Leaf and / or petal sample preparation method for spatial metabolomics analysis, sample pressing device and application thereof

Abstract

The present invention discloses a method for preparing leaf and / or petal samples for spatial metabolomics analysis, a sample pressing device and application, the preparation method comprising the following steps: fixing leaf and / or petal on a buffer material; placing the obtained buffer material with leaf and / or petal fixed between two hard materials; applying a program pressure to the obtained hard material with leaf and / or petal placed to reach a predetermined through-print target; taking out the buffer material with leaf and / or petal fixed, and completely peeling the leaf and / or petal from the buffer material; fixing the peeled leaf and / or petal on a sample slide, and finally obtaining leaf and / or petal samples for spatial metabolomics analysis. The plant leaf and petal sample mass spectrometry imaging pre-processing method of the present invention has wide universality, provides a simple and reliable pre-processing method for plant leaf and / or petal, and has wide application.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue sample processing, and relates to a leaf and / or petal sample preparation method for spatial metabolomics analysis, a sample pressing device and application. Background Art

[0002] Mass spectrometry imaging technology has been developed for more than a decade and has become relatively mature. Based on mass spectrometry imaging technology, the concept of spatial metabolomics has been developed and proposed, breaking through the bottleneck of loss of spatial information in traditional metabolomics research, and can analyze metabolites in different tissues and organs in three dimensions: qualitative, quantitative, and positional. Spatial metabolomics is widely used in life science fields such as clinical medicine, plant science, and drug development, providing a new visual perspective and multi-dimensional information depth for traditional metabolomics analysis.

[0003] However, most of the research progress related to spatial metabolomics reported so far is concentrated on research based on animal samples, and a small part is based on research on plant samples that can be sliced, such as rhizomes, seed kernels, etc. For samples such as plant leaves and petals that cannot be sliced ​​or directly imaged and scanned due to epidermal wax or villi, the current mainstream method is to image the plant imprints on membrane materials and conduct imaging research by scanning the imprinted membrane materials. The imprinting effect is hindered by problems such as the transfer efficiency of cell contents and the spatial distribution displacement. Therefore, the research progress of plant leaf imaging analysis that has been reported so far is far less than the obvious spatial distribution hierarchy effect provided by animal tissue imaging research. Therefore, it is necessary to invent a pretreatment method for plant leaves and petals for application in spatial metabolomics mass spectrometry imaging analysis. Summary of the invention

[0004] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a leaf and / or petal sample preparation method for spatial metabolomics analysis, a sample pressing device and application.

[0005] The present invention provides a pre-treatment method for simple and rapid mass spectrometry imaging of plant leaf and / or petal samples suitable for spatial metabolomics. The method for treating leaves and / or petals is simple and reliable, and can ensure full exposure of the cellular contents of the leaves and / or petals, and is completely unaffected by whether the leaves and / or petals have epidermal wax or villi, thereby achieving direct imaging scanning of the leaves and / or petals.

[0006] The present invention provides a pre-treatment method for simple and rapid mass spectrometry imaging of plant leaf and / or petal samples suitable for spatial metabolomics, comprising the following steps:

[0007] Step 1: Fix the leaves and / or petals on the porous material cushioning material;

[0008] Step 2: The cushioning material with the leaves and / or petals fixed thereon is placed between two hard materials that can cover the leaves and / or petals; the size of the hard material generally needs to be larger than the size of the cushioning material;

[0009] Step 3: Press the upper and lower hard materials with program pressure, and stop pressing after the outline of the leaves and / or petals is printed on the porous material buffer material; the printing means that the overall outline of the leaves and / or petals is printed on the buffer material, and the edges and main veins / veins are clear;

[0010] Step 4: Take out the cushioning material with the leaves and / or petals fixed after the pressing is completed, and completely separate the leaves and / or petals from the cushioning material;

[0011] Step 5: Fix the leaves and / or petals onto a sample slide to obtain leaf and / or petal samples for final spatial metabolomics analysis.

[0012] After obtaining the leaf and / or petal samples in step 5, AFADESI spatial imaging scanning analysis can be directly performed on the leaf and / or petal whose cells have been ruptured.

[0013] The type of buffer material and its own structure have a certain influence on the integrity of leaves and / or petals after processing. Generally speaking, the more holes and the denser the buffer material has, the more conducive it is to obtain leaves and / or petals samples with good effects after embossing. In the specific implementation process, the buffer material is recommended but not limited to the following porous material materials: cotton cloth, nylon mesh, porous polytetrafluoroethylene (PTFE), etc.

