阅读说明：本技术 一种样本afai-desi检测方法及其应用 (Sample AFAI-DESI detection method and application thereof ) 是由 付艳蕾 季佩佩 杨贤法 胡哲 陆嘉伟 舒烈波 于 2021-09-02 设计创作，主要内容包括：本发明提供一种样本AFAI-DESI检测方法,包括：将组织样本冰冻切片后获得的样片染色,采用AFAI-DESI进行检测,水平位移速率为0.03-0.05mm/s,垂直位移距离为0.01-0.03mm,雾化气流量为0.65-0.75MPa,喷雾溶剂流速为5.5-6.5μL/min,喷针伸出的长度为0.45-0.55mm,DESI喷针与载玻片之间的夹角为45-55°。本发明进一步提供上述方法在提高空间分辨率的用途。本发明提供的一种样本AFAI-DESI检测方法及其应用,提高了本技术的空间分辨率至20μm,同时保证了代谢物检测灵敏度和覆盖度。(The invention provides a sample AFAI-DESI detection method, which comprises the following steps: staining a sample obtained after the tissue sample is frozen and sliced, and detecting by adopting AFAI-DESI, wherein the horizontal displacement rate is 0.03-0.05mm/s, the vertical displacement distance is 0.01-0.03mm, the atomized gas flow is 0.65-0.75MPa, the flow rate of the atomized solvent is 5.5-6.5 muL/min, the extending length of the spray needle is 0.45-0.55mm, and the included angle between the DESI spray needle and the glass slide is 45-55 degrees. The invention further provides the use of the above method for increasing spatial resolution. The sample AFAI-DESI detection method and the application thereof provided by the invention improve the spatial resolution of the technology to 20 μm, and simultaneously ensure the detection sensitivity and coverage of metabolites.)

1. A sample AFAI-DESI detection method, comprising: staining a sample obtained after the tissue sample is frozen and sliced, and detecting by adopting AFAI-DESI, wherein the horizontal displacement rate is 0.03-0.05mm/s, the vertical displacement distance is 0.01-0.03mm, the atomized gas flow is 0.65-0.75MPa, the flow rate of the atomized solvent is 5.5-6.5 muL/min, the extending length of the spray needle is 0.45-0.55mm, and the included angle between the DESI spray needle and the glass slide is 45-55 degrees.

2. The method for detecting AFAI-DESI in a sample according to claim 1, wherein said cryosectioning of said tissue sample comprises: freezing the tissue sample, thawing, fixing, slicing, adhering to a glass slide, and freezing and storing the obtained sample for later use.

3. The method for detecting the AFAI-DESI of a sample according to claim 1, wherein said staining comprises the steps of:

1) immersing the sample wafer in methanol for fixation, immersing the sample wafer in pure water again, immersing the sample wafer in hematoxylin for carrying out primary dyeing to form bluing, and then immersing the sample wafer in hydrochloric acid ethanol for differentiation;

2) immersing the differentiated sample wafer in pure water, washing with water, immersing in eosin for dyeing for the second time, immersing in gradient ethanol for dehydration, immersing in xylene for permeabilization, and sealing with neutral gum.

4. The sample AFAI-DESI detection method of claim 1, wherein the AFAI-DESI detection conditions are: the spraying voltage is 0V; the voltage of a transmission tube is 0V; the scanning polarity is in a positive/negative ion mode; the capillary temperature is 350 ℃; the temperature of the auxiliary gas is 0 ℃; the scanning mode is a full scanning scan; the scanning range is 100-1000 Da; the resolution was 70000.

5. The AFAI-DESI detection method of claim 1, wherein when detecting the AFAI-DESI, the mass spectrometry imaging platform is scanning line by line at a scanning speed of 0.035-0.045 mm/s; the step interval between two adjacent scanning lines is 0.015-0.025 mm; the delay time after each line of scanning is 6-8 ms; the speed of returning to the start of scanning of the same line is 9-11 mm/s.

6. The AFAI-DESI assay of claim 1, wherein the AFAI-DESI assay is performed under conditions of spray point state: the flow rate of the atomized gas is 0.69-0.71 MPa; the flow rate of the spraying solvent is 5.9-6.1 mu L/min; the length d2 of the protruding needle is 0.49-0.51 mm.

