Analysis method employing imaging mass spectrometry

Abstract

An aspect of the present invention is an analysis method using MALDI-MS and LA-ICP-MS, both capable of imaging mass spectrometry, the method comprising: a step of setting a region A on a sample to which a matrix has been applied (S2); a step of executing mass spectrometry for each of a plurality of micro-regions within the region A using MALDI-MS (S3); a step of setting, on the sample, a region B that includes all of the region A and a range where the matrix exists that is different from the region A (S4); a step of executing mass spectrometry for each of a plurality of micro-regions within the region B using LA-ICP-MS (S5); a step of determining the presence or absence of an influence of a matrix-related component based on the data within the region A and the data in the range outside the region A obtained by LA-ICP-MS (S6); and a step of correcting the signal intensity in the data obtained in S5 as necessary based on the determination result (S7).

Description

TECHNICAL FIELD

[0001] The present invention relates to an analysis method for a sample using imaging mass spectrometry that visualizes the spatial distribution of components contained 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 advancing in various fields such as medicine and biochemistry. An imaging mass spectrometer using a Matrix-Assisted Laser Desorption / Ionization (MALDI) mass spectrometer (hereinafter also referred to as "MALDI-MS") can perform molecular imaging of various organic compounds such as proteins, amino acids, and lipids existing within a sample. On the other hand, a Laser Ablation-Inductively Coupled Plasma-Mass Spectrometer (hereinafter also referred to as "LA-ICP-MS"), which combines a laser ablation system with an inductively coupled plasma-mass spectrometer, can perform elemental imaging of various metal elements and the like existing within a sample (see Patent Literature 1, etc.). Therefore, by combining these two mass spectrometers, it is possible to acquire information on both molecular imaging and elemental imaging of substances existing within a sample and to perform an analysis that associates them.

[0003] For example, Non-Patent Literature 1 discloses a multimodal imaging system that displays a molecular imaging image and an elemental imaging image obtained from the two mass spectrometers mentioned above side-by-side on the same screen.PRIOR ART DOCUMENTS PATENT LITERATURE

[0004] Patent Literature 1: Japanese Unexamined Patent Application Publication No. 2019-190841NON-PATENT LITERATURE

[0005] Non-Patent Literature 1: "Multimodal Imaging System", [online], [searched on March 7, 2023], Shimadzu Corporation, Internet <URL: https: / / www. shimadzu.com / an / sites / shimadzu.com.an / files / pim / pim_document_file / brochures / 13216 / c146-e423_1.pdf>SUMMARY OF INVENTION PROBLEM TO BE SOLVED BY THE INVENTION

[0006] In LA-ICP-MS, most of the substances present on the sample surface are ablated by laser irradiation. Therefore, analysis by LA-ICP-MS is substantially a destructive analysis, and it is impossible to subject a sample that has been analyzed by LA-ICP-MS to analysis by MALDI-MS. On the other hand, in analysis by MALDI-MS, a MALDI matrix is applied to the surface of the sample. Therefore, if a sample analyzed by MALDI-MS is subjected to analysis by LA-ICP-MS, the analysis results may be affected by the matrix itself or by contaminants related to the process of applying the matrix. There is also a concern that the signal intensity may decrease due to laser irradiation marks formed during analysis by MALDI-MS. For these reasons, in the analysis using the conventional multimodal imaging system described above, the analysis by MALDI-MS and the analysis by LA-ICP-MS are performed on different samples, typically on different slice samples obtained by continuously slicing an object of analysis.

[0007] However, even with continuous slice samples from the same object of analysis, their shapes are not identical, and superimposing the molecular imaging image obtained by MALDI-MS and the elemental imaging image obtained by LA-ICP-MS requires a complicated registration process involving image deformation processing. Furthermore, since the distribution of contained components is not completely identical even in continuous slice samples in the first place, it is difficult to perform superimposition analysis of the molecular imaging image and the elemental imaging image with sufficiently high accuracy with the conventional method.

[0008] The present invention has been made to solve these problems, and its main object is to provide an analysis method using imaging mass spectrometry that can easily perform high-accuracy superimposition analysis without performing complicated registration processing of a molecular imaging image obtained by MALDI-MS and an elemental imaging image obtained by LA-ICP-MS.MEANS FOR SOLVING THE PROBLEM

[0009] One aspect of the 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, the method comprising: a first region setting step of setting a first analysis region on a sample to which a matrix has been applied; a first analysis execution step of executing mass spectrometry for each of a plurality of micro-regions within the first analysis region using the matrix-assisted laser desorption / ionization mass spectrometer to acquire mass spectrometry data; a second region setting step of setting, on the sample to which the matrix has been applied, a second analysis region that includes all of the first analysis region and a range where the matrix exists that is different from the first analysis region; a second analysis execution step of executing mass spectrometry for each of a plurality of micro-regions within the second analysis region using the laser ablation-inductively coupled plasma-mass spectrometer to acquire mass spectrometry data; a determination step of determining, based on the mass spectrometry data within the first analysis region and the mass spectrometry data in a range outside the first analysis region, acquired in the second analysis execution step, whether there is an influence of a component related to the matrix in the mass spectrometry in the second analysis execution step; and an information correction step of, when it is determined in the determination step that there is an influence of the matrix-related component, correcting signal intensity information in the mass spectrometry data obtained in the second analysis execution step as necessary.