[0014] The thickness of the porous polytetrafluoroethylene is 0.5 mm and the apparent density is 2.1-2.3 G / cm 3 , tensile strength ≥14.0 MPa / min, elongation at break ≥150%;

[0015] The count of the cotton cloth is 40 to 80; preferably, the count of the cotton cloth is 80;

[0016] The mesh number of the nylon mesh is 100-300 meshes; preferably, the mesh number of the nylon mesh is 150 meshes. In actual use, when the mesh number of the nylon mesh is greater than 150 meshes, the embossing effects of nylon meshes of different meshes are not much different, and the use of nylon meshes of 300 meshes is not limited.

[0017] In the actual implementation of the present invention, the embossing effect of the nylon mesh is relatively better.

[0018] In step 1, the leaves include hairy leaves, waxy leaves, leathery leaves, etc.;

[0019] The petals include fluffy petals, waxy petals, etc.

[0020] In step 1, the leaves and / or petals may be fixed on the buffer material by transparent or translucent adhesive materials, such as transparent adhesive, double-sided adhesive, etc.;

[0021] In step 2, the hard material includes an aluminum plate, an iron plate, a wooden plate, a plastic plate, etc.; the thickness of the aluminum plate and the iron plate is 3-5 mm, and the thickness of the plastic plate and the wooden plate is 5-8 mm;

[0022] In step 3, the printing pressure and time have an impact on the degree of cell rupture and integrity of the leaves and / or petals. The program pressure is recommended for different leaf types as follows but is not limited to the following programs, which can be further adjusted according to the actual state of the leaves and / or petals in use:

[0023] Villus leaves: initial pressure 2.5 MPa, maintain for 1-2 min, increase to 8 MPa at a rate of 2 MPa / min, maintain for 1-2 min, release the pressure immediately after penetration;

[0024] Wax leaves: initial pressure 2.5 MPa, maintain for 2-3 min, increase to 8 MPa at a rate of 2 MPa / min, maintain for 2-3 min, release the pressure immediately after penetration;

[0025] Leathery leaves: Initial pressure 3 MPa, maintain for 2-3 min, increase to 10 MPa at a rate of 2 MPa / min, maintain for 2-3 min, release pressure immediately after penetration;

[0026] For hairy petals and waxy petals: the initial pressure is 2.5 MPa, maintained for 2-3 min, then increased to 6 MPa at a rate of 2 MPa / min, maintained for 2-3 min, and the pressure is released immediately after printing;

[0027] The process of printing and pressing after printing the outline of leaves and / or petals on a porous material cushioning material is referred to as "rubbing".

[0028] The present invention also provides a leaf and / or petal sample pressing device, which can be used to implement the above-mentioned preparation method, and the sample pressing device comprises: a leaf and / or petal fixing mechanism, a leaf and / or petal clamping mechanism, a rubbing and pressing mechanism, a leaf and / or petal peeling mechanism, and a leaf and / or petal bonding mechanism;

[0029] The leaf and / or petal fixing mechanism is used to fix the leaf and / or petal on the buffer material through a transparent or translucent sticky material;

[0030] The blade and / or petal clamping mechanism is used to clamp the buffer material with blades and / or petals fixed thereon between two hard materials;

[0031] The rubbing and pressing mechanism is used to apply a program pressure to the hard material on which the leaves and / or petals are placed;

[0032] The leaf and / or petal peeling mechanism is used to peel the printed leaf and / or petal from the buffer material;

[0033] The leaf and / or petal bonding mechanism is used to bond the peeled leaf and / or petal to the sample slide.

[0034] The rubbing and stamping mechanism is a relatively important sample preparation mechanism in the present invention, and further comprises: a sample holding cavity formed by the side wall and the device bottom plate, a stamping cover, and a positioning block;

[0035] The sample holding cavity is used to place a hard material to be imprinted, which is clamped with a buffer material that fixes leaves and / or petals;

[0036] The interior of the side wall is provided with a continuous internal thread;

[0037] The bottom plate of the device is connected with a pressure sensor for detecting the rubbing pressure;

[0038] The upper surface of the bottom plate of the device is cross-shaped with two mutually perpendicular grooves;