7. The method as claimed in claim 1, wherein the spatial geometry of the AFAI-DESI is as follows: the included angle alpha 1 between the DESI spray needle and the glass slide is 49-51 degrees; the included angle alpha 2 between the ion transmission tube and the glass slide is 14-16 degrees; the vertical distance d1 between the spray needle and the glass slide is 1.9-2.1 mm; the horizontal distance d3 between the spray point and the ion transmission pipe is 1-3 mm; the distance d4 between the lower edge of the ion transmission tube and the slide is 0.98-1.02 mm.

8. Use of a method of sample AFAI-DESI detection according to any of claims 1-7 for spatial resolution enhancement in the analysis of spatial metabolic composition images of samples of micron size.

9. The use of claim 8, wherein the sample is selected from one or more of mouse eyeball tissue, mouse kidney glomerular tissue, or mouse embryonic tissue.

10. Use according to claim 8, wherein the spatial resolution of the sample is ≧ 20 μm.

Technical Field

The invention belongs to the technical field of mass spectrometry, relates to a sample AFAI-DESI detection method and application thereof, and particularly relates to a sample AFAI-DESI detection method and application thereof in small-size, micron-size sample space metabolome.

Background

Traditional non-targeted metabonomics mainly carry out high-throughput detection on metabolites in tissues by means of tissue homogenate, but the spatial information of a sample is lost by the tissue homogenate. The in-situ mass spectrometry realizes mass spectrometry after in-situ ionization of exogenous substances such as drugs and endogenous substances such as micromolecular metabolites, lipids, proteins and the like in frozen tissue slices by the in-situ ionization technology, and obtains the distribution characteristics of different substances in tissues by analyzing data. The in-situ mass spectrometry technology is the integration of in-situ ionization mass spectrometry technology and tissue section technology. The space metabolome is developed based on an in-situ mass spectrometry technology and a metabonomics data processing method, and because the space metabolome has simple pretreatment, no mark and high flux, the space metabolome can simultaneously present the distribution and expression conditions of thousands of molecules in different tissues, organs or tissue micro-regions, so the space metabolome is widely applied to the fields of tumor metabolism, drug screening, disease diagnosis, toxicological research and the like.

The most commonly used Mass spectrometric Imaging techniques at present are Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) and Electrospray Ionization-Mass Spectrometry Imaging (DESI-MSI). MALDI-MSI has the greatest advantage of high spatial resolution, which can reach 5 μm, but also has the disadvantages of needing to operate in vacuum, being inconvenient, having limited space, being not suitable for large-volume tissue samples, needing to add matrix, having matrix effect, and the like; the DESI-MSI has the advantages of being carried out under normal pressure, convenient to operate, suitable for different molecular analyses, free of adding matrixes, and low in spatial resolution and sensitivity. The AFAI-DESI mass spectrum imaging technology is optimized on the basis of DESI-MSI, improves the detection sensitivity by increasing the distance of an ion transmission pipe and air-assisted power, is suitable for collecting large-volume samples, but has low spatial resolution, the current optimal resolution can only reach 100 mu m, is not suitable for the resolution of fine micro-areas, such as fine tissue micro-areas of mouse embryos, mouse glomeruli and the like, and greatly limits the application of the AFAI-DESI technology in the fields of reproductive genetics, growth and development, cranial neuroscience and the like.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a sample AFAI-DESI detection method and its application, which is used to solve the problem of the lack of methods for ensuring the sensitivity and coverage of metabolite detection and the application of improving the spatial resolution of AFAI-DESI in the analysis of micro-scale small-size sample spatial metabolic composition image in the prior art.

To achieve the above and other related objects, a first aspect of the present invention provides a sample AFAI-DESI detection method, comprising: staining a sample obtained after the tissue sample is frozen and sliced, and detecting by adopting AFAI-DESI, wherein the horizontal displacement rate is 0.03-0.05mm/s, the vertical displacement distance is 0.01-0.03mm, the atomized gas flow is 0.65-0.75MPa, the flow rate of the atomized solvent is 5.5-6.5 muL/min, the extending length of the spray needle is 0.45-0.55mm, and the included angle between the DESI spray needle and the glass slide is 45-55 degrees.

The AFAI-DESI (air Flow Assisted Ionization desorption Ionization) is a mass spectrometry imaging technology of an aerodynamic Assisted Ionization desorption/Ionization spray ion source.

Preferably, the tissue sample cryosection comprises: freezing the tissue sample, thawing, fixing, slicing, adhering to a glass slide, and freezing and storing the obtained sample for later use.

