Open-type mass spectrum imaging analysis method integrating preparation of tissue slices on adhesive tape and adsorption platform device

Abstract

The invention belongs to the technical field of mass spectrum imaging, and aims to solve the problems that biological tissue slices are in the most common sample analysis form and need to be transferred to a carrier for analysis, and the process is time-consuming, labor-consuming and easy to lose. The invention provides an open-type mass spectrum imaging analysis method integrating a preparation technology of a sample adhered on an adhesive tape and a vacuum adsorption platform device, only a transparent adhesive tape is used as a carrier, and the adhesive tape adhered with the sample can be adsorbed and fixed through a vacuum adsorption mass spectrum imaging platform after the processes of tabletting treatment and freeze drying. Other fixed carriers and operation processes are not needed, and an open ion source is directly adopted for mass spectrum imaging analysis. The invention provides the simple and efficient mass spectrum imaging analysis method based on the AFADESI-MSI system, the mass spectrum imaging analysis of high flux and continuous slices of whole animals and dispersed organ tissues is easy to realize, the method has the technical characteristics of time saving, labor saving, low loss and environmental protection, and the detection signal intensity and the signal-to-noise ratio of the mass spectrum imaging analysis can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of mass spectrometry imaging technology, and specifically relates to a method and device for simple and efficient mass spectrometry imaging analysis of biological samples. Background Art

[0002] Mass spectrometry imaging is a molecular imaging technique that combines mass spectrometry analysis with image visualization. It requires no specific labeling and uses desorption probes to perform multi-point scanning detection of various molecules contained on the surface of samples such as biological tissue sections. This generates a multidimensional mass spectrometry data array showing the relationship between ion intensity and spatial position. This data is then reconstructed and visualized using data processing software, enabling simultaneous in situ visualization of multiple molecules. Currently, the most commonly used mass spectrometry imaging techniques include matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and open-type mass spectrometry imaging, represented by desorption electrospray ionization imaging (DESI-MSI). MALDI-MSI and DESI-MSI technologies have demonstrated good analytical performance and wide applicability in the in situ analysis of molecules such as proteins, peptides, carbohydrates, lipids, and exogenous drugs. Therefore, they play an increasingly important role in the field of biomedical research and greatly promote the study of molecular mechanisms of action and the development of spatial multi-omics. The inventors independently developed an air flow assisted desorption electrospray ionization mass spectrometry imaging technology method (Air Flow Assisted Desorption Electrospray Ionization Mass Spectrometry Imaging, AFADESI-MSI), which is based on the electrospray desorption principle of DESI and uses long-distance air power to assist the transmission of ions. By removing solvents and ion aggregation, the detection signal intensity of ions is improved. Due to the improvement of the operating space, it is more suitable for mass spectrometry imaging analysis of large-volume samples such as whole animal tissues.

[0003] Although mass spectrometry imaging technology has been widely used in the biomedical field for disease diagnosis, tumor metabolism, drug screening, and drug efficacy and toxicology studies, how to obtain biological tissue slice samples simply and efficiently, especially large and continuous tissue slice samples, to further meet the requirements of high-throughput analysis of mass spectrometry imaging methods, remains one of the major issues limiting the promotion and application of mass spectrometry imaging technology. Currently, the preparation of most small-volume biological tissue slice samples is to cut tissue slices from a frozen tissue module and thaw them on a non-detachable or conductive slide to complete the slice preparation. However, large-volume or dispersed biological tissue slice samples, such as whole animal tissue, need to be adhered to the frozen tissue module using special material tape due to the lack of necessary connections between organs and tissues. The cut tissue slices are then adhered to the tape to achieve in situ whole-body cutting of the whole animal tissue sample. The whole animal tissue sample is then transferred to a slide coated with AB glue adhesive. The special tape is separated from the tissue slice by ultraviolet light irradiation or low-temperature freezing conditions, and the tissue slice is adhered to the slide by adhesive. The slice sample is then freeze-dried and can be used for mass spectrometry imaging analysis. Since each slice must be made by hand, in addition to high requirements for cutting technology to ensure the integrity and continuity of the slices, the process of transferring the slices on the tape to the adhesive itself also has a high possibility of destroying the integrity and continuity, which brings challenges to the demand for unified and large-scale slice production. The fusion of adhesives and tissue slices also introduces new interference to the analysis of target molecules in the sliced tissue. Some studies have used double-sided tape or adhesive sprayed on the surface of the slide to fix the slice samples on the tape with a slide before imaging and scanning. However, careful adjustment is required to ensure that the smooth surface of the tape fits smoothly with the double-sided tape or adhesive on the slide, and bubbles need to be removed, which is time-consuming and labor-intensive. It is still difficult to completely avoid the generation of bubbles trapped between the tape and the slide, which will interfere with subsequent mass spectrometry imaging analysis. In addition, this treatment method not only produces discarded adhesive-loaded fragments that require additional processing, but also poses a risk of contamination and inhalation to the surrounding environment and personnel during the use of the spray glue. Summary of the Invention

