Analytic method using imaging quality analysis

Abstract

According to one aspect of the present invention, an analysis method using MALD I-MS and LA-ICP-MS both capable of performing imaging mass analysis comprises: a step (S2) for setting a region (A) on a sample coated with a substrate; a step (S3) for performing mass analysis on each of the plurality of minute regions in the region A using MALD I-MS; a step (S4) for setting, on the sample, a region (B) including the entire region (A) and a region in which the matrix is present, the region (B) being different from the region (A); a step (S5) for performing mass analysis on each of the plurality of minute regions in the region B using LA-ICP-MS; a step (S6) for determining the presence or absence of an influence of the matrix-related component on the basis of data within the region A and data of a range outside the region A generated by LA-ICP-MS; and a step (S7) of correcting, as necessary, the signal strength in the data obtained in (S5) on the basis of the determination result.

Description

Technical Field

[0001] The present invention relates to a sample analysis method using imaging mass analysis for visualizing the spatial distribution of components included in the sample. Background Art

[0002] In recent years, the use of imaging mass spectrometry, which visualizes the spatial distribution of components contained in a sample, has been rapidly developing in various fields such as medicine and biochemistry. In an imaging mass spectrometer (hereinafter sometimes referred to as "MALDI-MS") that utilizes a matrix-assisted laser desorption / ionization (MALDI) mass spectrometer, it is possible to perform molecular imaging of various organic compounds such as proteins, amino acids, and lipids present in a sample. On the other hand, in a laser ablation inductively coupled plasma mass spectrometer (hereinafter sometimes referred to as "LA-ICP-MS") that combines a laser ablation device with an inductively coupled plasma mass spectrometer, it is possible to perform elemental imaging of various metal elements, etc., present in a sample (see Patent Document 1, etc.). Therefore, by combining these two mass spectrometers, it is possible to obtain information on both molecular imaging and elemental imaging of substances present in a sample, and perform analysis that correlates them.

[0003] For example, Non-Patent Document 1 discloses a multimodal imaging system that displays molecular imaging images and elemental imaging images obtained from the two mass spectrometers side by side on the same screen.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2019-190841

[0007] Non-patent literature

[0008] Non-Patent Document 1: "Multimodal Imaging System," [Online], [retrieved March 7, 2023], Shimadzu Corporation, website link <URL:https: / / www.shimadzu.com / an / sites / shimadzu.com.an / files / pim / pim_document_file / brochures / 13216 / c146-e423_1.pdf> Summary of the Invention

[0009] Technical problem to be solved by the invention

[0010] In LA-ICP-MS, laser irradiation causes most of the substances present on the sample surface to disappear. Therefore, analysis based on LA-ICP-MS is essentially destructive, and samples analyzed by LA-ICP-MS cannot be subjected to analysis based on MALDI-MS. On the other hand, in MALDI-MS analysis, a matrix for MALDI-MS is applied to the sample surface. Therefore, when a sample analyzed by MALDI-MS is subjected to LA-ICP-MS analysis, there is a risk that the analysis results will be affected by the matrix itself or by inclusions associated with the matrix application process. Furthermore, there is a concern that signal intensity may be reduced due to laser irradiation marks formed during MALDI-MS analysis. Therefore, in analysis using the aforementioned conventional multimodal imaging systems, MALDI-MS analysis and LA-ICP-MS analysis are performed on different samples, typically different slice samples obtained by continuously slicing the object to be analyzed.

[0011] However, even consecutive sample slices from the same analyte have varying shapes. Overlaying molecular imaging images obtained by MALD I-MS with elemental imaging images obtained by LA-ICP-MS requires complex alignment tasks involving image deformation. Furthermore, even consecutive sample slices do not always have identical component distributions, making it difficult to accurately overlay molecular and elemental imaging images using conventional methods.

[0012] The present invention was completed to solve such technical problems. Its main purpose is to provide an analysis method using imaging mass spectrometry that can easily perform high-precision coincidence analysis without performing complicated alignment processing between the molecular imaging image obtained by MALD I-MS and the elemental imaging image obtained by LA-ICP-MS.

[0013] Solutions for solving the above technical problems

[0014] One embodiment of the imaging mass spectrometry analysis method of the present invention is an analysis method using a matrix-assisted laser desorption ionization mass spectrometer capable of imaging mass spectrometry and a laser ablation inductively coupled plasma mass spectrometer capable of imaging mass spectrometry, comprising:

[0015] a first region setting step of setting a first analysis region on the sample coated with the matrix;

[0016] a first analysis execution step of performing mass analysis on each of a plurality of micro-regions within the first analysis region using a matrix-assisted laser desorption ionization mass spectrometer to acquire mass analysis data;

[0017] a second region setting step of setting a second analysis region on the sample coated with the matrix, the second analysis region including the entire first analysis region and a region where the matrix exists, which is different from the first analysis region;

[0018] a second analysis execution step of performing mass analysis on each of a plurality of micro regions within the second analysis region using a laser ablation inductively coupled plasma mass spectrometer to acquire mass analysis data;

[0019] a determination step of determining whether or not the matrix-related components have an influence on the mass analysis in the second analysis step based on the mass analysis data within the first analysis region and the mass analysis data of a range outside the first analysis region acquired in the second analysis step;

[0020] The information correction step is a step of correcting the signal intensity information in the mass spectrometry data obtained in the second analysis execution step as needed when the determination step determines that there is an influence of the matrix-related component.