[0010] The second region setting step may be performed after the execution of the first analysis execution step, that is, after the execution of the imaging mass spectrometry for the first analysis region. Alternatively, the second region setting step may be executed before the execution of the first analysis execution step, and the first analysis execution step and the second analysis execution step may be sequentially executed in a state where both the first analysis region and the second analysis region have been determined.EFFECTS OF THE INVENTION

[0011] In the above aspect of the analysis method using imaging mass spectrometry according to the present invention, it is possible to ascertain whether or not a component contained in the matrix or a component (contaminant) mixed in during the process of matrix application affects the mass spectrometry data obtained by the laser ablation-inductively coupled plasma-mass spectrometer. That is, it is possible to ascertain the signal intensity information at the mass-to-charge ratio corresponding to the contaminant. Furthermore, for example, when the influence of the contaminant is large to an extent that it cannot be ignored, it is possible to correct the signal intensity information affected by the contaminant and create an elemental imaging image using higher-accuracy mass spectrometry data. As a result, according to this analysis method, it is possible to perform molecular imaging by MALDI-MS and elemental imaging by LA-ICP-MS using the same sample instead of using separate samples, and it is possible to easily perform high-accuracy superimposition analysis without performing complicated registration processing involving image deformation processing.BRIEF DESCRIPTION OF DRAWINGS

[0012] [Fig. 1] A schematic configuration diagram of an embodiment of a multimodal imaging system for carrying out the analysis method according to the present invention. [Fig. 2] A flowchart showing an example of an analysis procedure in the multimodal imaging system of the present embodiment. [Fig. 3] An explanatory diagram of an example of an operation for setting a region on a sample to be analyzed by MALDI-MS in the multimodal imaging system of the present embodiment. [Fig. 4] An explanatory diagram of an example of an operation for setting a region on a sample to be analyzed by LA-ICP-MS in the multimodal imaging system of the present embodiment. [Fig. 5] A schematic diagram of an operation for determining the influence of contaminants in the multimodal imaging system of the present embodiment. [Fig. 6] An explanatory diagram of another example of an operation for setting a region to be analyzed by LA-ICP-MS in the multimodal imaging system of the present embodiment. DESCRIPTION OF EMBODIMENTS

[0013] Hereinafter, an example of the analysis method according to the present invention will be described with reference to the accompanying drawings. Fig. 1 is a schematic configuration diagram of an embodiment of a multimodal imaging system for carrying out this analysis method.

[0014] As shown in Fig. 1, the multimodal imaging system of the present embodiment includes a matrix application device 1, an imaging mass spectrometer (MALDI-MS device) 2, a laser ablation-inductively coupled plasma-mass spectrometer (LA-ICP-MS device) 3, and a control and processing unit 4. The physical entity of the control and processing unit 4 is a personal computer (or a computer with higher performance), and dedicated control and processing software is installed in this personal computer. Connected to this control and processing unit 4 are an input unit 5 including a keyboard and the like, and a display unit 6 which is a display monitor.

[0015] The matrix application device 1 is a device that thinly applies a MALDI matrix to the surface of a sample 101 placed on a slide glass 100. As the matrix application device 1, for example, "iMLayer" or "iMLayer AERO" manufactured by Shimadzu Corporation can be used. The sample 101 is, for example, a slice sample obtained by thinly slicing an organ of a laboratory animal such as a mouse.

[0016] The MALDI-MS device 2 is an imaging mass spectrometer using a matrix-assisted laser desorption / ionization mass spectrometer. Within a range of a predetermined analysis region set on the sample 101 to which a matrix has been applied, it sequentially irradiates laser to positions separated by a predetermined distance, and collects mass spectrometry data by performing mass spectrometry on various ions generated from micro-regions including each position of the sample 101. Normally, in the imaging mass spectrometer 2, for each micro-region within the analysis region, an ion intensity signal over a predetermined mass-to-charge ratio (m / z) range can be acquired as mass spectrometry data. As the MALDI-MS device 2, for example, "iMScope ™< QT" manufactured by Shimadzu Corporation can be used.

