Method for preparing mass spectrum imaging sample and embedding medium

Abstract

The invention discloses a method for preparing a mass spectrum imaging sample and an embedding medium, and the method comprises the following steps: sample embedding: picking a plant tissue to be detected as a sample, and embedding the sample to be detected by using the embedding medium; when the complete sample form is not needed, clamping and fixing the to-be-detected sample by adopting a clamp, then putting the to-be-detected sample and the clamp into an embedding box, and adding an embedding medium until the to-be-detected sample is completely immersed in the embedding medium; when a complete sample form is needed, fixing the to-be-detected sample by adopting a sample preparation box, and then adding an embedding medium until the to-be-detected sample is completely immersed in the embedding medium; gradient freezing: freezing the embedded sample twice at different temperatures; and slicing. By means of the method, rapid preparation of mass spectrum imaging samples of different plant tissue samples can be achieved, and preparation of detection plane mass spectrum imaging samples of different layer cells, different tissue areas and different specific angles of different plant tissues can also be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of mass spectrometry imaging, and in particular to a method for preparing a mass spectrometry imaging sample and an embedding agent. Background Art

[0002] Metabolomics is the process of studying all small molecules in living organisms and their changes. It is widely regarded as the most important omics technology in the post-genomic era. Its application has rapidly penetrated into fields closely related to human survival, such as disease diagnosis, pharmaceutical research and development, nutrition and food science, toxicology, environmental science, and botany. Unlike animals, plants have the particularity of morphology, structure, and surface structure, as well as the particularity of leaf materials in physical properties such as toughness and water content. Existing spatial metabolomics sample preparation methods based on animal samples are difficult to use for plant samples.

[0003] However, existing methods for preparing plant spatial metabolome samples, especially for fine tissues such as leaves, flowers, fruit pods or roots, mostly use methods of transfer by pressing of adsorbable materials, or methods of frozen sectioning. Among them, transfer by pressing can lead to the overflow of intracellular substances after cell rupture and deformation of the overall spatial structure of thicker tissues, which in turn leads to contamination and superposition of spatial data, and it is impossible to obtain reliable spatial omics information. The frozen section method often makes it difficult to obtain qualified mass spectrometry imaging samples, and there are often problems such as embedding failure, section deformation, and difficulty in obtaining section materials at specific angles. In response to this, the present application proposes a method and embedding agent for preparing mass spectrometry imaging samples to solve such problems. Summary of the invention

[0004] Based on this, in order to solve the problems existing in the prior art, in a first aspect, the present application provides a method for preparing a mass spectrometry imaging sample, comprising:

[0005] S1. Sample embedding: Take the plant tissue to be tested as a sample, and use an embedding agent to embed the sample to be tested, wherein the embedding agent is composed of gelatin, polyvinyl alcohol and modified cellulose;

[0006] Embedding includes I or II as follows:

[0007] I) When the complete sample form is not required, a clamp is used to clamp and fix the sample to be tested, and then the sample to be tested together with the clamp is placed in an embedding box, and an embedding agent is added until the sample to be tested is completely immersed in the embedding agent;

[0008] II) When a complete sample morphology is required, a sample preparation box is used to fix the sample to be tested, and then an embedding agent is added until the sample to be tested is completely immersed in the embedding agent;

[0009] S2, gradient freezing: the embedded samples are frozen twice at different temperatures;

[0010] S3. Slicing: Slice the embedded sample after freezing to obtain a mass spectrometry imaging sample.

[0011] Preferably, the content of modified cellulose in the embedding agent is 3%, the content of polyvinyl alcohol is 2.5%, and the content of gelatin is 2.5%.

[0012] Preferably, the modified cellulose is carboxyethyl cellulose or methyl cellulose.

[0013] More preferably, the modified cellulose is carboxyethyl cellulose.

[0014] Preferably, the clamp comprises:

[0015] The main body includes two U-shaped frames and a connecting piece. The two U-shaped frames are connected to form the main body through the connecting piece. The inner side wall of each U-shaped frame is provided with at least one sliding groove;

[0016] The buckle is slidably connected with the slide groove. When the buckle is inserted into the slide groove, the buckle and the U-shaped frame clamp and fix the plant tissue.

