Embedding mold

Abstract

An embedding mold is provided. 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 utility model relates to the field of biomedical technology, specifically relates to a embedding mould. BACKGROUND

[0002] Coherent tomography technology (OCT) is a kind of high-resolution, non-contact three-dimensional imaging technology, and the XYZ three plane micron scale, axial millimeter level penetration depth imaging can be realized by measuring the interference signal of reference light and sample light, and the structure information of tissue is obtained, and it is widely used in biomedical field.At the same time, based on the birefringence characteristics of tissue, combined with polarization detection technology, polarization OCT imaging technology (PSOCT) can further detect the polarization characteristics of tissue, such as the birefringence characteristics of myelin in brain tissue, so that micron scale imaging of label-free tissue can be realized.For the tissue with size far larger than the imaging depth, combined with tissue section technology and continuous section imaging technology, it has become an important tool to obtain the overall situation of tissue.For this kind of continuous section imaging technology, vibration section technology is a suitable section method, which not only has lower requirements for sample pre-processing, but also can maximize the preservation of the original physiological characteristics and tissue structure of the tissue, for example, combined with vibration section and OCT imaging equipment, after completing the continuous section of the tissue, the OCT imaging equipment is used to obtain the section imaging of the section, until the section and section imaging of the whole brain are completed, then the image of each section is spliced to realize the three-dimensional reconstruction of the complete biological tissue.

[0003] Before sectioning, some support materials are usually used to embed the sample to ensure the integrity of the tissue morphology and structure during sectioning.However, the existing tissue embedding method, such as using agarose with a mass concentration of 4%, or adding a reagent (such as sodium borohydride) to increase the adhesion of the sample surface on this basis, since the agarose does not penetrate into the sample, only adheres to the sample surface, and the liquid agarose with a mass concentration of 4% has poor flowability, it is difficult to fill the concave and convex grooves, fine gaps on the surface of the tissue, so during sectioning, the sample surface and agarose are more likely to separate, affecting the section or section quality.Through experiments, if the concentration is reduced to improve the flowability of agarose, the solidified agarose becomes soft and cannot provide enough hardness and support for the tissue, at the same time, sodium borohydride is a commonly used material for hydrogen production, and there is a safety hazard in the operation process.

[0004] In order to obtain complete tissue slices or sections, the prior art also coagulates some embedding material (for example, agarose) as a bottom support of the embedding block before embedding, and then performs secondary coagulation by pouring embedding material again. The incomplete filling and secondary coagulation of the embedding material cause the embedding material and the tissue surface not to be closely integrated. In the process of multiple slicing, the knife blade applies a certain pressure to the section to be cut of the embedded sample with transverse vibration each time the knife blade is advanced. In the later slicing process of the tissue, the tissue sample is prone to be partially loosened or even completely detached from the embedding material, so that the slices or sections are extremely uneven, and even the complete imaging of the entire tissue cannot be obtained due to the abnormal imaging of the uneven thickness slices or uneven sections. Practical new type content

[0005] The utility model aims at least solve prior art or related technical problems in the prior art or related technical problems that embedding sample is prone to be partially loosened or even completely detached in the slicing process, so that the slices or sections are uneven, and even the complete imaging of the entire tissue cannot be obtained due to the abnormal imaging of the uneven thickness slices or uneven sections.

[0006] To achieve the above object, the first aspect embodiment of the utility model provides an embedding mold, wherein the embedding mold comprises a bottom plate, a side wall plate, a side baffle and a support, wherein the bottom plate is a support part, the side wall plate is arranged on the bottom plate and forms a containing cavity with a side opening by enclosing; the side baffle is detachably combined with the side wall plate at the side opening to form a shell structure with an embedding groove; the support is detachably installed on the shell structure and can extend from the outside of the shell structure to the embedding groove, for supporting the biological tissue to be embedded, so that the biological tissue to be embedded is suspended in the inside of the shell structure.

[0007] In some embodiments, the embedding mold further comprises a limiting piece, which is detachably connected with the shell structure and can extend into the embedding groove in the transverse direction and is located above the support, so that the biological tissue to be embedded can be limited between the support and the limiting piece.

[0008] In some embodiments, the side wall plate is formed with a plurality of support connecting holes on the opposite two sides, respectively, and the support is a plurality of support rods, both ends of the plurality of support rods can be inserted into the corresponding support connecting holes on the two sides of the side wall plate, respectively, to form a support surface in the embedding groove.

[0009] In some embodiments, the side wall plate is formed with a plurality of limiting holes on the two opposite sides respectively, the limiting holes are arranged above the support connecting holes, the limiting pieces are a plurality of limiting rods, and the two ends of the plurality of limiting rods can be inserted into the corresponding limiting holes on the two sides of the side wall plate respectively to limit the biological tissue to be embedded above the biological tissue to be embedded.

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

[0011] In some embodiments, one of the side wall plate and the side baffle is provided with a clamping groove, and the other of the side wall plate and the side baffle is provided with a clamping convex, the clamping convex can cooperate with the clamping groove, so that the two sides of the side baffle can be detachably connected with the side wall plate.