The sample slice is a glass slide adhered with the sample slice.

More preferably, the ice is stored in an ultra-low temperature refrigerator at-85 to-75 ℃.

More preferably, the thawing is performed in a refrigerator at-25 to-15 ℃ overnight.

More preferably, the fixing is to fix the tissue sample by adding embedding glue on the bottom support of the embedding box. The embedding glue is only required to completely cover the tissue sample.

Further preferably, the Embedding Gel is Cryo-Gel Embedding Gel (Leica Cryo-Gel Embedding Medium, Item No. 39475237).

More preferably, the sectioning is performed by sectioning the fixed tissue sample with a microtome.

More preferably, the slice has a thickness of 8-20 μm.

More preferably, the slide is a Superfrost Plus positive charge anti-shedding slide.

Preferably, the dyeing comprises the steps of:

1) immersing the sample wafer in methanol for fixation, immersing the sample wafer in pure water again, immersing the sample wafer in hematoxylin for carrying out primary dyeing to form bluing, and then immersing the sample wafer in hydrochloric acid ethanol for differentiation;

2) immersing the differentiated sample wafer in pure water, washing with water, immersing in eosin for dyeing for the second time, immersing in gradient ethanol for dehydration, immersing in xylene for permeabilization, and sealing with neutral gum.

More preferably, in step 1), the methanol is pre-cooled at-25 to-15 ℃, preferably-20 ℃.

Further preferably, the pre-cooling time is 1-2 h.

More preferably, in step 1), the immersion volume of the methanol is 200-300mL, preferably 250 mL.

More preferably, in step 1), the slices are immersed in methanol for a fixed time of 45 to 90s, preferably 60 s.

More preferably, in step 1), the slice is immersed in pure water for a period of 8 to 12 seconds, preferably 10 seconds.

More preferably, in step 1), the immersion volume of the pure water is 200-300mL, preferably 250 mL.

More preferably, in step 1), the immersion volume of the hematoxylin is 200-300mL, preferably 250 mL.

More preferably, in step 1), the time for the first dyeing is 5-7 min.

More preferably, in the step 1), the immersion volume of the ethanol hydrochloride is 200-300mL, and preferably 250 mL.

More preferably, in the step 1), the ethanol hydrochloride is obtained by dissolving 0.5-1% of hydrochloric acid in 70% of ethanol solution.

The differentiation refers to the removal of excess bound staining agent from the tissue sample.

More preferably, in step 1), the differentiation time is 1-3 s.

More preferably, in step 2), the washing with pure water is repeated for 2 to 3 times, and the washing time is 10 to 20s, preferably 15 s.

More preferably, in step 2), the immersion volume of the pure water washing is 200-300mL, preferably 250 mL.

More preferably, in step 2), the immersion volume of eosin is 200-300mL, preferably 250 mL.

More preferably, in step 2), the time for the second dyeing is 10-30 s.

More preferably, in the step 2), the dehydration is performed by sequentially using 100% ethanol, 95% ethanol and 75% ethanol.

The dehydration utilizes the rapid penetration speed of ethanol, and has obvious contraction effect on the sample tissue.

The 95% ethanol is ethanol water solution with the volume percentage of 95%. The 75% ethanol is 75% ethanol water solution by volume percentage.

More preferably, in the step 2), the immersion volumes of the 100% ethanol, the 95% ethanol and the 75% ethanol in the dehydration are 200-300mL, preferably 250mL respectively.

More preferably, in step 2), the dehydration time in the gradient ethanol is 25 to 35s, preferably 30 s.

More preferably, in step 2), the immersion volume of the xylene is 200-300mL, preferably 250 mL.

More preferably, in step 2), the time for permeabilization in xylene is from 9 to 11min, preferably 10 min.

The xylene permeabilization described above is the use of xylene to solubilize some of the adipose tissue of the sample.

More preferably, in step 2), the neutral gum is a natural gum dissolved in xylene.

Further preferably, the neutral gum is a 55-65 wt% xylene solution of natural gum, preferably a 60 wt% xylene solution of natural gum.

And (5) covering the sample wafer by the neutral gum, and sealing the wafer.

Preferably, the swatch remains wet during the dyeing. I.e. the slide to which the specimen slice is adhered, does not dry out during the entire experiment.

Preferably, during the detection of the AFAI-DESI, the data acquisition uses an Xcaliibur data acquisition and processing system.