[0004] In response to the above-mentioned problems, the present invention provides an open mass spectrometry imaging analysis method that integrates the preparation technology of biological tissue slices on tape (hereinafter referred to as slices on tape) and a vacuum adsorption platform device. This method does not require the use of professional tape, but only uses non-professional tape that is easily purchased on the market; there is no need for the transfer and pasting operation process, no need to use fixed carriers such as slides for pasting biological tissue slices, and no need to use fixing glue coated on the carrier to stick the tissue slices. Only transparent tape required for the tissue cutting process is used as the carrier. Since there is no need to remove the tape during the transfer and pasting process to separate the tape from the tissue slice and allow the slice to adhere to the fixing glue on the carrier, the integrity of large-volume or non-continuous organ tissue sample slices can be guaranteed to the greatest extent, thereby The method realizes the preparation of continuously cut slice samples and avoids the introduction of new interference to mass spectrometry imaging analysis due to the use of fixing glue. Furthermore, by using a vacuum adsorption platform device as a mass spectrometry imaging analysis platform, the slice samples on the tape can be quickly fixed and the position adjusted without the need for additional double-sided tape or fixing clips, or the smooth surface of the transparent tape is fixed to a carrier such as a slide with double-sided tape or spray glue before mass spectrometry imaging analysis. By switching the on-off valve of the vacuum adsorption platform, the mass spectrometry imaging analysis of different samples can be quickly replaced, thereby realizing simple and efficient mass spectrometry imaging analysis, and at the same time improving the mass spectrometry imaging detection signal intensity and signal-to-noise ratio of molecular ions of substances in biological samples, including background ions, exogenous drug molecular ions and endogenous metabolite molecular ions.

[0005] The first aspect of the present invention provides a method for preparing tissue sections suitable for high-throughput mass spectrometry imaging analysis, particularly for preparing large or dispersed tissue sample sections. The method comprises the following steps: freeze-fixing the large tissue sample; selecting a suitable 4-6 cm wide transparent tape that maintains adhesion at -20°C; pressing the cut tissue sections adhered to the tape; and then storing the bulk sample at -78°C and / or freeze-drying at -15°C.

[0006] The transparent tape is a commercial product that is easily available on the market and is not a product that is professionally used for transferring and sticking tissue sections and requires ultraviolet light irradiation.

[0007] The tissue sections on the tape are not limited to large-volume tissue samples or tissue samples that need to be cut continuously, but are more suitable for large-volume or organ tissue dispersed samples, such as whole animal tissue and / or intestinal tissue, and are not limited to species such as rats and / or mice.

[0008] The cutting thickness of whole animal tissue sections is 25 to 60 μm, usually using 3-5% sodium carboxymethyl cellulose solution as the freezing medium, using a Leica CM3600 large microtome, with a cutting temperature of -25 to -20°C and a cutting speed of 60-80 mm / s.

[0009] The cut tissue slices on the tape can be pressed without pretreatment or under low temperature conditions using a hydrophobic surface module or device made of polytetrafluoroethylene, but not limited to such materials. After the slices are pressed, the slice samples on the tape can be placed flat, or the adhesive side of the tape can be rolled inward so that the edges of the tape are overlapped and pasted to form a barrel shape. Then, 10-20 slices rolled into a barrel are collectively sealed and stored at -78°C for subsequent analysis.

[0010] Freshly prepared or tape-sectioned samples stored at -78°C need to be freeze-dried before mass spectrometry imaging analysis. The freezing temperature is -20 to -15°C and the drying time is 0.5 to 3 hours. Samples dried in a vacuum dryer and / or freeze dryer can be used for mass spectrometry imaging analysis directly or after covering the tape area around the tissue section sample with a smooth material such as paper or plastic film.

[0011] The second aspect of the technical solution of the present invention is to provide a vacuum adsorption platform device made of microporous ceramic material as a mass spectrometry imaging analysis platform, which can conveniently and quickly realize mass spectrometry imaging scanning of sliced samples on tape, while making the transparent tape carrying the tissue slices fit tightly with the hard ceramic plane. There is no bubble residue problem to consider, and no other processing process is required. The flatness of the surface of the sliced sample to be analyzed can be guaranteed, thereby ensuring the parallelism and stability of the system parameters when different or continuously cut samples are subjected to mass spectrometry imaging analysis.

[0012] The microporous ceramic vacuum adsorption platform of the present invention has no special requirements on size specifications. In a specific embodiment of the present invention, the size is 10 cm×20 cm×2 cm, and is composed of a microporous ceramic table top and an aluminum alloy metal frame sealed together.

[0013] The metal frame of the microporous ceramic vacuum adsorption platform described in the present invention is connected to the metal lining plate located below at the four top corners through four springs and four fixing screws (length 2 cm). The specifications of the metal lining plate in the specific embodiment of the present invention are 10cm×20cm×0.5cm. Screw holes are opened in the middle part of the metal lining plate, which is connected and fixed to the electric translation stage in the X-axis direction or Y-axis direction of mass spectrometry imaging through screws. The adsorption platform can be horizontally adjusted by adjusting the tightness of the four fixing screws and four springs.