[0039] The stamping cover comprises an upper and lower structure, the side wall of the lower structure is provided with an external thread matching the internal thread; the upper layer is a cylindrical structure with a slightly smaller diameter, the middle part of the cylindrical structure is hollowed out, reducing the weight and material requirements of the stamping cover, and the upper part of the upper layer of the cylindrical wall is provided with a cylindrical connection hole for connecting with an external rotating mechanism, and the stamping cover is rotated by the rotating structure to gradually screw the stamping cover into the sample holding cavity, fix it and slowly apply pressure to the bottom, so as to apply a program pressure to the sample placed in the sample holding cavity;

[0040] The upper part of the locking block is rectangular, and the lower part is trapezoidal, and its thickness matches the width of the groove on the bottom plate of the device. A locking structure matching the trapezoidal shape of the lower part of the locking block is provided in the groove; when the locking block is placed vertically in the groove, that is, when the long side of the locking block points to the center of the circle of the bottom plate of the device, it can slide freely in the groove. When the locking block needs to fix the sample placed in the sample holding cavity, the locking block moves along the groove to the edge of the sample and rotates 90° to fix the locking block in the groove, thereby fixing the sample.

[0041] The present invention also provides the sample preparation method or the use of the sample pressing device in the rapid preparation of spatial metabolomics leaf and / or petal samples.

[0042] The beneficial effects of the present invention include:

[0043] (1) The present invention provides a new method for preparing leaves and / or petals for spatial metabolomics, which is simple, reliable and stable. After the sample preparation is completed, the leaves and / or petals can be directly scanned, which can better reflect the true spatial distribution of metabolites in the leaves and / or petals, and can be used for spatial metabolomics mass spectrometry imaging of plant leaves and / or petals for detection and analysis.

[0044] (2) The present invention adopts a method of directly scanning the plant leaves and / or petals after rubbing, thereby avoiding the problem that the printing method is mainly due to the structure of the leaves and / or petals, and the veins are printed on the film material. In addition, the cells of the leaves and / or petals are not completely broken during the printing process, the efficiency of transferring the substances to the film material is uneven, and diffusion is prone to occur after the transfer, resulting in blurred contours of the imaged leaves and / or petals.

[0045] (3) The present invention selects a rubbing method to break up leaf and / or petal cells to expose cell contents, and selects a porous material to buffer the rubbing process, thereby protecting the integrity of the leaf while breaking up the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] 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 those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0047] Figure 1 This is a comparison chart of the effects of different buffer materials on the imaging effect of the blade at m / z=391.1065 in the present invention. a: The membrane material after porous polytetrafluoroethylene is used as the buffer material; b: The blade after 40-count white cotton cloth is used as the buffer material; c: The blade after 80-count white cotton cloth is used as the buffer material; d: The blade after porous polytetrafluoroethylene is used as the buffer material; e: The blade after 100-mesh nylon mesh is used as the buffer material; f: The blade after 150-mesh nylon mesh is used as the buffer material; g: The blade after 300-mesh nylon mesh is used as the buffer material.

[0048] Figure 2 This is a comparison chart of the effects of different pressures on the imaging effect of m / z=391.1066 in the leaves of Loropetalum chinensis when 150 mesh nylon mesh is used as the buffer material in the present invention. a: The pressure of 12 MPa is too high; b: The pressure of 8 MPa is moderate; c: The pressure of 4 MPa is too low.

[0049] Figure 3These are images of different types of leaves in the present invention. a: velvety leaves (from Loropetalum chinensis); b: waxy leaves (from Camphor tree); c: leathery leaves (from Osmanthus fragrans).

[0050] Figure 4 The present invention takes camphor tree leaves as an example of the leaf state diagram of the rubbing operation process. a: The leaf is stuck on the porous material buffer material by the translucent sticky material; b: The leaf after rubbing; c: The leaf is separated from the porous material buffer material and pasted on the double-sided tape; d: The leaf after the leaf imaging scan; e: The leaf outline is rubbing with white cotton cloth; f: The imaging scan of the leaf when m / z=425.4229.

[0051] Figure 5 It is a display diagram of imaging camphor tree and Loropetalum chinensis in the embodiment of the present invention.

[0052] Figure 6 This is a display diagram of imaged Solanum nigrum and Ophiopogon japonicus petals in an embodiment of the present invention.

[0053] Figure 7 The following are the effects of loose fixation of the sample and the glass slide during the implementation of the present invention: a: The sample is not firmly fixed and is scratched by the ion transfer tube; b: The blade debris enters the mass spectrometer cone.