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

[0013] According to the second aspect of the present application, a preparation method of 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 the side baffle and the side wall plate to form a shell structure with an embedding groove together with the bottom plate; mounting a support on the shell structure; placing a biological tissue to be embedded on the support so that the biological tissue to be embedded is suspended in the interior of the shell structure; pouring a gelatin solution into the embedding groove to immerse the biological tissue to be embedded; extracting the support when the gelatin solution is solidified to a semi-solid state, then, taking off the embedding mold after complete solidification, and then performing cross-linking treatment to obtain an embedded sample.

[0014] In some embodiments, the cross-linking treatment comprises: placing the embedded sample in a cross-linking agent for cross-linking treatment after taking off the embedding mold, so as to obtain an embedded sample in which the gelatin is denatured and hardened into a solid.

[0015] In some embodiments, the embedding mold further comprises a limiting piece, the support and the limiting piece are detachably connected with the shell structure respectively and extend into the embedding groove in the transverse direction, and the preparation method further comprises: connecting the limiting piece to the shell structure and locating above the biological tissue to be embedded after the biological tissue to be embedded is arranged on the support, and limiting the biological tissue to be embedded above the biological tissue to be embedded.

[0016] In some embodiments, the preparation method further comprises: when the gelatin solution is coagulated and reaches a semi-coagulated state, disassembling the limiting member from the shell structure.

[0017] According to a third aspect of the present application, a method for imaging a section of a biological tissue is provided, wherein the method comprises: embedding a biological tissue by using the preparation method to obtain an embedded sample; continuously sectioning and imaging the embedded sample; and reconstructing an image of the embedded sample according to data of each sectioned and imaged sample to obtain an overall image of the biological tissue.

[0018] According to the embedding mold, the preparation method of the embedded sample, and the imaging method of the biological tissue, the position of the biological tissue to be embedded is fixed before embedding by the support, and the position of the biological tissue to be embedded is fixed 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 horizontal space of the embedding groove, and space is reserved for smooth filling of the embedding material. Therefore, in the corresponding preparation method of the embedded sample, the embedding material can be filled without 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 by one-time filling, thereby forming a tightly structured and integrated embedded sample. The integrated embedded sample obtained by using the embedding mold is more stable in structure, and the biological tissue and the embedding material are tightly combined. When the embedded sample is sectioned or continuously sectioned and imaged, the embedded sample can be stably fixed in the embedding material during the sectioning process, so that the sectioning thickness or the sectioning surface can be uniform, and complete and clear tissue imaging can be obtained, thereby providing more reliable and accurate basis for subsequent pathological diagnosis, scientific research, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the present application or the prior art, the following will be described by taking the embedding of a mouse brain and the experimental verification of the embedding effect by using the PSOCT imaging technology as an embodiment or a drawing of an embedding example in the prior art. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other embodiments according to the drawings without creative labor.

[0020] Figure 1 is a structural schematic view of an embedding mold provided by the present application;

[0021] Figure 2 is a structural schematic view of a side baffle in the embedding mold provided by the present application;

[0022] Figure 3is a structure schematic view of the side wall and the bottom plate in the embedding mold provided by the utility model;

[0023] Figure 4 is a structure schematic view of the supporting piece in the embedding mold provided by the utility model;

[0024] Figure 5 is a structure schematic view of the limiting piece in the embedding mold provided by the utility model;

[0025] Figure 6 is a structure schematic view of the hole plug in the embedding mold provided by the utility model;

[0026] Figure 7 is a structure schematic view of the embedding mold when demolding provided by the utility model;

[0027] Figure 8 is a contrastive drawing of the PSOCT imaging of the section of the embedded mouse brain after slicing of the embedded mouse brain obtained by the embedding mold and the embedding method provided by the utility model and the embedded mouse brain obtained by the prior art, wherein A is the XY plane and the YZ plane of the PSOCT imaging of the section of the embedded mouse brain obtained by the prior art, and B is the XY plane and the YZ plane of the PSOCT imaging of the section of the embedded mouse brain obtained by the embedding mold and the embedding method provided by the utility model;

[0028] Figure 9 is an embedding schematic view of the embedded mouse brain after being embedded by the embedding mold and the embedding method provided by an embodiment of the utility model;

[0029] Figure 10 is a contrastive drawing of the reconstruction effect of the PSOCT imaging of the whole mouse brain obtained by the embedding mold and the embedding method provided by the utility model and the prior art, wherein A is the reconstruction effect drawing of the imaging of the embedded mouse brain sample obtained by the prior art, and B is the reconstruction effect drawing of the imaging of the embedded mouse brain sample using the utility model.

[0030] Reference signs:

[0031] 10, shell structure; 11, bottom plate; 111, mounting groove; 12, side baffle; 121, clamping groove; 13, side wall; 131, supporting piece connecting hole; 132, limiting hole; 133, limiting line; 134, clamping convex; 21, supporting piece; 22, limiting piece; 30, hole plug; 100, embedded sample. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the utility model clearer, the technical scheme in the utility model will be described clearly and completely in combination with the drawings in the utility model below. Obviously, the described embodiments are part of the embodiments of the utility model, rather than all the embodiments. Based on the embodiments in the utility model, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the utility model.

[0033] The orientation or positional relationship indicated by the terms "center", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "horizontal", "top", "bottom", "inner", "outer" and the like in the description and claims of the utility model is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the utility model, and does not indicate or imply that the indicated device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the utility model.

