A method for microscopic chemical identification of traditional Chinese medicine at single cell level

Abstract

The application discloses a single-cell level traditional Chinese medicine micro-chemical identification method, comprising the following steps: powder sealing and sheet preparation, medicinal material powder adding, medicinal material micro-identification, coordinate identification and positioning, LMJ-SSP extraction, extraction liquid concentration and sample spotting, and MALDI-MS detection. Microscopic information and chemical component information of target microstructure can be obtained through the above steps. The method provided by the application can obtain traditional Chinese medicine microscopic information through traditional micro-technology, and can also obtain chemical information corresponding to the microstructure by means of LMJ-SSP and mass spectrometry, so that the chemical information can be combined with the cross-verification of the microstructure to improve the reliability of traditional Chinese medicine identification.

Description

Technical Field

[0001] This invention belongs to the field of analysis and relates to the identification of traditional Chinese medicine, specifically to a single-cell level microchemical identification method for traditional Chinese medicine. Background Technology

[0002] Microscopic identification of traditional Chinese medicine (TCM) utilizes a microscope to identify the tissue, cell, or inclusion characteristics of crude drugs and prepared TCM formulations. It relies on specific microscopic features as the basis for identification and is one of the scientific methods for pharmacognosy identification. However, due to technical limitations, the chemical composition of powders can be destroyed after microscopic identification due to permeation with chloral hydrate or water. Furthermore, the microscopic features of medicinal materials lack cross-verification with other techniques and cannot be correlated with information about the chemical characteristics of the medicinal materials. Simultaneously, microscopic identification techniques require considerable experience and experimental skills, thus limiting their application.

[0003] Novel surface sampling and ionization techniques, such as the Liquid Microjunction Surface Sampling Probe (LMJ-SSP), offer a variety of tools for surface analysis and drug distribution analysis in animal tissues. The LMJ-SSP probe technology can be used in conjunction with mass spectrometry ion sources such as ESI or APCI. As an open-type surface desorption ionization technique, it boasts advantages such as simple structure, flexible operation, and time and resource savings, and has been applied in rapid sample surface analysis and mass spectrometry molecular imaging studies. The LMJ-SSP sampling technique primarily utilizes the surface tension of the liquid to form a droplet microjunction between the probe and the sample surface, thereby extracting and desorbing the analyte on the surface, and finally ionizing it through an ESI plasma source. This technology can be used for mass spectrometry analysis of drugs and proteins in dry and solution states; analysis of components separated on dye, ink, and reversed-phase C8 or C18 thin-layer chromatography plates; and detection of exogenous substances in tissue sections. Currently, research on this type of technology mainly focuses on the automated control of the probe-sample surface distance, sampling stability, and sample material properties.

[0004] If a method could be established that can obtain microscopic information of traditional Chinese medicine (TCM) using traditional microscopy techniques, and also obtain the corresponding chemical information of the microstructure using LMJ-SSP combined with mass spectrometry, the chemical information could be combined with the associated microstructure to cross-validate the identification results of TCM, thereby improving the reliability of TCM identification. Currently, no such method has been reported.

[0005] Based on the research and development results of the above methods, this invention is hereby proposed. Summary of the Invention

[0006] The present application aims to overcome the deficiencies of the prior art, and provides a single-cell level traditional Chinese medicine microscopic chemical identification method.

[0007] The above-mentioned object of the present application is achieved by the following technical solutions.

[0008] A single-cell level traditional Chinese medicine microscopic chemical identification method comprises the following steps:

[0009] Step S1, powder sealing preparation: an appropriate amount of liquid gelatin aqueous solution with a mass fraction of 10%-15% is added to a glass slide, and a gelatin layer with uniform thickness and without air bubbles and impurities is formed after the gelatin is solidified, thereby obtaining a powder sealing;

[0010] Step S2, medicinal material powder addition: an appropriate amount of traditional Chinese medicine powder is carefully and uniformly added to the gelatin layer of the powder sealing, and the traditional Chinese medicine powder is naturally dried or vacuum dried after being firmly adhered to the gelatin;

[0011] Step S3, medicinal material microscopic identification: a plurality of marks are dispersed on the back of the dried powder sealing, and low-power field observation is performed under a low-power microscope, and after the target microscopic structure is found, high-power field observation of the target microscopic structure is further performed by using a high-power microscope, and the low-power field picture containing the marks and the target microscopic structure and the high-power field picture containing the target microscopic structure are recorded respectively;