[0017] Preferably, the main body and the buckle are made of a material having good toughness at low temperatures, such as red wax, silicone resin or titanium alloy.

[0018] Preferably, the buckle is U-shaped, including a U-shaped main body, and a limiting component is provided between the buckle and the slide groove, and the limiting component is used to limit the buckle from sliding in the slide groove.

[0019] Preferably, 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 slide groove, and the second limiting block is arranged on the side of the buckle close to the slide groove, when the buckle and the U-shaped frame clamp and fix the plant tissue, the first limiting block limits the second limiting block.

[0020] Preferably, a first pressing block is provided at the outer bottom of the U-shaped body, a first protrusion is provided at the inner bottom of the U-shaped frame, and a first groove matching the shape and size of the first protrusion is provided at the bottom of the first pressing block.

[0021] Preferably, after the buckle and the U-shaped frame clamp and fix the plant tissue, the upper end of the buckle protrudes from the slide groove.

[0022] Preferably, the sample preparation box comprises a box body, and a plurality of second protrusions arranged in an array are provided on the bottom surface of the box body;

[0023] II) adding an embedding agent until the sample to be tested is completely immersed in the embedding agent, and the specific steps include:

[0024] First, add a small amount of embedding agent to the sample preparation box until it just covers the second protrusion, put the sample to be tested into the sample preparation box, add embedding agent again until it completely covers the sample to be tested, and finally press the block lightly into the sample preparation box until there is obvious resistance;

[0025] Preferably, the pressed block is an embedding medium ice block frozen at -20°C, and its size is adapted to the sample preparation box, and is used to be inserted into the box body and cooperate with the box body to clamp and fix the plant tissue.

[0026] Preferably, after the embedding of the plant tissue is completed, the method further comprises:

[0027] Use a pipette to remove air bubbles adhering to the sample surface.

[0028] Preferably, the gradient freezing step comprises:

[0029] The embedded plant tissue is first frozen at -40°C for 10-20 minutes, and then frozen at -20°C for 20 minutes.

[0030] In a second aspect, the present application provides an embedding agent, which is composed of an aqueous solution of gelatin, polyvinyl alcohol and modified cellulose.

[0031] Preferably, the content of modified cellulose in the embedding agent is 3%, the content of polyvinyl alcohol is 2.5%, and the content of gelatin is 2.5%.

[0032] Preferably, the modified cellulose is carboxyethyl cellulose or methyl cellulose.

[0033] More preferably, the modified cellulose is carboxyethyl cellulose.

[0034] Beneficial effects: The method of the present application can not only realize the rapid preparation of mass spectrometry imaging samples of different plant tissue samples, but also realize the preparation of mass spectrometry imaging samples of different layers of cells, different tissue regions and detection planes of different specific angles of different plant tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0036] Figure 1 A schematic diagram of a process for preparing a mass spectrometry imaging sample according to the present invention;

[0037] Figure 2A schematic diagram of the overall structure of a fixture for the method of preparing a mass spectrometry imaging sample according to the present invention;

[0038] Figure 3 This is a schematic diagram of the main structure of the method for preparing a mass spectrometry imaging sample of the present invention;

[0039] Figure 4 A schematic diagram of the buckle structure of the method for preparing a mass spectrometry imaging sample of the present invention;

[0040] Figure 5 An exploded view of a fixture for the method of preparing a mass spectrometry imaging sample according to the present invention;

[0041] Figure 6 A schematic diagram of the cross-sectional structure of a fixture for the method of preparing a mass spectrometry imaging sample according to the present invention;

[0042] Figure 7 A schematic diagram of the overall structure of a sample preparation box for the method of preparing a mass spectrometry imaging sample of the present invention;

[0043] Figure 8 A schematic diagram of the cross-sectional structure of a sample preparation box of the method for preparing a mass spectrometry imaging sample of the present invention;

[0044] Fig. 9 This is a schematic diagram of the comparison of the effects of embedding agents of different proportions according to the present invention;

[0045] Fig.10 This is a schematic diagram of the results of using OCT embedding medium without clamping the sample in the present invention;