Preferably, the detection conditions of the AFAI-DESI are shown in Table 1 as follows: the spraying voltage is 0V; the voltage of a transmission tube is 0V; the scanning polarity is in a positive/negative ion mode; the capillary temperature is 350 ℃; the temperature of the auxiliary gas is 0 ℃; the scanning mode is a full scanning scan; the scanning range is 100-1000 Da; the resolution was 70000.

TABLE 1AFAI ion Source and Mass Spectroscopy scanning common parameter Table

Parameter(s)	Value of
		Spray voltage	0V
Voltage of transmission tube	0V
		Scanning polarity	Positive/negative
Capillary temperature	350℃
		Temperature of auxiliary gas	0
Scanning mode	Full scan
		Scanning range	100-1000Da
Resolution ratio	70000

Preferably, when the AFAI-DESI is detected, the mass spectrometry imaging platform adopts a line-by-line scanning mode, and the scanning speed (horizontal displacement rate) is 0.035-0.045(Vx, mm/s), preferably 0.04(Vx, mm/s); the step interval (vertical displacement distance) between two adjacent scanning lines is 0.015 to 0.025(Dy, mm), preferably 0.02(Dy, mm); the delay time after each line of scanning is 6-8(Dt, ms), preferably 7(Dt, ms); the speed of returning to the start of scanning of the same line is 9-11mm/s, preferably 10 mm/s.

When the mass spectrum imaging platform is used for data acquisition, an X axis and a Y axis of a scanning area are determined in a progressive scanning mode according to the size of a tissue sample wafer determined in the early stage, the X axis direction is set as the scanning speed (Vx, mm/s), the Y axis direction is set as the scanning length (mm), and the step distance between two adjacent scanning lines is set on the Y axis. After each line of scanning is finished, in order to keep synchronization with the acquisition of mass spectrum data, a certain delay time (Dt, s) is generally required to be set, then the platform quickly returns to the scanning starting point of the same line to avoid pollution and damage to undetected lines, then the next line of scanning is started according to the steps after the set displacement distance is stepped along the Y axis, and the scanning is circulated until all sample areas are scanned, and the scanning mode ensures the accuracy and reliability of mass spectrum imaging results.

Preferably, when the AFAI-DESI is detected, the spray point state conditions are as follows: the atomized gas flow is 0.69-0.71MPa, preferably 0.7 MPa; the flow rate of the spraying solvent is 5.9-6.1 muL/min, preferably 6 muL/min; the length d2 of the protruding needle is 0.49-0.51mm, preferably 0.5 mm.

Among the three, atomizing air flow is bigger, and the atomization effect is better, and the spraying solvent velocity of flow is bigger, and is also higher to the efficiency of sample extraction desorption, but can lead to the atomization effect to reduce, and high velocity of flow can lead to liquid to spread, and the formation of image effect is poor scheduling problem, and the length that the spray needle stretches out is longer, and the atomization effect is poor, consequently needs to obtain the optimal combination.

Preferably, when the AFAI-DESI is detected, the spatial geometry parameters are: the included angle alpha 1 between the DESI spray needle and the glass slide is 49-51 degrees, preferably 50 degrees; the included angle alpha 2 between the ion transmission tube and the glass slide is 14-16 degrees, preferably 15 degrees; the vertical distance d1 between the spray needle and the glass slide is 1.9-2.1mm, preferably 2.0 mm; the horizontal distance d3 of the spray point from the ion transport tube is 1-3mm, preferably 2 mm; the distance d4 between the lower edge of the ion transport tube and the slide is 0.98-1.02mm, preferably 1 mm.

The AFAI-DESI described above is tested by generating a focused, coaxially aligned spray, as determined by the relationship between the solvent flow, the atomizing gas flow, the nozzle projection, the distance from the nozzle to the emitter tip, and the high voltage applied. Once the nebulizer parameters are optimized, the final task is to optimize the geometrical parameters, i.e. height of the nebulizer above the surface and distance between the nebulizer and the inlet capillary, etc.

The second aspect of the invention provides the application of the sample AFAI-DESI detection method in improving the spatial resolution in the analysis of the spatial metabolic composition image of the micron-sized small-size sample.

Preferably, the sample includes, but is not limited to, mouse eyeball tissue, mouse kidney glomerular tissue, mouse embryonic tissue.