[0012] Step S4, coordinate identification and positioning: the low-power field picture is input into software with a microscopic structure identification function, the pixel point coordinate values of the target microscopic structure and at least one of the marks on the picture are output by the software, one of the marks is selected as a positioning point, the positioning point is set as a relative zero point, and the accurate positioning of the target microscopic structure is obtained according to the pixel point coordinate values of the positioning point and the target microscopic structure;

[0013] Step S5, LMJ-SSP extraction: a droplet micro-connection surface sampling probe is used to extract the target microscopic structure in situ according to the accurate positioning of the target microscopic structure, and an extraction liquid is collected;

[0014] Step S6, concentration and spotting of the extraction liquid: the extraction liquid is concentrated, spotted on an ITO glass slide, and a matrix is added;

[0015] Step S7, MALDI-MS detection: the ITO glass slide is placed on a target plate, the target plate is inserted into the target, and the software is opened to determine the chemical components in the extract according to the general operation method of the MALDI mass spectrometer;

[0016] Through the above steps, the microscopic information and chemical component information of the target microscopic structure can be obtained.

[0017] Preferably, the mark in step S3 is an "X" mark.

[0018] Preferably, in step S6, the aminoacridine is used as the negative ion detection matrix, and the a-p-hydroxy cinnamic acid is used as the positive ion detection matrix.

[0019] Preferably, in step S6, the method for adding the matrix is a matrix reconstitution point sample method or a matrix spray method.

[0020] Advantages:

[0021] 1. The method provided by the present application can obtain microscopic information of traditional Chinese medicine through traditional microscopic techniques, and can also obtain chemical information corresponding to the microscopic structure by means of LMJ-SSP and mass spectrometry, and then the chemical information can be combined with the cross-verification of the microscopic structure to improve the reliability of the identification of traditional Chinese medicine.

[0022] 2. The method provided by the present application is not a simple combination of traditional microscopic techniques and LMJ-SSP-mass spectrometry techniques. The present application uses a gelatin layer coated powder mounting, which can not only fix the traditional Chinese medicine powder, but also enhance the targeting extraction effect of LMJ-SSP on the traditional Chinese medicine powder, and will not pollute the mass spectrometry. More specifically:

[0023] When obtaining the microscopic structure characteristics, the gelatin has high transparency and can fix the traditional Chinese medicine powder well without producing artifacts after mounting; compared with the polyacrylamide gel which is firstly used in the experiment, the polyacrylamide gel will produce serious diffusion after the traditional Chinese medicine powder is screened in, which affects the fixing and positioning effect of the traditional Chinese medicine powder; compared with the transparent tape commonly used to fix the traditional Chinese medicine powder, the transparent tape has wrinkles after imaging, which affects the observation;

[0024] When LMJ-SSP extraction, researchers found that when the LMJ-SSP extraction probe contacts the gelatin layer, the dynamic pressure at the extraction probe increases and is adsorbed on the gelatin layer, which can effectively reduce the diffusion of extraction droplets on the mounting (such as Figure 4 ), and thus increase the extraction resolution; when using polyacrylamide gel, this phenomenon is not observed;

[0025] In addition, compared with polyacrylamide gel and the like, the compounds in the extraction liquid will not diffuse into the gelatin layer to cause component loss; and the chemicals in the gelatin will not be released into the extraction liquid to cause mass spectrometry pollution.

[0026] 3. The method provided by the present application uses mass spectrometry method to deeply analyze the chemical information of single cell structure, which fills the gap that microscopic identification techniques cannot be verified by other techniques, and reduces the destructive effect on the chemical information of medicinal material powder; chemical component identification is a favorable support for microscopic identification, which reduces the difficulty of work and the requirement of identification experience. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1The flow chart of the single-cell level traditional Chinese medicine micro-chemical identification method of the present application, wherein A is a general glass slide, B is a powder sealing glass slide with added gelatin, C is powder screening, D is micro-identification of the medicinal material and coordinate identification of the powder, E is LMJ-SSP control and extraction, and F is extraction liquid concentration, sample spotting and MALDI-MS detection;

[0028] Figure 2 The powder sealing glass slide preparation, solidification and drying process chart, Fritillaria cirrhosa powder sealing glass slide, and the observed micrographs of the representative traditional Chinese medicine safflower pollen grains and Fritillaria cirrhosa starch grains; wherein A is a glass slide with a gelatin layer formed after adding gelatin, B is a powder sealing glass slide after drying the gelatin, C is the finished product of the powder sealing glass slide after screening the Fritillaria cirrhosa medicinal material powder (the "X" is a positioning mark on the back side of the glass slide), D is an example of Fritillaria cirrhosa starch grains observed under a microscope, and E is an example of safflower pollen grains observed under a microscope.