[0054] Figure 8 This is a sample preparation schematic diagram and detection imaging diagram in the tea imprint imaging report published in 2019 in the prior art.

[0055] Fig. 9 It is the imprint effect diagram and imaging effect diagram in the leaf imprint related article published in 2022 in the prior art.

[0056] Fig.10 This is a schematic diagram of imprinting and imaging in a related patent for leaf imprinting using a nanoimprinting substrate, which was published in 2020 with the number CN 111239238 A.

[0057] Fig.11 It is a schematic diagram of leaf imprints and imaging in a patent related to Chinese medicinal plant imprints with publication number CN 115824749 A in 2023 in the prior art.

[0058] Fig.12 It is a schematic diagram of the structure of the leaf and / or petal rubbing and pressing mechanism in the present invention.

[0059] Fig.13 It is a top view of the sample holding cavity in the rubbing and imprinting mechanism of the present invention.

[0060] Fig.14 It is a three-dimensional diagram of the sample containing cavity in the rubbing and imprinting mechanism of the present invention.

[0061] Fig.15 It is a cross-sectional view of the sample containing cavity in the rubbing and imprinting mechanism of the present invention.

[0062] Fig.16 It is a side view of the stamping cover in the rubbing and stamping mechanism of the present invention.

[0063] Fig.17 It is a top view of the stamping cover in the rubbing and stamping mechanism of the present invention.

[0064] Fig.18 It is a three-dimensional diagram of the stamping cover in the rubbing stamping mechanism of the present invention.

[0065] Fig.19 It is a cross-sectional view of the stamping cover in the rubbing and stamping mechanism of the present invention.

[0066] Fig. 20 It is a front view of the positioning block in the rubbing and stamping mechanism of the present invention.

[0067] Fig.21 It is a three-dimensional diagram of the positioning block in the rubbing and stamping mechanism of the present invention.

[0068] Fig. 22 It is a top view of the positioning block in the rubbing and stamping mechanism of the present invention.

[0069] Fig.23 It is a schematic cross-sectional structure diagram of the stamping cover in the rubbing stamping mechanism of the present invention being gradually screwed into the sample containing cavity.

[0070] Figure 13-Figure 23 In the figure, 1-side wall, 2-device bottom plate, 3-stamping cover, 4-positioning block, 5-internal thread, 6-groove, 7-external thread, 8-cylindrical structure, 9-connecting hole. DETAILED DESCRIPTION

[0071] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0072] The present invention proposes a method for rapidly preparing leaf and / or petal samples for spatial metabolomics analysis, comprising the following steps: fixing leaf and / or petal on a buffer material; placing the obtained buffer material with leaf and / or petal fixed between two hard materials that can cover leaf and / or petal; applying program pressure to the obtained hard material with leaf and / or petal placed to reach a predetermined through-print target; taking out the buffer material with leaf and / or petal fixed, and completely peeling the leaf and / or petal from the buffer material; fixing the peeled leaf and / or petal on a sample slide, and finally obtaining leaf and / or petal samples for spatial metabolomics analysis. The plant leaf and petal sample mass spectrometry imaging pretreatment method of the present invention has wide universality, changes direction in the conventional imprint pretreatment method, does not image the membrane material, and directly images the leaf and / or petal, which can ensure the comprehensive exposure of leaf and / or petal cell contents, remove the influence of leaf epidermal wax hair, etc., has a wide range of buffer material selection, and provides a simple and reliable pretreatment method for plant leaf and / or petal, which can be applied to all types of mass spectrometry imagers commercialized on the market.

[0073] Example 1

[0074] 1. Instrument

[0075] AFA-DESI imaging platform (Victor); Thermo Fisher Q-Orbitrap HFX liquid chromatography-mass spectrometry (Thermo).

[0076] 2. Leaf and / or petal sample pretreatment

[0077] Fix the leaves and / or petals on a 150-mesh nylon mesh and place them between two hard materials that can cover the leaves and / or petals. Press the hard materials with programmed pressure to make the leaves and / or petals have their outlines printed on the 150-mesh nylon mesh. Stop the printing and pressing, and separate the leaves and / or petals from the 150-mesh nylon mesh smoothly. Transfer and fix the leaves and / or petals on a sample slide, and use AFADESI mass spectrometry imaging technology to directly perform spatial imaging scanning and analysis on the leaves and / or petals with ruptured cells.

[0078] 3. Mass spectrometry imaging detection

[0079] Spray: solvent acetonitrile: water = 80: 20 (V / V), flow rate 4 μL / min.