Preferably, the spatial resolution of the sample is ≧ 20 μm, specifically such as 20-90 μm, 20-30 μm, 30-60 μm, 60-90 μm, preferably 20 μm.

As mentioned above, the sample AFAI-DESI detection method and the application thereof greatly improve the spatial resolution of the technology to 20 μm by optimizing the space geometric parameters of the AFAI-DESI, the displacement platform moving speed and other parameters. The method simultaneously ensures the detection sensitivity and the coverage of the metabolites, successfully applies the spatial resolution to the spatial metabolic composition image analysis of micron-sized small-size samples such as mouse kidney glomeruli, mouse embryos, mouse eyeballs and the like, and provides a new method for the subsequent application to the related researches such as genetic reproduction, tissue organ development, brain science research and the like.

Drawings

FIG. 1 shows HE staining patterns of mouse eyeball of the present invention.

FIG. 2 shows mass spectrometric images of mouse eyeball sections of the present invention under different parameter combinations 2a, 2b, 2c, wherein FIG. 2a employs parameter combination a; FIG. 2b uses parameter combination b; fig. 2c uses a parameter combination c.

FIG. 3 shows the imaging effect of the mouse brain tissue section of the present invention FIGS. 3a, 3b, 3c, 3d, wherein FIG. 3a uses combination parameter 1; FIG. 3b employs combination parameter 3; FIG. 3c employs combination parameter 2; fig. 3d uses the combination parameter 4.

FIG. 4 shows a comparison of mass spectra mean intensity within the collection region for mouse brain tissue sections of the present invention FIGS. 4a, 4b, 4c, wherein FIG. 4a employs combination parameter 1; FIG. 4b employs combination parameter 2; fig. 4c uses the combination parameter 3.

FIG. 5a shows HE staining of mouse eyeball of the present invention.

FIG. 5b shows a 20 μm resolution mass spectrometry image of the mouse eyeball of the present invention.

FIG. 5c shows a graph of mouse embryo crystal violet staining according to the present invention.

FIG. 5d shows 20 μm resolution mass spectrometry images of mouse embryos of the present invention.

FIG. 5e shows a graph of HE staining of the renal cortex of a mouse of the invention.

FIG. 5f shows 20 μm resolution mass spectrometry images of mouse renal cortex of the present invention.

FIG. 6a shows an accurate image of 20 μm resolution mass spectrometry of the mouse eyeball of the present invention.

FIG. 6b is a diagram showing the peak detection of the metabolite spectrum of the mouse eye at 20 μm.

FIG. 7a shows an accurate image of 20 μm resolution mass spectral imaging of mouse embryos of the present invention.

FIG. 7b is a graph showing the peak detection of the metabolite spectrum of the mouse embryo of the present invention at 20 μm.

FIG. 8a shows an accurate image of 20 μm resolution mass spectral imaging of mouse renal cortex of the present invention.

FIG. 8b is a graph showing the peak detection of the metabolite spectrum of the mouse kidney cortex at 20 μm.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The reagents and equipment used in the following examples are as follows:

1. reagent

Distilled water (drochen); hematoxylin, eosin (Sigma); methanol, 100% ethanol, 95% ethanol, 75% ethanol, xylene (michelin); ethanol hydrochloride (alatin); acetonitrile (siemmerfet); Cryo-Gel Embedding Gel (Leica Cryo-Gel Embedding Medium, Item No. 39475237).

The kidney, eyeball and embryo of the mouse are obtained from Shanghai animal center of Chinese academy of sciences.

2. Instrument for measuring the position of a moving object

AFAI-DESI mass spectrometry imager [ Vico To (Beijing) science and technology Limited ]; embedding cassette (Jiangsu Shitai laboratory instruments Co., Ltd.).

Example 1

1. Sample processing

Freezing the tissue sample in-80 deg.C ultra-low temperature refrigerator, taking out, and thawing in-20 deg.C refrigerator overnight. And adding Cryo-Gel embedding glue on the bottom support of the embedding box, wherein the Cryo-Gel embedding glue completely covers the tissue sample to fix the thawed tissue sample. And (3) slicing the fixed tissue sample by using a microtome, and adhering and fixing the sliced tissue sample on a Superfrost Plus positive charge anti-shedding glass slide to obtain a sample 1 #. The thickness of the slices was 14 μm. And then placing the sample wafer No. 1 into an ultra-low temperature refrigerator with the temperature of minus 80 ℃ for freezing storage for subsequent imaging analysis.