[0017] The LA-ICP-MS device 3 is a combination of a laser ablation system and an inductively coupled plasma-mass spectrometer. Within a range of a predetermined analysis region set on the sample 101, while scanning the irradiation position of the laser linearly, at every predetermined time interval, that is, every time the laser irradiates a range of a predetermined length on the sample 101, it can acquire an ion intensity signal at one or more preset m / z values as mass spectrometry data. As the LA-ICP-MS device 3, a combination of "ICPMS-2030" manufactured by Shimadzu Corporation and a laser ablation system can be used.

[0018] The control and processing unit 4 includes, as functional blocks, a data storage unit 40, a matrix influence determination unit 41, a matrix influence correction unit 42, an imaging image creation unit 43, an integrated spectrum data creation unit 44, a multivariate analysis processing unit 45, a misregistration correction unit 46, a region setting unit 47, a display processing unit 48, and a control unit 49.

[0019] Next, an example of a characteristic analysis using the multimodal imaging system of the present embodiment will be described with reference to Fig. 1, as well as Figs. 2 to 5. Fig. 2 is a flowchart showing the procedure of this analysis. Fig. 3 is an explanatory diagram of an example of an operation for setting a region on the sample 101 to be analyzed by MALDI-MS. Fig. 4 is an explanatory diagram of an example of an operation for setting a region on the sample 101 to be analyzed by LA-ICP-MS. Fig. 5 is a schematic diagram of an operation for determining the influence of contaminants based on the collected mass spectrometry data.

[0020] First, a user places the sample 101 on the slide glass 100, sets it in the matrix application device 1, and performs a predetermined operation. Thereby, the matrix application device 1 thinly applies a matrix onto the sample 101 (Step S1). Since the matrix is applied substantially uniformly over a wide area on the slide glass 100 including the entire sample 101, the matrix is directly applied onto the slide glass 100 outside the sample 101. Therefore, on the slide glass 100, a portion where both the sample 101 and the matrix exist and a portion where the sample 101 does not exist and only the matrix exists are formed. As is well known, various compounds are used as the matrix and are appropriately selected according to the type of the sample 101, the substance to be analyzed, and so on.

[0021] Next, the user sets the slide glass 100 on which the matrix has been applied at a predetermined position in the MALDI-MS device 2, and sets an analysis region A on the sample 101 where molecular imaging is desired (Step S2). Generally, this analysis region A is called a Region of Interest (ROI).

[0022] As a specific procedure in Step S2, the user performs microscopic observation of the upper surface of the sample 101 using a microscope attached to the MALDI-MS device 2, and indicates the range of the analysis region A on a microscopic observation image displayed on the screen of the display unit 6, as shown in Fig. 3(A). The indication of this analysis region A can be performed by the user operating a pointing device such as a mouse on the microscopic observation image to change the size and position of a region designation frame, under the control of the region setting unit 47. In the example shown in Fig. 3(A), the shape of the region designation frame is rectangular, but this shape may be made arbitrarily changeable.

[0023] When the setting of the analysis region A is completed and the user performs a predetermined operation with the input unit 5, the MALDI-MS device 2 executes imaging mass spectrometry within the analysis region A set on the sample 101 under the control of the control unit 49 (Step S3).

[0024] Specifically, in the MALDI-MS device 2, while moving the slide glass 100 stepwise by predetermined distances in the X-axis direction and the Y-axis direction, which are orthogonal to each other in a plane parallel to the upper surface of the slide glass 100, it irradiates the sample 101 within the analysis region A with a pulsed laser beam focused to a small diameter, collects ions generated from the vicinity of the irradiation position, and executes mass spectrometry. Mass spectrometry data (mass spectrum data) over a predetermined m / z range can be obtained by a single laser irradiation. However, in the MALDI method, the amount of ions generated by a single laser irradiation is not necessarily large. Therefore, the same position or very close positions on the sample 101 may be irradiated with the laser beam multiple times, and the mass spectrometry data obtained for each laser irradiation may be integrated. This method of imaging mass spectrometry for a predetermined analysis region A on the sample 101 is completely the same as the analysis method in a conventional general imaging mass spectrometer.

[0025] In Step S3, mass spectrum data over a predetermined m / z range is obtained at each of the numerous micro-regions (laser irradiation positions) within the analysis region A as shown in Fig. 3(B). The large amount of data thus collected in the MALDI-MS device 2 is sent to the control and processing unit 4 and temporarily stored in the data storage unit 40.

[0026] Next, the user removes the analyzed slide glass 100 from the MALDI-MS device 2 and sets it at a predetermined position in the LA-ICP-MS device 3. Then, the user sets an analysis region B for LA-ICP-MS analysis on the sample 101, which includes the entire analysis region A (Step S4).

