Clamp for preparing mass spectrum imaging sample
By combining a U-shaped frame clamp with a modified embedding agent, the problems of embedding failure and section deformation in plant sample preparation were solved, achieving efficient mass spectrometry imaging sample preparation.
Patent Information
- Application Number
- CN202520076971.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2026-01-30
- Estimated Expiration
- 2035-01-14
AI Technical Summary
Existing plant sample preparation methods often fail to produce qualified mass spectrometry imaging samples, resulting in problems such as embedding failure, slice deformation, and difficulty in obtaining slice material at specific angles.
A clamp comprising a U-shaped frame and clips was designed to fix plant tissues by limiting components, and combined with an improved embedding agent and gradient freezing procedure, to achieve efficient fixation and sectioning of plant samples.
High-quality preparation of plant samples was achieved, obtaining samples with complete cell morphology and detection planar mass spectrometry imaging at specific angles, solving the problems of embedding failure and section deformation.
Smart Images
Figure CN223856829U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model relates to mass spectrum imaging technical field especially relates to a kind of preparation mass spectrum imaging sample's fixture. BACKGROUND
[0002] Metabolomics is the study of all small molecules in the body and their changes, widely regarded as the most important omics technology in the post-genomic era, its application rapidly penetrates into disease diagnosis, pharmaceutical research and development, nutrition food science, toxicology, environmental science, botany and other fields closely related to human survival. Compared with animal samples, plant samples have huge differences in tissue structure characteristics, making it difficult to use existing spatial metabolomics sample preparation methods based on animal samples for plant samples.
[0003] Existing plant spatial metabolomics sample preparation methods, especially for fine tissues such as leaves, flowers, fruits, and root systems, often use adsorbable materials for tablet transfer or frozen section methods. However, these methods often fail to produce qualified mass spectrum imaging samples, often resulting in embedding failure, section deformation, and difficulty in obtaining specific angle section materials. To address these issues, the present application provides a fixture for preparing mass spectrum imaging samples. SUMMARY
[0004] Therefore, to solve the problems in the prior art, the present application provides a fixture for preparing mass spectrum imaging samples, characterized in that it comprises:
[0005] A main body comprising two U-shaped frames and a connecting piece, the two U-shaped frames are connected by the connecting piece to form the main body, and at least one sliding slot is provided on the inner side wall of each U-shaped frame.
[0006] A buckle is slidably connected with the sliding slot, and when the buckle is inserted into the sliding slot, the buckle and the U-shaped frame clamp and fix the plant tissue.
[0007] Further, the buckle is a U-shaped body, and a limiting assembly is provided between the buckle and the sliding slot to limit the sliding of the buckle in the sliding slot.
[0008] Further, the limiting assembly comprises a first limiting block and a second limiting block, the first limiting block is arranged on the inner wall of the sliding slot, and the second limiting block is arranged on the side of the buckle close to the sliding slot, and when the buckle and the U-shaped frame clamp and fix the plant tissue, the first limiting block limits the second limiting block.
[0009] Further, the bottom of the buckle is provided with a pressing block, the bottom of the U-shaped frame is provided with a protrusion, and a groove matching the shape and size of the protrusion is formed in the bottom of the pressing block.
[0010] Further, the protrusion is in the shape of a straight line or a wave.
[0011] Further, the connecting piece is integrally formed with the two U-shaped frames.
[0012] Further, when the buckle and the U-shaped frame clamp and fix the plant tissue, the upper end of the buckle protrudes from the sliding groove.
[0013] Further, the main body and the buckle are made of a material having good toughness at low temperature, and the material includes red wax, silicone or titanium alloy material.
[0014] Beneficial effects: through the clamp designed in this way, the plant material can be fixed at the required angle, meanwhile, the bottom of the U-shaped frame can make the bottom surface of the plant leaf or stem have a gap with the bottom of the sample preparation box, so that the plant leaf or stem can be fully wrapped by the embedding agent, and the sample plane for mass spectrum imaging detection meeting the target requirements can be obtained. BRIEF DESCRIPTION OF DRAWINGS
[0015] In order to more clearly illustrate the technical scheme in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or the prior art description, and obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained according to the structures shown in the drawings without creative labor.