[0021] In addition, the second area setting step can be performed after the execution of the first analysis execution step, that is, after the execution of the imaging quality analysis of the first analysis area, but the second area setting step can also be performed before the execution of the first analysis execution step, and the first analysis execution step and the second analysis execution step can be performed in sequence when both the first analysis area and the second analysis area are determined.

[0022] Effects of the Invention

[0023] In the above-described embodiment of the analysis method using imaging mass analysis according to the present invention, it is possible to determine whether components (inclusions) contained in the matrix or introduced during the matrix coating process affect the mass analysis data obtained by a laser ablation inductively coupled plasma mass spectrometer. Specifically, it is possible to determine the signal intensity information corresponding to the mass-to-charge ratio of the inclusions. Furthermore, for example, if the influence of the inclusions is too significant to be ignored, it is possible to correct the signal intensity information affected by the inclusions, allowing the generation of elemental imaging images using more accurate mass analysis data. Thus, according to this analysis method, molecular imaging using MALDI-MS and elemental imaging using LA-I CP-MS can be performed using the same sample, rather than using separate samples. This allows for simple, high-precision coincidence analysis without the need for complex alignment processing associated with image distortion. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the configuration of one embodiment of a multimodal imaging system for implementing the analysis method according to the present invention.

[0025] Figure 2 This is a flowchart showing an example of an analysis procedure in the multimodal imaging system according to the present embodiment.

[0026] Figure 3 This is an explanatory diagram of an example of the operation of setting a region to be analyzed by MALDI-MS in the multimodal imaging system according to the present embodiment.

[0027] Figure 4 This is an explanatory diagram of an example of the operation of setting a region to be analyzed by LA-ICP-MS in the multimodal imaging system according to the present embodiment.

[0028] Figure 5 This is a schematic diagram of the operation of determining the influence of inclusions in the multimodal imaging system of this embodiment.

[0029] Figure 6 This is an explanatory diagram of another example of the operation of setting the region to be analyzed by LA-ICP-MS in the multimodal imaging system according to the present embodiment. DETAILED DESCRIPTION

[0030] Hereinafter, an example of the analysis method according to the present invention will be described with reference to the accompanying drawings.

[0031] Figure 1 This is a schematic diagram of an embodiment of a multimodal imaging system for implementing the analysis method.

[0032] like Figure 1 As shown, the multimodal imaging system of this embodiment includes a matrix coating device 1, an imaging mass spectrometer (MALD I-MS device) 2, a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS device) 3, and a control / processing device 4. The control / processing device 4 is implemented as a personal computer (or higher-performance computer) with dedicated control / processing software installed. Connected to the control / processing device 4 are an input unit 5 including a keyboard and a display unit 6 serving as a display monitor.

[0033] The matrix coating apparatus 1 is an apparatus for thinly coating the surface of a sample 101 placed on a glass slide 100 with a matrix for MALD I. For example, "i MLayer" or "iMLayerAERO" manufactured by Shimadzu Corporation can be used as the matrix coating apparatus 1. The sample 101 is, for example, a slice specimen obtained by thinly slicing an organ of an experimental animal such as a mouse.

[0034] The MALDI-MS device 2 is an imaging mass spectrometer using a matrix-assisted laser desorption ionization mass spectrometer. It sequentially irradiates laser light at positions spaced apart by predetermined intervals within a predetermined analysis region set on a sample 101 coated with a matrix, thereby performing mass analysis on various ions generated from microscopic regions at various positions of the sample 101, thereby collecting mass analysis data. Typically, in the imaging mass spectrometer 2, it is possible to obtain ion intensity signals covering a predetermined mass-to-charge ratio (m / z) range as mass analysis data for each microscopic portion within the above-mentioned analysis region. As the MALDI-MS device 2, for example, the "i MScope" manufactured by Shimadzu Corporation can be used. TM QT”.

[0035] The LA-ICP-MS apparatus 3 is a combination of a laser ablation apparatus and an inductively coupled plasma mass spectrometer. It can linearly scan the laser irradiation position within a predetermined analysis region set on the sample 101. At each predetermined time interval, i.e., each time the laser irradiates a predetermined length of the sample 101, it acquires ion intensity signals at one or more predetermined m / z values ​​as mass analysis data. The LA-ICP-MS apparatus 3 can be a combination of the "I CPMS-2030" manufactured by Shimadzu Corporation and a laser ablation apparatus.