[0034] In three-dimensional imaging technology, the section after slicing is the basic unit for constructing a complete tissue image. Each section after slicing 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 section after slicing is not planar and reaches or exceeds the slice thickness, it means that a large amount of imaging information has been lost during the slicing process. Specifically, the non-planar section will not be able to completely cover the tissue in some areas, resulting in information loss in the Z direction. This information loss cannot be compensated for by simple interpolation or repair in subsequent image reconstruction, because the lost information is substantial and cannot be recovered from other section imaging.

[0036] For complete and intact imaging of the tissue, 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 can also lead to misunderstanding or misjudgment of the tissue structure, thereby negatively affecting scientific research or clinical diagnosis.

[0037] In order to obtain an image of a complete biological tissue, the inventors of the utility model found that the problem can be solved by forming a more stable and better combined embedding sample.

[0038] According to the embedding mold, the biological tissue in the embedding sample is completely attached to the embedding material, the biological tissue can be stably fixed in the embedding material during a slicing process, and the probability of biological tissue falling off is reduced, so that after a sample slice is obtained by vibration slicing technology each time, the section is uniform and complete, and the imaging of all tissues is ensured.

[0039] According to the first aspect of the utility model, provide a kind of embedding mold, wherein, as shown in Figures 1 to 6 As shown, the embedding mold includes a bottom plate 11, a side wall plate 13 and a side baffle 12. The side wall plate 13 is arranged on the bottom plate 11 and encloses a receiving cavity with a side opening. The side baffle 12 is detachably combined with the side wall plate 13 at the side opening to block the receiving cavity and form a shell structure 10 with an embedding groove. A support 21 is detachably mounted on the shell structure 10 and can extend from the outside of the shell structure 10 into the embedding groove with a predetermined distance from the bottom plate 11. The support 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 in the embodiments of the utility model, the support 21 is arranged 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. The biological tissue to be embedded can be suspended in the shell structure 10 without completely occupying the horizontal space of the embedding groove, leaving space for smooth infusion of the embedding material. In the corresponding preparation method of the embedding sample 100, the embedding material can be injected without the biological tissue to be embedded, and the embedding material can smoothly enter the embedding groove at the bottom of the biological tissue to be embedded to completely embed (cover) the biological tissue to be embedded by one-time infusion, thereby forming a compact and integrated embedding sample 100. Compared with the method of using part of the embedding material to solidify as a base in the conventional method, the utility model does not need to use part of the embedding material to solidify as a base, thereby avoiding the problem of loose combination between the base and the subsequent embedding material. The integrated embedding sample 100 obtained by the embedding mold is more stable in structure, and the biological tissue is tightly combined with the embedding material. During slicing or continuous section imaging after slicing of the embedding sample, the biological tissue can be stably fixed in the embedding material during slicing, thereby ensuring the uniformity of the slice thickness or section, and further obtaining complete and clear tissue imaging, which provides more reliable and accurate basis for subsequent pathological diagnosis, scientific research, etc.

[0041] In the embodiment of the utility model, the biological tissue to be embedded refers to the biological tissue sample which has been treated by fixation. Specifically, the fresh biological tissue can be soaked in a fixative (such as 4% paraformaldehyde) for a certain time (3-12 hours) to maintain the morphology and structure of the biological tissue. Here, the paraformaldehyde can dissolve in lipid substances (such as the phospholipid bilayer of cells) and chemically cross-link with the amino groups in the cells to form a cross-linked network, thereby maintaining the three-dimensional structure of the cells and tissues and preventing the organelles from dissolving, autolysis or deforming.

[0042] In the embodiment of the utility model, the biological tissue to be embedded has a size much larger than the imaging depth of imaging. Since the imaging depth is limited, it is unrealistic to directly image the large biological tissue (the biological tissue with a size much larger than the imaging depth). Therefore, the biological tissue sample needs to be continuously sliced by using the biological tissue slicing technology, and the continuous sections after slicing are imaged to sequentially splice each section image, thereby gradually constructing a three-dimensional structure image of the entire tissue sample.

[0043] In the embodiment of the utility model, the embedding mold comprises a bottom plate 11, a side wall plate 13 and a side baffle 12. The bottom plate 11 serves as the basic part of the embedding mold and can provide a stable support surface to ensure the stability of the entire mold during use. The side wall plate 13 is arranged on the edge of the bottom plate 11 and extends upward. The side wall plate 13 and the bottom plate 11 can be enclosed to form a containing cavity with a side opening. The containing cavity can be blocked or opened to form an embedding groove for containing the biological tissue to be embedded and the embedding agent. Specifically, the side baffle 12 is used to detachably combine with the side wall plate 13 at the side opening. In this way, 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 containing cavity, thereby providing a closed environment for the embedding material in a fluid state and ensuring the smooth perfusion of the embedding material in a fluid state. When the side baffle 12 is removed, the side opening of the containing cavity can be opened, so that the embedded sample can be smoothly and conveniently separated from the shell structure, thereby avoiding the difficulty in taking out the embedded sample due to its small size.

[0044] In the embodiment of the utility model, the shell structure 10 has embedding groove, here, the shell structure 10 is the structure that is jointly formed by bottom plate 11, side wall 13 and side baffle 12, the shell structure 10 can form any shape, as an example, the shell structure 10 can be cubic or cylindrical, the utility model does not make too much limitation to this. Embedding groove refers to the accommodation space for filling embedding material, the volume size of embedding groove is not less than the volume of the biological tissue to be embedded, specifically not less than 1.5 times of 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, to avoid problems such as incomplete embedding of biological tissue and deformation of biological tissue, further improve the embedding effect. As a specific example, the volume of the biological tissue to be embedded is 15x7x11mm 3 , the size of embedding groove is 20x20x25mm 3 .

