A method of preparing a mass spectrometry imaging sample and an embedding agent

Abstract

The application discloses a method for preparing a mass spectrometry imaging sample and an embedding agent, and relates to the field of sample preparation, and specifically discloses a method for preparing a mass spectrometry imaging sample and an embedding agent, which comprises the following steps: sample embedding: taking a plant tissue to be detected as a sample, embedding the sample to be detected by using an embedding agent; when a complete sample shape is not needed, clamping and fixing the sample to be detected by using a clamp, then putting the sample to be detected together with the clamp into an embedding box, and adding the embedding agent until the sample to be detected is completely immersed in the embedding agent; when a complete sample shape is needed, fixing the sample to be detected by using a sample preparation box, and then adding the embedding agent until the sample to be detected is completely immersed in the embedding agent; gradient freezing: freezing the embedded sample twice at different temperatures; and slicing. By the method, the mass spectrometry imaging sample of different plant tissue samples can be rapidly prepared, and the mass spectrometry imaging sample of different layers of cells, different tissue regions and different detection plane angles of different plant tissues can be prepared.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass spectrometry imaging technology, in particular to a method for preparing a mass spectrometry imaging sample and an embedding agent. BACKGROUND

[0002] Metabolomics is a study of all small molecules and their changes in the living body, and is widely regarded as the most important omics technology in the post-genomic era. Its application has rapidly penetrated into the fields closely related to human survival such as disease diagnosis, pharmaceutical research and development, nutrition and food science, toxicology, environmental science, botany, etc. Unlike animals, plants have special morphological, structural and surface structural characteristics, as well as special physical properties such as toughness and water content of leaf materials. The existing spatial metabolomics sample preparation methods based on animal samples cannot be used for plant samples.

[0003] However, the existing plant spatial metabolomics sample preparation methods, especially for fine tissues such as leaves, flowers, fruits, pods or root systems, mostly use adsorbable material pressing and transferring or freezing sectioning. Among them, pressing and transferring can cause cell rupture, overflow of intracellular substances and deformation of the overall spatial structure of thick tissues, resulting in pollution and superposition of spatial data, and thus cannot obtain reliable spatial omics information. The freezing sectioning method often fails to obtain qualified mass spectrometry imaging samples, and often has problems such as embedding failure, section deformation and difficulty in obtaining section materials at specific angles. To solve these problems, the present application provides a method for preparing a mass spectrometry imaging sample and an embedding agent. SUMMARY

[0004] Therefore, in order to solve the problems 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: taking a plant tissue to be tested as a sample, embedding the sample to be tested with an embedding agent, the embedding agent being composed of gelatin, polyvinyl alcohol and modified cellulose;

[0006] The embedding includes I or II as follows:

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

[0008] 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;

[0009] S2, gradient freezing: freezing the embedded sample at two different temperatures;

[0010] S3, slicing: slicing the embedded sample after freezing to obtain the mass spectrometry imaging sample.

[0011] Preferably, the content of the modified cellulose in the embedding agent is 3%, the content of the polyvinyl alcohol is 2.5%, and the content of the 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 comprises 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 groove is arranged on the inner side wall of each U-shaped frame;

[0016] The buckle is in sliding connection with the sliding groove, and when the buckle is inserted into the sliding groove, the buckle and the U-shaped frame clamp and fix the plant tissue.

[0017] Preferably, the material of the main body and the buckle is a material with good toughness at low temperature, such as red wax, silicone or titanium alloy, etc.

[0018] Preferably, the buckle is U-shaped and comprises a U-shaped main body, and a limiting assembly is arranged between the buckle and the sliding groove, and the limiting assembly is used to limit the sliding of the buckle in the sliding groove.

[0019] Preferably, 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 groove, and the second limiting block is arranged on the side of the buckle close to the sliding 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.

[0020] Preferably, the outer side bottom of the U-shaped main body is provided with a first pressing block, the inner side bottom of the U-shaped frame is provided with a first protrusion, and a first recess with a shape and size matching the first protrusion is formed in the bottom of the first pressing block.

[0021] Preferably, when the buckle and the U-shaped frame clamp and fix the plant tissue, the upper end of the buckle protrudes out of the sliding groove.

