen from Figure A that the mouse brain sample embedded in agarose in Comparative Example 1 has cracks generated during the slicing process at positions "I" and "II", and there are gaps in the position "III" caused by incomplete filling of the uneven surfaces of the sample surface. After measurement, the YZ image in Figure A shows that the unevenness that occurred during the slicing process was as high as 200 microns, which is equivalent to the set slice thickness. This degree of unevenness means that a large amount of imaging information has been discarded at the slice section, which poses a severe challenge to the redundant imaging requirements of later images in the Z direction. For research that pursues intact and lossless imaging of tissues, this defect will undoubtedly be fatal, and it will directly damage the integrity and accuracy of the imaging data.

[0109] As can be seen from FIG. B , the sliced ​​surface of the slices obtained from the embedded sample of the embodiment is flat, and there are no embedding voids in the XY plane, cracks generated during the slicing process, etc.

[0110] Fig. 9 It is a schematic diagram of a mouse brain after embedding using the embedding mold and embedding method provided by an embodiment of the present invention.

[0111] Fig.10 The following is a comparison of the PSOCT imaging reconstruction effects of the whole mouse brain obtained using the embedding molds and embedding methods provided in the present invention and the prior art. A is the imaging reconstruction effect of the embedded mouse brain sample in Comparative Example 1, and B is the imaging reconstruction effect of the embedded mouse brain sample in the embodiment of the present application. The image display resolution is 10x10mm 2 .

[0112] from Fig.10 As can be seen in Figure A, the agarose-embedded mouse brain sample in Comparative Example 1 caused imaging information loss in multiple locations, seriously damaging the integrity and accuracy of the imaging data. As can be seen from Figure B, the reconstruction of the entire mouse brain tissue is complete, with no surface structural loss.

[0113] Through 3D reconstruction technology, researchers can observe the structure of the mouse brain from multiple angles, which is crucial for understanding the relationship between the three-dimensional structure and function of the brain, and provides technical support for research in fields such as neuroscience and brain science.

[0114] In summary, the embedding mold and method provided by the present invention significantly improve the imaging quality of mouse brain samples, avoid the common cracks and gaps in traditional embedding methods, ensure the integrity of the tissue and the accuracy of imaging, which is of great significance for subsequent image reconstruction and analysis, and can provide more reliable data support. The embedding process provided by the present invention is simple and clear to operate, and the embedding success rate is extremely high.

[0115] Although the embodiments of the present application have been described in detail above, those skilled in the art may make various modifications and variations to the embodiments of the present application without departing from the spirit and scope of the present application. However, it should be understood that, in the opinion of those skilled in the art, these modifications and variations will still fall within the spirit and scope of the embodiments of the present application defined by the claims.

Claims

1. An embedding mold, characterized in that: The embedding mold comprises: Bottom plate (11); A side panel (13) is arranged on the bottom panel (11) and encloses a receiving cavity with a side opening; A side baffle (12), the side baffle (12) being detachably combined with the side enclosure plate (13) at the side opening to form a shell structure (10) having an embedded groove; A support member (21) is detachably mounted on the shell structure (10) and can extend from the outside of the shell structure (10) to the embedding groove, and is used to support the biological tissue to be embedded so that the biological tissue to be embedded is suspended inside the shell structure (10).

2. The embedding mold according to claim 1, characterized in that: The embedding mold further comprises a limiting member (22), which is detachably connected to the shell structure (10), can extend laterally into the embedding groove, and is located above the supporting member (21), so that the biological tissue to be embedded can be limited between the supporting member (21) and the limiting member (22).

3. The embedding mold according to claim 2, characterized in that: The side panel (13) is formed with a plurality of support member connection holes (131) on two opposite side surfaces, and the support member (21) is a plurality of support rods, and the two ends of the plurality of support rods can be respectively inserted into the support member connection holes (131) corresponding to the two side surfaces of the side panel (13) to form a support surface in the embedding groove.

4. The embedding mold according to claim 3, characterized in that: The side panel (13) is formed with a plurality of limiting holes (132) on two opposite side surfaces, respectively. The limiting holes (132) are arranged above the support member connection hole (131). The limiting member (22) is a plurality of limiting rods, and the two ends of the plurality of limiting rods can be respectively inserted into the limiting holes (132) corresponding to the two side surfaces of the side panel (13), so as to limit the biological tissue to be embedded above the biological tissue to be embedded.

5. The embedding mold according to claim 4, characterized in that: The embedding mold further comprises a plurality of hole plugging plugs (30), which can cooperate with the support member connecting holes (131) and the limiting holes (132) to block unused support member connecting holes (131) and the limiting holes (132).

6. The embedding mold according to claim 1, characterized in that: A card slot (121) is provided on one of the side panel (13) and the side baffle (12), and a card protrusion (134) is provided on the other of the side panel (13) and the side baffle (12). The card protrusion (134) can cooperate with the card slot (121), so that both sides of the side baffle (12) can be detachably connected to the side panel (13).

7. The embedding mold according to claim 1, characterized in that: The shell structure (10) is made of silica gel, the bottom plate (11) is provided with a mounting groove (111), and the lower end of the side baffle (12) can be inserted into the mounting groove (111).

8. A method for preparing an embedded sample, characterized in that: The preparation method utilizes the embedding mold according to claim 1, and the preparation method comprises: The side baffle plate (12) and the side enclosure plate (13) are combined and enclosed with the bottom plate (11) to form a shell structure (10) having an embedded groove; Installing a support member (21) on the housing structure (10); Placing the biological tissue to be embedded on the support member (21) so that the biological tissue to be embedded is suspended inside the shell structure (10); Filling the embedding tank with a gelatin solution to submerge the biological tissue to be embedded; When the gelatin solution solidifies to a semi-solidified state, the support member (21) is pulled out, and then, when it is completely solidified, the embedding mold is removed, and a cross-linking treatment is performed to obtain an embedded sample.

9. The preparation method according to claim 8, characterized in that: The cross-linking treatment comprises: placing the embedded sample in a cross-linking agent for cross-linking treatment, so that the gelatin forms a fixed insoluble cross-linked product, the cross-linking agent is selected from at least one of paraformaldehyde, formaldehyde and glutaraldehyde, the concentration of the cross-linking agent is 3%-6%, and the cross-linking treatment time is 24h-48h, thereby obtaining an embedded sample (100) wrapped in gelatin that is denatured and hardened to be solid.

10. The preparation method according to claim 8, characterized in that: The embedding mold further comprises a stopper (22), the support member (21) and the stopper (22) are respectively detachably connected to the housing structure (10) and extend laterally into the embedding groove, and the preparation method further comprises: After the biological tissue to be embedded is arranged on the support member, the limiting member (22) is connected to the shell structure (10) and is located above the biological tissue to be embedded, so as to limit the biological tissue to be embedded above the biological tissue to be embedded.

11. The preparation method according to claim 10, characterized in that: The preparation method further comprises: When the gelatin solution solidifies and reaches a semi-solidified state, the limiting component is removed from the shell structure.

12. A method for imaging biological tissue, characterized in that: The method: Embedding the biological tissue using the preparation method according to any one of claims 8 to 11 to obtain an embedded sample; Performing continuous section imaging or section imaging on the embedded sample; The image of the embedded sample is reconstructed according to each imaging data to obtain the overall imaging of the biological tissue.

Images