[0036] The control / processing device 4 includes a data storage unit 40, a matrix effect determination unit 41, a matrix effect correction unit 42, an imaging image generation unit 43, an integrated spectrum data generation unit 44, a multivariate analysis processing unit 45, a position offset correction unit 46, an area setting unit 47, a display processing unit 48 and a control unit 49 as functional blocks.

[0037] Then, except Figure 1 In addition, refer to Figures 2 to 5 An example of characteristic analysis using the multimodal imaging system according to this embodiment will be described. Figure 2 Flowchart showing the steps of this analysis. Figure 3 1 is an explanatory diagram of an example of a process for setting an analysis target region on a sample 101 by MALD I-MS. Figure 4 1 is an explanatory diagram of an example of a process for setting an analysis target region on a sample 101 by LA-ICP-MS. Figure 5 This is a schematic diagram of the process of determining the impact of inclusions based on the collected quality analysis data.

[0038] First, the user places sample 101 on slide glass 100, is arranged on matrix coating device 1 and carries out specified operation.Thus, matrix coating device 1 is thinly coated with matrix (step S1) on sample 101.Matrix is ​​roughly evenly coated on the larger range on the slide glass 100 that comprises sample 101 whole, so outside of sample 101, matrix is ​​directly coated on slide glass 100.Therefore, on slide glass 100, form the part where sample 101 and matrix exist simultaneously and sample 101 does not have the part where only matrix exists.In addition, as well-known, various compounds can be used as matrix, and can be suitably selected according to the kind of sample 101 or the material of analysis target etc.

[0039] Next, the user places the matrix-coated slide 100 at a predetermined position of the MALD I-MS apparatus 2 and sets an analysis region A for molecular imaging on the sample 101 (step S2). This analysis region A is generally called a region of interest (ROI).

[0040] As a specific step in step S2, the user uses a microscope attached to the MALD I-MS device 2 to perform microscopic observation on the surface of the sample 101, such as Figure 3 As shown in (A), the range of the analysis area A is indicated on the microscopic observation image displayed on the screen of the display unit 6. Under the control of the area setting unit 47, the user can operate the microscopic observation image with a pointing device such as a mouse to change the size and position of the area designation frame to indicate the analysis area A. Figure 3 In the example shown in (A) of FIG. , the shape of the area designation frame is a rectangle, but this shape may be changed arbitrarily.

[0041] When the setting of the analysis area A is completed and the user performs a predetermined operation through the input unit 5 , the MALDI-MS apparatus 2 executes imaging mass analysis in the analysis area A set on the sample 101 under the control of the control unit 49 (step S3 ).

[0042] Specifically, in the MALDI-MS apparatus 2, while the slide 100 is moved in steps along mutually orthogonal X- and Y-axis directions within a plane parallel to the upper surface of the slide 100 by a predetermined distance, a laser beam narrowed to a small diameter is pulsed into the analysis region A on the sample 101. Ions generated near the irradiation position are collected and mass analyzed. With a single laser irradiation, mass analysis data (mass spectrum data) covering the specified m / z range can be obtained. However, the amount of ions generated by a single laser irradiation in the MALDI method is not necessarily large. Therefore, the laser beam can be irradiated multiple times at the same position on the sample 101, or at very close positions, and the mass analysis data obtained from each laser irradiation can be accumulated. This method of imaging mass analysis of a specified analysis region A on the sample 101 is identical to the analysis method used in conventional imaging mass spectrometers.

[0043] In step S3, Figure 3 Mass spectrometric data within a predetermined m / z range is obtained for each of the numerous microscopic regions (laser irradiation positions) within the analysis region A shown in FIG (B). The large amount of data collected in this manner by the MALD I-MS apparatus 2 is transmitted to the control / processing device 4 and temporarily stored in the data storage unit 40.

[0044] Next, the user removes the analyzed slide 100 from the MALD I-MS apparatus 2 and sets it in a predetermined position in the LA-ICP-MS apparatus 3. An analysis region B, which includes the entire analysis region A and is the target of LA-ICP-MS analysis, is then set on the sample 101 (step S4).

[0045] As a specific step in step S4, the user first uses the microscope provided in the LA-ICP-MS device 3 (strictly speaking, a laser ablation device) to perform microscopic observation of the upper surface of the sample 101. Figure 4 As shown in (A), the laser irradiation marks produced by the MALD I-MS analysis can be clearly observed. Therefore, the user identifies the boundary of the analysis area A based on the laser irradiation marks and sets the analysis area B so that it includes a portion that is wider than the boundary of the analysis area A and deviates from the sample 101 and directly places the matrix on the slide 100 (see Figure 4 (B)). The analysis region B can also be designated by the user operating a pointing device such as a mouse on the microscopic observation image to change the size and position of the region designation frame under the control of the region setting unit 47.