[0080] Needle size: 20 μm.

[0081] Imaging parameters: The scanning parameters were set by the mass spectrometry imaging platform control program software, see Table 1 below.

[0082]

[0083] Mass spectrometry parameters: Data acquisition was performed using the Xcalibur data acquisition and processing system, and the data acquisition sequence was set according to the sample size, step spacing, scanning speed, etc. The AFADESI ion source parameters and mass spectrometry data acquisition parameters are detailed in Table 2 below.

[0084]

[0085] 4. Data Processing

[0086] The tools provided by Thermo acquisition software were used to convert the original mass spectrometry data (.raw format) into a .cdf format file through Converter, and then imported into Massimaging. The sample size, number of scan lines and other information were input to generate an analysis plan, perform background subtraction, peak alignment, peak screening, normalization, smoothing, etc., and finally image the data.

[0087] Example 2 Application of the method

[0088] The parameter method of Example 1 was used to perform mass spectrometry imaging detection on camphor tree leaves, red flower loropetalum leaves, Solanum nigrum petals, and Ophiopogon japonicus petals. The results are shown in Figure 5 , Figure 6 The detection information of the top 100 responding substances is shown in Table 3 below, among which 11,277 substances were detected in Cinnamomum camphora leaves, and 799 substances had a response greater than 1,000; 11,217 substances were detected in Loropetalum chinensis leaves, and 477 substances had a response greater than 1,000; 9,541 substances were detected in Solanum nigrum petals, and 899 substances had a response greater than 1,000; 7,642 substances were detected in Ophiopogon japonicus petals, and 934 substances had a response greater than 1,000.

[0089]

[0090]

[0091]

[0092] The present invention investigates the buffer materials, including porous polytetrafluoroethylene, 40-count white cotton cloth, 80-count white cotton cloth, 100-mesh nylon mesh cloth, 150-mesh nylon mesh cloth, and 300-mesh nylon mesh cloth. It is found that porous material materials with high counts or meshes, including hydrophilic and hydrophobic materials, can be used as buffers for rubbing. Compared with scanning film materials, the component distribution structure exposed by scanning leaves is more real and clear. The characteristic m / z of Loropetalum chinensis at 391.1065 is used as the display data. For details, see Figure 1 ,according to Figure 1 The image of the membrane material after the porous polytetrafluoroethylene is used as the buffer material is printed. The effect is obvious that the cell contents are transferred and diffused, and the transfer is incomplete. Figure 1 The image of the blade after the porous polytetrafluoroethylene is used as the buffer material. Figure 1 The a-cell contents in the image are not diffused obviously, but there is a clear inconsistency in the degree of cell fragmentation. Figure 1 b in Figure 1 The c in the figure corresponds to the leaf images printed with 40-count and 80-count white cotton cloth as the buffer material, respectively. Figure 1 Compared with d in the figure, the imaging structure of the leaf is clearer and the contents of the cell fragments are more evenly exposed. Figure 1 The e / f / g in the figure correspond to the leaf images printed with 100 mesh, 150 mesh and 300 mesh nylon mesh as the buffer material. Figure 1 The b / c blade imaging structure is clearer and more complete, so nylon mesh is better as a buffer material, and the effect is better above 150 mesh.

[0093] The research process of the present invention compares the same 150 mesh nylon mesh as the buffer material. The final pressure applied is 12 MPa, which is too large ( Figure 2 a), 8 MPa moderate ( Figure 2 b) in the figure, 4 MPa is too small ( Figure 2 c) on the leaf imprint, with the characteristic m / z of Loropetalum chinensis at 391.1065 as the display data, see Figure 2 ,obvious Figure 2 The b pressure of 8 MPa is moderate, the overall outline of the leaf imaging structure is clear, and the veins are complete.

[0094] The present invention uses 150 mesh nylon mesh as a buffer material to process samples of different types of leaves, including velvety red loblolly leaves (a), waxy camphor leaves (b), and leathery osmanthus leaves (c). The analysis results of the spatial imaging scan are partially shown in FIG. Figure 3 , Figure 3 The images of 12 substances, 133.0159, 191.0546, 213.1493, 383.0908, 391.1060, 441.2505, 447.0945, 483.2741, 515.1491, 593.1528, 601.2156, and 639.1584, were displayed for three types of leaves, covering 100-700 m / z. It can be seen intuitively in the figure that the three types of leaves have obvious material differences, and all have characteristic distribution detected substances. Some substances are obviously distributed at the edge of the leaves, some substances are close to the veins, and most substances are evenly distributed on the leaves. Figure 8~Figure 11The imaging results reported in the prior art show that the leaf imaging structure is obviously clearer and more complete. The results show that the leaf pretreatment method invented in this study is simple and reliable, and fully exposes the spatial distribution of leaf components. The differences in the spatial distribution of components of three different types of leaves are intuitively displayed in the imaging images. It is a pretreatment method that is very suitable for spatial metabolomics analysis of leaf / petal samples.

