Imaging quality analysis system and analysis method using imaging quality analysis

Abstract

The imaging mass analysis system of the present invention comprises: an imaging mass analysis unit (100) for collecting data by performing mass analysis on a plurality of micro-areas within a measurement area set on a target sample, and obtaining an image showing a distribution of signal intensity based on the data; a quantitative analysis unit (300) for performing analysis on the target sample or a similar sample by a prescribed analysis method showing higher quantitativeness than the mass analysis performed by the imaging mass analysis unit, and obtaining a quantitative value using the analysis result; and a processing unit (400) for obtaining a relationship between the signal intensity and the quantitative value based on the quantitative value and the signal intensity, and using the relationship to estimate a quantitative value at an arbitrary position within the signal intensity distribution. Thus, it is possible to reduce complicated and labor-intensive operations and obtain a high-precision concentration image corresponding to an MS imaging image.

Description

Technical Field

[0001] The invention relates to an imaging mass analysis system and an analysis method using imaging mass analysis. Background Art

[0002] In the imaging mass spectrometers described in Patent Document 1, Non-Patent Document 1, etc., the two-dimensional intensity distribution of ions with a specific mass-to-charge ratio m / z in the surface of a sample such as a biological tissue slice can be measured while observing the surface morphology through an optical microscope. By plotting the intensity distribution by specifying the mass-to-charge ratio of characteristic ions for a specific compound, an image showing the distribution of the specific compound in the sample can be obtained (hereinafter sometimes referred to as a "mass analysis imaging image" or "MS imaging image"). In such a mass spectrometer, generally speaking, a matrix-assisted laser desorption / ionization (hereinafter referred to as "MALDI") method is used as the ionization method.

[0003] Usually, the MS imaging image obtained by the imaging mass spectrometer is a signal intensity (ion intensity) distribution image, but depending on the purpose and use of the analysis, it is sometimes necessary to obtain the concentration (amount of existence) of the substance of interest to the user at a specific position on the sample or the two-dimensional distribution of the concentration. In an imaging mass spectrometer using the MALDI method, even if the concentration of a certain substance is the same, the obtained signal intensity often varies greatly depending on the state of the sample and the state of the device. In order to reduce the deviation of the quantitative value that depends on the state of the device, the following method is usually used: while measuring the target slice sample as the analysis object, a standard sample prepared by mixing a standard substance containing a target substance with a known concentration with a matrix is ​​also measured, and the signal intensity obtained for the target slice sample is converted into a concentration using the measurement result of the standard sample (hereinafter referred to as the "In-solution method").

[0004] The sample preparation method of the standard sample in the above-mentioned In-solution method is different from that of the target slice sample. Therefore, the sample state such as the mixing state of the standard and the matrix is ​​different in the standard sample and the target slice sample, and the influence of the deviation of the quantitative value depending on the sample state remains. Therefore, in order to eliminate this influence, there is also the following method: the standard is placed on a simulated slice sample that does not contain the target substance similar to the target slice sample, and the matrix is ​​attached in the same way as the target slice sample to prepare the standard sample (hereinafter referred to as the "On-tissue method"). Furthermore, there is also the following method: after temporarily crushing the simulated slice sample and adding the standard, a simulated thin sheet sample containing a target substance with a known concentration is prepared by molding it into the same shape as the slice sample (hereinafter referred to as the "In-tissue method").

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2018 / 037491

[0008] Patent Document 2: International Publication No. 2015 / 053039

[0009] Patent Document 3: International Publication No. 2019 / 186999

[0010] Patent Document 4: International Publication No. 2019 / 229897

[0011] Non-patent literature

[0012] Non-patent document 1: Axel Walch et al., “MALDI imaging mass spectrometry for direct tissue analysis: a new frontier in molecular histology”, Journal of Histochemistry and Cell Biology, Vol. 130, Article No. 421, 2008 (online, retrieved on March 16, 2020, website<URL:https: / / link.springer.com / article / 10.1007 / s00418-008-0469-9> ) Summary of the invention

[0013] Technical problem to be solved by the invention

[0014] In the above-mentioned On-tissue method, although the reduction in quantification caused by the state of the sample can be reduced, since the target substance is only placed on the simulated slice sample, the extraction efficiency of the ions generated when the laser is irradiated is different from that of the target slice sample. Therefore, compared with the In-solution method, the quantification is improved, but it is difficult to ensure a high quantification. On the other hand, in the In-tissue method, since the extraction efficiency of the ions generated when the laser is irradiated on the target slice sample and the simulated thin-sheet sample containing the standard is basically the same, the quantification is further improved compared with the On-tissue method. However, in the In-tissue method, the work of making the simulated thin-sheet sample containing the standard is quite cumbersome and complicated, and this operation is almost manual and therefore inefficient.