[0027] As a specific procedure in Step S4, the user first performs microscopic observation of the upper surface of the sample 101 using a microscope provided in the LA-ICP-MS device 3 (strictly speaking, the laser ablation system). On the microscopic observation image at this time, laser irradiation marks from the MALDI-MS analysis are clearly observable, as shown in Fig. 4(A). Therefore, the user recognizes the boundary of the analysis region A from the laser irradiation marks and sets the analysis region B to be wider than it and to include a portion where the matrix is directly on the slide glass 100 outside the sample 101 (see Fig. 4(B)). The indication of this analysis region B can also be performed by the user operating a pointing device such as a mouse on the microscopic observation image to change the size and position of a region designation frame, under the control of the region setting unit 47.

[0028] Here, since imaging mass spectrometry is performed on the same sample 101 on the same slide glass 100 with the MALDI-MS device 2 and the LA-ICP-MS device 3, if the reference positions for setting the slide glass 100 in both devices 2 and 3 are uniquely determined and the correspondence between the two reference positions is clear, no misregistration occurs between the sample image in the microscopic observation image obtained by the MALDI-MS device 2 and the sample image in the microscopic observation image obtained by the LA-ICP-MS device 3. In other words, there is no need to perform special registration processing. Even if the reference positions for setting the slide glass 100 in both devices 2 and 3 are not uniquely determined or the correspondence between the two reference positions is not established, since the shape of the sample image is the same, the misregistration correction unit 46 can correct the misregistration between the two images by a simple process (movement and rotation operations in the X-Y plane) using the sample image in the microscopic observation image obtained by the MALDI-MS device 2 and the sample image in the microscopic observation image obtained by the LA-ICP-MS device 3.

[0029] When the setting of the analysis region B in Step S4 is completed and the user performs a predetermined operation with the input unit 5, the LA-ICP-MS device 3 executes imaging mass spectrometry within the analysis region B set on the sample 101 under the control of the control unit 49 (Step S5).

[0030] Unlike the MALDI-MS device 2, the LA-ICP-MS device 3 does not collect all ion intensity data over a predetermined m / z range, but selectively collects ion intensity data at one or more specific m / z values designated in advance. Usually, this specific m / z value is determined according to the element that the user is interested in, that is, wishes to observe. For example, if the user wants to observe the distribution of iron (Fe) in the sample 101, the m / z value corresponding to Fe is designated as the m / z value to be observed.

[0031] Also, the LA-ICP-MS device 3 does not move the sample 101 stepwise, but moves the sample 101 continuously in the X-axis direction within the analysis region B. At this time, the laser is irradiated substantially continuously onto the sample 101, and for each predetermined laser irradiation time, the intensity signal for ions having a specific m / z value collected within that laser irradiation time is integrated for each m / z value. Therefore, for each predetermined micro-region on the sample 101 (a region of a predetermined length in the direction along the X-axis), ion intensity data at a specific m / z value is obtained as mass spectrometry data. Here, the interval between laser irradiation positions in the MALDI-MS device 2 and the size of the micro-region in the LA-ICP-MS device 3 are set so that the spatial resolution of imaging mass spectrometry in the MALDI-MS device 2 and the spatial resolution of imaging mass spectrometry in the LA-ICP-MS device 3 are substantially the same.

[0032] In Step S5, ion intensity data at a specific m / z value is obtained in each of the numerous micro-regions within the analysis region B as shown in Fig. 4(C). The data collected by the imaging mass spectrometry in this Step S5 is also sent to the control and processing unit 4 and temporarily stored in the data storage unit 40.

[0033] When the imaging mass spectrometry by the LA-ICP-MS device 3 is completed, the matrix influence determination unit 41 in the control and processing unit 4 executes a process to determine the presence or absence of the influence of the matrix using a part of the data collected in Step S5 (Step S6).

[0034] The presence or absence of the influence of the matrix can be determined as follows. As shown in Fig. 4(C), in the imaging mass spectrometry by the LA-ICP-MS device 3, ion intensity data of a micro-region where only the matrix exists on the slide glass 100 is also acquired. Therefore, as shown in Fig. 5, within the analysis region B, there exist a first portion B1 where the matrix exists on the sample 101 and analysis by the MALDI-MS device 2 has been performed (that is, laser has been irradiated by the MALDI-MS device 2), a second portion B2 where the matrix exists on the sample 101 and analysis by the MALDI-MS device 2 has not been performed, and a third portion B3 where only the matrix exists (the sample 101 does not exist).

[0035] The mass spectrometry data obtained from the micro-regions in the third portion B3 reflects components (elements) originally contained in the matrix and components mixed in during the process of applying the matrix with the matrix application device 1, for example, components that have leached out from the piping through which the matrix flows (hereinafter, these components are collectively referred to as "matrix-related components"). In contrast, the mass spectrometry data obtained from the micro-regions in the first portion B1 and the second portion B2 reflects both the matrix-related components and the components contained in the sample 101. However, in the first portion B1, some of the matrix-related components and the components contained in the sample 101 may have been lost due to the imaging mass spectrometry by the MALDI-MS device 2.