[0014] The sealed cavity of the microporous ceramic vacuum adsorption platform described in the present invention is connected to an air valve via a threaded opening located in the middle. The valve can be connected to a vacuum pump or vacuum pipeline via a quick-connect connector and a flexible vacuum exhaust tube. The adsorption platform can move freely with the electronically controlled translation stage while maintaining the adsorption state. There are no special requirements for the size of the air valve and hose. In the specific embodiment of the present invention, a No. 6 pipeline is used. By opening or closing the air valve, the platform's vacuum adsorption and normal pressure non-adsorption states can be quickly switched. The smooth surface of the tape with the biological tissue section sample attached is brought into contact with the platform surface to achieve sample fixation. The position of the tape sample on the platform surface can also be freely adjusted.

[0015] The microporous ceramic vacuum adsorption platform of the present invention is not only suitable for tape samples, but also for sample carriers with smooth or flat surfaces such as slides and paper. It can quickly adsorb sample carriers for fixation and adjust the fixed position of the carrier or sample on the platform.

[0016] The tabletop of the microporous ceramic vacuum adsorption platform of the present invention can be used to directly mark the coordinate axis scale and scanning starting point on the platform surface with a pencil, which can be changed and erased according to different sample sizes and analysis requirements. Alternatively, the scale and / or marks can be drawn or printed on paper, and then the paper is adsorbed on the platform surface as a background pattern to meet the positioning and range determination requirements of sample mass spectrometry imaging analysis, while maintaining the adsorption efficiency of the adsorption platform.

[0017] The present invention uses a microporous ceramic vacuum adsorption platform for mass spectrometry imaging analysis, and can directly place the freeze-dried slice sample with its smooth side facing down within the range defined by the adsorption platform. The position is adjusted as needed so that the edge and starting point of the rectangular area where the tissue slice is located coincide with the coordinate axis scale line and starting point. There is no need to pre-delineate the area to be scanned for each slice sample, thereby reducing the preparation and replacement time for sample analysis and improving the throughput of mass spectrometry imaging analysis.

[0018] The open mass spectrometry imaging analysis using a vacuum adsorption mass spectrometry imaging platform described in the present invention is not limited to the aerodynamically assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) method, but can be applied to desorption electrospray ionization mass spectrometry imaging (DESI-MSI) or desorption electrospray / secondary photoionization mass spectrometry imaging (DESI-PI-MSI), etc., a variety of open mass spectrometry imaging methods that require the use of a mass spectrometry imaging platform for sample analysis.

[0019] The present invention utilizes a vacuum adsorption mass spectrometry imaging platform for AFADESI-MSI analysis. Based on pre-defined scale positioning lines and scanning starting points on the adsorption platform, the spatial positioning parameters of the imaging system's scanning probe and ion transmission are adjusted. The edges and starting points of the rectangular region containing the tissue slice are aligned with the positioning lines and starting point. Scanning method parameters are then set, and mass spectrometry imaging data acquisition and analysis is performed. When analyzing consecutive slices, the relevant parameters and platform position remain fixed, allowing for rapid sample exchange and continuous mass spectrometry imaging analysis. This ensures that each slice maintains the same data acquisition starting position and instrument system status, facilitating the acquisition of mass spectrometry images from multiple consecutive slices.

[0020] The third aspect of the technical solution of the present invention is to provide a specific application of the tape slice preparation technology described in the first aspect of the integrated technical solution and the vacuum adsorption mass spectrometry imaging platform method described in the second aspect in AFADESI-MSI analysis. The ion transfer tube length of the AFADESI-MSI ion source device used is 45-55 cm, the angle between the ion transfer tube and the horizontal plane is 15-20 °, the distance between the lower edge of the ion transfer tube inlet and the slice sample is 0.5-1.0 mm, the auxiliary air power flow rate is 25-40 L / min, the electrospray needle atomization gas line pressure is 0.6-0.8 MPa; the spray solvent flow rate is 5-10 μL / min; the spray solvent is a conventional solvent suitable for open desorption electrospray mass spectrometry imaging method (water, methanol, acetonitrile or other solvents not limited thereto), preferably The solvent conditions for the elution were a mixed solvent consisting of acetonitrile and water in a volume ratio of 8:2; the voltage applied to the solvent flow path was 5000-7000 V; the length of the electrospray needle capillary extended 0.5-1 mm; the angle between the electrospray needle and the horizontal plane was 55-65°; the vertical distance between the electrospray needle tip and the slice was 1.0-4.0 mm; the horizontal distance between the electrospray needle tip and the outer edge of the ion transfer tube inlet was 1-3 mm; mass spectrometry imaging data acquisition was achieved by moving the adsorption platform where the sample was located, using a line-by-line scanning method; the lengths of the scanning area in the X-axis and Y-axis directions were determined according to the size of the tissue sample, the X-axis direction was set as a line-by-line horizontal scanning direction, and the scanning speed was 0.01-0.4 mm / s; the Y-axis direction was set as the stepping direction of the scanning line, and the stepping distance was 0.1-0.5 mm.