[0046] Fig.11 This is a schematic diagram of the results of the present invention without clamping and gradient freezing;

[0047] Fig.12 This is a schematic diagram of the clamping result without using a clamp in the present invention;

[0048] Fig.13 This is a schematic diagram of the results of the present invention without using the sample preparation box of the present application;

[0049] Fig.14 It is a schematic diagram of the preparation method results of the present invention;

[0050] Fig.15 A sample diagram prepared by the preparation method of the present invention;

[0051] In the figure: 1, main body; 11, U-shaped frame; 111, slide groove; 112, first limit block; 113, first protrusion; 12, connecting piece; 2, buckle; 21, U-shaped main body; 22, second limit block; 23, first pressure block; 231, first groove; 3, box body; 31, second protrusion; 4, second pressure block.

[0052] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0055] In addition, the descriptions of "first", "second", etc. in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, "and / or" in the full text includes three solutions. Taking A and / or B as an example, it includes technical solution A, technical solution B, and technical solution that satisfies both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0056] like Figure 1 As shown, the embodiment of the present application provides a method for preparing a mass spectrometry imaging sample, comprising:

[0057] S1. Sample embedding: Take the plant tissue to be tested as a sample and use an embedding agent to embed the sample to be tested.

[0058] The embedding medium is composed of gelatin, polyvinyl alcohol and modified cellulose.

[0059] Embedding includes I or II as follows:

[0060] I) When the complete sample form is not required, a clamp is used to clamp and fix the sample to be tested, and then the sample to be tested together with the clamp is placed in an embedding box, and an embedding agent is added until the sample to be tested is completely immersed in the embedding agent;

[0061] II) When a complete sample morphology is required, a sample preparation box is used to fix the sample to be tested, and then an embedding agent is added until the sample to be tested is completely immersed in the embedding agent.

[0062] Before embedding, find the sample plane first, clamp and fix the sample using the above I or II according to the different sample shape requirements, and check whether the sample is level.

[0063] During embedding, the embedding agent is slowly injected until the plant tissue is completely submerged. Preferably, a pipette can be used to eliminate bubbles attached to the surface of the sample to ensure that it is fully embedded.

[0064] S2, gradient freezing: The embedded samples are frozen twice at different temperatures.

[0065] The steps of gradient freezing are: first freeze the embedded plant tissue at -40°C for 10-20 minutes, and then freeze it at -20°C for 20 minutes.

[0066] The inventors of the present application have discovered that this freezing method can achieve rapid and complete freezing of embedded samples, allowing the samples to better maintain tissue integrity, and can also allow the clamp or sample preparation box to be easily separated from the frozen samples, thereby achieving the purpose of rapidly preparing mass spectrometry imaging samples.

[0067] S3. Slicing: Slice the embedded sample after freezing to obtain a mass spectrometry imaging sample.

[0068] When slicing, take the frozen sample out of the clamp or sample preparation box, then place it on the stage and fix it, put in the freezing head and adjust the angle, and use the corresponding blade to slice according to the required thickness, so as to obtain mass spectrometry imaging samples of different layers of cells of different plant tissues, different tissue regions and detection planes at different specific angles.

[0069] The inventors of this application found in their research that due to the particularity of plant tissue in morphology, structure, surface structure, and physical properties such as hydrophobicity, toughness, and water content, when conventionally embedding plant tissue, the plant tissue cannot fully infiltrate the embedding agent and the position is difficult to fix, resulting in embedding failure, deformation during slicing, and difficulty in obtaining slicing materials at specific angles. In order to overcome the above problems, this application has developed a new plant mass spectrometry imaging sample preparation method by optimizing the embedding agent, freezing program, developing and designing an embedding device, etc., which can conveniently and efficiently prepare high-quality mass spectrometry imaging samples with complete cell morphology and detection planes that meet expectations.

[0070] The method of the present application can not only realize the rapid preparation of mass spectrometry imaging samples of different plant tissue samples, but also realize the preparation of mass spectrometry imaging samples of different layers of cells, different tissue regions and detection planes of different specific angles of different plant tissues.