[0036] As an example, assume that two m / z values, m / z M1 and m / z M2, are the target m / z values for imaging mass spectrometry in Step S5, and that the ion intensity data at these two m / z values are obtained as shown in Fig. 5. In the mass spectrometry data obtained from the micro-regions in the third portion B3, if the ion intensity at the target m / z value is substantially zero (in the case of m / z M2 in Fig. 5), it can be determined that there is no influence of matrix-related components at that m / z value. In practice, for example, if the ion intensity at that m / z value is below a predetermined threshold, it can be determined that there is no influence of matrix-related components at that m / z value. On the other hand, if the ion intensity at the target m / z value is not substantially zero (in the case of m / z M1 in Fig. 5), it can be determined that there is an influence of matrix-related components at that m / z value.

[0037] It is possible to identify the elements contained in the matrix by analyzing only the matrix in advance (i.e., by a preliminary experiment). However, in that case, it is not possible to identify contaminants mixed in during the application process using the matrix application device 1. The situation of such contaminant mixing also changes depending on the usage conditions of the device 1. In contrast, by analyzing the matrix actually applied to the slide glass 100 on which the sample 101 is placed, there is an advantage that ion intensity data reflecting the state of the matrix applied to the sample 101 can be obtained.

[0038] If it is determined that there is an influence of a matrix-related component at the target m / z value, the matrix influence correction unit 42 performs a process to correct the ion intensity at the same m / z value obtained from the micro-regions in the analysis region A, that is, in the first portion B1 (Step S7). In the example shown in Fig. 5, the ion intensity at m / z M2 is not corrected, and the ion intensity at m / z M1 is corrected. As one of the simplest correction methods in Step S7, a calculation may be performed to subtract the ion intensity Im at the same m / z value obtained from the micro-regions in the third portion B3 from the ion intensity Is at the same m / z value obtained from each micro-region in the first portion B1. At this time, as the ion intensity Im, it is preferable to use the average of the ion intensities at the same m / z value obtained from a plurality of micro-regions in the third portion B3. This can substantially eliminate or at least reduce the influence of the matrix-related components on the ion intensity.

[0039] The simplest correction method described above does not take into account the possibility that the amount of matrix-related components may decrease during the mass spectrometry by the MALDI-MS device 2. In other words, if the decrease is negligible compared to the original amount of the component, the simplest method described above can be used. On the other hand, if the decrease in the amount of matrix-related components during the mass spectrometry by the MALDI-MS device 2 cannot be ignored, it is advisable to perform a process that reflects that decrease during the correction.

[0040] Specifically, in micro-regions that are close to each other in the first portion B1 and the second portion B2, it can be estimated that the amount of components contained in the sample 101 before analysis is almost the same. Therefore, the difference in ion intensity at the target m / z value obtained from those micro-regions can be estimated to correspond to the amount of decrease during the mass spectrometry by the MALDI-MS device 2. That is, using the ion intensity data obtained from the micro-regions in the first portion B1 and the ion intensity data obtained from the micro-regions in the second portion B2, it is possible to calculate the ion intensity Id at the target m / z value that is estimated to have decreased due to the mass spectrometry by the MALDI-MS device 2. Therefore, a process may be executed to subtract the decreased ion intensity Id from the ion intensity Im at the same m / z value obtained from the micro-regions in the third portion B3, and to consider this Im-Id as the influence of the matrix-related component in the ion intensity Is obtained from the micro-regions in the first portion B1 and subtract it, that is, to execute the calculation of Is - (Im - Id).

[0041] When executing the processes of Steps S6 and S7, it is necessary to recognize the first to third portions B1 to B3 within the analysis region B. Since the first portion B1 coincides with the analysis region A, there is no problem, but the identification of the second portion B2 and the third portion B3 requires recognition of the presence or absence of the sample 101. This recognition can be performed, for example, by the user visually confirming the microscopic observation image when setting the analysis region B or when executing Step S6. Alternatively, instead of relying on the user's judgment, the second portion B2 and the third portion B3 may be identified automatically using image recognition processing.

[0042] As described above, here, from the mass spectrometry data obtained from the micro-regions existing respectively in the first portion B1 and the third portion B3, or from the mass spectrometry data obtained from the micro-regions existing respectively in the first portion B1, the second portion B2, and the third portion B3, it is possible to determine the presence or absence of the influence of matrix-related components at the target m / z value. Then, if it can be determined from the result of the determination that the influence of the matrix-related components cannot be ignored, the influence of the matrix-related components can be reduced by correcting the ion intensity at the target m / z value. As a result, even if the sample 101 used for mass spectrometry by the MALDI-MS device 2 is used as it is for mass spectrometry by the LA-ICP-MS device 3, a highly accurate result with reduced influence of matrix-related components can be obtained as the mass spectrometry result from the LA-ICP-MS device 3.