Then, the sample No. 1 was immersed in 250mL of methanol pre-cooled at-20 ℃ for 2 hours to fix for 60s, further immersed in 250mL of pure water for 10s, further immersed in 250mL of hematoxylin to perform the first staining for 6min to form bluing, and further immersed in 250mL of hydrochloric acid ethanol to differentiate for 2 s. The differentiated sample 1# was immersed in 250mL of pure water and washed with water 2 times for 15 seconds. Immersing in 250mL of eosin for secondary dyeing for 20s, immersing in 250mL of 100% ethanol, 250mL of 95% ethanol and 250mL of 75% ethanol in sequence for gradient ethanol dehydration, immersing in 250mL of dimethylbenzene for permeabilization, covering the sample with 60 wt% of natural gum dimethylbenzene solution as neutral gum, and sealing to obtain sample No. 1 to be tested.

2. Instrumental detection

And (3) detecting the sample 1# to be detected by adopting AFAI-DESI, wherein the detection conditions of the AFAI-DESI are shown in table 1, and the data acquisition is realized by using an Xcaliibur data acquisition and processing system.

When AFAI-DESI is detected, the mass spectrum imaging platform adopts a line-by-line scanning mode, and the scanning speed (horizontal displacement rate) is 0.04(Vx, mm/s); the step pitch (vertical displacement distance) between two adjacent scanning lines is 0.02(Dy, mm); the delay time after the end of each line of scanning is 7(Dt, ms); the speed of returning to the start of scanning of the same line was 10 mm/s.

The spraying point state conditions are as follows: the flow rate of the atomized gas is 0.7 MPa; the flow rate of the spray solvent is 6 mu L/min; the length d2 of the protruding needle is 0.5 mm.

The space geometric parameters are as follows: the included angle alpha 1 between the DESI spray needle and the glass slide is 50 degrees; the included angle alpha 2 between the ion transmission tube and the glass slide is 15 degrees; the vertical distance d1 between the spray needle and the glass slide is 2.0 mm; the horizontal distance d3 from the spray point to the ion transport tube was 2 mm; the distance d4 between the lower edge of the ion transport tube and the slide is 1 mm.

Example 2

The mouse eyeball was subjected to sample treatment in step 1 of example 1 to obtain a mouse eyeball section sample stained as shown in FIG. 1.

Then, the mouse eyeball slice sample is subjected to AFAI-DESI detection according to the step 2 in the example 1, wherein the scanning speed (horizontal displacement rate) and the stepping distance (vertical displacement distance) between two adjacent scanning lines are 2 parameter setting parameter combinations a, b and c, and the specific numerical values are shown in the table 2. The figure of the mass spectrum image under different parameter combinations is shown in figure 2.

TABLE 2

Parameter(s)	Parameter combination a	Parameter combination b	Parameter combination c
				X-axis scanning speed (Vx, mm/s)	0.08	0.04	0.02
Y-axis step spacing (Dy, mm)	0.04	0.02	0.01

As can be seen from fig. 2a, 2b and 2c, the tissue micro-regions can be finely divided by the parameter combination b, the parameter combination a has a wider profile than the image of b, the image of the combination c has obvious horizontal stripes and the tissue region is not clear, so that the spatial resolution of 20 μm can be realized when the parameter combination b, i.e. the X-axis scanning speed is 0.04mm/s and the Y-axis step pitch (Dy, mm) is 0.02 mm.

Example 3

Mouse brain tissue was subjected to sample treatment according to step 1 of example 1 to obtain mouse brain tissue section samples.

And then, carrying out AFAI-DESI detection on the mouse brain tissue slice sample according to the step 2 in the embodiment 1, wherein a combination parameter 1, a combination parameter 2, a combination parameter 3 and a combination parameter 4 are respectively set. The figure of the mass spectrum image under different parameter combinations is shown in figure 3.