[0029] Figure 3 The schematic diagram of the LMJ-SSP device; wherein 1 is a three-axis table with fixed probes, 2 is a three-way series LMJ-SSP probe, 3 is a self-built sample table and sample glass slide, 4 is an electric two-axis table, 5 is a micro-sampler, 6 is a syringe pump, 7 is a vacuum control valve, 8 is a vacuum pump access valve, and 9 is a self-built vacuum collection box.

[0030] Figure 4 The gelatin sealing glass slide can effectively reduce the diffusion area of the LMJ-SSP droplets under three flow rates;

[0031] Figure 5 The software operation process and result output of the Fritillaria cirrhosa intracellular content starch grains located by MicroMS, and the coordinate information of the single dispersed starch grains can be obtained; wherein A is the result of the software identifying the starch grains in the field of view under a low-power microscope, B is the threshold view of the A microscopic image, C is the A microscopic image and the corresponding size-number bar chart of each starch grain, and D is the image information and starch grain coordinate output of A;

[0032] Figure 6 The representative spectrum of the Fritillaria cirrhosa single starch grain, containing the appearance characteristics of the single intracellular content starch grain;

[0033] Figure 7 The medicinal material powders of other three kinds of pseudo-products of Fritillaria cirrhosa, the starch grain morphology and spectrum comparison. DETAILED DESCRIPTION

[0034] The substantial content of the present application will be specifically introduced below in combination with examples, but the protection scope of the present application is not limited by this.

[0035] The method of microscopic chemical identification of traditional Chinese medicine at single cell level includes the following steps: powder slide preparation, powder screening, microscopic identification, coordinate identification of powder, LMJ-SSP control and extraction, extraction liquid concentration and spotting, and MALDI-MS determination. Figure 1 The specific operations of each step are as follows:

[0036] (1) Powder slide preparation: using the selected gelatin as the slide material, a piece of ordinary glass slide is placed on a horizontal plane, then the prepared liquid 10%-15% gelatin aqueous solution is dropped on the glass slide by using a dropper, about 1 ml, then waiting or slightly spreading the gelatin solution with the dropper, the effective gelatin slide is obtained after the gelatin is solidified and the thickness is uniform, without bubbles or other impurities (the ice cooling method can be used).

[0037] For the slide material, we screened a variety of common laboratory gel materials such as gelatin, biological gel, and transparent double-sided tape commonly used in botanical microscopy, and the results are as follows:

[0038] Biological gel: for example, polyacrylamide gel, which produces serious diffusion after screening powder.

[0039] Adhesive tape: there are wrinkles on the surface after imaging, which affects observation.

[0040] Gelatin can be quickly solidified at room temperature during preparation, and has the advantages of good elasticity, viscosity, easy availability, fast preparation, no mass spectrum interference, no imaging interference, rapid drying after solidification, good transparency, and no material diffusion.

[0041] In order to verify that gelatin slide preparation can reduce the droplet diffusion of LMJ extraction probe, we prepared a blank gelatin slide, and used sunset yellow pigment to represent the droplet diffusion range, as shown in Figure 4 When the vacuum degree of the collection tank is 31 kpa, compared with ordinary glass slide, the droplet diffusion of gelatin slide is reduced at three liquid flow rates of 62, 60, and 58 μL / min.

[0042] (2) Powder screening: prepare the powder of the medicine to be tested and a No. 5 sieve, use a medicine spoon to gently screen the trace amount of medicine powder through the No. 5 sieve and onto the prepared glass slide containing solidified gelatin, wait for the powder to be firmly adhered to the solid gelatin, then wait for further drying of the glass slide, during which microscopic identification can be performed. At this time, the gelatin slide containing the medicine powder is completed, the trace amount of medicine powder is evenly screened and adhered to the gelatin, and the glass slide can be inverted or lightly tapped on the back to shake off the medicine powder that is not completely adhered. Then the gelatin slide is placed for drying (vacuum drying box can be used for drying, but attention should be paid to prevent the gelatin from melting. The gelatin will slowly lose water during drying, and attention should be paid to avoid airtight placement. (As shown in Figure 2 ) The powder slide prepared without bubbles and impurities is preferred.