[0043] Returning to the flowchart of Fig. 2, the explanation will be continued. After the process of Step S7 is completed, the imaging image creation unit 43 creates a molecular imaging image at a predetermined m / z value corresponding to the analysis region A, using the imaging mass spectrometry data acquired by the MALDI-MS device 2 and stored in the data storage unit 40 (Step S8). Also, the imaging image creation unit 43 creates an elemental imaging image at a predetermined m / z value corresponding to the analysis region A, using the imaging mass spectrometry data acquired by the LA-ICP-MS device 3 and corrected in Step S7 (Step S9). The m / z value for each imaging image can be appropriately specified by the user.

[0044] The display processing unit 48 displays the molecular imaging image and the elemental imaging image thus created side-by-side or superimposed on the screen of the display unit 6 (Step S10).

[0045] The integrated spectrum data creation unit 44 acquires, for each micro-region within the analysis region A, the mass spectrometry data from the MALDI-MS device 2 (mass spectrum data over a predetermined m / z range) and the mass spectrometry data from the LA-ICP-MS device 3 (ion intensity data at a predetermined m / z value) obtained from that micro-region or its corresponding position, and obtains data integrated on a single m / z axis (Step S11).

[0046] Generally, the m / z values of ions derived from compounds detected by the MALDI-MS device 2 and ions derived from metal elements detected by the LA-ICP-MS device 3 do not overlap. Therefore, the data can be integrated by simply arranging both data on the m / z axis. However, if there is a large difference in detection sensitivity between the MALDI-MS device 2 and the LA-ICP-MS device 3, it is advisable to investigate the difference in detection sensitivity in advance and correct either or both signal intensities so that the difference becomes smaller before integration. If the m / z values of ions derived from compounds detected by the MALDI-MS device 2 and ions derived from metal elements detected by the LA-ICP-MS device 3 overlap, either one (usually the one with higher sensitivity) may be adopted, or both may be added after multiplying each signal intensity by an appropriate coefficient.

[0047] The multivariate analysis processing unit 45 executes multivariate analysis processing based on the data integrated in Step S11 (Step S12). For example, the data is organized into the format of a data matrix showing the relationship between m / z values and ion intensities for each micro-region, and by performing multivariate analysis such as Principal Component Analysis (PCA) or Partial Least Squares (PLS) analysis on that data matrix, a region where a specific component is concentrated and distributed is searched for. Alternatively, by performing Hierarchical Cluster Analysis (HCA), images with similar distributions among a large number of m / z values are searched for.

[0048] The display processing unit 48 displays the result of such multivariate analysis on the screen of the display unit 6 in a predetermined format (Step S13).

[0049] Of course, the order of the processing of Steps S8 to S10 and the processing of Steps S11 to S12 can be changed as appropriate.

[0050] In the above example, the analysis region B was set by extending the analysis region A outward, but this is just one example and can be changed as follows. Fig. 6 is an explanatory diagram of another example of the operation of setting the analysis region B in the multimodal imaging system of the above-described embodiment.

[0051] As shown in Fig. 6(A), when 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 so as to expand this analysis region A, the area of the analysis region B other than the analysis region A becomes quite large. As is clear from the above description, the mass spectrometry data obtained from the micro-regions existing in the analysis region B other than the analysis region A is only used for determining the presence or absence of the influence of matrix-related components and for correcting that influence, and mass spectrometry data from a large number of micro-regions is not necessary. Rather, if the area of the analysis region B other than the analysis region A is large, the time required to perform mass spectrometry on that portion with the LA-ICP-MS device 3 becomes long, leading to a decrease in analysis efficiency. Therefore, when the analysis region A is set only in a very small part of the interior of the sample 101, for example, as shown in Figs. 6(B) and 6(C), an analysis region C may be defined at a position separated from the analysis region A, and the analysis region A and the analysis region C may be combined to form the analysis region B.

[0052] Fig. 6(B) is an example of a case where the analysis region C is defined so as to straddle a portion where the sample 101 exists (the second portion B2 described above) and a portion where it does not exist (the third portion B3 described above). On the other hand, Fig. 6(C) is an example of a case where the analysis region C is defined only in a portion where the sample 101 does not exist. In the case of Fig. 6(B), as described above, by using the mass spectrometry data from the micro-regions included in the analysis region B other than the analysis region A, it is possible to estimate the degree of decrease of the matrix-related components due to the mass spectrometry performed by the MALDI-MS device 2 in the analysis region A. In this way, the position and size of the analysis region B can be appropriately determined as long as it includes the entire analysis region A and includes a portion where the matrix exists other than the analysis region A.

[0053] The above embodiment is merely an example of the present invention, and it is clear that appropriate modifications, additions, and corrections made within the spirit of the present invention are included in the scope of the claims of the present application.[Various Aspects]

[0054] Those skilled in the art will understand that the exemplary embodiments described above are specific examples of the following aspects.