[0016] Figure 1 The flowchart of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0017] Figure 2 The overall structure diagram of the clamp of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0018] Figure 3 The main body structure diagram of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0019] Figure 4 The buckle structure diagram of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0020] Figure 5 The exploded view of the clamp of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0021] Figure 6 The cross-sectional structure diagram of the clamp of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0022] Figure 7 The overall structure diagram of the sample preparation box of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0023] Figure 8 The cross-sectional structure diagram of the sample preparation box of the method for preparing the mass spectrum imaging sample of the present application is shown in the figure.
[0024] Figure 9 The effect comparison result schematic diagram of different proportions of embedding agent of the utility model;
[0025] Figure 10 The result schematic diagram of not clamping sample using OCT embedding agent of the utility model;
[0026] Figure 11 The result schematic diagram of not using clamp clamping and not gradient freezing of the utility model;
[0027] Figure 12 The result schematic diagram of not using clamp clamping of the utility model;
[0028] Figure 13 The result schematic diagram of not using the sample box of the utility model of the application of the utility model;
[0029] Figure 14 The result schematic diagram of preparation method of the utility model;
[0030] Figure 15 The sample diagram prepared by the preparation method of the utility model;
[0031] In the figure: 1, main body;11, U-shaped frame;111, sliding groove;112, first limit block;113, first protrusion;12, connecting piece;2, buckle;21, U-shaped main body;22, second limit block;23, first pressing block;231, first recess;3, box body;31, second protrusion;4, second pressing block.
[0032] The realization, functional characteristics and advantages of the utility model will be further described with reference to the accompanying drawings in combination with embodiments. DETAILED DESCRIPTION
[0033] The technical solutions in the embodiments of the utility model will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the utility model. Obviously, the described embodiments are only 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 fall within the protection scope of the utility model.
[0034] It should be noted that all directionality indications (such as up, down, left, right, front, back, etc.) in the embodiments of the utility model are only used to explain the relative position relationship, movement condition, etc. between components in a certain specific posture (as shown in the drawings), if the specific posture changes, the directionality indications also change accordingly.
[0035] In addition, the description of "first", "second" and the like in the present application is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the technical features or implicitly indicating the number of the technical features indicated. Therefore, the features limited by "first", "second" can be explicitly or implicitly included at least one of the features. In addition, "and / or" throughout the text includes three schemes, taking A and / or B as an example, including A technical solution, B technical solution, and A and B technical solution that meet at the same time; in addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the realization of ordinary skilled in the art, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor in the protection scope required by the present application.
[0036] As shown in Figure 1 The application embodiment provides a method for preparing a mass spectrometry imaging sample, comprising:
[0037] S1, sample embedding: taking the plant tissue to be tested as a sample, embedding the sample to be tested with an embedding agent.
[0038] The embedding agent is composed of gelatin, polyvinyl alcohol and modified cellulose.
[0039] The embedding includes I or II as follows:
[0040] I) when the complete sample morphology is not required, clamps are used to fix the sample to be tested, and then the sample to be tested is placed in an embedding box together with the clamps, and the embedding agent is added until the sample to be tested is completely immersed in the embedding agent;
[0041] II) when the complete sample morphology is required, a sample preparation box is used to fix the sample to be tested, and then the embedding agent is added until the sample to be tested is completely immersed in the embedding agent.
[0042] Before embedding, the sample plane is found, and according to different sample morphology requirements, the sample is fixed by I or II, and whether the sample is horizontal is checked.
[0043] During embedding, the embedding agent is slowly injected until it completely covers the plant tissue, and preferably, a pipette gun can also be used to eliminate the bubbles attached to the surface of the sample to make it fully embedded.
[0044] S2, gradient freezing: the embedded sample is frozen twice at different temperatures.
[0045] The steps of gradient freezing are: first, the plant tissue after embedding treatment is frozen at-40℃ for 10-20 minutes, and then frozen at-20℃ for 20 minutes.
[0046] The inventors of this utility model have discovered that this freezing method can achieve rapid and thorough freezing of embedded samples, allowing the samples to maintain their tissue integrity, and also allows the clamps or sample preparation boxes to be easily separated from the frozen samples, thereby achieving the goal of rapidly preparing mass spectrometry imaging samples.
[0047] S3. Slicing: The frozen embedded sample is sliced to obtain a mass spectrometry imaging sample.