[0095] The present invention studies the same pretreatment of the upper and lower surfaces of camphor tree leaves and red loblolly vine leaves, and then performs spatial imaging scanning and analysis. It is found that there are more imaging substances on the lower surface, and the lower surface of the upper leaf is more likely to expose the cell contents as a rubbing surface. For details, see Figure 4 , Figure 5 , Figure 4 Take camphor tree leaves as an example to show the leaf status diagram of the rubbing operation process. Figure 5 The imaging of the embodiment shows the camphor tree and the red flower Loropetalum, Figure 5 The first row corresponds to the imaging of the three substances on the upper and lower surfaces of the camphor tree leaves. It is obvious that the imaging intensity of the lower surface is higher and clearer. The second row corresponds to the imaging of the three substances on the lower and upper surfaces of the loblolly vine leaves. It also shows that the imaging intensity of the lower surface is higher and clearer.

[0096] During the research process of the present invention, it was found that the unstable fixation of the sample on the sample slide may cause the following hazards: vacuum adsorption of the ion transfer tube, fluctuation of the sample, touching the transfer tube, generating debris, and the debris entering the mass spectrometer cone, blocking the mass spectrometer ion transfer tube, see Figure 7 ; Figure 7 The a in the figure shows that the sample is not firmly fixed and is scratched by the ion transfer tube; Figure 7 Panel b shows blade debris entering the mass spectrometer cone.

[0097] Comparison of this invention with published articles and patents can be found in Figure 8~Figure 11 ,from Figure 8 You can see that the 2019 Tea Imprint article [1] The imprinting process is clearly shown, but the leaves on the imprinted film have obviously spread, resulting in unclear outlines of the tea leaves and unclear imaging spatial distribution characteristics of various substances; Leaf imprinting articles published in 2022 [2] ,from Fig. 9 It can be seen that there is an obvious problem of poor efficiency in transferring cell contents after leaf crushing; the drawings in the patent application text of leaf imprinting with nanoimprinting substrate with publication number CN111239238 A in 2020 and the drawings in the patent application text of Chinese medicinal plant imprinting with publication number CN115824749 A in 2023, from Fig.10 , Fig.11 It can be seen that the leaf veins are mainly imprinted, and the problem of the efficiency of cell content transfer after imprinting remains. Figure 5 , Figure 6 This is the effect of direct imaging of plant leaves and petals after being processed and printed by the method of the present invention. Since there is no influence of transfer and diffusion, it is only necessary to consider whether the cells are broken and whether the contents are fully exposed. The integrity of the overall imaging of the leaves and petals and the sensitivity of substance detection are significantly improved.

[0098] In order to achieve the effect of rubbing, the research process of this invention designs a rubbing and pressing mechanism, see Fig.12 ; Fig.12 A shows the overall structure of the rubbing and pressing device and the blade and / or petal clamping mechanism being inserted into the device and fixed by the clamping block during pressing; Fig.12 The bottom structure of the B rubbing and pressing device has a hollow groove at the bottom, into which a positioning block can be placed; Fig.12 The C-card block plane structure in.

[0099] Based on the above results, it can be seen that the method of the present invention optimizes the pretreatment of leaves and / or petals, selects plant leaves and / or petals after rubbing, rather than membrane materials after printing, so that it is not affected by whether the leaves and / or petals have epidermal wax or villi, and can directly perform spatial imaging scanning and analysis on the leaves and / or petals, providing a simple and reliable sample pretreatment method for spatial metabolomics mass spectrometry imaging analysis of plant leaves and / or petals.