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

[0023] The specific steps of adding the embedding agent into the II) until the sample to be tested is completely immersed in the embedding agent include:

[0024] First, add a small amount of embedding agent to the second protrusion in the sample preparation box, put the sample to be tested into the sample preparation box, and then add embedding agent to the sample to be tested, and finally press the briquette in the sample preparation box until there is obvious resistance;

[0025] Preferably, the briquette is an embedding agent ice block frozen at-20℃. Its size is matched with the sample preparation box for insertion into the box body and clamping fixation of the plant tissue.

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

[0027] Use a pipette gun to eliminate bubbles attached to the surface of the sample.

[0028] Preferably, the gradient freezing step comprises:

[0029] First, freeze the plant tissue after embedding treatment at-40℃ for 10-20 minutes, and then at-20℃ for 20 minutes.

[0030] In a second aspect, the application provides an embedding agent, which is composed of a water 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 effect: The method of the 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 different specific angle detection planes of different plant tissues. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained according to the structures shown in these drawings without creative labor.

[0036] Figure 1 Flowchart of the method for preparing mass spectrometry imaging samples of the application;

[0037] Figure 2The overall structure of the clamp for the method for preparing a mass spectrometry imaging sample of the present application is shown in the schematic diagram;

[0038] Figure 3 The main body structure of the method for preparing a mass spectrometry imaging sample of the present application is shown in the schematic diagram;

[0039] Figure 4 The buckle structure of the method for preparing a mass spectrometry imaging sample of the present application is shown in the schematic diagram;

[0040] Figure 5 The exploded view of the clamp of the method for preparing a mass spectrometry imaging sample of the present application is shown in the schematic diagram;

[0041] Figure 6 The cross-sectional structure of the clamp of the method for preparing a mass spectrometry imaging sample of the present application is shown in the schematic diagram;

[0042] Figure 7 The overall structure of the sample preparation box of the method for preparing a mass spectrometry imaging sample of the present application is shown in the schematic diagram;

[0043] Figure 8 The cross-sectional structure of the sample preparation box of the method for preparing a mass spectrometry imaging sample of the present application is shown in the schematic diagram;

[0044] Figure 9 The effect comparison results of different proportions of embedding agents of the present application are shown in the schematic diagram;

[0045] Figure 10 The results of using an OCT embedding agent without clamping the sample of the present application are shown in the schematic diagram;

[0046] Figure 11 The results of not using a clamp to clamp and not gradient freezing of the present application are shown in the schematic diagram;

[0047] Figure 12 The results of not using a clamp to clamp of the present application are shown in the schematic diagram;

[0048] Figure 13 The results of not using the sample preparation box of the present application are shown in the schematic diagram;

[0049] Figure 14 The results of the preparation method of the present application are shown in the schematic diagram;

[0050] Figure 15 The sample prepared by the preparation method of the present application is shown in the schematic diagram;

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

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

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

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

[0055] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the term "and / or" throughout the text includes three solutions; taking A and / or B as an example, it includes technical solution A, technical solution B, and a technical solution that simultaneously satisfies A and B. Furthermore, the technical solutions of various embodiments can be combined with each other, but this must be based on the ability of a person skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0056] like Figure 1 As shown in the embodiments of this application, a method for preparing a mass spectrometry imaging sample is provided, including:

[0057] S1. Sample embedding: The plant tissue to be tested is taken as a sample and embedded in the sample using an embedding agent.

[0058] The embedding agent consists of gelatin, polyvinyl alcohol, and modified cellulose.

[0059] Embedding includes the following I or II:

[0060] I) When the complete sample morphology is not required, use a clamp to hold and fix the sample to be tested, and then put the sample to be tested together with the clamp into the embedding box, and add embedding agent until the sample to be tested is completely immersed in the embedding agent;

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

[0062] Before embedding, the sample plane is found, and according to different sample morphology requirements, the sample is clamped and fixed using I or II above, and whether the sample is horizontal is checked.

[0063] During embedding, the embedding agent is slowly injected until the plant tissue is completely immersed, and preferably, a pipette gun can also be used to eliminate bubbles attached to the surface of the sample to ensure sufficient embedding.