[0021] The beneficial technical effects of the present invention are embodied in the following aspects:

[0022] 1. The present invention provides a technology for preparing slice samples adhered to transparent tape, which is a key step in mass spectrometry imaging analysis. Compared with the method of obtaining slice samples by transfer adhesion, the technology does not require the use of special tape for slicing, does not require the transfer adhesion process and the fixing glue for transfer adhesion, and does not require the use of carriers such as slides for carrying and adhering tissue slices, thereby simplifying the sample preparation process. When the samples on the tape are frozen and stored in batches, they are rolled inward into a cylindrical shape with the smooth surfaces in contact, which makes it easy to store them in batches and avoid cross contamination. Since there is no need to separate the tape from the tissue slice, damage to the slice tissue surface and tissue loss caused by the tape removal process is avoided, the original appearance of the slice tissue surface can be retained to the greatest extent, the loss rate of the slice sample in the production process is reduced, and continuously cut slices are easy to obtain; the use of disposable consumables such as fixatives and slides is avoided, the experimental cost and impact on the environment are reduced, the manpower and time consumed in manually applying fixatives on the slide surface and the resulting operational errors between samples are avoided, the phenomenon of cracking of the tissue slice caused by the curing process of the fixative is avoided, and the competitive inhibition or enhancement of the ionization of molecules in the sample by highly ionizable substances in the fixative is avoided.

[0023] 2. The present invention provides a method for mass spectrometry imaging analysis using a vacuum adsorption imaging platform. Compared with the method of fixing the tape sample by using double-sided tape or non-drying spray adhesive, the method avoids the problem that the tissue section is difficult to adjust after the position is fixed, and avoids the problem that bubbles are easily wrapped in the tape and cannot be completely flatly attached during the bonding process between the tape and the carrier. At the same time, it avoids the pollution and inhalation risks brought to the environment and personnel by spray adhesive and waste carriers. The method of fixing the sample using a vacuum adsorption platform can arbitrarily adjust the fixed position of the sample on the plane on the tape, keep the parameters of the mass spectrometry imaging analysis system unchanged, quickly replace different samples, and easily perform mass spectrometry imaging analysis of continuous slice samples, thereby improving the throughput of mass spectrometry imaging analysis. The use of a vacuum adsorption platform can also provide a hard interface for desorption electrospray ionization technology that can be maintained continuously during the imaging scan, without loss and reusable, providing favorable conditions for the instantaneous ion exchange and transfer process required by this sampling method, while improving the detection signal intensity and signal-to-noise ratio of the molecular ions of substances in most tissue samples. The present invention provides a simple and efficient mass spectrometry imaging analysis platform device and method based on the AFADESI-MSI imaging system, which provides a research strategy and technical means for in situ flux analysis and continuous sample analysis of biological tissue samples, and provides technical support to meet the rapidly growing demand for mass spectrometry imaging analysis in fields such as biomedicine and life sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Schematic diagram of an open mass spectrometry imaging analysis method provided by the present invention that integrates tissue section preparation on tape and an adsorption platform device. Figure 1 Serial number: tissue section on tape 1, pressing module 2, ion transfer tube 3, aerodynamically assisted ionization ion source interface 4, vacuum adsorption platform 5, gas path valve 6, electrically controlled translation stage 7, metal lining 8, positioning scale lines drawn on the platform surface 9, desorption electrospray probe 10.

[0025] Figure 2 This is a comparison of the optical images of the transfer adhesive sample 2a and the sample on the tape 2b after freeze-drying provided in Example 1 of the present invention. Figure 3 This is a comparison chart showing the effect of the tableting process on the drying results of frozen section samples on tape provided in Example 1 of the present invention, wherein: Figure 3 a is the optical image of the frozen section without compression treatment, Figure 3 c is an optical image of the slice after drying;

[0026] Figure 3 b is the optical image of the frozen section after compression processing. Figure 3 d is the optical image of the slice after drying. Figure 4 A comparison of the characteristic ion signal intensities of the blue stripe sample printed on photographic paper and the whole animal tissue section sample adhered to tape provided in Example 2 of the present invention, obtained using a conventional imaging platform and a vacuum adsorption imaging platform, respectively. Figure 4 a shows the schematic diagram of the starting position and scanning mode for mass spectrometry data acquisition for the blue stripe sample; Figure 4 b shows an example of an extracted ion chromatogram of basic blue characteristic ions obtained by this method and corresponding to the corresponding stripe area; Figure 4 c shows the average value of basic blue characteristic ion intensity and RSD comparison between samples collected under two platform conditions; Figure 4 d shows the corresponding positions and scanning methods for mass spectrometry data acquisition of whole animal tissue slice samples; Figure 4 e shows the distribution of choline in the mass spectrum image of the slice; Figure 4 f shows an example of an extracted ion chromatogram of choline characteristic ions obtained using a reciprocating scanning method; Figure 4 g shows the average values of choline characteristic ion intensities collected under the two platform conditions and the RSD comparison between samples.

[0027] Figure 5Shown are optical images of three continuously cut whole animal tissue sections (numbered A, B and C) from a dosed mouse provided in Example 3 of the present invention and mass spectrometry images of characteristic ions obtained in positive ion and negative ion modes.

[0028] Figure 6 Shown is a comparison chart of the average values of characteristic background ion signal intensities in the positive ion mass spectrometry images of three slices A, B and C provided in Example 3 of the present invention.

[0029] Figure 7 Shown are the positive and negative ion mass spectrometry images of slices A, B, and C provided in Example 3 of the present invention, and a comparison of the intensity data of the distribution of positive and negative ions of drug molecules and 8 specific endogenous metabolite molecular ions in the five organ tissues of the brain, stomach, spleen, lung, and liver.