[0100] The present invention provides a leaf and / or petal sample pressing device, which can realize the leaf and / or petal sample preparation method proposed in the present invention. The sample pressing device comprises: a leaf and / or petal fixing mechanism, a leaf and / or petal clamping mechanism, a rubbing and pressing mechanism, a leaf and / or petal peeling mechanism, and a leaf and / or petal bonding mechanism;

[0101] The leaf and / or petal fixing mechanism is used to fix the leaf and / or petal on the buffer material through a transparent or translucent sticky material;

[0102] The blade and / or petal clamping mechanism is used to clamp the buffer material with blades and / or petals fixed thereon between two hard materials;

[0103] The rubbing and pressing mechanism is used to apply a program pressure to the hard material on which the leaves and / or petals are placed;

[0104] The leaf and / or petal peeling mechanism is used to peel the printed leaf and / or petal from the buffer material;

[0105] The leaf and / or petal bonding mechanism is used to bond the peeled leaf and / or petal to the sample slide.

[0106] like Figure 13-Figure 23As shown, the rubbing and pressing mechanism comprises: a sample holding cavity formed by a side wall 1 and a device bottom plate 2, a pressing cover 3, and a positioning block 4;

[0107] The sample holding cavity is used to place a hard material to be imprinted, which is a buffer material holding and fixing a leaf and / or petal sample;

[0108] The stamped cover 3 is used to cooperate with the sample holding cavity to apply a program pressure to the sample;

[0109] The positioning block 4 is used to position and fix the sample in the sample holding cavity.

[0110] The interior of the side wall 1 is provided with a continuous internal thread 5;

[0111] The device bottom plate 2 is connected to a pressure sensor for detecting the rubbing pressure;

[0112] The upper surface of the device bottom plate 2 is cross-shaped with two mutually perpendicular grooves 6; and / or,

[0113] The stamping cover 3 includes an upper and lower structure, the side wall of the lower structure is provided with an external thread 7 matching the internal thread 5; the upper layer is a cylindrical structure 8, the middle part of the cylindrical structure 8 is hollowed out, and the upper part of the cylindrical wall of the cylindrical structure 8 is provided with a cylindrical connecting hole 9 for connecting with an external rotating mechanism, and the stamping cover 3 is rotated by the rotating structure to gradually screw the stamping cover 3 into the sample holding cavity, so as to apply a program pressure to the sample placed in the sample holding cavity; and / or,

[0114] The upper part of the locking block 4 is a rectangular parallelepiped, and the lower part is a trapezoidal structure. The thickness of the locking block matches the width of the groove 6 on the bottom plate 2 of the device, and the groove 6 is provided with a locking structure that matches the trapezoidal shape of the lower part of the locking block 4; when the locking block 4 is placed vertically in the groove 6, it can slide freely in the groove 6, and the sample can be fixed by rotating the locking block 4 90° in the groove 6.

[0115] References

[0116] [1]Liao Y, Fu 10.1016 / j.foodchem.2019.04.055. Epub 2019 Apr 16. PMID: 31054666.

[0117] [2]Wu L, Qi K, Liu C, Hu Y, Xu M, Pan Y. Enhanced Coverage andSensitivity of Imprint DESI Mass Spectrometry Imaging for Plant Leaf Metabolites by Post-photoionization. Anal Chem. 2022 Nov 1;94(43):15108-15116. doi: 10.1021 / acs.analchem.2c03329. Epub 2022 Oct 6. PMID: 36201321.

[0118] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A method for preparing leaf and / or petal samples for spatial metabolomics analysis, characterized in that: The preparation method comprises the following steps: Step 1: Fix the leaves and / or petals on the cushioning material; Step 2: placing the cushioning material with the leaves and / or petals fixed thereon obtained in step 1 between two hard materials that can cover the leaves and / or petals; Step 3: applying a program pressure to the hard material with leaves and / or petals placed thereon obtained in step 2 so that the leaves and / or petals are printed through on the buffer material; The program pressure is set according to the type of leaves and / or petals: Villus leaves: initial pressure 2.5 MPa, maintain for 1-2 min, increase to 8 MPa at a rate of 2 MPa / min, maintain for 1-2 min, release the pressure immediately after penetration; Wax leaves: initial pressure 2.5 MPa, maintain for 2-3 min, increase to 8 MPa at a rate of 2 MPa / min, maintain for 2-3 min, release the pressure immediately after penetration; Leathery leaves: Initial pressure 3 MPa, maintain for 2-3 min, increase to 10 MPa at a rate of 2 MPa / min, maintain for 2-3 min, release pressure immediately after penetration; For hairy petals and waxy petals: the initial pressure is 2.5 MPa, maintained for 2-3 min, then increased to 6 MPa at a rate of 2 MPa / min, maintained for 2-3 min, and the pressure is released immediately after printing; Step 4: Take out the cushioning material with the leaves and / or petals fixed thereon, and completely peel off the leaves and / or petals from the cushioning material; Step 5: Fix the peeled leaves and / or petals onto a sample slide to obtain leaf and / or petal samples for final spatial metabolomics analysis.