[0046] Here, imaging mass analysis is performed on the same sample 101 on the same slide 100 by the MALDI-MS apparatus 2 and the LA-ICP-MS apparatus 3. Therefore, if the reference position for setting the slide 100 is uniquely determined in each of the two apparatuses 2 and 3, and the correspondence between the two reference positions is clear, there will be no positional offset between the sample image in the microscopic observation image obtained by the MALDI-MS apparatus 2 and the sample image in the microscopic observation image obtained by the LA-ICP-MS apparatus 3. In other words, no special alignment processing is required. Furthermore, even if the reference position for setting the slide 100 is not uniquely determined in each of the two apparatuses 2 and 3, or the reference positions do not correspond, since the sample images have the same shape, the positional offset correction unit 46 can use the sample image in the microscopic observation image obtained by the MALD I-MS apparatus 2 and the sample image in the microscopic observation image obtained by the LA-ICP-MS apparatus 3 to correct the positional offset between the two images through simple processing (translation and rotation operations within the XY plane).

[0047] If the setting of the analysis area B in step S4 is completed and the user performs the prescribed operation through the input unit 5, then under the control of the control unit 49, the LA-I CP-MS device 3 performs imaging mass analysis within the analysis area B set on the sample 101 (step S5).

[0048] Unlike the MALD I-MS apparatus 2, the LA-ICP-MS apparatus 3 does not collect ion intensity data across a specified m / z range. Instead, it selectively collects ion intensity data at one or more pre-specified specific m / z values. Typically, these specific m / z values ​​are determined based on the element that the user is interested in, or wishes to observe. For example, if the user wishes to observe the distribution of iron (Fe) in sample 101, the m / z value corresponding to Fe is designated as the m / z value of interest.

[0049] Furthermore, the LA-I CP-MS apparatus 3 does not move the sample 101 in a step-like manner, but rather moves the sample 101 continuously along the X-axis within analysis region B. In this case, the sample 101 is irradiated with laser light substantially continuously. At each predetermined laser irradiation time, the intensity signal for ions with a specific m / z value collected during that laser irradiation time is integrated for each m / z value. Thus, for each predetermined microregion (a region of a predetermined length along the X-axis) on the sample 101, ion intensity data at a specific m / z value is obtained as mass analysis data. The interval between laser irradiation positions in the MALD I-MS apparatus 2 and the size of the microregions in the LA-I CP-MS apparatus 3 are set so that the spatial resolution of the imaging mass analysis in the MALD I-MS apparatus 2 is approximately the same as that in the LA-I CP-MS apparatus 3.

[0050] In step S5, Figure 4 Ion intensity data at specific m / z values ​​are obtained for each of the numerous microregions within the analysis region B shown in (C). The data collected by the imaging mass analysis in step S5 is also sent to the control / processing device 4 and temporarily stored in the data storage unit 40.

[0051] When the imaging mass analysis in the LA-ICP-MS apparatus 3 is completed, the matrix influence determination unit 41 in the control / processing apparatus 4 performs a process for determining whether or not there is matrix influence using part of the data collected in step S5 (step S6).

[0052] Whether or not there is an influence of the matrix can be determined as follows.

[0053] like Figure 4 As shown in (C), in the imaging mass analysis based on the LA-I CP-MS device 3, ion intensity data of a small area where only the matrix exists on the slide 100 is also obtained. Figure 5 As shown, within the analysis region B, there are: a first portion B1, where a matrix is ​​present on the sample 101 and analysis is performed using the MALD I-MS device 2 (i.e., laser irradiation is performed by the MALD I-MS device 2); a second portion B2, where a matrix is ​​present on the sample 101 and analysis is not performed using the MALD I-MS device 2; and a third portion B3, where only a matrix is ​​present (no sample 101 is present).

[0054] The mass spectrometry data obtained from the microscopic region located at the third site B3 reflects components (elements) originally contained in the matrix, as well as components introduced during the matrix coating process in the matrix coating apparatus 1, such as components eluted from the tube through which the matrix flows (hereinafter collectively referred to as "matrix-related components"). In contrast, the mass spectrometry data obtained from the microscopic regions located at the first site B1 and the second site B2 reflects both matrix-related components and components contained in the sample 101. However, in the first site B1, due to imaging mass spectrometry performed by the MALD I-MS apparatus 2, some matrix-related components and components contained in the sample 101 may be lost.

[0055] As an example, let m / z M1 and m / z M2 be the m / z values ​​to be analyzed in the imaging mass analysis in step S5. The ion intensity data at these two m / z values ​​are as follows: Figure 5 In the mass spectrometry data obtained from the micro region located at the third part B3, the ion intensity in the m / z value of the analysis object is substantially zero ( Figure 5 In the case of m / z M2 in the analysis, it can be determined that there is no influence of matrix-related components in the m / z value. In fact, for example, when the ion intensity at the m / z value is below a predetermined threshold, it can be determined that there is no influence of matrix-related components in the m / z value. On the other hand, when the ion intensity at the m / z value of the analysis object is not substantially zero ( Figure 5 In the case of m / z M1 in the above example, it can be determined that there is an influence of the matrix-related component in the m / z value.