[0030] Figure 8 Shown are the mass spectrogram images of ion m / z 747.5186 in negative ion mode for slices A, B, and C provided in Example 3 of the present invention, along with the extracted ion current chromatogram in the .raw format mass spectrometry data on line 33, as well as the chromatographic peaks of the ion in the regions corresponding to brain, thymus, and lung tissues, and the automatic integration peak area and signal-to-noise ratio comparison diagram.

[0031] Figure 9 Shown is a mass spectrometry image of the spatiotemporal distribution of the drug in the whole intestinal tissue obtained by the mass spectrometry imaging analysis method provided by the present invention, as provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0033] Example 1

[0034] Preparation of whole-mount mouse tissue sections adhered to scotch tape

[0035] A whole mouse, sacrificed at a specific time point after drug administration, was placed in right lateral decubitus and frozen in liquid nitrogen. After removal, the animal was placed in a freezing mold and covered with a colloid formed from a 3% to 5% sodium carboxymethylcellulose solution. After freezing in a -80°C ultra-low temperature freezer to form a frozen block (5 cm × 10 cm × 5 cm), the frozen block was removed and placed in a -20°C to -25°C cryo-microtome (Leica CM3600, Germany) along the tail-to-head direction. After 1–2 h of freezing, the frozen block was automatically cut layer by layer until the layer with the most organs was exposed. A piece of tape, approximately 5 cm × 15 cm, was cut and tightly adhered to the cut whole tissue and the surrounding embedding gel layer using a roller and a plastic pressure plate. Continuous 30 μm thick whole-animal tissue sections were then cut manually. Take two consecutive slices, one of which is transferred and glued to a slide pre-evenly coated with AB glue under freezing conditions. The slice is tightly attached to the slide by a roller, and then placed in a -80℃ ultra-low temperature refrigerator overnight. The next day, it is placed in a -20℃~-25℃ freezing microtome. After 20 minutes, the transparent tape on the surface is carefully removed to obtain the transferred whole animal tissue slice. It is then dried in a -20℃ vacuum desiccator for 2 hours, then moved to room temperature and continued to dry for 2 hours to obtain a slice sample that can be used for mass spectrometry imaging analysis ( Figure 2 a). Place the other slice sample attached to the tape between two polytetrafluoroethylene blocks (10 cm × 10 cm × 3 cm) under freezing conditions. After applying appropriate pressure, remove the slice and freeze-dry it for 0.5 h to obtain a slice sample that can be used for mass spectrometry imaging analysis ( Figure 2 b) Use a scanner to record the optical images of the two slices ( Figure 2 ), it can be seen that the tissue slices obtained by the transfer-sticking method, compared with the tissue slices adhered to the tape, have larger cracks in the brain, liver, kidney and other tissues and between different organ tissues after the drying process, while the slice samples adhered to the tape can better preserve the in situ state and tissue appearance of the tissue organs.

[0036] In order to compare the effect of the compression process on maintaining the in situ state of the sliced sample on the tape, another animal was selected for administration and the same tissue slice preparation method was used to select two consecutively cut whole animal tissue slices ( Figure 3 a and 3b), one of which is left untreated ( Figure 3 a), the other piece was processed by tableting ( Figure 3 b), then the two slices were dried for 0.5 h at the same time, and the two slices were adsorbed and fixed on the adsorption platform for comparison ( Figure 3c and 3d), it can be seen that the slices that have not been pressed have more shrinkage and cracking on the brain, liver, kidney, heart, lung, intestine, muscle and other organ tissues ( Figure 3 c), while the slices processed by pressing have clearer outlines of each organ and tissue after drying, less wrinkling and cracking, and a smoother tissue surface ( Figure 3 d) The tissue slice preparation method provided by the present invention can easily obtain large-volume tissue samples, continuously cut tissue slices with clear organ contours, and retain the in situ state and tissue appearance of the tissue organs.

[0037] Example 2

[0038] Comparison of mass spectrometry imaging analysis using a vacuum adsorption imaging platform and mass spectrometry imaging analysis using a conventional imaging platform

[0039] In order to compare the effect of vacuum adsorption imaging platform and conventional imaging platform on the ion signal intensity of desorbed sample in sample mass spectrometry imaging analysis, the blue stripe sample ( Figure 4 a), and intestinal tissue sections of whole animal tissue sections adhered to tape ( Figure 4 d) Data were collected using a QTRAP5500 mass spectrometer (AB SCIEX, USA) in both MRM (Multiple Reaction Monitoring) mode (m / z 479.6 → m / z 435.5) and EPI (Enhanced Product Ion Scanning) mode (m / z 104.1). Because the streak samples were prepared using a color inkjet printer, the dye component (basic blue, m / z 479.6) was evenly distributed throughout each streak sample. The choline component (m / z 104.1) contained in the intestinal tissue of the whole animal tissue section also had a relatively even distribution ( Figure 4 e) The part selected by the red dotted circle, two types of samples can be used for evaluation.