[0045] In the embodiment of the utility model, the shell structure 10 has bottom wall and side wall, here, the bottom wall is formed by the bottom plate, the side wall is composed of the side baffle 12 and the side wall 13, the support 21 is detachably installed on the side wall of the shell structure 10, specifically, the support 21 is detachably installed on the side wall 13, and can extend transversely into the embedding groove and have a predetermined distance between the bottom plate 11, in this way, when the biological tissue to be embedded can be suspended in the shell structure 10 and does not completely occupy the transverse space of the embedding groove, space is reserved for smooth infusion of embedding material. In this way, the embedding material can be smoothly infused from the bottom wall upwards, so as to facilitate the formation of an integrated embedding sample.

[0046] In some embodiments, the support 21 is detachably installed on the shell structure 10. Before the embedding material is in a semi-solid state, the support 21 is used to support the biological tissue to be embedded, to ensure that the biological tissue maintains the correct position and shape in the embedding groove until the embedding material gradually enters the semi-solid state. During the process from semi-solid to complete solidification, since the biological tissue to be embedded can be lifted by the embedding material in the semi-solid state, the support 21 needs to be withdrawn in time during the transition of the embedding material from the semi-solid state to the complete solidification state, which can avoid becoming a foreign matter in the embedding sample 100 and affecting the subsequent slicing effect.

[0047] According to the utility model, the size of the support 21 should be as small as possible, in this way, to minimize the impact on the quality of the embedding sample 100 during the later withdrawal process.

[0048] In some embodiments, as Figure 3As shown, the support 21 is detachably mounted on the side wall 13 of the housing structure 10. The side wall 13 is formed with a plurality of support connecting holes 131 on the opposite sides, specifically, the plurality of support connecting holes 131 are arranged in the horizontal direction, and the support 21 is a plurality of support rods, the two ends of the plurality of support rods can be inserted into the corresponding support connecting holes 131 on the two sides of the side wall 13, respectively, to form a support surface in the embedding groove. The support surface has a predetermined distance from the bottom plate and the slot of the embedding groove, so that not only can the biological tissue to be embedded be stably suspended in the middle of the housing structure 10 during the embedding process, but also the problem of uneven embedding or difficulty in slicing caused by the tissue being too close to the bottom plate or the slot in the obtained embedding sample 100. In addition, using a plurality of support rods with a diameter of not greater than 3mm to form the support surface as the support 21 can minimize the interference with the embedding sample 100 during the later extraction process, and the structure is simple, easy to install and detach. During the gradual solidification of the embedding material, the operator can easily extract the support rods to avoid them becoming foreign matter in the embedding sample 100, thereby ensuring the smooth progress of the subsequent slicing and imaging steps, and providing strong support for obtaining high-quality and integrated embedding samples 100.

[0049] According to the utility model, the support rod itself is small in size, so the interference with the embedding sample 100 during the later extraction process will be minimized, thereby ensuring the high-quality embedding of the tissue. As a specific example, the support rod is adapted to the support connecting hole 131, and the support rod is columnar, specifically a stainless steel column or a titanium column with a diameter of 0.5-1.5mm. The stainless steel column or titanium column with such a size is small in size, so the interference with the embedding process during the later extraction process will be minimized, thereby ensuring the high-quality embedding of the embedding sample 100. In addition, the stainless steel column or titanium column has good corrosion resistance and high strength, and can effectively support the biological tissue to be embedded to ensure its positional stability. In addition, the surface of the stainless steel column or titanium column is smooth, which can facilitate the extraction of the support and avoid affecting the integrity of the embedding sample 100.

[0050] In histological research, the tissues to be analyzed are different, and for tissues with a larger volume and an average density greater than 10% gelatin solution, only the support 21 is needed to stably position the tissue in the housing structure 10. The support area and shape of the support 21 can be set based on the shape and size of the tissue to ensure that the tissue does not displace or deform during the embedding process. For tissues with a small volume (generally with a length, width and height of several millimeters) and an average 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 the small tissues during the embedding process, in some embodiments, the embedding mold further comprises a limiting piece 22, which is detachably connected with the side wall 13, can extend into the embedding groove in the transverse direction and is located above the support piece 21, so that the biological tissue to be embedded can be limited between the support piece 21 and the limiting piece 22.

[0052] In these embodiments, by arranging the limiting piece 22, more sizes of tissues can be adapted, so that effective limiting of tissues of different sizes can be realized, and it is ensured that the step of filling the embedding material will not affect the position of the tissues, thereby not only ensuring the stability of the position of the tissues in a single experiment, but also maintaining the relative consistency of the position of the tissues 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 relative fixation of the position of the tissues can make it easier to find corresponding structures and features between different samples, thereby helping to establish an accurate comparison relationship between different samples, thereby revealing the differences in tissue structure and function, and drawing the most repeatable conclusion, thereby providing strong support for further research. In addition, since the limiting piece 22 ensures the position stability of the tissues in the embedded sample 100, when imaging, the target structure can be more easily found and observed, and the experimental efficiency can be improved.

[0053] According to the utility model, the size of the limiting piece 22 should be as small as possible, so as to minimize the impact on the quality of the embedded sample 100 during the later extraction process.