[0056] Alternatively, by performing mass analysis of only the matrix in advance (i.e., through preliminary experiments), it is possible to determine the elements contained in the matrix. However, this method cannot determine the inclusions introduced during the coating process using the matrix coating apparatus 1. The extent of such inclusions also varies depending on the usage of the apparatus 1. In contrast, analyzing the matrix applied to the actual slide 100 carrying the sample 101 provides the advantage of obtaining ion intensity data reflecting the state of the matrix applied to the sample 101.

[0057] When it is determined that the m / z value of the analyte is affected by the matrix-related components, the matrix effect correction unit 42 corrects the ion intensity (step S7) by correcting the ion intensity at the same m / z value obtained from the analysis region A, that is, from the microregion located in the first site B1. Figure 5In the example shown, the ion intensity of m / z M2 is not corrected, but rather the ion intensity of m / z M1. The simplest correction method in step S7 is to subtract the ion intensity Im from the ion intensity Is, which is the ion intensity at the same m / z value obtained from each microregion located in the first site B1, and the ion intensity Im, which is the ion intensity at the same m / z value obtained from the microregion located in the third site B3. In this case, the ion intensity Im is preferably the average of the ion intensities at the same m / z value obtained from multiple microregions located in the third site B3. This can substantially eliminate or at least reduce the influence of matrix-related components on the ion intensity.

[0058] The simplest calibration method described above does not take into account the potential decrease in the amount of matrix-related components during mass analysis using the MALD I-MS apparatus 2. In other words, if this decrease is negligible compared to the original component amount, the simplest method described above can be used. On the other hand, if the decrease in the amount of matrix-related components during mass analysis using the MALD I-MS apparatus 2 is not negligible, processing can be performed to reflect this decrease during calibration.

[0059] Specifically, in the closely located microregions of the first and second sites B1 and B2, it is estimated that the amounts of components contained in the sample 101 before analysis are approximately the same. Therefore, it is estimated that the difference in ion intensities among the m / z values ​​of the target analyte obtained from these microregions corresponds to the reduction in mass analysis by the MALD I-MS apparatus 2. Specifically, the ion intensity data obtained from the microregion located in the first site B1 and the ion intensity data obtained from the microregion located in the second site B2 can be used to calculate the ion intensity Id of the m / z value of the target analyte estimated to have been reduced by mass analysis by the MALD I-MS apparatus 2. Therefore, the ion intensity Id of this reduction can be subtracted from the ion intensity Im, which is the ion intensity at the same m / z value obtained from the microregion located in the third site B3. This Im-Id is then treated as the influence of matrix-related components from the ion intensity Is obtained from the microregion located in the first site B1, and subtracted. In other words, the calculation of Is-(Im-Id) can be performed.

[0060] Furthermore, when executing steps S6 and S7, it is necessary to identify the first, second, and third sites B1 to B3 within analysis region B. Since first site B1 coincides with analysis region A, there is no problem. However, in order to identify second and third sites B2 and B3, it is necessary to verify the presence of sample 101. For example, during the setting of analysis region B or during step S6, the user can perform this identification by visually checking the microscopic image. Alternatively, the second and third sites B2 and B3 can be automatically identified through image recognition processing, without relying on user judgment.

[0061] As described above, the presence or absence of matrix-related components in the m / z values ​​of the target analyte can be determined based on mass analysis data obtained from microscopic regions present in the first site B1 and the third site B3, or based on mass analysis data obtained from microscopic regions present in the first site B1, the second site B2, and the third site B3. Furthermore, if this determination indicates that the influence of matrix-related components cannot be ignored, the influence of matrix-related components can be mitigated by correcting the ion intensity of the target analyte's m / z values. Consequently, even if the sample 101 used for mass analysis by the MALD I-MS apparatus 2 is directly used for mass analysis by the LA-ICP-MS apparatus 3, a highly accurate mass analysis result with the influence of matrix-related components mitigated can be obtained by the LA-ICP-MS apparatus 3.

[0062] return Figure 2 , continue to explain.

[0063] After the processing in step S7 is completed, the imaging image generator 43 uses the imaging mass spectrometry data acquired by the MALDI-MS apparatus 2 and stored in the data storage unit 40 to generate a molecular imaging image at a predetermined m / z value corresponding to the analysis region A (step S8). Furthermore, the imaging image generator 43 uses the imaging mass spectrometry data acquired by the LA-ICP-MS apparatus 3 and corrected in step S7 to generate an elemental imaging image at a predetermined m / z value corresponding to the analysis region A (step S9). The m / z value of each imaging image can be appropriately specified by the user.