[0040] The comparison method used is to first adsorb and fix the same sample on a vacuum adsorption platform for data acquisition in mass spectrometry imaging mode, and then use double-sided tape to fix the sample on a conventional mass spectrometry imaging platform, and then continue to collect data in mass spectrometry imaging mode. In both cases, the same sample desorption method and condition parameters are used for data acquisition. In this embodiment, the electrospray solvent uses a mixed solvent of acetonitrile: water = 8:2, with a flow rate of 5μL / min. A line-by-line scanning method is used, and the X-axis movement speed of the translation stage is set to 0.4mm / s, and the Y-axis step distance is 0.5mm. The scanning method used is to fix the X-axis scanning distance. When the landing point of the electrospray on the sample surface sweeps across the sample area and reaches the scanning end point after walking the set scanning distance, the platform where the sample is located is translated and stepped forward in the Y-axis direction according to the set distance. Then the electrospray landing point returns to the starting area along the opposite direction of the X-axis (see the scanning process). Figure 4 (a Scanning path diagram), the data collected during a zigzag reciprocating process is stored in a data file. In the case of two fixed samples, three reciprocating processes are scanned continuously. The characteristic ion chromatographic peak is extracted from the data collected during each reciprocating process (see the example Figure 4 b and Figure 4 f), then perform automatic integration processing, calculate the peak area value, and further calculate the average ion intensity and RSD value of the three reciprocating processes, which are used as the average indicator of the sample ion signal intensity obtained under the sampling conditions. For the blue stripe sample, since the stripes are evenly distributed and there are many of them, only the forward scanning data are compared. For the whole animal tissue section sample adhered to the tape, in order to reduce the influence of tissue differences, the sample ion signal intensity collected in one reciprocating process is averaged and then compared. The data comparison results of the two types of samples obtained under the two sample fixing platform conditions are shown in Figure 4 c and 4g, it can be seen that the use of vacuum adsorption mass spectrometry imaging platform for imaging analysis can obtain higher sample ion detection signal intensity and smaller RSD value.

[0041] Example 3

[0042] Comparison of whole animal mass spectrometry imaging analysis results under two conditions: the sample was fixed with double-sided tape and the sample was fixed with adsorption platform. Three consecutive slices (numbered A, B and C, whose optical images are shown in Figure 1) were randomly selected from the continuously cut 30μm thick whole animal tissue slices (2.6cm×5.8cm). Figure 5), serial sections were cut from a single dosed mouse fixed in the supine position, exposing organs and tissues such as the brain, lungs, thymus, stomach, liver, spleen, and intestines. After drying, the three sections were scanned to obtain optical images. Two of the sections (numbered A and B) were then affixed to a glass slide (5 cm × 10 cm) using double-sided tape. A rectangular frame denoting the intended scanning range and the scanning starting point was drawn on the back of the slide using a marker. The slide was then mounted on a conventional imaging translation stage. The AFADESI-MSI method system parameters were set, using the same desorption electrospray solvent conditions and translation stage movement parameters as in Example 2. Mass spectrometry imaging data was acquired from the two whole animal tissue sections using a positive-negative ion switching full scan mode. After that, the conventional translation stage was switched to a vacuum adsorption stage, the adsorption stage switch was turned on, and sample C was adsorbed and fixed in the rectangular frame area delineated on the stage. The position and starting point of the sample were adjusted to be close to or overlapped with the rectangular frame. The height of the plane where the sliced sample C was located was adjusted to be consistent with the height of the plane where the sliced samples A and B were imaged and analyzed. The same AFADESI-MSI analysis conditions and method parameters as those for samples A and B were used to collect the whole animal mass spectrometry imaging data of sample C. The obtained raw data .raw file was converted to a .cdf format file using the instrument's built-in Xcalibur software, and then imported into the independently developed MassImager software for image reconstruction and background signal subtraction. The mass spectrum image of the characteristic ion was obtained. Taking the positive ion m / z 215.2234 and the negative ion m / z 747.5186 as examples, their mass spectrum images are shown in Figure 5 Based on the sample's optical image and the specific distribution profile of specific ions in each organ in its mass spectrum image, circle the region of interest (ROI) in the mass spectrum image with the mouse. The average mass spectrum data of the pixels contained in the ROI can be extracted and the corresponding data can be saved in a .txt or .xls file for further data processing.

[0043] In the positive ion mass spectrometry images of slices A, B and C, three positions ( Figure 6 The yellow box area in the background of the mass spectrum image) and the selected position on each image is the same (for example, the position at the 26th minute of the third row of the scan), 3×3 pixels are selected at each position, and the average ion intensity data contained in the 9 pixels are obtained. The top ten ions in terms of ion intensity are selected from the data, and the average ion intensity of these ions obtained at the three positions of the three images is calculated as the intensity data of the characteristic ion. These ion intensity data are further compared through bar graphs (see Figure 6From the bar graph, it can be seen that the intensities of seven background ions (m / z 85.0809, m / z 149.0239, m / z 157.0841, m / z 191.1048, m / z 279.1587, m / z 295.1523, and m / z 363.233) in the mass spectrum image of slice C are significantly higher than those in the mass spectrum images of sample slices A and B, and the intensities of the other three background ions are basically close (m / z 407.2412, m / z 535.3254, and m / z 257.175).