[0048] During slicing, the frozen sample is removed from the clamp or sample preparation box, then placed on the stage and fixed. The freezing head is inserted and the angle is adjusted. The appropriate blade is used to slice the sample according to the required thickness. This will produce mass spectrometry imaging samples of different layers of cells, different tissue regions, and detection planes at different specific angles in different plant tissues.
[0049] The inventors of this utility model have discovered that, due to the unique morphology, structure, and surface structure of plant tissues, as well as their special physical properties such as hydrophobicity, toughness, and water content, conventional embedding methods for plant tissues often result in insufficient infiltration of the embedding agent and difficulty in fixing the position, leading to embedding failure, deformation in sections, and difficulty in obtaining section material at specific angles. To overcome these problems, this application has developed a novel method for preparing plant mass spectrometry imaging samples by optimizing the embedding agent, freezing procedure, and designing an embedding device. This method can conveniently and efficiently prepare high-quality mass spectrometry imaging samples with intact cell morphology and a detection plane that meets expectations.
[0050] The method described in this application not only enables the rapid preparation of mass spectrometry imaging samples of different plant tissues, but also enables the preparation of planar mass spectrometry imaging samples of different cell layers, different tissue regions, and different specific angles of different plant tissues.
[0051] In one embodiment, the encapsulating agent contains 3% carboxyethyl cellulose, 2.5% polyvinyl alcohol, 2.5% gelatin, and the remainder is water.
[0052] like Figures 2-6 As shown, in one embodiment, the clamp includes:
[0053] The main body 1 includes two U-shaped frames 11 and a connector 12. The two U-shaped frames 11 are connected by the connector 12 to form the main body 1. Each U-shaped frame 11 has at least one sliding groove 111 on its inner side wall.
[0054] Clip 2 is slidably connected to slide groove 111. When clip 2 is inserted into slide groove 111, clip 2 and U-shaped frame 11 clamp and fix the plant tissue.
[0055] In the embodiment, the connecting piece 12 is H-shaped and arranged horizontally, the two U-shaped racks 11 are arranged in parallel and vertically, the two U-shaped racks 11 are arranged on the two sides of the connecting piece 12 respectively, the connecting piece 12 has four connecting end points, each two connecting end points correspond to the U-shaped rack 11 on one side, and are fixedly connected with the two ends of the U-shaped rack 11 respectively, and the connecting piece 12 and the two U-shaped racks 11 on the two sides are integrally formed by 3D printing, the number of the sliding grooves 111 of each U-shaped rack 11 is two, and the two sliding grooves 111 are arranged vertically on the inner walls on the two sides of the U-shaped rack 11 respectively, wherein the material of the main body 1 and the buckle 2 is a material having good toughness at low temperature, and the material of the main body 1 and the buckle 2 is red wax, silicone or titanium alloy.
[0056] When the plant tissue is clamped, the plant leaves or stems to be prepared for slicing are taken out using tweezers, and then the taken-out plant leaves or stems are placed with the front surface downward or at a specific angle as required, flattened in the clamp, and the two ends of the plant leaves or stems are located on the bottom surfaces of the two U-shaped racks 11 respectively, and finally the buckle 2 is carefully inserted into the sliding groove 111, when the distance between the bottom of the buckle 2 and the inner bottom surface of the U-shaped rack 11 is less than the thickness of the plant leaves or stems, the buckle 2 and the U-shaped rack 11 complete the clamping and fixing of the plant leaves or stems, and then the clamp with the clamped plant leaves or stems is placed in the embedding box, at this time, the thickness of the bottom material of the U-shaped rack 11 causes a gap between the bottom surface of the plant leaves or stems and the bottom of the embedding box, so that the plant leaves or stems are fully wrapped by the embedding agent.
[0057] Through the clamp designed in this way, the plant material can be clamped and fixed at the required angle, and the bottom of the U-shaped rack 11 causes a gap between the bottom surface of the plant leaves or stems and the bottom of the embedding box, so that the plant leaves or stems are fully wrapped by the embedding agent, and a sample plane for mass spectrometry imaging detection meeting the target requirements is obtained.
[0058] In one embodiment, the buckle 2 is U-shaped and includes a U-shaped body 21, and a limiting assembly is arranged between the buckle 2 and the sliding groove 111, and the limiting assembly is used to limit the sliding of the buckle 2 in the sliding groove 111.