[0064] The display processing unit 48 displays the molecular imaging image and elemental imaging image generated in this manner side by side or superimposed on the screen of the display unit 6 (step S10 ).

[0065] The integrated spectrum data generating unit 44 obtains, for each micro-region within the analysis region A, the mass analysis data (mass spectrum data covering a specified m / z range) generated by the MALD I-MS device 2 and the mass analysis data (ion intensity data at a specified m / z value) generated by the LA-I CP-MS device 3 obtained from the micro-region or its corresponding position, and obtains data that integrates them on a single m / z axis (step S11).

[0066] Typically, the m / z values ​​of the compound-derived ions detected by the MALD I-MS apparatus 2 and the metal-derived ions detected by the LA-I CP-MS apparatus 3 do not overlap. Therefore, data integration can be achieved by simply arranging the data on the m / z axis. However, if there is a significant difference in detection sensitivity between the MALD I-MS apparatus 2 and the LA-I CP-MS apparatus 3, this difference in detection sensitivity can be investigated in advance, and integration can be performed after correcting the signal intensities of one or both to minimize the difference. If the m / z values ​​of the compound-derived ions detected by the MALD I-MS apparatus 2 and the metal-derived ions detected by the LA-I CP-MS apparatus 3 overlap, either value (generally the one with higher sensitivity) can be used, or the signal intensities of both values ​​can be multiplied by appropriate coefficients and then added together.

[0067] The multivariate analysis processing unit 45 performs multivariate analysis processing based on the data integrated in step S11 (step S12 ).

[0068] For example, data can be organized into a data matrix showing the relationship between m / z values ​​and ion intensities for each microregion. Multivariate analysis such as principal component analysis (PCA) and partial least squares analysis (PLS) can be performed on this data matrix to search for regions where specific components are concentrated. Alternatively, hierarchical cluster analysis (HCA) can be performed to search for images with similar distributions across a large number of m / z values.

[0069] The display processing unit 48 displays the result of the multivariate analysis in a predetermined format on the screen of the display unit 6 (step S13 ).

[0070] Of course, the order of the processing of steps S8 to S10 and the processing of steps S11 to S12 can be appropriately replaced.

[0071] In the above example, the analysis region B is set by expanding the analysis region A outward, but this is only an example and can be changed as follows. Figure 6 This is an explanatory diagram of another example of the operation of setting the analysis region B in the multimodal imaging system according to the above-described embodiment.

[0072] like Figure 6As shown in (A), in the case where the analysis region A is set only in a very small part of the interior of the sample 101, if the analysis region B is set in a manner that expands the analysis region A, the area of ​​the analysis region B outside the analysis region A becomes quite large. As can be seen from the above description, the mass analysis data obtained from the tiny region in the analysis region B outside the analysis region A is only used to determine whether the matrix-related components have an influence and to correct the influence, and mass analysis data from a large number of tiny regions are not required. On the contrary, if the area of ​​the analysis region B outside the analysis region A is large, the time required for mass analysis of this part by the LA-I CP-MS device 3 becomes longer, resulting in a decrease in the analysis efficiency. Therefore, in the case where the analysis region A is set only in a very small part of the interior of the sample 101, for example, Figure 6 As shown in (B) and (C), analysis region C can be determined at a position separated from analysis region A, and analysis region A and analysis region C can be combined to form analysis region B.

[0073] Figure 6 (B) is an example of a case where the analysis region C is determined so as to span the portion where the sample 101 exists (the second portion B2) and the portion where the sample 101 does not exist (the third portion B3). Figure 6 (C) is an example of a case where the analysis region C is determined only in the portion where the sample 101 does not exist. Figure 6 In case (B), as described above, the degree of reduction of matrix-related components caused by mass analysis performed in analysis region A by the MALD I-MS apparatus 2 can be estimated using mass analysis data from a micro region included in analysis region B outside analysis region A.

[0074] As described above, as long as the analysis region B includes the entire analysis region A and includes portions other than the analysis region A where the matrix is ​​present, the position and area of ​​the analysis region B can be appropriately determined.

[0075] The above-described embodiment is merely an example of the present invention, and any changes, additions, or modifications appropriately made within the scope of the gist of the present invention are clearly encompassed by the scope of the claims of the present application.

[0076] [Various options]

[0077] Those skilled in the art should understand that the above exemplary embodiments are specific examples of the following schemes.