As can be seen from FIGS. 3a, 3b, 3c, and 3d, the imaging effect of the brain tissue slice is optimized by using the combination parameter 1 (the flow rate of the atomized gas is 0.7MPa, the flow rate of the spray solvent is 6 μ L/min, the extension d2 of the nozzle needle is 0.5mm, the horizontal included angle of the nozzle needle is 50 °, and the vertical distance from the nozzle needle to the slide glass is 2 mm). By adopting the combination parameter 2 (the flow rate of atomized gas is 0.6MPa, the extending length of the spray needle is higher than 1mm or the flow rate of the spray solvent is higher than 6 muL/min, the horizontal included angle of the spray needle is 50 degrees, and the vertical distance between the spray needle and the glass slide is 2mm), the incomplete atomization is easy to occur, the solvent diffusion is caused, and the boundary of the tissue micro-area is not clear. By adopting the combination parameter 3 (the flow rate of atomized gas is 0.8MPa, the flow rate of the atomizing solvent is 3 muL/min, the extension length d2 of the spray needle is 0.5mm, the horizontal included angle of the spray needle is 50 degrees, and the vertical distance between the spray needle and the glass slide is 2mm), the desorption efficiency of metabolites is low and the sensitivity is reduced easily. By adopting the combination parameters 4 (the flow rate of atomized gas is 0.7MPa, the flow rate of spray solvent is 6 muL/min, the extension length d2 of the spray needle is 0.5mm, the horizontal included angle of the spray needle is too large (more than 50 degrees) or too small (less than 35 degrees), and the vertical distance between the spray needle and a glass slide is 2mm), a striped imaging picture is easy to appear.

Meanwhile, the mass spectrum average intensity (abundance) of choline (m/z, 104.1072) under different parameter combinations is compared as shown in fig. 4. As can be seen from fig. 4a, 4b, 4c, the sensitivity with combination parameter 1 is significantly better than combination parameters 2 and 3.

In addition, the number of mass spectra peaks of choline (m/z, 104.1072) were compared for different combinations of parameters, and the results are shown in Table 3. As can be seen from table 3, the coverage with combination parameter 1 is also significantly better than combination parameters 2 and 3. Therefore, by adopting the combination parameter 1, the choline abundance is highest, the mass spectrum peak number is the largest, and in the state, the AFAI-DESI can ensure the mass spectrum imaging effect and the metabolite detection coverage is wide while realizing the space high resolution.

TABLE 3 number of peaks in mass spectrum in scan region under different combination parameters

	Combination parameter 1	Combination parameter 2	Combination parameter 3
				Number of mass spectral peaks	27632	20350	10896

Example 4

Three micron-sized small-size samples of mouse eyeballs, mouse embryos and mouse kidneys are subjected to spatial metabonomics analysis, and the samples are processed according to the step 1 in the embodiment 1 to obtain tissue slice samples. The staining patterns of mouse eyeball, mouse embryo and mouse kidney are shown in fig. 5a, 5c and 5e respectively.

The tissue slice samples were then subjected to AFAI-DESI detection as in step 2 of example 1, and the detailed imaging profiles of the mass spectra are shown in FIGS. 5b, 5d, and 5 f. The imaging result shows that the method can well distinguish micron-sized tissue micro-regions, the resolution can reach 20 mu m, the detection sensitivity can be ensured, and tens of thousands of mass spectrum peaks can be detected simultaneously.

The metabolites detected by the micro-scale small-size samples of mouse eyeball, mouse embryo and mouse kidney at the resolution of 20 μm are shown in Table 4, and FIGS. 6a, 6b, 7a, 7b, 8a and 8 b.

TABLE 4 Mass Spectroscopy Peak detection of tissue sections

	Mouse eyeball	Mouse embryo	Mouse renal cortex
				Number of mass spectral peaks	14035	20440	25034

Therefore, when the speed of the displacement platform is optimized to the horizontal displacement speed of 0.04mm/s, the vertical displacement distance is 0.02mm, the geometric parameters are optimized to the flow of the spray solvent of 6 mu L/min, the atomization air pressure is 0.7MPa, the extension length of the spray needle is 0.5mm, and the horizontal included angle of the spray needle is 50 degrees, the AFAI-DESI spatial resolution can reach 20 mu m, the metabolite detection sensitivity and the coverage degree are ensured, the spatial resolution is successfully applied to the spatial metabolic composition image analysis of micron-sized small-size samples such as mouse kidney glomeruli, mouse embryos, mouse eyeballs and the like, and a new method is provided for the subsequent application to the related researches such as genetic reproduction, tissue organ development, brain science research and the like.

In conclusion, the sample AFAI-DESI detection method and the application thereof provided by the invention improve the spatial resolution of the technology to 20 μm, and simultaneously ensure the detection sensitivity and coverage of metabolites. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.