[0059] In the embodiment, the buckle 2 has elasticity, and the end of the buckle 2 can be elastically deformed, and through the limiting assembly arranged between the buckle 2 and the sliding groove 111, when the buckle 2 and the U-shaped rack 11 complete the clamping and fixing of the plant leaves or stems, the limiting assembly makes the buckle 2 and the U-shaped rack 11 keep clamping and fixing the plant leaves or stems.
[0060] In one embodiment, the limiting component includes a first limiting block 112 and a second limiting block 22. The first limiting block 112 is disposed on the inner wall of the slide groove 111, and the second limiting block 22 is disposed on the outer side of the U-shaped body 21. When the buckle 2 and the U-shaped frame 11 clamp and fix the plant tissue, the first limiting block 112 limits the second limiting block 22.
[0061] In this embodiment, the first limiting block 112 is set on the bottom surface of the slide groove 111. When the buckle 2 and the U-shaped frame 11 clamp and fix the plant leaves or stems, the two sides of the buckle 2 are inserted into the slide groove 111 and the upper end of the buckle 2 is pinched. At this time, the buckle 2 is deformed, which reduces the gap at the upper end of the buckle 2. At this time, the second limiting block 22 on the buckle 2 will avoid the first limiting block 112 moving downward from above the second limiting block 22 during the downward movement. After the buckle 2 and the U-shaped frame 11 have completed clamping and fixing the plant leaves or stems, the end of the buckle 2 is released to allow the buckle 2 to return to its original position. At this time, the second limiting block 22 is inserted into the slide groove 111 and is located below the first limiting block 112. At this time, the first limiting block 112 will resist the second limiting block 22 to prevent the buckle 2 from sliding upward in the slide groove 111, thereby achieving the purpose of clamping and fixing the plant leaves or stems.
[0062] In one embodiment, a first pressing block 23 is provided on the outer bottom of the U-shaped body 21, a first protrusion 113 is provided on the inner bottom of the U-shaped frame 11, and a first groove 231 matching the shape and size of the first protrusion 113 is provided on the bottom of the first pressing block 23.
[0063] In this embodiment, the number of first protrusions 113 can be set to several. The setting of the first pressing block 23 can increase the contact area between the bottom of the buckle 2 and the plant leaves or stems, making the clamping and fixing of the plant leaves or stems more stable. By setting the first protrusion 113 and the first groove 231, wherein the shape of the protrusion is straight or wavy, the friction can be increased. The cooperation of the first protrusion 113 and the first groove 231 can effectively ensure that the sample does not change its position or spatial shape in the clamping state and during the freezing process.
[0064] In one embodiment, after the buckle 2 and the U-shaped frame 11 clamp and fix the plant tissue, the upper end of the buckle 2 protrudes into the sliding groove 111.
[0065] In this embodiment, this configuration serves as a point of leverage for the operator to apply force to the clip 2 when the plant tissue is frozen and detached from the clamp, facilitating the disassembly of the clip 2.
[0066] like Figures 7-8 As shown, in one embodiment, the sample preparation box includes:
[0067] Box 3, with several arrayed second protrusions 31 arranged on the bottom surface inside the box 3;
[0068] The second pressing block 4 is used for being inserted into the box body 3 and clamping and fixing the plant tissue together with the box body 3.
[0069] In the embodiment, the cross-sectional shape of the second protrusion 31 is triangular, rhombic or other irregular shape, and one of the sharp corners is arranged upward. By arranging a plurality of second protrusions 31, the complete plant tissue can be clamped and fixed, so that the complete tissue morphology can be obtained.
[0070] When clamping and fixing the plant tissue, first, add the embedding agent with half height in the embedding box of the same size and model, and place it in the freezing chamber of the slicing machine to freeze until it is solidified, so as to obtain the pressing block matched with the sample box. When embedding, first, add the embedding agent to the sample box to fill the bottom space until the height of the sharp corner protrusion is covered, then use tweezers to pick the plant leaves / stems to be prepared for slicing and place them in the sample box, then add the embedding liquid again to cover the sample tissue so that it is completely wrapped by the embedding agent, and finally use the pressing block to press in the sample box until there is obvious resistance.
[0071] The embedding agent provided by the embodiment of the application is composed of a water solution of gelatin, polyvinyl alcohol and modified cellulose, wherein the content of the modified cellulose is 3%, the content of the polyvinyl alcohol is 2.5%, and the content of the gelatin is 2.5%.