[0078] (Item 1) One embodiment of an analysis method using imaging mass spectrometry according to the present invention is an analysis method using a matrix-assisted laser desorption ionization mass spectrometer capable of imaging mass spectrometry and a laser ablation inductively coupled plasma mass spectrometer capable of imaging mass spectrometry, comprising:

[0079] a first region setting step of setting a first analysis region on the sample coated with the matrix;

[0080] a first analysis execution step of performing mass analysis on each of a plurality of micro-regions within the first analysis region using a matrix-assisted laser desorption ionization mass spectrometer to acquire mass analysis data;

[0081] a second region setting step of setting a second analysis region on the sample coated with the matrix, the second analysis region including the entire first analysis region and a region where the matrix exists, which is different from the first analysis region;

[0082] a second analysis execution step of performing mass analysis on each of a plurality of micro regions within the second analysis region using a laser ablation inductively coupled plasma mass spectrometer to acquire mass analysis data;

[0083] a determination step of determining whether or not the matrix-related components have an influence on the mass analysis in the second analysis step based on the mass analysis data within the first analysis region and the mass analysis data of a range outside the first analysis region acquired in the second analysis step;

[0084] The information correction step is a step of correcting the signal intensity information in the mass spectrometry data obtained in the second analysis execution step as needed when the determination step determines that there is an influence of the matrix-related component.

[0085] The analysis method described in item 1 can determine whether components contained in the matrix or components introduced during the matrix coating process (inclusions) affect mass analysis data obtained using a laser ablation inductively coupled plasma mass spectrometer. Specifically, signal intensity information regarding the mass-to-charge ratio corresponding to the inclusions can be determined. For example, if the inclusions' influence is significant enough to be negligible, correction can be performed to correct the signal intensity information affected by the inclusions, allowing elemental imaging images to be generated using more accurate mass analysis data. Therefore, this analysis method allows molecular imaging using MALD I-MS and elemental imaging using LA-I CP-MS to be performed using the same sample, rather than using separate samples. This allows for simple, high-precision coincidence analysis without requiring complex alignment processing associated with image distortion.

[0086] (Item 2) In the analysis method described in Item 1, the second analysis region can be set as a region obtained by expanding the first analysis region outward.

[0087] According to the analysis method described in item 2, when a user manually sets the second analysis region, for example, they can simply expand the range indicating the first analysis region or the marker corresponding to the first analysis region outward. This allows the second analysis region to be appropriately set through a simple operation, reducing the user's workload and reducing setting errors.

[0088] (Item 3) In the analysis method described in Item 1, the range where the matrix exists in the second analysis region can be set as a region separate from the first analysis region.

[0089] That is, in the analysis method described in item 2, the area outside the first analysis region where the matrix is ​​present is continuous with the first analysis region, but it does not have to be continuous. If the first analysis region is an extremely narrow area near the center of the sample, if the second analysis region is defined by extending the first analysis region to a location where the matrix is ​​present but the sample is absent, the second analysis region becomes considerably larger than the first analysis region. A larger second analysis region increases the time required for imaging mass analysis using LA-I CP-MS.

[0090] In contrast, the analysis method described in item 3 allows the second analysis region to be determined by adding the minimum range required to determine the presence or absence of matrix effects to the first analysis region. This prevents excessively prolonged imaging mass analysis time using LA-I CP-MS. Consequently, the time required for imaging mass analysis using LA-I CP-MS can be reduced, allowing for efficient analysis.

[0091] (Item 4) The analysis method described in any one of Items 1 to 3 may further include an image generation and display step, generating an imaging image corresponding to the first analysis area based on the mass analysis data obtained in the first analysis execution step, and generating an imaging image corresponding to the first area based on the mass analysis data corrected in the information correction step, and displaying the two imaging images side by side or overlapping.

[0092] According to the analysis method described in item 4, a molecular imaging image corresponding to a first analysis region on a sample and an elemental imaging image can be displayed on the same display screen. The elemental imaging image is an image corresponding to the same first analysis region on the same sample and shows the highly precise distribution of a predetermined element. This makes it possible, for example, to accurately compare the distribution of molecules and elements.

[0093] (Item 5) In the analysis method described in any one of Items 1 to 4, in the information correction step, when it is determined by the judgment step that there is an influence of the matrix-related component, it is possible to perform a process of subtracting the intensity equivalent to the contribution of the matrix-related component from the mass analysis data obtained by the second analysis execution step.

[0094] According to the analysis method described in Item 5, even when the target element whose spatial distribution is to be confirmed by imaging mass analysis based on LA-ICP-MS overlaps with elements derived from matrix-related components, the influence of the matrix-related components can be reduced, and the intensity information of the target element can be obtained more accurately.

[0095] (Item 6) In the analysis method described in any one of Items 1 to 5, it can further include a data integration step, for substantially the same position on the sample, generating a mass spectrum that integrates intensity information at the same mass-to-charge ratio based on the mass analysis data obtained in the first analysis execution step and the mass analysis data obtained in the second analysis execution step.