[0055] (Aspect 1) An aspect of the 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, the method comprising: a first region setting step of setting a first analysis region on a sample to which a matrix has been applied; a first analysis execution step of executing mass spectrometry for each of a plurality of micro-regions within the first analysis region using the matrix-assisted laser desorption / ionization mass spectrometer to acquire mass spectrometry data; a second region setting step of setting, on the sample to which the matrix has been applied, a second analysis region that includes all of the first analysis region and a range where the matrix exists that is different from the first analysis region; a second analysis execution step of executing mass spectrometry for each of a plurality of micro-regions within the second analysis region using the laser ablation-inductively coupled plasma-mass spectrometer to acquire mass spectrometry data; a determination step of determining, based on the mass spectrometry data within the first analysis region and the mass spectrometry data in a range outside the first analysis region, acquired in the second analysis execution step, whether there is an influence of a component related to the matrix in the mass spectrometry in the second analysis execution step; and an information correction step of, when it is determined in the determination step that there is an influence of the matrix-related component, correcting signal intensity information in the mass spectrometry data obtained in the second analysis execution step as necessary.

[0056] In the analysis method according to Aspect 1, it is possible to ascertain whether or not a component contained in the matrix or a component (contaminant) mixed in during the process of matrix application affects the mass spectrometry data obtained by the laser ablation-inductively coupled plasma-mass spectrometer. That is, it is possible to ascertain the signal intensity information at the mass-to-charge ratio corresponding to the contaminant, and, for example, when the influence is large to an extent that it cannot be ignored, it is possible to correct the signal intensity information affected by the contaminant and create an elemental imaging image using higher-accuracy mass spectrometry data. Therefore, according to this analysis method, it is possible to perform molecular imaging by MALDI-MS and elemental imaging by LA-ICP-MS using the same sample instead of using separate samples, and it is possible to easily perform high-accuracy superimposition analysis without performing complicated registration processing involving image deformation processing.

[0057] (Aspect 2) In the analysis method according to Aspect 1, the second analysis region may be a region obtained by expanding the first analysis region outward.

[0058] According to the analysis method of Aspect 2, for example, when a user manually sets the second analysis region, it is only necessary to perform an operation of expanding a marker indicating the range of the first analysis region or corresponding to the first analysis region outward. This makes it possible to appropriately set the second analysis region with a simple operation, which can reduce the user's workload and reduce setting errors.

[0059] (Aspect 3) In the analysis method according to Aspect 1, the range where the matrix exists in the second analysis region may be a region separate from the first analysis region.

[0060] That is, in the analysis method according to Aspect 2, the range where the matrix exists outside the first analysis region is continuous with the first analysis region, but it does not have to be continuous. If the first analysis region is a very narrow range near the center of the sample, and one attempts to define the second analysis region by expanding the first analysis region to a position where no sample exists and the matrix exists, the second analysis region becomes considerably larger than the first analysis region. If the second analysis region is large, the time required for imaging mass spectrometry by LA-ICP-MS becomes long.

[0061] In contrast, according to the analysis method of Aspect 3, it is possible to determine the second analysis region by adding the minimum necessary range to the first analysis region in order to determine the presence or absence of the influence of the matrix, so the time required for imaging mass spectrometry by LA-ICP-MS is not unnecessarily prolonged. Therefore, the time required for imaging mass spectrometry by LA-ICP-MS can be suppressed, and the analysis can be performed efficiently.

[0062] (Aspect 4) The analysis method according to any one of Aspects 1 to 3 may further comprise an image creation and display step of creating an imaging image corresponding to the first analysis region based on the mass spectrometry data obtained in the first analysis execution step, creating an imaging image corresponding to the first region based on the mass spectrometry data corrected in the information correction step, and displaying both imaging images side-by-side or superimposed.

[0063] According to the analysis method of Aspect 4, a molecular imaging image corresponding to the first analysis region on a sample and a highly accurate elemental imaging image showing the distribution of a predetermined element corresponding to the same first analysis region on the same sample can be displayed on the same display screen. This allows, for example, for a highly accurate comparison of the distribution of molecules and elements.

[0064] (Aspect 5) In the analysis method according to any one of Aspects 1 to 4, in the information correction step, when it is determined in the determination step that there is an influence of a matrix-related component, a process of subtracting an intensity corresponding to the contribution of the matrix-related component from the mass spectrometry data obtained in the second analysis execution step may be performed.

[0065] According to the analysis method of Aspect 5, even when the target element whose spatial distribution is to be confirmed by imaging mass spectrometry by LA-ICP-MS overlaps with an element derived from a component related to the matrix, the influence of the component related to the matrix can be reduced, and the intensity information of the target element can be obtained more accurately.