[0043] (3) Microscopic identification of medicinal materials: Draw an "X" mark on the back of the dried powder, place it under an optical microscope or other microscope for bright field observation. First, use a low-power lens to observe, and then further use a high-power lens to observe the microscopic structure of traditional Chinese medicine after finding the microscopic structure. Record the low-power lens field containing the "X" mark and the microscopic structure, and the high-power lens field containing the microscopic structure characteristics. Specifically, you can compare the microscopic identification atlas of traditional Chinese medicine, select the tissue fragments, cells and intracellular inclusions with recognition degree. In the operation, single cells and intracellular inclusions should be selected for determination to obtain high-precision chemical component identification and avoid the average effect. Single cells and intracellular inclusions should be selected with clear edges and clear and detailed characteristic structures consistent with the characteristics of the microscopic atlas. After placing the gelatin mark under the microscope, use the scanning imaging software to set a certain overlap range to obtain a larger low-power lens field containing the "X" mark and the microscopic structure.

[0044] The control method of the microscopic structure is as follows: manual control or screening can refer to "Chinese Pharmacopoeia" or "Microscopic Identification Atlas of Traditional Chinese Medicine" for identification. Scanning screening (using MicroMS as an example), for scanning of Fritillariae cirrhosae starch granules, set parameters minimum size: 50, threshold: 160-175, image channel: 2. This software is not suitable for larger fragments or opaque structures. For software recognition, it needs to be reviewed or manually labeled.

[0045] (4) Coordinate identification of medicinal material powder: Input the low-power lens microscopic picture into a software that can identify cells or microscopic substances, such as open-source MicroMS software 8. This software can identify starch granules and pollen particles and output the pixel point coordinates on the picture. This coordinate can be calculated by the pixel length contained in the scale line when the magnification of the microscope is constant, i.e. scale pixel length / scale actual length = pixel number / actual length in this magnification. Further, the "X" mark under the low-power lens can produce intersecting corners to determine the positioning point. Set this positioning point as the relative zero point, subtract the coordinates of the two points to obtain the precise positioning coordinates of the target analyte. In this experiment, single dispersed cells and intracellular inclusions are used as the research object.

[0046] (5) LMJ-SSP control and extraction: The structure of LMJ-SSP control instrument is as follows Figure 3, comprising a controller, an xy two-axis table, an iron bracket, a three-axis table fixed tee, and a probe, a droplet probe, the droplet probe further takes coaxial capillary as the main body, the equipment selects capillary, the inner tube parameters: 150 μm / 362 μm, the outer tube parameters: 536 μm / 659 μm, which is fixed by tee nesting, the side of tee is connected to a constant flow pump, the upper end of the inner tube is connected to a vacuum box (containing an instrument panel and connected to a vacuum pump), the vacuum box is a self-programmed rotary sliding table, the collection rack, the glass fixing module, and the connection module in the device are all made of acrylic plates cut by 3D printing or laser cutting machine, the probe extraction process is monitored in real time by a micro camera, and the coordinates of the target analyte are obtained, for example, a commercial controller is used to control the xy two-axis table positioning.

[0047] LMJ-SSP extraction: the parameters that need to be adjusted for LMJ-SSP extraction are: injection pump parameters, including solvent, extraction volume and flow rate, relative height of probe and glass, collection container of collection box (for example: 1.5 ml ep tube, sample vial, 300 microliter inner cannula), microscope camera parameter focusing, etc. After observing the stable formation of liquid junction through the video recording of the microscope camera, the device can be operated, each extraction includes washing, extraction, and washing steps, for example, in this experiment, 80% methanol is used as the solvent, the washing volume is 100 microliters, the extraction volume is 350 microliters, and the extract of the quantitatively accumulated cells and contents is obtained.

[0048] (6) Concentration and spotting of the extract: the extract is concentrated by evaporation under a nitrogen blowing instrument, and is redissolved with a volume of 10 microliters to improve the signal collection of mass spectrometry in single cells and contents. For example, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis requires spotting on an indium-tin oxide (ITO) coated glass slide and adding a matrix, and the matrix composition is, for example, amino acridine (9-AA) for negative ion detection matrix and a-p-hydroxy cinnamic acid (a-CHCA) for positive ion detection matrix.

[0049] The spotting experiment is divided into two kinds of loading methods combined with matrix addition: matrix redissolving spotting method and matrix spraying method. The matrix redissolving spotting method directly redissolves the dried sample extract with 10 microliters of prepared matrix solution; the matrix spraying method redissolves 10 microliters of pure solvent, and then uniformly deposits the matrix on the ITO glass slide through a matrix spraying instrument.