[0054] In some embodiments, as shown in Figure 3 The side wall 13 is formed with a plurality of limiting holes 132 on the opposite two side surfaces, and the plurality of limiting holes 132 are arranged in the horizontal direction, and the limiting holes 132 are arranged above the support piece connecting hole 131. The limiting piece 22 is a plurality of limiting rods, and the two ends of the plurality of limiting rods can be inserted into the corresponding limiting holes 132 on the two side surfaces of the side wall 13, so as to limit the biological tissue to be embedded above the biological tissue to be embedded. When the limiting rod is inserted in place, the movement of the biological tissue to be embedded during the embedding process is limited, so that the position of the tissue in the embedding groove is relatively fixed, and problems such as difficulty in slicing or decrease in sample quality caused by position deviation of the tissue during the embedding process are avoided.

[0055] According to the utility model, the limiting rod is small in size, so that the interference to the embedding sample 100 during the later extraction process is reduced to the minimum, thereby ensuring the high quality of the sample. As a specific example, the limiting rod with a diameter of not greater than 3 mm is used to form the pressing surface as the limiting piece 22, so that the interference to the embedding sample 100 during the later extraction process is minimal, and the structure is simple, easy to install and disassemble. During the gradual solidification of the embedding material, the operator can conveniently extract the limiting rod to avoid the limiting rod becoming a foreign matter in the embedding sample 100, thereby ensuring the smooth progress of the subsequent slicing and imaging steps and providing strong support for obtaining the high-quality and integrated embedding sample 100.

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

[0057] In some embodiments, the embedding mold further comprises a plurality of hole plugs 30, which can be matched with the support piece connecting hole 131 and the limiting hole 132, and are used to plug the unused support piece connecting hole 131 and limiting hole 132 before the embedding material (for example, gelatin) is injected.

[0058] As a specific example, the support piece connecting hole 131 and the limiting hole 132 are circular through holes. Correspondingly, as shown in Figure 6 the outer shape of the hole plug 30 is set to be stepped, including a first column segment and a second column segment. The first column segment can be embedded in the circular through hole and tightly fit the hole wall of the circular through hole to form effective plugging. The second column segment can cover the outside of the circular through hole to plug the circular through hole, avoiding the problem of difficulty in removal due to excessive embedding, and improving the reliability and durability of the hole plug 30. In addition, the second column segment can also be used as a gripping part for user operation during use.

[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, and the diameter of the first column segment is 0.5 mm-1.5 mm, and the diameter of the second column segment can be 3 mm-4 mm. As a specific example, as shown in Figure 6As shown, the first column segment has a size of φ1mm and a length of 2mm, and the second column segment has a size of φ3mm and a length of 3mm. The first column segment is inserted into the support connecting hole 131 and the limiting hole 132 from the outside of the side wall 13, and the second column segment covers the outside of the support connecting hole 131 and the limiting hole 132 to seal the support connecting hole 131 and the limiting hole 132.

[0060] In some embodiments, one of the side wall 13 and the side baffle 12 is provided with a clamping groove 121, and the other is provided with a clamping protrusion 134. The clamping protrusion 134 can cooperate with the clamping groove 121, so that the two sides of the side baffle 12 can be detachably connected with the side wall 13.

[0061] Referring to FIGS. 1 to Figure 3 The side baffle 12 is provided with a clamping groove 121, and the side wall 13 is provided with a clamping protrusion 134. The clamping protrusion 134 can cooperate with the clamping groove 121, so that the two sides of the side baffle 12 can be detachably connected with the side wall 13. The side wall 13 is formed as a U-shaped frame, composed of three vertical plates and designed integrally, and is integrally formed on the upper surface of the bottom plate to form a surrounding structure with a side opening with the bottom plate, and the clamping protrusion 134 is arranged on the edge of the two side plates.

[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 wall 13 and gently push it in, so that the side wall 13 and the side baffle 12 can be connected in the first direction. This connection method not only is simple and easy to operate, but also ensures the close fit between the side baffle 12 and the side wall 13, effectively preventing the leakage of embedding material that may occur during embedding. 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 side walls of the side baffle 12, and the side baffle 12 is installed on the side wall 13 along the horizontal direction.

[0063] In some embodiments, the shell structure 10 is made of silica gel, which has good flexibility and durability. Peeling the shell structure 10 outward facilitates the demolding of the embedding block, while ensuring that the embedding block can be demolded without damage after solidification, maintaining the integrity of the embedding block.

[0064] To further improve the sealing and stability of the detachable connection between the side wall plate 13 and the side baffle 12, in some embodiments, 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. On the one hand, the sealing of the detachable connection between the side wall plate 13 and the side baffle 12 can be ensured, and on the other hand, the side wall plate 13 and the side baffle 12 can be limited from a second direction different from the first direction, so that the stability and reliability of the detachable connection between the side wall plate 13 and the side baffle 12 can be ensured.

[0065] In some embodiments, the embedding mold further comprises a limiting line 133 extending in the horizontal direction. As shown in the figure, the limiting line 133 is arranged on the side wall plate 13 and above the limiting hole 132 of the mounting limiting piece 22. Thus, when the embedding material is poured, the limiting line 133 can remind the operator to complete the pouring operation, so as to ensure that the embedding material can completely cover the biological tissue to be embedded, and the integrity of the embedding sample 100 is ensured. Figure 3

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

[0067] Step S101, the side baffle 12 and the side wall plate 13 are combined and enclosed with the bottom plate 11 to form a shell structure 10 with an embedding groove.