[0044] Further referring to the outlines of the five organ tissues of brain, stomach, spleen, lung and liver in the three slice optical images A, B and C, the mass spectrum data within the organ outlines were circled and extracted in their positive and negative ion mass spectrum images respectively. The drug molecular ions in positive and negative ion modes and the ion intensity data of the other 8 endogenous metabolite molecular ions (4 positive ions and 4 negative ions) specifically distributed within the five organ outlines were extracted from the data. The ion intensities were then compared using bar graphs (see Figure 7 a and 7b), it can be seen that in the positive and negative ion modes, the average intensity of the drug molecular ions distributed in the five organ tissues is close to that in the liver tissue of slice B in the negative ion mode ( Figure 7 b), which are significantly higher in the mass spectrum of slice C than in the mass spectrum of slices A and B, and the distribution intensity of the drug in the five organs and tissues of slices A and B is basically similar. The other 8 specific ions (m / z 156.042, m / z 307.044, m / z 541.263, m / z 796.525, m / z 146.046, m / z 267.073, m / z 327.233, m / z 619.289, their corresponding intensity distributions are shown in Figure 7 c), it can be seen that, except for the case where the ion intensities are similar, the vast majority of these specific ions in the five organ tissues in slice C exhibit higher ion intensities than in slices A and B. This indicates that the mass spectrometry imaging method using samples on tape and an adsorption platform is beneficial for improving the desorption of substance molecules in the sample and their ionization intensity, thereby improving the sensitivity of mass spectrometry imaging analysis and detection.

[0045] In addition, the mass spectrometry imaging analysis method using the sample on tape and the adsorption platform has a clearer outline in the mass spectrometry images generated by the organ-specific distribution of ions. This may be related to the ability to generate a higher signal-to-noise ratio of the ion signal. The mass spectrometry images of the ion m / z 747.5186 in the negative ion mode of slices A, B, and C are used as an example to illustrate. The mass spectrometry images of the three slices were reconstructed from 70 lines of .raw format files. The middle row of the .raw format file, which can simultaneously include the brain, thymus, and lung tissue, and the 33rd line of the .raw format file, were selected (see Figure 8 The mass spectrum was represented by the dotted line in the figure), and the corresponding files were opened using the Xcalibar software that comes with the Q Exactive mass spectrometer to extract the ion current chromatogram of ion m / z 747.5186. Since the ion m / z 747.5186 has specific distribution and clear outlines in the brain, thymus, and lung tissue regions in the image, the extracted ion current chromatogram peaks in the time period can be automatically integrated and the signal-to-noise ratio calculated based on the time period in which the specific ion is collected. The chromatographic peaks appearing in the brain, thymus, and lung tissues can be obtained. The corresponding time periods are 0.1-0.5 min, 0.95-1.15 min, and 1.3-1.45 min, respectively. The signal-to-noise ratios of the extracted ion current chromatogram peaks in the three time periods are shown in the 33rd row of mass spectrometry data for the three slices, respectively, and are marked in their corresponding extracted ion current chromatograms. It can be seen that the signal-to-noise ratios in slices A and B are relatively close, with some highs and some lows, but both are significantly lower than the signal-to-noise ratio in slice C. A high signal-to-noise ratio makes it easier to distinguish the specific ion signal from the surrounding background signal or noise signal, thereby presenting a clearer organ-specific distribution outline in the mass spectrometry image of slice C.

[0046] Example 4

[0047] Mass spectrometry imaging analysis of whole mouse intestinal tissue sections adhered to scotch tape

[0048] The whole intestinal tissues of two mice were dissected out after being killed at 0.5 h and 4 h after administration. After washing with 0°C physiological saline, the intestinal tissues were arranged in a zigzag pattern from the small intestine to the colon and fixed on a cardboard plane. Then, the same freezing method and tape sectioning sample preparation method as in Example 1 were used to prepare the intestinal tissue sections (see Figure 9 a and 9b) were tableted and freeze-dried, and then mass spectrometry imaging analysis was performed using the same AFADESI-MSI system and method conditions as in Example 3. The mass spectrometry image of the drug molecule characteristic product ion (m / z 135.0950) was obtained as shown in FIG. Figure 9c and 9d, it can be seen that 0.5 h after administration, the drug is mainly distributed in the middle part of the small intestine, and 4 h after administration, the drug has reached the colon area. This mass spectrometry imaging analysis method provides a simple and efficient technical means for obtaining information on the spatiotemporal distribution of drugs in the entire intestinal tissue.

[0049] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. An open mass spectrometry imaging analysis method that integrates the sample preparation technology of biological tissue sections adhered to transparent tape and a vacuum adsorption mass spectrometry imaging platform device.

2. The open mass spectrometry imaging analysis method according to claim 1, characterized in that: The biological tissue section sample preparation technology adhered to transparent tape directly uses the adhesive tape required for adhering tissue sections when cutting biological tissue samples for frozen sections as a carrier, without the need for transferring and adhering to glass slides or other carriers. The tape uses transparent tape that is easily available on the market, eliminating the need for special tape suitable for the transfer process. After the sheeting and batch freeze-drying processes, the sample can be used for subsequent experiments or batch cryopreservation.

3. The open mass spectrometry imaging analysis method according to claim 1, wherein: The vacuum adsorption mass spectrometry imaging platform device is a device that fixes a microporous ceramic vacuum adsorption platform on a three-dimensional electrically controlled mobile platform. The on-off valve of the adsorption platform is connected to the vacuum exhaust pipeline. The adsorption platform moves freely with the electrically controlled translation stage while maintaining the adsorption state. The sample can be fixed by fitting the smooth surface of the tape with the biological tissue section sample attached to the platform surface.