[0064] S2, gradient freezing: the embedded sample is frozen at two different temperatures.

[0065] The gradient freezing step is: first, the plant tissue after embedding treatment is frozen at -40°C for 10-20 minutes, and then frozen at -20°C for 20 minutes.

[0066] The present inventors have found that this freezing method can not only achieve rapid and sufficient freezing of the embedded sample, so that the sample can better maintain the integrity of the tissue, but also make it easier for the clamp or sample preparation box to separate from the frozen sample, thereby achieving the purpose of rapidly preparing mass spectrometry imaging samples.

[0067] S3, slicing: the embedded sample after freezing is sliced to obtain a mass spectrometry imaging sample.

[0068] During slicing, the frozen sample is removed from the clamp or sample preparation box, then it is fixed on the sample stage, placed in the cryohead and adjusted the angle, and then sliced according to the required thickness using the corresponding blade, so that different layer cells, different tissue regions and different specific angle detection planes of different plant tissues can be obtained.

[0069] The present inventors have found in research that because of the particularity of plant tissue in morphology, structure, surface structure, and the particularity of physical properties such as hydrophobicity, toughness, and water content, etc., when the plant tissue is conventionally embedded, the plant tissue cannot be fully infiltrated with the embedding agent and the position is difficult to fix, resulting in embedding failure, deformation during slicing, and difficulty in obtaining specific angle slicing materials. In order to overcome the above problems, the present application optimizes the embedding agent, the freezing program, and develops a new embedding device, and develops a new plant mass spectrometry imaging sample preparation method, which can conveniently and efficiently prepare high-quality mass spectrometry imaging samples with complete cell morphology and meet the desired detection plane.

[0070] The method of the present application not only enables rapid preparation of different plant tissue sample mass spectrometry imaging samples, but also enables preparation of different layer cells, different tissue regions and different specific angle detection plane mass spectrometry imaging samples of different plant tissues.

[0071] In one embodiment, the embedding agent contains 3% carboxymethyl cellulose, 2.5% polyvinyl alcohol, 2.5% gelatin, and the rest is water.

[0072] As shown in the drawings, in one embodiment, the clamp comprises: Figures 2-6 The main body 1 comprises two U-shaped frames 11 and a connecting piece 12, the two U-shaped frames 11 are connected by the connecting piece 12 to form the main body 1, and the inner side wall of each U-shaped frame 11 is provided with at least one sliding groove 111;

[0073] The buckle 2 is in sliding connection with the sliding groove 111, and when the buckle 2 is inserted into the sliding groove 111, the buckle 2 clamps and fixes the plant tissue together with the U-shaped frame 11.

[0074] In this embodiment, the connecting piece 12 is H-shaped and horizontally arranged, the two U-shaped frames 11 are arranged in parallel and vertically, the two U-shaped frames 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 one side of the U-shaped frame 11, and are fixedly connected with the two ends of the U-shaped frame 11, and the connecting piece 12 and the two U-shaped frames 11 on the two sides are integrally formed by 3D printing, the number of the sliding grooves 111 of each U-shaped frame 11 is two, and the two sliding grooves 111 are vertically arranged on the inner side walls of the two sides of the U-shaped frame 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.

[0075] When clamping the plant tissue, the plant leaves or stems to be prepared for slicing are picked up using tweezers, and then the picked 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 surface of the two U-shaped frames 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 side 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 the clamp with the clamped plant leaves or stems is placed into the embedding box, at this time, the thickness of the bottom 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 beneficial to the full wrapping of the plant leaves or stems by the embedding agent.

[0076] 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 frame 11 will cause 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 can be fully wrapped by the embedding agent, and a sample plane for mass spectrometry imaging detection meeting the target requirements can be obtained.

[0077]

[0078] ​In one embodiment, the buckle 2 is U-shaped, comprising 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.

[0079] In the embodiment, the buckle 2 is elastic, and the end of the buckle 2 can be elastically deformed. When the buckle 2 and the U-shaped frame 11 complete the clamping and fixing of the plant leaves or stems, the limiting assembly makes the buckle 2 and the U-shaped frame 11 keep clamping and fixing the plant leaves or stems.