[0071] In one embodiment, in the embedding agent, the content of carboxyethyl cellulose is 3%, the content of polyvinyl alcohol is 2.5%, the content of gelatin is 2.5%, and the rest is water.

[0072] like Figure 2-6 As shown, in one embodiment, the clamp includes:

[0073] The main body 1 includes two U-shaped frames 11 and a connecting piece 12. The two U-shaped frames 11 are connected to form the main body 1 by the connecting piece 12. The inner side wall of each U-shaped frame 11 is provided with at least one slide groove 111;

[0074] The buckle 2 is slidably connected to the slide groove 111. When the buckle 2 is inserted into the slide groove 111, the buckle 2 and the U-shaped frame 11 clamp and fix the plant tissue.

[0075] In this embodiment, the connecting member 12 is H-shaped and horizontally arranged, the two U-shaped frames 11 are relatively parallel and vertically arranged, the two U-shaped frames 11 are respectively arranged on both sides of the connecting member 12, the connecting member 12 has four connection end points and each two connection end points correspond to the U-shaped frame 11 on one side, and are respectively fixedly connected to the two ends of the U-shaped frame 11, optionally, the connecting member 12 and the U-shaped frames 11 on both sides are integrally formed by 3D printing, the number of the slide grooves 111 of each U-shaped frame 11 is two, and they are respectively vertically arranged on the inner walls of the two sides of the U-shaped frame 11, wherein the material of the main body 1 and the buckle 2 is a material with good toughness at low temperature, optionally, the material of the main body 1 and the buckle 2 is red wax, silicone resin or titanium alloy;

[0076] When clamping the plant tissue, use tweezers to pick the plant leaves or stems to be sliced, then place the picked plant leaves or stems face down or at a specific angle as required, flatten them in the clamp, and make the two ends of the plant leaves or stems respectively located on the bottom surfaces of the two U-shaped frames 11, and finally carefully insert the buckle 2 into the slide groove 111. When the distance between the bottom of the buckle 2 and the inner bottom surface of the U-shaped frame 11 is less than the thickness of the plant leaves or stems, the buckle 2 and the U-shaped frame 11 complete the clamping and fixing of the plant leaves or stems, and then put the clamp holding the plant leaves or stems into the embedding box. At this time, the thickness of the bottom material of the U-shaped frame 11 will cause a gap between the bottom surface of the plant leaves or stems and the bottom of the embedding box, which is conducive to the embedding agent to fully wrap the plant leaves or stems;

[0077] By using such a designed clamp, the plant material can be clamped and fixed at the desired angle. At the same time, the bottom of the U-shaped frame 11 will create a gap between the bottom surface of the plant leaves or stems and the bottom of the embedding box, so that the embedding agent can fully wrap the plant leaves or stems, and obtain a mass spectrometry imaging detection sample plane that meets the target requirements.

[0078] In one embodiment, the buckle 2 is U-shaped, including a U-shaped body 21 , and a limiting component is provided between the buckle 2 and the slide groove 111 , and the limiting component is used to limit the buckle 2 from sliding in the slide groove 111 .

[0079] In this embodiment, the buckle 2 is elastic and the end of the buckle 2 can produce elastic deformation. By setting a limit assembly between the buckle 2 and the slide groove 111, when the buckle 2 and the U-shaped frame 11 complete the clamping and fixing of the plant leaves or stems, the limit assembly enables the buckle 2 and the U-shaped frame 11 to maintain the clamping and fixing of the plant leaves or stems.

[0080] In one embodiment, the limiting assembly includes a first limiting block 112 and a second limiting block 22. The first limiting block 112 is arranged on the inner wall of the slide groove 111, and the second limiting block 22 is arranged 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.

[0081] In this embodiment, the first limit block 112 is arranged 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, so that the spacing between the upper ends of the buckle 2 is reduced. At this time, the second limit block 22 on the buckle 2 will avoid the first limit block 112 moving downward from the second limit block 22 during the sliding process. When the buckle 2 and the U-shaped frame 11 complete the clamping and fixing of the plant leaves or stems, the end of the buckle 2 is released to restore the buckle 2. At this time, the second limit block 22 is inserted into the slide groove 111 and is located below the first limit block 112. At this time, the first limit block 112 will resist the second limit 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.