[0068] Step S102, the support 21 is mounted on the shell structure 10.

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

[0070] Step S104, the gelatin solution is poured into the embedding groove at one time to immerse the biological tissue to be embedded. Specifically, the gelatin solution is poured until the limiting line 133 of the shell structure.

[0071] Step S105, when the gelatin solution is solidified to a semi-solid state, the support 21 is extracted, then the embedding mold is removed after complete solidification, and then a cross-linking treatment is performed to obtain the embedding sample 100.

[0072] The preparation method of the embedding sample 100 provided by the embodiment of the present application can be obtained by pouring the flowing embedding material (gelatin) at one time and then solidifying. This operation mode simplifies the embedding step, and at the same time, helps to form integrated solidification of the tissue and the sample, and improves the embedding effect. After pouring the gelatin, it is placed in a paraformaldehyde solution for cross-linking, and the obtained solidified embedding sample 100 can meet the requirements for subsequent slicing and section PSOCT imaging after slicing.

[0073] ​In addition, the gelatin has better fluidity than other materials such as agarose. During embedding, the gelatin is easy to fill the small gaps, concave and convex grooves on the surface of the tissue, so as to better adapt to the concave and convex texture of the tissue surface, realize the complete adhesion between the tissue sample and the embedding material, and improve the embedding quality. The tissue can be stably placed in the embedding material during the whole sectioning process, the probability of tissue falling off is reduced, the uniformity and integrity of the section are ensured, and the imaging of the whole tissue is ensured.

[0074] In some embodiments, the cross-linking treatment includes placing the embedded sample 100 in a paraformaldehyde solution for reaction. By the amine aldehyde condensation reaction between paraformaldehyde and gelatin, chemical cross-linking between gelatin molecules can be formed. Specifically, the embedded sample is placed in a cross-linking agent for cross-linking treatment, so that the gelatin forms insoluble cross-linking material. 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%, for example, 4%, and the cross-linking treatment time is 24h-48h, so that the embedded sample is wrapped by the hardened gelatin. Through the cross-linking treatment, the stability and hardness of the embedded sample can be enhanced, and the sample can be prevented from deforming or breaking during subsequent sectioning. In addition, by performing the cross-linking treatment by paraformaldehyde and the like, the initial embedded sample 100 can be fixed again, so that the embedded sample and the gelatin form a more stable and tight combination, which is suitable for sectioning, so as to ensure the flatness of the section, and then obtain imaging of each complete section.

[0075] In some embodiments, the embedding mold further comprises a limiting piece 22, the supporting piece 21 and the limiting piece 22 are respectively detachably connected with the shell structure 10 and extend into the embedding groove in the transverse direction, and the preparation method further comprises: after the biological tissue to be embedded is arranged on the supporting piece, the limiting piece 22 is connected to the shell structure 10 and located above the biological tissue to be embedded.

[0076] In some embodiments, the preparation method further comprises: when the gelatin solution is solidified to a semi-solid state, the limiting piece 22 is detached from the shell structure 10. That is, when the gelatin is strong enough to support the biological tissue to be embedded, but has not been completely solidified, the limiting piece 22 is detached from the shell structure 10, so as to ensure that the detachment of the limiting piece 22 does not damage the tissue or the gelatin, thereby ensuring that the detachment of the limiting piece 22 does not affect the quality of the embedded sample.

[0077] According to the utility model, the embedding block containing the sample is placed in a refrigerator at 3-10℃ to solidify the gelatin solution until the gelatin is completely solidified.

[0078] According to the utility model, the mass concentration of gelatin solution is 10% to 15%, the gelatin solution in the concentration range can gelate and form gelatin, and the gelatin state can provide a stable support environment for the biological sample and effectively prevent the sample from moving or deforming during processing and imaging. Secondly, as a natural polymer material, gelatin has good biocompatibility and biodegradability and has less influence on the physiological activity of the biological sample.

[0079] According to a third aspect of the utility model, an imaging method of biological tissue is provided, wherein the method: embeds biological tissue by the preparation method to obtain an embedded sample; performs continuous section surface imaging or section imaging on the embedded sample; reconstructs an image of the embedded sample according to data of each section surface imaging to obtain overall imaging of the biological tissue.

[0080] Taking continuous section surface imaging as an example, performing continuous section surface imaging on the embedded sample includes imaging a first preset thickness of the embedded sample by using an imaging device to obtain first section surface imaging, then performing a sectioning operation to remove a surface layer of the embedded sample smaller than a second preset thickness. Then, a second preset thickness is imaged to obtain second section surface imaging, and the next sectioning operation is performed to remove the surface layer of the embedded sample smaller than the first preset thickness. In this way, until the required number of sections or the biological tissue sample is completely sectioned, 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 section is smaller than the imaging thickness of the current imaging, the first section surface imaging and the second section surface imaging have a certain overlapping area, which facilitates image reconstruction to obtain overall imaging of the biological tissue.

[0081] Specifically, the data of each section surface imaging includes 1st, 2nd, …, Nth target section surface imaging, wherein N is a positive integer. Based on the 1st, 2nd, …, Nth target section surface imaging, image reconstruction of the embedded sample is realized by using image processing software to obtain overall imaging of the biological tissue.