[0050] (7) MALDI-MS detection: take the commercial MALDI mass spectrometer UltrafleXtreme MALDI TOF / TOF MS as an example, place the ITO glass on the target plate, put the target plate into the target, open the software and follow the general operation method of the MALDI mass spectrometer to determine the chemical components in the extract. After the foregoing extraction process, a good signal can be obtained. The determined chemical substances combined with the affiliated microstructure are the chemical component information in this microstructure. Based on this information, the pharmacognosy identification results can be cross-verified to identify fake and study Chinese herbal plant single cells.

[0051] The above method is further introduced below in combination with the microchemical identification of Fritillariae Cirrhosae Bulbus starch granules.

[0052] Fritillariae Cirrhosae Bulbus is one of the most representative Sichuan native and precious medicinal materials, which has the effects of clearing heat and moistening lung, reducing phlegm and relieving cough, resolving and dissipating, and eliminating abscess. It is used for lung heat and dry cough, dry cough with little phlegm, yin deficiency and laborious cough, blood in sputum, scrofula, mastitis, and lung abscess. With long circulation in the market, the supply of Fritillariae Cirrhosae Bulbus resources is short, the quality evaluation and control is weak, the technical standards are missing, the terminal products are lacking, and the policy support is insufficient, which restricts the healthy development of the current Fritillariae Cirrhosae Bulbus industry. Through the investigation of common counterfeits on the market, we determined to detect three kinds of counterfeits, namely flour, sweet potato starch, and mountain bitter fungus.

[0053] Through the research on Fritillariae Cirrhosae Bulbus in Chinese Pharmacopoeia and chemical component detection, we determined that the main substances contained in Fritillariae Cirrhosae Bulbus are steroidal alkaloids, represented by peimine B and sipeimine, and that LMJ-SSP extraction is adopted with chloroform solvent according to the extraction process of the Pharmacopoeia.

[0054] (1) Fritillariae Cirrhosae Bulbus single cell level powder sealing slide preparation, the specific steps are as follows:

[0055] Place the clean glass slide on the tin foil paper and cool it in the ice box. Take the prepared 15% gelatin aqueous solution, which has been swollen and placed at room temperature in liquid state before use. Select a dropper to absorb about 1 ml of gelatin solution, drop it in the center of the glass slide, and spread it from the dropper. The gelatin spreads on the glass slide in a transparent layer. Use forceps to touch and confirm whether it is solidified.

[0056] Take a small amount (about 0.1-0.3 mg) of Fritillariae Cirrhosae Bulbus powder when it is solidified, and evenly sieve it on the surface of the gelatin with a No. 5 sieve. After the gelatin is solidified, take the glass slide and gently tap the back side of the glass slide. Confirm that no powder falls off and it can be used for microscopic observation. If a mixed powder sample is determined, the powders of Fritillariae Cirrhosae Bulbus and counterfeits are mixed in equal amounts before being sieved onto the surface of the gelatin with a No. 5 sieve, that is, a mixed powder gelatin slide.

[0057] (2) According to the Chinese Pharmacopoeia and the Microscopic Identification Atlas of Traditional Chinese Medicine and literature (State Pharmacopoeia Commission. Chinese Pharmacopoeia [M]. Beijing: China Medical Science and Technology Press, 2020; Zhao Zhizhen, Chen Huiruo. Microscopic Identification Atlas of Traditional Chinese Medicine [M]. Fuzhou: Fujian Science and Technology Press, 2016; Chen Yuxiu, Li Yuyan. Research on the identification method of Fritillaria cirrhosa [J]. Agricultural Medical Science, 2021, 43(06): 528-531), the powder of Fritillaria cirrhosa was placed on the sample stage of the microscope and fixed, and the starch granules were identified. Under bright field conditions, the low power lens was used to scan the area containing single dispersed Fritillaria cirrhosa starch granules, and the scanned picture was stored in TIF format. The positioning operation is shown in "(4) Coordinate identification of medicinal material powder". Figure 5 This shows how to set parameters for positioning and identification software represented by MicroMS to obtain the precise coordinates of single starch granules.