[0082] In one embodiment, a first pressing block 23 is disposed at the outer bottom of the U-shaped body 21 , a first protrusion 113 is disposed at 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 formed at the bottom of the first pressing block 23 .

[0083] In this embodiment, the number of first protrusions 113 can be set to several. The setting of the first pressure 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 force can be increased. The cooperation between the first protrusion 113 and the first groove 231 can effectively ensure that the sample will not change its position and spatial shape in the clamping state and during the freezing process.

[0084] 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 out of the slide groove 111 .

[0085] In this embodiment, through such an arrangement, when the plant tissue is completely frozen and separated from the clamp, the buckle 2 is used as a force point for the operator to apply force to the buckle 2, which facilitates the disassembly of the buckle 2.

[0086] like Figure 7-8 As shown, in one embodiment, the sample preparation box includes:

[0087] The box body 3 has a plurality of second protrusions 31 arranged in an array on the inner bottom surface of the box body 3;

[0088] The second pressing block 4 is used to be inserted into the box body 3 and cooperate with the box body 3 to clamp and fix the plant tissue.

[0089] In this embodiment, the cross-sectional shape of the second protrusion 31 is a triangle, a rhombus or other irregular shapes, and one of the sharp corners is arranged upward. By providing a plurality of second protrusions 31, the complete plant tissue can be clamped and fixed, so that a complete tissue morphology can be obtained;

[0090] When clamping and fixing plant tissues, first add half the embedding agent to an embedding box of the same size and place it in the freezer of the slicer to freeze until solidified, and obtain a pressing block that fits the sample box. When embedding, first add an appropriate amount of embedding agent to fill the bottom space of the sample box until it is above the height of the sharp protrusions, then use tweezers to pick the plant leaves / stems to be sliced, place them in the sample box, add 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 gently press in the sample box until there is obvious resistance.

[0091] The embodiment of the present application provides an embedding agent, which is composed of gelatin, polyvinyl alcohol and an aqueous solution of modified cellulose, wherein the content of the modified cellulose is 3%, the content of polyvinyl alcohol is 2.5%, and the content of gelatin is 2.5%.

[0092] Preferably, the modified cellulose is carboxyethyl cellulose or methyl cellulose.

[0093] More preferably, the modified cellulose is carboxyethyl cellulose.

[0094] In this embodiment, gelatin, polyvinyl alcohol and modified cellulose can be configured into a completely mixed and dissolved aqueous solution according to the above concentrations, and the internal bubbles can be removed, for example, by using a vacuum pump to evacuate the bubbles.

[0095] The inventors of the present application have found that the improved embedding agent has a low viscosity before freezing and can well infiltrate the epidermal hairs of plant tissues, thereby greatly increasing the surface infiltration area of ​​the tissues; and can more closely combine the plant tissues with the cellulose skeleton; and has high tensile strength and flexibility, which greatly improves the toughness of the embedding agent in the frozen state, improves the degree of adhesion to the plant tissues, avoids deformation and breakage due to different water contents in different tissue parts during the freezing process of the plant tissues, and is more conducive to obtaining complete and uniform slices.

[0096] In one embodiment, the embedding agent of the present application comprises 3% carboxyethyl cellulose, 2.5% polyvinyl alcohol, and 2.5% gelatin.

[0097] Application Effect Embodiment

[0098] Example 1 Comparison of the effects of embedding agents in different proportions