[0082] In the following, the beneficial effects of the utility model will be described in conjunction with specific examples.

[0083] In order to further reflect the significant effect of the embedding mold and the embedding method provided by the utility model, the mouse brain samples embedded by different methods are sectioned under the same sectioning parameters, and the sectioned sections are imaged by PSOCT using the same imaging parameters, so as to illustrate the beneficial effects of the embedding mold and the embedding method provided by the utility model.

[0084] The method of preparing the embedded sample comprising a mouse brain in the example group was performed 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 clamping protrusions 134 on the side plates of the side wall plate 13 with the clamping grooves 121 of the side baffle 12 to form a shell structure 10 with an embedding groove.

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

[0087] Step 203) Insert a plurality of support rods into the corresponding support connecting holes 131 on the two side plates of the side wall plate 13 in sequence, and place the biological tissue to be embedded, ensuring that the tissue is suspended in the shell structure 10 and does not contact the inner wall of the shell structure 10, then insert a plurality of limiting rods into the corresponding limiting holes 132 on the two side plates of the side wall plate 13 in sequence to limit the biological tissue to be embedded above the biological tissue to be embedded; use a hole plug to plug the unused support connecting holes 131 and limiting holes 132.

[0088] Step 204) Pour the prepared gelatin as an embedding material along one corner of the embedding mold until the limiting line 133 on the side plate of the side wall plate 13.

[0089] Step 205) Move the embedding mold and the sample to a 4° refrigerator to accelerate the gelatin solidification, and remove the support rods and limiting rods when the gelatin reaches a semi-solid state.

[0090] Step 206) Place the mold containing the embedding block with the sample in a 4° refrigerator again until the embedding block is completely solidified, remove the side baffle 12, and as shown in Figure 7 , gently pry the two side plates of the side wall plate 13 outward so that the side plates are separated from the peripheral solidified gelatin, and then take out the initial embedding sample containing the mouse brain.

[0091] Step 207) Soak the removed initial embedding sample containing the mouse brain in a PFA solution with a concentration of 4% at room temperature for cross-linking treatment for 2 days to obtain an embedding sample 100.

[0092] Step 208) Take out the embedding sample 100 in PFA and soak it in a container containing 1XPBS, and place the soaking container on a shaker for cleaning for one day.

[0093] Step 209) After cleaning with 1XPBS, the embedding sample 100 is then soaked in new 1XPBS.

[0094] Step 210) The embedding sample 100 containing the mouse brain is adhered to the slice buffer tank using 502 glue, the brain tissue is coronally sectioned at a sectioning speed of 0.1 mm / s and a sectioning thickness of 200 microns using the Leica VT 1200s and the self-designed knife holder, the sectioned surface of the obtained embedding sample is imaged using PSOCT, and the PSOCT imaging of the tissue surface at the same sectioning position is as shown in FIG. 1B. Figure 8

[0095] The method of preparing the embedded sample comprising a mouse brain of Comparative Example 1 was performed by the following steps:

[0096] Step 301) The shell structure 10 with an embedding tank in the example group is used, and the hole plug is used to block the support hole 131 and the limiting hole 132.

[0097] Step 302) The agarose with a weight concentration of 4% is solidified to form a base support.

[0098] Step 303) The agarose with a mass-volume concentration of 4% is slowly poured along one corner of the embedding mold until the limiting line 133 of the side plate of the side wall plate 13.

[0099] Step 304) The embedding mold and the sample are moved to a 4° refrigerator to accelerate the solidification of the agarose. After the embedding block is completely solidified, the side baffle 12 is removed, as shown in FIG. 2B, the two side plates of the side wall plate 13 are gently pried outward, so that the side plates are separated from the peripheral solidified agarose, and then the initial embedding sample containing the mouse brain is obtained. Figure 7

[0100] Step 305) The embedding sample 100 containing the mouse brain is adhered to the slice buffer tank using 502 glue, the brain tissue is coronally sectioned at a sectioning speed of 0.1 mm / s and a sectioning thickness of 200 microns using the Leica VT 1200s and the self-designed knife holder, the sectioned surface of the obtained embedding sample is imaged using PSOCT, and the PSOCT imaging of the tissue surface at the same sectioning position is as shown in FIG. 1B. Figure 8

[0101] The method of preparing the embedded sample comprising a mouse brain of Comparative Example 2 was performed by the following steps:

[0102] The embedding material in the example group is replaced with agarose with a weight concentration of 4%, and the embedding sample of Comparative Example 2 is formed using the same manufacturing method as the example group.

[0103] The method of preparing the embedded sample comprising a mouse brain of Comparative Example 3 was performed by the following steps:

[0104] Steps of cross-linking treatment are not performed (i.e., steps 207-209 are not performed), and the embedding sample of Comparative Example 3 is formed using the same manufacturing method as the example group.

[0105] ​​​Conclusion: Through multiple verifications, the inventors discovered that the mold of this invention, combined with gelatin as the embedding material, resulted in embedded samples from multiple embodiments that, under the same slicing parameters, were less prone to cracking and had fewer voids, producing smooth cross-sections and thus ensuring tissue integrity. In contrast, the embedded samples in Comparative Examples 1, 2, and 3, under the same slicing parameters, exhibited cracks and voids in their slices, resulting in poor tissue integrity.