[0066] (Aspect 6) The analysis method according to any one of Aspects 1 to 5 may further comprise a data integration step of creating a mass spectrum in which intensity information at the same mass-to-charge ratio is integrated, using the mass spectrometry data obtained in the first analysis execution step and the mass spectrometry data obtained in the second analysis execution step for substantially the same position on the sample.

[0067] In the analysis method according to Aspect 6, for example, in the first analysis execution step, mass spectrum data over a predetermined m / z range is obtained for each micro-region within the first analysis region, and in the second analysis execution step, ion intensity data at a specific m / z value for each position substantially corresponding to that micro-region is obtained. Therefore, for example, for each corresponding micro-region, by adding the ion intensity data obtained in the second analysis execution step at the specific m / z value to the mass spectrum data obtained in the first analysis execution step, both data can be integrated on a single m / z axis. This makes it possible to handle two different types of mass spectrometry data together.

[0068] (Aspect 7) The analysis method according to Aspect 6 may further comprise an analysis processing step of executing a multivariate analysis process on the mass spectrum data integrated in the data integration step.

[0069] As described above, according to the analysis method of Aspect 6, since both data are integrated on a single m / z axis for each micro-region on the sample, data pairing m / z values and signal intensity values can be obtained for each micro-region. According to the analysis method of Aspect 7, by subjecting the data thus obtained to multivariate analysis, it is possible to automatically and objectively perform analyses that were conventionally mainly confirmed by the user's visual inspection, such as extracting m / z values with high similarity in spatial distribution, or extracting m / z values showing a distribution similar to the spatial distribution of a specific element.

[0070] (Aspect 8) In the analysis method according to any one of Aspects 1 to 7, the range where the matrix exists may be a region where only the matrix is applied.

[0071] According to the analysis method of Aspect 8, since no sample exists 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 application process, and to determine the signal intensity due to those components.REFERENCE SIGNS LIST

[0072] 1Matrix application device 2Imaging mass spectrometer (MALDI-MS device) 3Laser ablation-inductively coupled plasma-mass spectrometer (LA-ICP-MS device) 4Control and processing unit 40 Data storage unit 41 Matrix influence determination unit 42 Matrix influence correction unit 43 Imaging image creation unit 44 Integrated spectrum data creation unit 45 Multivariate analysis processing unit 46 Misregistration correction unit 47 Region setting unit 48 Display processing unit 49... Control unit 5 Input unit 6 Display unit 100 Slide glass 101 Sample

Claims

1. An analysis method using imaging mass spectrometry, 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, the method comprising: a first region setting step of setting a first analysis region on a sample to which a matrix has been applied; a first analysis execution step of executing mass spectrometry for each of a plurality of micro-regions within the first analysis region using the matrix-assisted laser desorption / ionization mass spectrometer to acquire mass spectrometry data; a second region setting step of setting, on the sample to which the matrix has been applied, a second analysis region that includes all of the first analysis region and a range where the matrix exists that is different from the first analysis region; a second analysis execution step of executing mass spectrometry for each of a plurality of micro-regions within the second analysis region using the laser ablation-inductively coupled plasma-mass spectrometer to acquire mass spectrometry data; a determination step of determining, based on the mass spectrometry data within the first analysis region and the mass spectrometry data in a range outside the first analysis region, acquired in the second analysis execution step, whether there is an influence of a component related to the matrix in the mass spectrometry in the second analysis execution step; and an information correction step of, when it is determined in the determination step that there is an influence of the matrix-related component, correcting signal intensity information in the mass spectrometry data obtained in the second analysis execution step as necessary.

2. The analysis method using imaging mass spectrometry according to claim 1, wherein the second analysis region is a region obtained by expanding the first analysis region outward.

3. The analysis method using imaging mass spectrometry according to claim 1, 5the range where the matrix exists in the second analysis region is a region separate from the first analysis region.

4. The analysis method using imaging mass spectrometry according to claim 1, further comprising an image creation and display step of creating an imaging image corresponding to the first analysis region based on the mass spectrometry data obtained in the first analysis execution step, creating an imaging image corresponding to the first region based on the mass spectrometry data corrected in the information correction step, and displaying both imaging images side-by-side or superimposed.

5. The analysis method using imaging mass spectrometry according to claim 1, wherein in the information correction step, when it is determined in the determination step that there is an influence of a matrix-related component, a process of subtracting an 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 mass spectrometry according to claim 1, further comprising a data integration step of creating a mass spectrum in which intensity information at the same mass-to-charge ratio is integrated, using the mass spectrometry data obtained in the first analysis execution step and the mass spectrometry data obtained in the second analysis execution step for substantially the same position on the sample.

7. The analysis method using imaging mass spectrometry according to claim 6, further comprising an analysis processing step of executing a multivariate analysis process on the mass spectrum data integrated in the data integration step.

8. The analysis method using imaging mass spectrometry according to claim 1, wherein the range where the matrix exists is a region where only the matrix is applied.

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