[0080] In one embodiment, the limiting assembly comprises a first limiting block 112 and a second limiting block 22, the first limiting block 112 is arranged on the inner wall of the sliding 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 tissues, the first limiting block 112 limits the second limiting block 22.

[0081] In the embodiment, the first limiting block 112 is arranged on the bottom surface of the sliding 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 sliding groove 111, and the upper end of the buckle 2 is pinched. At this time, the buckle 2 is deformed, so that the distance between the upper end of the buckle 2 is reduced. At this time, the second limiting block 22 on the buckle 2 slides downward to avoid the first limiting block 112 moving downward from the upper side of the second limiting block 22. 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 limiting block 22 is inserted into the sliding groove 111 and located below the first limiting block 112. At this time, the first limiting block 112 abuts against the second limiting block 22 to prevent the buckle 2 from sliding upward in the sliding groove 111, so as to achieve the purpose of keeping clamping and fixing the plant leaves or stems.

[0082] In one embodiment, the outer bottom of the U-shaped body 21 is provided with a first pressing block 23, the inner bottom of the U-shaped frame 11 is provided with a first protrusion 113, and the bottom of the first pressing block 23 is provided with a first recess 231 matched with the shape and size of the first protrusion 113.

[0083] In the embodiment, the number of the first protrusions 113 can also be set to several, and the setting of the first pressing block 23 can increase the contact area of the bottom of the buckle 2 with the plant leaves or stems, so that the clamping and fixing of the plant leaves or stems is more stable. The first protrusion 113 and the first recess 231 are set, wherein the shape of the protrusion is linear or wavy, so as to increase the friction. The cooperation of the first protrusion 113 and the first recess 231 can effectively ensure that the sample does not change the position and the spatial form in the clamping state and the freezing process.

[0084] In one embodiment, when the buckle 2 is clamped and fixed to the plant tissue with the U-shaped bracket 11, the upper end of the buckle 2 protrudes from the sliding groove 111.

[0085] In this embodiment, when the plant tissue is frozen and the clamp is released, the buckle 2 is convenient to disassemble as the force point of the operator.

[0086] As shown in the drawings, in one embodiment, the sample preparation box comprises: Figures 7-8 The box body 3 is internally provided with a plurality of arrayed second protrusions 31 on the bottom surface;

[0087] The second pressing block 4 is used for inserting into the box body 3 and clamping and fixing the plant tissue in cooperation with the box body 3.

[0088] In this 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 intact plant tissue can be clamped and fixed, so that the complete tissue morphology can be obtained.

[0089] When clamping and fixing the plant tissue, first add half the height of the embedding agent in the embedding box of the same size and put it into the freezing chamber of the microtome to freeze until it is solidified, and obtain the pressing block suitable for the sample preparation box. When embedding, first add an appropriate amount of embedding agent to fill the bottom space in the sample preparation box until the height of the sharp corner protrusion is reached, then use tweezers to pick the plant leaves / stems to be prepared for slicing and place them in the sample preparation box, then 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 press in the sample preparation box until there is obvious resistance.

[0090] The embedding agent provided in 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%.

[0091] Preferably, the modified cellulose is carboxyethyl cellulose or methyl cellulose.

[0092] More preferably, the modified cellulose is carboxyethyl cellulose.

[0093] In this embodiment, the gelatin, polyvinyl alcohol and modified cellulose can be configured into a completely mixed and dissolved water solution according to the above concentrations, and internal bubbles are removed, for example, a vacuum pump vacuum removal method can be used to remove the bubbles.

[0094]

[0095] ​The present application discloses a plant tissue embedding agent and a preparation method thereof. The embedding agent comprises carboxyethyl cellulose, polyvinyl alcohol and gelatin. The embedding agent has low viscosity before freezing, can well infiltrate the epidermal hairs of plant tissues, can greatly improve the tissue surface infiltration area, can more closely realize the combination of plant tissues and the cellulose framework, has high tensile strength and flexibility, greatly improves the toughness of the embedding agent in the frozen state, improves the close degree of adhesion to the plant tissues, avoids the deformation and fragmentation of the plant tissues caused by different water contents of different tissue parts during the freezing process, and is more conducive to obtaining complete and uniform sections.