[0072] Preferably, the modified cellulose is carboxyethyl cellulose or methyl cellulose.
[0073] More preferably, the modified cellulose is carboxyethyl cellulose.
[0074] In the embodiment, the gelatin, polyvinyl alcohol and modified cellulose can be configured into a completely mixed and dissolved water solution according to the above-mentioned concentrations, and the internal bubbles are removed, for example, the vacuum pump vacuum removal method can be used to remove the bubbles.
[0075] The application discloses an embedding agent for plant tissue, which is composed of a water solution of gelatin, polyvinyl alcohol and modified cellulose, wherein the content of the modified cellulose is 3%, the content of the polyvinyl alcohol is 2.5%, and the content of the gelatin is 2.5%.
[0076] In one embodiment, the embedding agent of the application is composed of carboxyethyl cellulose 3%, polyvinyl alcohol 2.5% and gelatin 2.5%.
[0077] Application effect embodiment
[0078] Example 1 Effect comparison of different proportions of embedding agent
[0079] The leaf sample of the plant was taken with tweezers, and the tissue region needed for mass spectrometry imaging analysis was selected. The plant leaf was placed on the base of the clamp, and the tissue region needed for analysis was placed at the center of the base of the clamp. The sample was fixed on the base of the clamp using the clamp parts, and the tissue region needed for analysis was horizontally arranged in space. The clamp holding the sample was placed in the embedding box, and embedding agent 1 (embedding agent composition: carboxyethyl cellulose 3%, polyvinyl alcohol 2.5%, gelatin 2.5%), embedding agent 2 (embedding agent composition: carboxyethyl cellulose 4%, polyvinyl alcohol 2%, gelatin 1.5%), and embedding agent 3 (embedding agent composition: carboxyethyl cellulose 5%, polyvinyl alcohol 1.5%, gelatin 1%) were added to the embedding box, so that the embedding agent covered the sample and embedded for 5 minutes. Finally, a small amount of air bubbles around the sample was removed using a pipette gun, and the sample was fully combined with the embedding liquid. The embedding box was quickly placed in -40°C for 10-20 minutes, and then it was taken out and placed in the freezing area (-20°C) of the microtome. After being frozen for about 20 minutes, the clamp was separated from the embedding box together with the sample, and then the clamp was separated from the sample, obtaining the frozen and embedded sample. The sample was placed on the sample stage of the frozen section, and the sample was sectioned using a 8 μm section thickness and observed.
[0080] As shown in Figure 9 , the experimental results show that by naked eye, it can be observed that the sample embedded with embedding agent 1 (i.e. the embedding agent of the present application) is combined most closely with the embedding liquid, avoiding the sample from curling when cut by the blade, and the obtained section is smooth and flat. The samples embedded with embedding agent 2 and embedding agent 3 curl to different degrees when cut by the blade, especially the loss of the cilia part is more serious, and the cilia morphology is basically not obtained.
[0081] Example 2 Effect comparison of sample preparation
[0082] Four comparative examples were set to compare the preparation method of the present application with the experimental results. The steps and experimental results of the preparation method of the present application and the comparative examples are as follows:
[0083] Comparative Example 1: OCT embedding agent was used without clamping the sample
[0084] The leaf sample of the plant is taken with tweezers, and the tissue region to be studied is selected. The removed plant leaf is placed in an embedding box, and the OCT embedding agent (optimal cutting temperature compound) is added to embed the plant leaf for 5 minutes. The spatial position of the leaf in the embedding box is adjusted using tweezers, so that the region to be detected and analyzed remains horizontal in space. The embedding box is quickly placed in a-40℃ environment for 10-20 minutes, and then taken out and placed in the freezing area (-20℃) of the sectioning machine for about 20 minutes. After the sample is frozen, it is placed on the stage of the sectioning machine, and the sample is sectioned with a thickness of 8μm.
[0085] As shown in Figure 10 , the observed sample section is found to have bubbles or gaps due to the inability of the leaf to closely adhere to the embedding agent during embedding, resulting in curling of the sample during sectioning, causing the leaf to be pulled and broken due to not being tightly fixed, and unable to obtain complete sections, unable to perform subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations.