[0099] Use tweezers to take the leaf sample of the plant, select the tissue area that needs to be analyzed by mass spectrometry imaging, place the removed plant leaf on the clamp base, place the tissue area that needs to be analyzed on the center of the clamp base, and then use the clamp parts to fix the sample on the clamp base so that the tissue area that needs to be analyzed is horizontal in space. Place the clamp holding the sample in the embedding box, and use embedding agent 1 (embedding agent composition is carboxyethyl cellulose 3%, polyvinyl alcohol 2.5%, gelatin 2.5%), embedding agent 2 (embedding agent composition is carboxyethyl cellulose 4%, polyvinyl alcohol 2%, gelatin 1.5%), embedding agent 3 (embedding agent composition is carboxyethyl cellulose 5%, polyvinyl alcohol 1.5%, gelatin 1%) to add to the embedding box, so that the embedding agent covers the sample and embeds for 5 minutes, and finally use a pipette to remove a small amount of bubbles around the sample to fully combine the sample with the embedding solution. The embedding box was quickly placed at -40°C for 10-20 minutes, then taken out and placed in the freezing area (-20°C) of the microtome. After freezing for about 20 minutes, the clamp and the sample were separated from the embedding box, and then the clamp was separated from the sample to obtain the frozen embedded sample. The sample was placed on the frozen section stage, and the sample was sliced ​​and observed using a slice thickness of 8μm.

[0100] like Fig. 9 As shown, the experimental results show that it can be observed with the naked eye that the samples embedded in embedding agent 1 (i.e., the embedding agent of the present application) are most tightly combined with the embedding liquid, thereby preventing the samples from curling due to blade cutting, and the obtained slices are smooth and flat, while the samples embedded in embedding agent 2 and embedding agent 3 curled to varying degrees when cut by the blade, especially the ciliary part suffered more serious loss, and the ciliary morphology could basically not be obtained.

[0101] Example 2 Sample Preparation Effect Comparison

[0102] Four comparative examples were set up for experimental comparison with the preparation method of the present application. The steps and experimental results of the preparation method of the present application and the comparative examples are as follows:

[0103] Comparative Example 1: Using OCT embedding medium, without clamping the sample

[0104] Use tweezers to take plant leaf samples, select the tissue area that needs to be sliced ​​and studied, place the removed plant leaves in an embedding box, add OCT embedding agent (optimal cutting temperature compound) to embed the plant leaves for 5 minutes, and use tweezers to adjust the spatial position of the leaves in the embedding box so that the area that needs to be tested and analyzed remains horizontal in the space. Quickly place the embedding box at -40°C for 10-20 minutes, then take it out and place it in the freezing area (-20°C) of the microtome for about 20 minutes. Place the frozen sample on the microtome stage and slice the sample with a slice thickness of 8μm.

[0105] like Fig.10 As shown, it was found from the observation of the sample slices that: since the leaves and the embedding agent could not fit tightly during the embedding process, bubbles or gaps existed, causing the samples to curl during slicing. As a result, the leaves were obviously pulled and broken during the slicing process because they were not tightly fixed. It was impossible to obtain complete slices, and it was impossible to perform subsequent microscopic observations, mass spectrometry imaging, or transcriptome detection and other operations.

[0106] Comparative Example 2: No clamping and no gradient freezing

[0107] Use tweezers to take plant leaf samples, select the tissue area that needs to be sliced ​​for research, place the removed plant leaves in an embedding box, add the embedding agent of the present application to embed the plant leaves for 5 minutes, and use tweezers to adjust the spatial position of the leaves in the sample preparation box so that the area that needs to be tested and analyzed remains horizontal in the space, and then place the sample together with the embedding box in the -20℃ low temperature area of ​​the freezing microtome for 40 minutes. Place the frozen sample on the stage of the microtome and slice the sample with a slice thickness of 8μm.

[0108] like Fig.11 As shown, observation of the sample slices revealed that: due to uneven agglomeration during the freezing process, the combined areas of different tissues broke and fragmented during the sample slicing process, making it impossible to obtain complete slices and perform subsequent microscopic observations, mass spectrometry imaging, or transcriptome detection.

[0109] Comparative Example 3: No clamping is used

[0110] Take mature leaf samples of the sundew plant with tweezers, select the tissue area that needs to be sliced ​​for research, place the removed plant leaves in an embedding box, add the embedding agent of the present application to embed the plant leaves for 5 minutes, and use tweezers to adjust the spatial position of the leaves in the embedding box so that the area that needs to be tested and analyzed remains horizontal in the space, quickly place the embedding box at -40°C for 10-20 minutes, and quickly pre-cool at low temperature to achieve rapid and sufficient freezing of the embedded sample, then take it out and place it in the freezing area (-20°C) in the slicer, and freeze it for about 20 minutes. Then place the sample on the stage of the frozen section and slice the sample with a section thickness of 8μm.