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

[0097] Application effect embodiment

[0098] Example 1: Effect comparison of embedding agents with different proportions

[0099] The leaf sample of the plant is taken out with a pair of tweezers, and the tissue region that needs to be analyzed by mass spectrometry imaging is selected. The plant leaf taken out is placed on the base of the clamp, and the tissue region that needs to be analyzed is placed at the center of the base of the clamp. Then, the sample is fixed on the base of the clamp by using the clamp parts, so that the tissue region that needs to be analyzed is in a horizontal state in space. The clamp holding the sample is placed in the embedding box, and embedding agent 1 (the embedding agent comprises 3% carboxyethyl cellulose, 2.5% polyvinyl alcohol and 2.5% gelatin), embedding agent 2 (the embedding agent comprises 4% carboxyethyl cellulose, 2% polyvinyl alcohol and 1.5% gelatin), and embedding agent 3 (the embedding agent comprises 5% carboxyethyl cellulose, 1.5% polyvinyl alcohol and 1% gelatin) are added into the embedding box, so that the embedding agent covers the sample and is embedded for 5 minutes. Finally, a small amount of air bubbles around the sample is removed by using a pipette gun, so that the sample is fully combined with the embedding liquid. 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 the microtome. After being frozen for about 20 minutes, the clamp together with the sample is separated from the embedding box, and then the clamp is separated from the sample, so that the sample embedded after freezing is obtained. The sample is placed on the sample stage of the frozen section, and the sample is sectioned and observed by using a section thickness of 8 μm.

[0100] As shown in Figure 9 the experimental results show that, by naked eye, it can be observed that the sample embedded by using the embedding agent 1 (i.e. the embedding agent of the present application) is most closely combined with the embedding liquid, avoids curling of the sample caused by the cutting of the blade, and the obtained section is smooth and flat. The samples embedded by using the embedding agent 2 and the embedding agent 3 are curled to different degrees during the cutting of the blade, especially the loss of the cilia part is more serious, and the cilia morphology is basically not obtained.

[0101] Example 2: Effect comparison of sample preparation

[0102] By setting four comparative examples and the preparation method of the present application for experimental comparison, the preparation method of the present application and the steps and experimental results of the comparative examples are as follows:

[0103] Comparative Example 1 uses OCT embedding agent, and the sample is not clamped

[0104] The leaf sample of the plant is taken with tweezers, and the tissue region to be studied is selected. The 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°C environment for 10-20 minutes, and then taken out and placed in the freezing area (-20°C) of the sectioning machine for about 20 minutes. The sample after freezing is placed on the stage of the sectioning machine, and the sample is sectioned with a section thickness of 8 μm.

[0105] 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 the lack of close fixation during sectioning. Complete sections cannot be obtained, and subsequent microscopic observation, mass spectrometry imaging, or transcriptome detection operations cannot be performed.

[0106] Comparative Example 2 does not use a clamp to clamp and does not use gradient freezing

[0107] The leaf sample of the plant is taken with tweezers, and the tissue region to be studied is selected. The 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 sample together with the embedding box is placed in the-20°C low temperature area of the freezing sectioning machine for 40 minutes. The sample after freezing is placed on the stage of the sectioning machine, and the sample is sectioned with a section thickness of 8 μm.

[0108] 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 the lack of close fixation during sectioning. Complete sections cannot be obtained, and subsequent microscopic observation, mass spectrometry imaging, or transcriptome detection operations cannot be performed.

[0109] Comparative Example 3 does not use a clamp to clamp

[0110] 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. The sample is then placed on the sample stage of the frozen section, and the sample is sliced using a 8 pm section thickness.

[0111] 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, resulting in that the sample slices cannot be completely planar or at the required angle during the slicing process, affecting subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations, etc.

[0112] Comparative Example Four does not use the sample preparation box of the application

[0113] 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 lightly until you feel obvious resistance. The embedding box is quickly placed in -40°C for 10-20 minutes, and then taken out and placed in the freezing area (-20°C) of the microtome. The sample is then placed on the sample stage of the frozen section, and the sample is sliced using a 8 pm section thickness.