[0058] (3) The operation of LMJ is shown in "(5) LMJ-SSP control and extraction". According to the extraction method in the Pharmacopoeia, chloroform was used as the extraction solvent, placed in a 15 ml centrifuge tube, the extraction volume was 350 microliters each time, and 100 microliters of solvent was used for washing before and after each extraction. Each group of six starch granules was extracted, the controller was turned on, the circuit was connected, and the positioning coordinates were converted and input into the controller. The two-axis stage was used for positioning extraction.

[0059] Supplement: According to the pre-experiment, the balance relationship between the flow rate and the vacuum degree of the instrument was recorded at different gradients. When the liquid was stable, it could be used as a reference parameter under the same instrument conditions.

[0060] (4) The extract was concentrated by nitrogen blowing concentrator, 10 ml of 5 mg / ml CHCA matrix solution was used for redissolution, vortexed for 30 seconds, and then spotted on the ITO glass. Each time, 0.5 microliters of sample was spotted, and about 10 times of the same spot were repeated to obtain a larger compound response. The glass can be heated during the spotting process to speed up the evaporation of the solution on the glass.

[0061] (5) The extract was analyzed by MALDI-MS, and the operation was according to the instructions of the MALDI-MS instrument.

[0062] (6) The mass spectrum obtained in step (2) was combined with the high power microscope picture to obtain the microscopic and chemical information of single starch granules. Similarly, by repeating the operations from (3) to (5) on the mixed powder glass, we obtained the morphological characteristics and chemical spectrum information of the starch granules from the mixed species. Based on these information, it was determined that m / z 430 was the characteristic compound of Fritillaria cirrhosa (verified by using the standard of fritillarinin), and whether the characteristic peak of 430 existed could identify the possible source of the starch granules. Thus, the identification of medicinal material powder was realized. Figure 6 、 7It can be seen that the appearance and morphology and the starch granule state of the three kinds of adulterants of Fritillaria cirrhosa and flour, sweet potato starch and mountain yam cannot be obviously distinguished, but the specific fritillary mother liquor ethyl alkaloid component of Fritillaria cirrhosa can be well distinguished, and the chemical information difference is based on the single starch granule morphology.

[0063] The above examples serve to specifically introduce the substantial content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

Claims

1. A method for microscopic chemical identification of traditional Chinese medicine at single cell level, characterized in that, The method comprises the following steps: Step S1, powder sealing preparation: take a proper amount of liquid gelatin solution with a mass fraction of 10%-15% and add it on a glass slide, and then wait for the gelatin to solidify to form a gelatin layer with uniform thickness, no bubbles and no impurities, and the powder sealing is obtained; Step S2, medicinal material powder addition: take a proper amount of traditional Chinese medicine powder and carefully and uniformly add it on the gelatin layer of the powder sealing, and then wait for the traditional Chinese medicine powder to be firmly adhered to the gelatin, and then naturally dry or vacuum dry; Step S3, medicinal material microscopic identification: make several marks on the back of the dried powder sealing, and then place it under a low-power microscope for bright field observation, and then further use a high-power microscope to observe the target microscopic structure after the target microscopic structure is found, and then record the low-power microscope field picture containing the marks and the target microscopic structure and the high-power microscope field picture containing the target microscopic structure; Step S4, coordinate identification and positioning: input the low-power microscope field picture into software with microscopic structure identification function, output the pixel point coordinate values of the target microscopic structure and at least one of the marks on the picture through the software, select one mark as a positioning point, set the positioning point as a relative zero point, and then obtain the accurate positioning of the target microscopic structure according to the pixel point coordinate values of the positioning point and the target microscopic structure; Step S5, LMJ-SSP extraction: use a droplet microjunction surface sampling probe to extract the target microscopic structure in situ according to the accurate positioning of the target microscopic structure, and collect the extraction liquid; Step S6, concentration and spotting of the extraction liquid: concentrate the extraction liquid, spot it on an ITO glass slide, and add a matrix; Step S7, MALDI-MS detection: place the ITO glass slide on a target plate, put the target plate into the target, open the software, and then determine the chemical components in the extract according to the general operation method of the MALDI mass spectrometer; Through the above steps, the microscopic information and chemical component information of the target microscopic structure can be obtained.

2. The method according to claim 1, characterized in that: The marks in step S3 are "X" shaped marks.

3. The method according to claim 1, characterized in that: In step S6, aminoacridine is used as a negative ion detection matrix, and a-p-hydroxy cinnamic acid is used as a positive ion detection matrix.

4. The method according to claim 1, characterized in that: In step S6, the method for adding the matrix is a matrix redissolving spotting method or a matrix spraying method.

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