[0111] like Fig.12 As shown, it was found that the sample slices obtained by observation: due to the lack of fixation, even if the leaves were constantly adjusted with tweezers during the freezing process, they still showed irregular curvature after freezing and were not spatially parallel to the bottom of the embedding box, resulting in the inability to complete the plane or slice at the required angle during the sample slicing process, affecting subsequent microscopic observation, mass spectrometry imaging or transcriptome detection and other operations.

[0112] Comparative Example 4: No sample preparation kit of this application was used

[0113] Add half the height of the embedding agent of the present application to embedding box 1 and place it in the freezer of the microtome to freeze until solidified to prepare embedding agent ice cubes. Add a small amount of embedding agent to embedding box 2 so that it completely covers the bottom and set aside. Then use tweezers to take the leaf sample of the plant, select the tissue area that needs to be sliced ​​and study, place it in the spare embedding box 2, add the embedding agent of the present 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 cube in embedding box 2 and press lightly until obvious resistance is felt, quickly place the embedding box at -40°C for 10-20 minutes, then take it out and place it in the freezing area (-20°C) in the microtome, and freeze it for about 20 minutes. Then place the sample on the stage of frozen section and slice the sample using a slice thickness of 8μm.

[0114] like Fig.13 As shown, observation of the sample slices revealed that: even if the sample was fixed using the pre-cooled embedding agent method, the surface of the embedding agent freezing block was still not flat during the freezing process, resulting in the leaves being bent toward the bottom of the embedding box after freezing and not being spatially parallel to the bottom of the embedding box, resulting in the inability to obtain complete plane or required angle slices during the sample slicing process, affecting subsequent microscopic observation, mass spectrometry imaging, or transcriptome detection and other operations.

[0115] Preparation method of the present application 1 using a clamp to clamp

[0116] Use tweezers to take the leaf sample of the plant, select the tissue area that needs to be sliced ​​and studied, place the removed plant leaf on the clamp base, place the tissue area that needs to be analyzed in the center of the clamp base, and then use the clamp parts to fix the sample on the clamp base so that the tissue area that needs to be analyzed is horizontal in space. Place the clamp holding the sample in the embedding box, add the embedding agent of the present application into the embedding box, make the embedding agent cover the sample and embed for 5 minutes, so that the sample and the embedding agent are fully combined. Then quickly place the embedding box at -40°C for 10-20 minutes, then take it out and place it in the freezing area (-20°C) of the slicer. After freezing for 20 minutes, separate the embedding box, and then separate the clamp and the sample to obtain the frozen embedded sample. Place the sample on the stage for frozen sectioning and slice the sample with a slice thickness of 8μm.

[0117] like Fig.14 As shown, by observing the slices prepared by the preparation method of the present application, it was found that: the sample prepared by the preparation method of the present application is tightly combined with the embedding agent, and the obtained slices are smooth and flat; the sample is fixed by the clamp and always maintains a relative level with the bottom plane of the embedding box, so that the obtained slice sample is always parallel to the surface of the leaf, and subsequent microscopic observation, mass spectrometry imaging or transcriptome detection and other operations can be carried out with high quality.

[0118] Preparation method 2 of this application uses a sample preparation box to clamp

[0119] Add half the height of the embedding agent of the present application to the embedding box 1 and place it in the freezer of the slicer to freeze until solidified to prepare the embedding agent ice cube. Add a small amount of embedding agent to the sample box so that it completely covers the triangular protrusion at the bottom and set aside. Take the leaf sample of the plant with tweezers, select the tissue area that needs to be sliced ​​and studied, place it in the sample box so that the tissue area to be analyzed is horizontal in space, add the embedding agent again and add it to the sample box, add the embedding agent of the present 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 cube in the sample box and press it lightly until you feel obvious resistance, quickly place the sample box at -40°C for 10-20 minutes, then take it out and place it in the freezing area (-20°C) in the slicer, and freeze it for about 20 minutes. Then place the sample on the stage of frozen section and slice the sample with a slice thickness of 8μm.