2. The preparation method according to claim 1, characterized in that The leaves include hairy leaves, waxy leaves, and leathery leaves; The petals include fluffy petals and waxy petals.

3. The preparation method according to claim 1, characterized in that: The buffer material is a porous material, including porous polytetrafluoroethylene, cotton cloth, and nylon mesh; wherein, The thickness of the porous polytetrafluoroethylene is 0.5 mm and the apparent density is 2.1-2.3 G / cm 3 , tensile strength ≥14.0MPa / min, elongation at break ≥150%; The count of the cotton cloth is 40 to 80; The mesh number of the nylon mesh is 100-300 meshes.

4. The preparation method according to claim 1, characterized in that: In step 1, the leaves and / or petals are fixed on the cushioning material by means of a transparent or translucent adhesive material.

5. The preparation method according to claim 1, characterized in that: In step 2, the hard material includes an aluminum plate, an iron plate, a wooden plate, or a plastic plate; and / or, The thickness of the aluminum plate or iron plate is 3-5 mm, and the thickness of the wooden plate or plastic plate is 5-8 mm.

6. A leaf and / or petal sample pressing device, characterized in that: The sample pressing device can realize the preparation method according to any one of claims 1 to 5, and the sample pressing device comprises: a leaf and / or petal fixing mechanism, a leaf and / or petal clamping mechanism, a rubbing and pressing mechanism, a leaf and / or petal peeling mechanism, and a leaf and / or petal bonding mechanism; The leaf and / or petal fixing mechanism is used to fix the leaf and / or petal on the buffer material through a transparent or translucent sticky material; The blade and / or petal clamping mechanism is used to clamp the buffer material with blades and / or petals fixed thereon between two hard materials; The rubbing and pressing mechanism is used to apply a program pressure to the hard material on which the leaves and / or petals are placed; The leaf and / or petal peeling mechanism is used to peel the printed leaf and / or petal from the buffer material; The leaf and / or petal bonding mechanism is used to bond the peeled leaf and / or petal to the sample slide.

7. The sample compression device according to claim 6, characterized in that: The rubbing and pressing mechanism comprises: a sample containing cavity formed by a side wall (1) and a device bottom plate (2), a pressing cover (3), and a positioning block (4); The sample holding cavity is used to place a hard material to be imprinted, which is a buffer material holding and fixing a leaf and / or petal sample; The stamped cover (3) is used to cooperate with the sample holding cavity to apply a program pressure to the sample; The positioning block (4) is used to position and fix the sample in the sample containing cavity.

8. The sample compression device according to claim 7, characterized in that: A continuous internal thread (5) is provided inside the side wall (1); The bottom plate of the device is connected with a pressure sensor for detecting the rubbing pressure; The upper surface of the device bottom plate (2) is provided with two mutually perpendicular grooves (6) in a cross shape; and / or, The stamping cover (3) comprises an upper and lower structure, wherein the side wall of the lower structure is provided with an external thread (7) matching the internal thread (5); the upper layer is a tubular structure (8), the middle part of the tubular structure (8) is hollowed out, and the upper part of the tubular wall of the tubular structure (8) is provided with a cylindrical connection hole (9) for connecting with an external rotating mechanism, and the stamping cover (3) is rotated by the rotating mechanism to gradually screw the stamping cover (3) into the sample holding cavity, so as to apply a programmed pressure to the sample placed in the sample holding cavity; and / or, The upper portion of the locking block (4) is a rectangular parallelepiped, and the lower portion is a trapezoidal structure. The thickness of the locking block matches the width of the groove (6) on the bottom plate (2) of the device. The groove (6) is provided with a locking structure that matches the trapezoidal shape of the lower portion of the locking block (4). When the locking block (4) is placed vertically in the groove (6), it can slide freely in the groove (6). The sample is fixed by rotating the locking block (4) 90 degrees in the groove (6).

9. Use of the sample preparation method according to any one of claims 1 to 5, or the sample pressing device according to any one of claims 6 to 8, in the rapid preparation of leaf and / or petal samples for spatial metabolomics.

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