[0086] Comparative Example Two does not use a clamp to hold and does not use gradient freezing
[0087] The leaf sample of the plant is taken with tweezers, and the tissue region to be studied is selected. The removed plant leaf is placed in an embedding box, and the OCT embedding agent (optimal cutting temperature compound) is added to embed the plant leaf for 5 minutes. The spatial position of the leaf in the embedding box is adjusted using tweezers, so that the region to be detected and analyzed remains horizontal in space. The embedding box is quickly placed in a-40℃ environment for 10-20 minutes, and then taken out and placed in the freezing area (-20℃) of the sectioning machine for about 20 minutes. After the sample is frozen, it is placed on the stage of the sectioning machine, and the sample is sectioned with a thickness of 8μm.
[0088] As shown in Figure 11 , the observed sample section is found to have bubbles or gaps due to the inability of the leaf to closely adhere to the embedding agent during embedding, resulting in curling of the sample during sectioning, causing the leaf to be pulled and broken due to not being tightly fixed, and unable to obtain complete sections, unable to perform subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations.
[0089] Comparative Example Three does not use a clamp to hold
[0090] With forceps to take the mature leaf samples of the plant, select the tissue area that needs to be sliced, place the plant leaves taken out in the embedding box, add the embedding agent of the application to embed the plant leaves for 5 minutes, and use forceps to adjust the spatial position of the leaves in the embedding box, so that the area to be detected and analyzed remains horizontal in space. The embedding box is quickly placed in -40°C for 10-20 minutes to achieve rapid and sufficient freezing of the embedded sample by low-temperature rapid precooling. Then it is taken out and placed in the freezing area (-20°C) of the microtome. After about 20 minutes, the sample is placed on the sample stage of the frozen section, and the sample is sliced with a thickness of 8 μm.
[0091] As shown in Figure 12 , the observed sample slices found that due to the lack of fixation, even if the leaves are constantly adjusted with forceps during the freezing process, the leaves still exhibit irregular bending after freezing, and cannot be parallel to the bottom surface of the embedding box in space, which leads to the inability to completely slice the sample in a flat or desired angle during the slicing process, affecting subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations.
[0092] Comparative Example Four does not use the sample preparation box of the application
[0093] In the embedding box 1, add half the height of the embedding agent of the application and place it in the freezing chamber of the microtome to freeze to solidification to prepare an embedding agent ice block. A small amount of embedding agent is added to the embedding box 2 so that it completely covers the bottom standby. Then take the leaf sample of the plant with forceps, select the tissue area that needs to be sliced, and place it in the standby embedding box 2. Add the embedding agent of the application again to cover the sample tissue so that it is completely wrapped by the embedding agent, and embed for 5 minutes. Then place the embedding agent ice block in the embedding box 2 and press it gently until you feel obvious resistance. Quickly place the embedding box in -40°C for 10-20 minutes, then take it out and place it in the freezing area (-20°C) of the microtome. Freeze for about 20 minutes. Then place the sample on the sample stage of the frozen section, and slice the sample with a thickness of 8 μm.
[0094] As shown in Figure 13 , the observed sample slices found that even if the sample is fixed using the method of precooling the embedding agent, the surface of the embedding agent freezing block is still not flat during the freezing process, which leads to the bending of the leaves towards the bottom surface of the embedding box after freezing, and the inability to be parallel to the bottom surface of the embedding box in space, which leads to the inability to completely slice the sample in a flat or desired angle during the slicing process, affecting subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations.
[0095] The preparation method one of the application uses a clamp to hold
[0096] A leaf sample of a plant is taken with tweezers, and a tissue region to be studied is selected. The leaf sample is placed on the base of the clamp, with the tissue region to be analyzed in the center of the base. The sample is fixed on the base of the clamp using clamp parts, so that the tissue region to be analyzed is horizontally arranged in space. The clamp holding the sample is placed in an embedding box, and the embedding agent of the application is added to the embedding box, so that the embedding agent covers the sample and embedding is performed for 5 minutes, so that the sample is fully combined with the embedding agent. Then the embedding box is quickly placed in a freezer at -40°C for 10-20 minutes, and then taken out and placed in the freezing area (-20°C) of a microtome, and frozen for 20 minutes. The embedding box is then separated, and the clamp and sample are separated, to obtain the frozen and embedded sample. The sample is placed on the stage of the cryosectioning machine, and the sample is sectioned using a section thickness of 8 μm.