[0096] In the analysis method described in Item 6, for example, in the first analysis execution step, mass spectrum data across a predetermined m / z range is obtained for each microregion within the first analysis region, and in the second analysis execution step, ion intensity data at a specific m / z value is obtained for each position substantially corresponding to the microregion. Therefore, for example, by adding the mass spectrum data obtained in the first analysis execution step to the ion intensity data at the specific m / z value obtained in the second analysis execution step for each corresponding microregion, both data can be integrated on a single m / z axis. This allows for simultaneous processing of two different types of mass analysis data.

[0097] (Item 7) The analysis method described in Item 6 can further include an analysis processing step of performing multivariate analysis processing on the mass spectrum data integrated in the data integration step.

[0098] As described above, the analysis method described in Item 6 integrates both data on a single m / z axis for each microscopic region on the sample, thereby obtaining data that pairs the m / z value with the signal intensity value for each microscopic region. The analysis method described in Item 7, by subjecting the thus obtained data to multivariate analysis, enables automated and objective analysis, such as extracting m / z values ​​with high spatial distribution similarity or extracting m / z values ​​that exhibit a distribution similar to that of a specific element, which has traditionally been primarily performed by visual inspection by the user.

[0099] (Item 8) In the analysis method described in any one of Items 1 to 7, the range where the matrix exists can be set to a region where only the matrix is ​​applied.

[0100] According to the analysis method described in item 8, since the sample does not exist in the range where the matrix exists, it is possible to accurately grasp the components contained in the matrix or mixed in during the matrix coating operation and determine the signal intensity of the components.

[0101] Description of Reference Numerals

[0102] 1. Matrix coating device

[0103] 2 Imaging mass spectrometry device (MALDI-MS device)

[0104] 3Laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS device)

[0105] 4Control / processing device

[0106] 40 Data storage unit

[0107] 41 Matrix Influence Determination Unit

[0108] 42 Matrix Effect Correction Unit

[0109] 43 Imaging image generation unit

[0110] 44 Integrated spectrum data generation unit

[0111] 45 Multivariate analysis processing unit

[0112] 46 Position offset correction unit

[0113] 47 Area Setting Department

[0114] 48 Display processing unit

[0115] 49 Control Department

[0116] 5 Input section

[0117] 6 Display unit

[0118] 100 slides

[0119] 101 samples.

Claims

1. An analysis method using imaging mass spectrometry, comprising: a matrix-assisted laser desorption ionization mass spectrometer capable of performing imaging mass spectrometry; and a laser ablation inductively coupled plasma mass spectrometer capable of performing imaging mass spectrometry, wherein: include: a first region setting step of setting a first analysis region on the sample coated with the matrix; a first analysis execution step of performing mass analysis on each of a plurality of micro-regions within the first analysis region using a matrix-assisted laser desorption ionization mass spectrometer to acquire mass analysis data; a second region setting step of setting a second analysis region on the sample coated with the matrix, the second analysis region including the entire first analysis region and a region where the matrix exists, which is different from the first analysis region; a second analysis execution step of performing mass analysis on each of a plurality of micro regions within the second analysis region using a laser ablation inductively coupled plasma mass spectrometer to acquire mass analysis data; a determination step of determining whether or not the matrix-related components have an influence on the mass analysis in the second analysis step based on the mass analysis data within the first analysis region and the mass analysis data of a range outside the first analysis region acquired in the second analysis step; The information correction step is a step of correcting the signal intensity information in the mass spectrometry data obtained in the second analysis execution step as needed when the determination step determines that there is an influence of the matrix-related component.

2. The analysis method using imaging quality analysis according to claim 1, wherein: The second analysis region is a region obtained by extending the first analysis region outward.

3. The analysis method using imaging quality analysis according to claim 1, wherein: The range where the matrix exists in the second analysis region is a region separated from the first analysis region.

4. The analysis method using imaging quality analysis according to claim 1, wherein: It further includes an image generation and display step, which generates an imaging image corresponding to the first analysis area based on the mass analysis data obtained in the first analysis execution step, and generates an imaging image corresponding to the first area based on the mass analysis data corrected in the information correction step, and displays the two imaging images side by side or overlapping.

5. The analysis method using imaging quality analysis according to claim 1, wherein: In the information correction step, when it is determined in the determination step that there is an influence of the matrix-related component, a process of subtracting the intensity corresponding to the contribution of the matrix-related component from the mass spectrometry data obtained in the second analysis execution step is performed.

6. The analysis method using imaging quality analysis according to claim 1, wherein: The method further includes a data integration step of generating a mass spectrum integrating intensity information at the same mass-to-charge ratio for substantially the same position on the sample based on the mass analysis data obtained in the first analysis execution step and the mass analysis data obtained in the second analysis execution step.

7. The analysis method using imaging quality analysis according to claim 6, wherein: The method further includes an analysis processing step of performing multivariate analysis on the mass spectrum data integrated in the data integration step.

8. The analysis method using imaging quality analysis according to claim 1, wherein: The range where the matrix exists is a region where only the matrix is ​​coated.

Images