[0015] The present invention is completed to solve such technical problems, and its purpose is to provide an imaging mass analysis system and an analysis method using imaging mass analysis, which can obtain high-precision quantitative results in specified areas in MS imaging images while minimizing tedious, complex and labor-intensive operations, or obtain an image showing high-precision concentration (existence) distribution corresponding to the entire MS imaging image or a part of it.

[0016] Solutions for solving the above technical problems

[0017] In order to solve the above-mentioned technical problems, an imaging mass analysis system of the present invention is provided, which comprises:

[0018] An imaging mass analysis unit collects data by performing mass analysis on a plurality of micro-areas within a measurement area set on a target sample, and acquires an image showing a distribution of signal intensity with respect to a specific mass-to-charge ratio or a range of mass-to-charge ratios based on the data;

[0019] a quantitative analysis unit for performing analysis by a predetermined analysis method showing higher quantitativeness than the mass analysis performed by the imaging mass analysis unit on a sample collected as described below, wherein the sample is a sample collected from a predetermined portion within the measurement area or a substantially corresponding measurement area for the target sample or a similar sample different from the target sample but having a substance distribution considered to be substantially the same as that of the target sample, and obtaining a quantitative value using the analysis result;

[0020] The processing unit calculates the relationship between the signal intensity obtained by the imaging mass analysis unit and the quantitative value obtained by the quantitative analysis unit based on the quantitative value of the sample relative to the specified part obtained in the quantitative analysis unit and the signal intensity at the position corresponding to the specified part in the signal intensity distribution obtained by the imaging mass analysis unit, and uses the relationship to infer the quantitative value of any position in the signal intensity distribution.

[0021] In order to solve the above technical problems, a solution of an analysis method using the imaging mass analysis system of the present invention is completed, which has the following features:

[0022] A first analysis execution step of performing imaging quality analysis on a measurement area on a target sample to obtain an image showing a distribution of signal intensity with respect to a specific mass-to-charge ratio or a mass-to-charge ratio range;

[0023] a second analysis execution step of executing analysis by a predetermined analysis method showing higher quantitativeness than the analysis performed in the first analysis execution step on a sample collected as described below, wherein the sample is a sample collected from a predetermined portion within the measurement area or a substantially corresponding measurement area with respect to the target sample or a similar sample different from the target sample but having a substance distribution considered to be substantially the same as that of the target sample, and obtaining a quantitative value using the analysis result;

[0024] A processing step, based on the quantitative value of the sample at the fixed position obtained in the second analysis execution step and the signal intensity at the position corresponding to the specified position in the signal intensity distribution obtained in the first analysis execution step, calculates the relationship between the signal intensity obtained by the imaging mass analysis and the quantitative value obtained by the analysis in a specified analysis method, and uses the relationship to infer the quantitative value of any position in the signal intensity distribution.

[0025] In the above scheme of the present invention, various methods commonly used for quantitative analysis can be used as the above-mentioned "prescribed analysis method". For example, any one of liquid chromatography, liquid chromatography mass analysis, gas chromatography, gas chromatography mass analysis, Raman spectrometry, infrared spectrometry, fluorescence analysis, staining quantitative analysis, etc. can be used. In addition, in MALDI mass analysis, instead of using the sample preparation method of spraying or coating the matrix solution on the sample surface commonly used in imaging mass analysis, each sample is prepared in the sample plate hole according to a very common sample preparation method such as the dried-droplet method, and the sample is irradiated with laser for mass analysis. Such a mass analysis method has higher quantitativeness than imaging mass analysis. Therefore, MALDI mass analysis performed by this method can also be used as the above-mentioned prescribed analysis method.

[0026] In the above scheme of the present invention, "a similar sample that is different from the target sample but in which the distribution of substances is considered to be substantially the same as that of the target sample" refers to, for example, other slice samples that are adjacent or close to the cut slice sample in the thickness direction when a biological tissue is cut into a thinner slice and the slice sample is used as a sample.