4. The open mass spectrometry imaging analysis method according to claim 1, wherein: Mass spectrometry imaging analysis can use aerodynamic-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI), desorption electrospray ionization mass spectrometry imaging (DESI-MSI), or desorption electrospray / secondary photoionization mass spectrometry imaging (DESI-PI-MSI) ion sources. According to the scale positioning line and scanning starting point pre-delineated on the vacuum adsorption platform, the spatial position parameters of the imaging system scanning probe and ion transmission are adjusted, the edge and starting point of the rectangular area where the tissue section is located are adjusted to coincide with the positioning line and starting point, and the scanning method parameters are set to perform mass spectrometry imaging data acquisition and analysis.

5. The open mass spectrometry imaging analysis method according to any one of claims 1 and 4, characterized in that: In the mass spectrometry imaging method, the length of the ion transfer tube of the AFADESI-MSI ion source device is 45-55 cm, the angle between the ion transfer tube and the horizontal plane is 15-20 degrees, the distance between the lower edge of the ion transfer tube inlet and the slice sample is 0.5-1.0 mm, the auxiliary air power flow rate is 25-40 L / min, the electrospray needle atomization gas line pressure is 0.6-0.8 MPa; the spray solvent flow rate is 5-10 μL / min; the spray solvent is a conventional solvent suitable for open desorption electrospray mass spectrometry imaging method (water, methanol, acetonitrile or other solvents not limited thereto), and the optimized solvent condition is a mixed solvent consisting of acetonitrile:water with a volume ratio of 8:2; a voltage is applied to the solvent flow path The voltage is 5000-7000V; the length of the electrospray needle capillary is 0.5-1mm; the angle between the electrospray needle and the horizontal plane is 55-65°; the vertical distance between the electrospray needle tip and the slice is 1.0-4.0mm; the horizontal distance between the electrospray needle tip and the outer edge of the ion transfer tube inlet is 1-3mm; mass spectrometry imaging data acquisition is achieved by moving the adsorption platform where the sample is located, using a line-by-line scanning method; the lengths of the scanning areas in the X-axis and Y-axis directions are determined according to the size of the tissue sample, the X-axis direction is set as a line-by-line horizontal scanning direction, and the scanning speed is 0.01-0.4mm / s; the Y-axis direction is set as the stepping direction of the scanning line, and the stepping distance is 0.1-0.5mm.

6. The open mass spectrometry imaging analysis method according to claim 1, wherein: The preparation technology of biological tissue slice samples adhered to transparent tape is as follows: embedding the tissue sample in a colloidal solution prepared from a 3-5% sodium carboxymethyl cellulose aqueous solution, freezing it at -78°C, thawing it in a microtome at -20°C to -25°C, and fixing it on the machine bed of the microtome; using a commercially available 4-6 cm wide transparent tape with good viscosity for slicing, the cut slices are adhered to the tape, the preferred thickness of the whole animal tissue slices is 25-60 μm, and using a pressurizing module or device with a polytetrafluoroethylene surface but not limited thereto for tableting. After tableting, the adhesive side of the tape can be rolled inward into a barrel shape. 10-20 slices can be stored in a sealed container and placed at -78°C for standby use. Alternatively, batch slices can be directly freeze-dried for 0.5-3 hours. The dried samples can be used for mass spectrometry imaging analysis directly or after covering the tape area around the tissue slice sample with paper or plastic film glossy material.

7. The open mass spectrometry imaging analysis method according to claim 1, wherein: The biological tissue slices include the preparation of large batches of continuously cut tissue slices, the preparation of large-volume solid and non-solid dispersed organ tissues, and are not limited to the preparation of rat and / or mouse whole animal tissues and continuous intestinal tissue slices.

8. The open mass spectrometry imaging analysis method according to any one of claims 1 and 4, characterized in that: The transparent tape carrying the tissue sections can be directly placed and fixed on the vacuum adsorption platform, and the angle and position can be freely adjusted on the platform surface as needed. By switching the air valve on and off the vacuum adsorption platform, rapid replacement of different samples for mass spectrometry imaging analysis can be achieved.

9. The open mass spectrometry imaging analysis method according to any one of claims 1 and 3, characterized in that: The composition of the vacuum adsorption mass spectrometry imaging platform device is not limited to microporous ceramic materials. The adsorption platform device includes but is not limited to: an adsorption table made of microporous ceramic material, a valve switch and a quick-plug interface for opening and closing the adsorption state, a metal outer frame made of aluminum alloy, a metal support plate for connecting the electric-controlled translation stage and fixing the microporous ceramic adsorption platform, an elastic screw knob for adjusting the platform level, and a soft vacuum exhaust tube that can move freely with the platform.

10. The open mass spectrometry imaging analysis method according to any one of claims 1 and 3, characterized in that: The microporous ceramic table of the adsorption platform can be marked with scales and scanning starting points in pencil according to the size and placement of the sample, and can be repeatedly erased and reused; and / or, the surface of the adsorption platform can be covered with paper with drawn or printed scale or positioning points. The scanning starting position and scanning range of the sample can be determined based on the marks on the adsorption platform plane or paper through the perspective effect of a slide or tape.

Images