[0114] 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, resulting in that the leaves exhibit bending towards the bottom surface of the embedding box after freezing, and cannot be parallel to the bottom surface of the embedding box in space, resulting in that the sample slices cannot be completely planar or at the required angle during the slicing process, affecting subsequent microscopic observation, mass spectrometry imaging or transcriptome detection operations, etc.

[0115] The preparation method one of the application uses a clamp to hold

[0116] 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.

[0117] As shown in Figure 14 , observation of the section prepared by the method of the application shows that: the sample prepared by the preparation 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 in a state 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.

[0118] The second preparation method of the application uses a sample preparation box to hold

[0119] 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.

[0120] As shown in Figure 15 , observation of the section prepared by the method of the application shows that: the sample prepared by the preparation method of the application has the following advantages: the sample is closely combined with the embedding agent, avoiding curling of the sample due to cutting by the blade, and the obtained section is smooth and flat; the sample always maintains a relative horizontal state with the bottom plane of the sample preparation box, and does not bend to one side, so that the obtained section sample is always in a state parallel to the surface of the leaf.

[0121] The above merely describes the preferred embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent structural changes made according to the content of the present application specification and drawings, or direct / indirect application in other related technical fields, are included in the patent protection scope of the present application.

Claims

1. A method of preparing a mass spectrometry imaging sample, characterized in that, The application relates to a plant tissue embedding agent for preparing a mass spectrum imaging sample. The embedding agent 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%. The embedding comprises I or II as follows: I) when the complete sample morphology is not required, a clamp is used to fix the sample, then the sample is put into an embedding box together with the clamp, and embedding agent is added until the sample is completely immersed in the embedding agent; the clamp comprises a main body and a buckle, the main body comprises two U-shaped frames and a connecting piece, the two U-shaped frames are connected through the connecting piece to form the main body, and at least one sliding groove is arranged on the inner side wall of each U-shaped frame; the buckle is in sliding connection with the sliding groove, and when the buckle is inserted into the sliding groove, the buckle and the U-shaped frame clamp the plant tissue; II) when the complete sample morphology is required, a sample preparation box is used to fix the sample, then embedding agent is added until the sample is completely immersed in the embedding agent; the sample preparation box comprises a box body, a plurality of second protrusions are arranged on the inner bottom surface of the box body in an array, a small amount of embedding agent is first added into the sample preparation box until the second protrusions are just covered, the sample is put into the sample preparation box, embedding agent is added again until the sample is completely covered, and finally a pressing block is lightly pressed in the sample preparation box until obvious resistance is generated. S2, gradient freezing: the embedded sample is frozen twice at different temperatures; S3, slicing: the embedded sample after freezing is sliced to obtain a mass spectrum imaging sample.

2. The method of preparing a mass spectrometry imaging sample according to claim 1, wherein, The buckle is in the shape of U and comprises a U-shaped main body, a limiting assembly is arranged between the buckle and the sliding groove, and the limiting assembly is used for limiting the sliding of the buckle in the sliding groove.

3. The method of preparing a mass spectrometry imaging sample according to claim 2, wherein, 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 groove, and the second limiting block is arranged on the side of the buckle close to the sliding groove; when the buckle and the U-shaped frame clamp the plant tissue, the first limiting block limits the second limiting block.

4. The method of preparing a mass spectrometry imaging sample of claim 2, wherein, The outer bottom of the U-shaped main body is provided with a first pressing block, the inner bottom of the U-shaped frame is provided with a first protrusion, and a first groove matching the shape and size of the first protrusion is formed in the bottom of the first pressing block.

5. The method of preparing a mass spectrometry imaging sample of claim 2, wherein, When the buckle and the U-shaped frame clamp the plant tissue, the upper end of the buckle protrudes out of the sliding groove.

6. The method of preparing a mass spectrometry imaging sample of claim 1, wherein, The pressing block is an embedding agent ice block frozen at-20 DEG C.

7. The method of preparing a mass spectrometry imaging sample of claim 1, wherein, The gradient freezing step comprises: the plant tissue after embedding treatment is first frozen at-40 DEG C for 10-20 minutes, and then frozen at-20 DEG C for 20 minutes.

8. An embedding agent, characterized in that, The embedding agent is applied to the method for preparing a mass spectrum imaging sample in any one of claims 1-7, and the embedding agent 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%.

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