[0120] like Fig.15 As shown, by observing the slices prepared by the method of the present application, it was found that the samples prepared by the preparation method of the present application have the following advantages: the sample is tightly combined with the embedding solution, which avoids the sample from curling due to cutting by the blade, and the obtained slices are smooth and flat; the sample always remains relatively level with the bottom plane of the sample preparation box, and there is no bending to one side, so that the obtained slice sample is always parallel to the surface of the blade.

[0121] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for preparing a mass spectrometry imaging sample, characterized in that: include: S1. Sample embedding: Take the plant tissue to be tested as a sample, and use an embedding agent to embed the sample to be tested, wherein the embedding agent is composed of an aqueous solution of gelatin, polyvinyl alcohol and modified cellulose; The embedding includes the following I or II: I) When the complete sample form is not required, a clamp is used to clamp and fix the sample to be tested, and then the sample to be tested together with the clamp is placed in an embedding box, and an embedding agent is added until the sample to be tested is completely immersed in the embedding agent; II) When a complete sample morphology is required, a sample preparation box is used to fix the sample to be tested, and then an embedding agent is added until the sample to be tested is completely immersed in the embedding agent; S2, gradient freezing: the embedded samples are frozen twice at different temperatures; S3. Slicing: Slice the embedded sample after freezing to obtain a mass spectrometry imaging sample.

2. The method for preparing a mass spectrometry imaging sample according to claim 1, characterized in that: The content of modified cellulose in the embedding agent is 3%, the content of polyvinyl alcohol is 2.5%, and the content of gelatin is 2.5%.

3. The method for preparing a mass spectrometry imaging sample according to claim 1, characterized in that: The fixture comprises: A main body, the 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 the inner side wall of each U-shaped frame is provided with at least one sliding groove; A buckle is slidably connected to the slide groove. When the buckle is inserted into the slide groove, the buckle and the U-shaped frame clamp and fix the plant tissue.

4. The method for preparing a mass spectrometry imaging sample according to claim 3, characterized in that: The buckle is U-shaped and includes a U-shaped main body. A limiting component is arranged between the buckle and the slide groove, and the limiting component is used to limit the buckle from sliding in the slide groove.

5. The method for preparing a mass spectrometry imaging sample according to claim 4, characterized in that: The limiting assembly includes a first limiting block and a second limiting block, wherein the first limiting block is arranged on the inner wall of the slide groove, and the second limiting block is arranged on a side of the buckle close to the slide groove, and when the buckle and the U-shaped frame clamp and fix the plant tissue, the first limiting block limits the second limiting block.

6. The method for preparing a mass spectrometry imaging sample according to claim 4, characterized in that: A first pressing block is arranged at the outer bottom of the U-shaped body, a first protrusion is arranged at the inner bottom of the U-shaped frame, and a first groove matching the shape and size of the first protrusion is opened at the bottom of the first pressing block.

7. The method for preparing a mass spectrometry imaging sample according to claim 4, characterized in that: When the buckle and the U-shaped frame clamp and fix the plant tissue, the upper end of the buckle protrudes from the slide groove.

8. The method for preparing a mass spectrometry imaging sample according to claim 1, characterized in that: The sample preparation box comprises a box body, and a plurality of second protrusions arranged in an array are arranged on the inner bottom surface of the box body; The specific steps of adding the embedding agent in step II until the sample to be tested is completely immersed in the embedding agent include: first adding a small amount of embedding agent to the sample preparation box until it just covers the second protrusion, placing the sample to be tested in the sample preparation box, adding the embedding agent again until it completely covers the sample to be tested, and finally lightly pressing the pressing block in the sample preparation box until there is obvious resistance; The compressed block is an embedding medium ice block frozen at -20°C.

9. The method for preparing a mass spectrometry imaging sample according to claim 1, characterized in that: The gradient freezing step comprises: The embedded plant tissue is first frozen at -40°C for 10-20 minutes, and then frozen at -20°C for 20 minutes.

10. An embedding agent, characterized in that: The embedding agent is composed of gelatin, polyvinyl alcohol and an aqueous solution of 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%.

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