[0097] As shown in Figure 14 , observation of the section prepared by the method of the application shows that: the sample prepared by the method of the application is closely combined with the embedding agent, and the obtained section is smooth and flat; the sample is fixed by the clamp and always maintains a relative horizontal state with the bottom plane of the embedding box, so that the obtained section sample is always parallel to the surface of the leaf, and subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations can be carried out with high quality.
[0098] The second preparation method of the application uses a sample preparation box to hold
[0099] Half the height of the embedding agent of the application is added to the embedding box 1 and placed in the freezing chamber of the microtome to freeze until solidification, to prepare an embedding agent ice block. A small amount of embedding agent is added to the sample preparation box so that it completely covers the bottom triangular protrusion, and is ready for use. A leaf sample of a plant is taken with tweezers, and a tissue region to be studied is selected. The sample is placed in the sample preparation box, so that the tissue region to be analyzed is horizontally arranged in space. The embedding agent is added again and placed in the sample preparation box. The embedding agent of the application is added again to cover the sample tissue, so that it is completely wrapped by the embedding agent, and embedding is performed for 5 minutes. Subsequently, the embedding agent ice block is placed in the sample preparation box and lightly pressed until a significant resistance is felt. The sample preparation box is quickly placed in a freezer at -40°C for 10-20 minutes, and then taken out and placed in the freezing area (-20°C) of a microtome, and frozen for about 20 minutes. Subsequently, the sample is placed on the stage of the cryosectioning machine, and the sample is sectioned using a section thickness of 8 μm.
[0100] As shown in Figure 15 , observation of the section prepared by the method of the application shows that: the sample prepared by the method of the application is closely combined with the embedding agent, and the obtained section is smooth and flat; the sample is fixed by the clamp and always maintains a relative horizontal state with the bottom plane of the embedding box, so that the obtained section sample is always parallel to the surface of the leaf, and subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations can be carried out with high quality.
[0101] The above merely describes preferred embodiments of the present application, and is not intended to limit the patent scope of the present application, and any equivalent structural transformation or direct / indirect application in other related technical fields under the inventive concept of the present application, as described in the present application specification and drawings, is included in the patent protection scope of the present application.
Claims
1. A clamp for preparing a mass spectrometry imaging sample, characterized in that, The utility model relates to a plant tissue clamping device, including: A main body, the main body includes two U-shaped frames and a connecting piece, two U-shaped frames are connected through the connecting piece and form the main body, and the inner side wall of each U-shaped frame is provided with at least one sliding slot; A buckle is slidably connected with the sliding slot, and when the buckle is inserted into the sliding slot, the buckle and the U-shaped frame clamp and fix plant tissues.
2. The clamp for preparing a mass spectrometry imaging sample according to claim 1, wherein, The buckle is a U-shaped body, a limiting assembly is arranged between the buckle and the sliding slot, and the limiting assembly is used for limiting the sliding of the buckle in the sliding slot.
3. The clamp for preparing a mass spectrometry imaging sample according to claim 2, characterized in that, The limiting assembly includes a first limiting block and a second limiting block, the first limiting block is arranged on the inner wall of the sliding slot, the second limiting block is arranged on the side of the buckle close to the sliding slot, and when the buckle and the U-shaped frame clamp and fix plant tissues, the first limiting block limits the second limiting block.
4. The clamp for preparing a mass spectrometry imaging sample according to claim 1, wherein, The bottom of the buckle is provided with a pressing block, the bottom of the U-shaped frame is provided with a plurality of protrusions, and the bottom of the pressing block is provided with a groove matched with the shape and size of the protrusions.
5. The clamp for preparing a mass spectrometry imaging sample according to claim 4, characterized in that, The shape of the protrusions is linear or wavy.
6. The clamp for preparing a mass spectrometry imaging sample of claim 1, wherein, The connecting piece is integrally formed with the two U-shaped frames.
7. The clamp for preparing a mass spectrometry imaging sample of claim 1, wherein, After the buckle and the U-shaped frame clamp and fix plant tissues, the upper end of the buckle protrudes from the sliding slot.
8. The clamp for preparing a mass spectrometry imaging sample according to claim 1, wherein, The main body and the buckle are made of red wax, silicone or titanium alloy.