[0106] The imaging of the embedded samples obtained in one of the embodiments and Comparative Example 1 after slicing will be described in detail below.

[0107] like Figure 8 As shown in Figures A and B, A is a cross-sectional image of the embedded mouse brain sample in Comparative Example 1, and B is a similar cross-sectional image of the embedded mouse brain sample in the embodiment of this invention. The figures show imaging in both the XY and YZ directions, with an image resolution of 10x10x10mm. 3 .

[0108] from Figure 8 As shown in Figure A, the mouse brain sample embedded in agarose in Comparative Example 1 exhibits cracks generated during the slicing process at locations "I" and "II," and gaps resulting from incomplete filling of the sample surface irregularities at location "III." Measurements in Figure A's YZ images reveal an unevenness of up to 200 micrometers during slicing, comparable to the set slice thickness. This level of unevenness means that a significant amount of imaging information is lost at the slice cross-section, posing a serious challenge to the redundancy required for subsequent imaging in the Z direction. For research aiming for non-destructive imaging of tissue integrity, this defect is undoubtedly fatal, directly compromising the integrity and accuracy of the imaging data.

[0109] As can be seen from Figure B, the slices obtained from the embedded samples in the embodiment have a flat surface, and there are no embedding gaps or cracks in the XY plane.

[0110] Figure 9 This is a schematic diagram of a mouse brain embedded using the embedding mold and embedding method provided in this embodiment of the invention.

[0111] Figure 10 These are comparative images of whole-brain PSOCT imaging reconstructions of mouse brains obtained using the embedding molds and methods provided in this invention and existing technologies. In comparison, A shows the imaging reconstruction of the embedded mouse brain sample from Comparative Example 1, and B shows the imaging reconstruction of the embedded mouse brain sample from the embodiment of this invention. The image resolution is 10x10mm. 2 .

[0112] from Figure 10As can be seen in A, the use of agarose-embedded mouse brain samples in Comparative Example 1 results in missing imaging information at multiple locations, severely compromising the integrity and accuracy of the imaging data. As can be seen in B, the reconstruction of the entire mouse brain tissue is complete, with no missing surface structures.

[0113] Through three-dimensional reconstruction technology, researchers can observe the structure of the mouse brain from multiple angles, which is crucial for understanding the three-dimensional structure and functional relationship of the brain, providing technical support for research in the fields of neuroscience, brain science, etc.

[0114] In summary, the embedding mold and method provided by the present application significantly improve the imaging quality of mouse brain samples, avoiding the common problems of cracks and voids in traditional embedding methods, ensuring the integrity of the tissue and the accuracy of the 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 application is simple and easy 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 can 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 these modifications and variations will still fall within the spirit and scope of the embodiments of the present application as defined by the claims.

Claims

1. An investment mold characterized by comprising: The embedding mold comprises: a bottom plate (11); a side plate (13) arranged on the bottom plate (11) and enclosing a containing cavity with a side opening; a side stop plate (12) detachably combined with the side plate (13) at the side opening to form a shell structure (10) with an embedding groove; a support (21) detachably mounted on the shell structure (10) and capable of extending into the embedding groove from the outside of the shell structure (10) for supporting the biological tissue to be embedded so that the biological tissue to be embedded is suspended inside the shell structure (10).

2. The investment mold according to claim 1, characterized by The embedding mold further comprises a limiting piece (22) detachably connected with the shell structure (10) and capable of extending into the embedding groove in the transverse direction and located above the support (21) so that the biological tissue to be embedded can be limited between the support (21) and the limiting piece (22).

3. The investment mold according to claim 2, wherein The side plate (13) is formed with a plurality of support connecting holes (131) on opposite sides, and the support (21) is a plurality of support rods, the two ends of which can be inserted into the corresponding support connecting holes (131) on the two sides of the side plate (13) to form a support surface in the embedding groove.

4. The investment mold according to claim 3, wherein The side plate (13) is formed with a plurality of limiting holes (132) on opposite sides, which are arranged above the support connecting holes (131), and the limiting piece (22) is a plurality of limiting rods, the two ends of which can be inserted into the corresponding limiting holes (132) on the two sides of the side plate (13) to limit the biological tissue to be embedded above.

5. The investment mold according to claim 4, wherein The embedding mold further comprises a limiting line (133) extending in the horizontal direction, which is arranged on the side plate (13) and above the limiting holes (132) for mounting the limiting piece (22).

6. The investment mold of claim 4, wherein The embedding mold further comprises a plurality of hole plugs (30) capable of cooperating with the support connecting holes (131) and the limiting holes (132) for plugging unused support connecting holes (131) and limiting holes (132).

7. The investment mold of claim 4, wherein The diameter of the limiting rod is 1-3 mm, and the length is 20-50 mm; and / or, the diameter of the support rod is 1-3 mm, and the length is 20-50 mm.

8. The investment mold of claim 4, wherein The limiting rod is a stainless steel column or a titanium column; and / or, the support rod is a stainless steel column or a titanium column.

9. The investment mold of claim 1, wherein One of the side plate (13) and the side stop plate (12) is provided with a clamping groove (121), and the other is provided with a clamping convex (134), which can cooperate with the clamping groove (121) so that the two sides of the side stop plate (12) can be detachably connected with the side plate (13).

10. The investment mold of claim 1, wherein 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).

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