[0027] Effects of the Invention

[0028] According to the above scheme of the present invention, compared with quantitative analysis performed by the In-tissue method, etc., it is possible to obtain a highly accurate quantitative result of a specific substance in a specified part of an MS imaging image while reducing cumbersome, complicated and labor-intensive operations. In addition, it is also possible to obtain an image showing a highly accurate concentration (existence amount) distribution of a specified substance corresponding to the entire MS imaging image corresponding to the measurement area on the target sample or a part of the image. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic block diagram of an imaging mass analysis system according to one embodiment of the present invention.

[0030] Figure 2 This is a diagram showing the configuration of the main parts of the imaging mass spectrometer included in the system of this embodiment.

[0031] Figure 3 This is a diagram showing the configuration of the main parts of a liquid chromatograph-mass spectrometer included in the system of this embodiment.

[0032] Figure 4 This is a diagram showing the configuration of the main parts of the laser microdissection device included in the system of this embodiment.

[0033] Figure 5 is used for Figure 4 The schematic cross-sectional view shown is for explaining the procedure of sample collection by the hot melt LMD method using a laser microdissection apparatus.

[0034] Figure 6 This is a perspective view for explaining sample collection and sample pretreatment in a laser microdissection device.

[0035] Figure 7 This is a flowchart showing an example of a processing procedure for acquiring a density image in the system of this embodiment.

[0036] Figure 8 This is a flowchart showing another example of the processing procedure for acquiring a density image in the system of this embodiment.

[0037] Fig. 9 1 is a diagram showing the relationship between a small area on an MS imaging image and a collection site of the LMD device in the system of this embodiment.

[0038] Fig.10 It is a figure which shows the specific example of the MS imaging image and the sample collection site for quantitative analysis.

[0039] Fig.11This is a diagram showing an example of processing when images of the same tissue on images of slice samples that are continuous in the thickness direction are superimposed. DETAILED DESCRIPTION

[0040] Hereinafter, an imaging mass analysis system according to an embodiment of the present invention will be described with reference to the drawings.

[0041] [System Configuration of the Present Embodiment]

[0042] Figure 1 : is a schematic block diagram of the imaging mass analysis system of this embodiment. Figure 2 This is a diagram showing the main components of the imaging mass analysis device included in this system. Figure 3 This is a diagram showing the main components of the liquid chromatography mass spectrometer included in this system. Figure 4 This is a diagram showing the main components of the laser microdissection device included in this system.

[0043] like Figure 1 As shown, the imaging mass analysis system of the present embodiment includes an imaging mass analysis device 100, a laser microdissection device (hereinafter sometimes referred to as "LMD device") 200, a liquid chromatography mass analysis device (hereinafter sometimes referred to as "LC-MS device") 300, a data processing unit 400, a main control unit 500, an input unit 600, and a display unit 700. The data processing unit 400 includes a display processing unit 401, an image deformation processing unit 402, a collection site determination unit 403, a concentration conversion information generation unit 404, and a concentration image generation unit 405 as functional blocks in addition to the functional blocks described later.

[0044] like Figure 2 As shown, the imaging quality analysis device 100 includes: a measuring unit 110, an analysis control unit 140, a data processing unit 400, a main control unit 500, an input unit 600, and a display unit 700. The data processing unit 400, the main control unit 500, the input unit 600, and the display unit 700 are respectively connected to Figure 1 The data processing unit 400, main control unit 500, input unit 600 and display unit 700 shown are shared.

[0045] The measurement unit 110 is an atmospheric pressure MALDI ion trap time-of-flight mass spectrometer, and includes an ionization chamber 120 whose interior is an atmosphere close to atmospheric pressure, and a vacuum chamber 130 which is evacuated by a vacuum pump (not shown).

[0046] The interior of the ionization chamber 120 is configured with: a sample stage 121 that can slide freely in the two-axis directions of X and Y in the figure, a laser irradiation unit 123 that irradiates a sample 122 mounted on the sample stage 121 with a laser to ionize a substance (compound) in the sample 122, and a microscopic photography unit 124 that obtains an optical microscopic image of the sample 122 on the sample stage 121' moved to a specified position.

[0047] The interior of the ionization chamber 120 and the interior of the vacuum chamber 130 are connected through a capillary 131. The interior of the vacuum chamber 130 is provided with an ion guide 132, an ion trap 133, a time-of-flight mass separator 134, and an ion detector 135. In this example, the ion trap 133 is a three-dimensional quadrupole type, and the time-of-flight mass separator 134 is a reflection type, but it is not limited thereto. In addition, it is obvious that the interior of the vacuum chamber 130 is divided into a plurality of sub-chambers, and a multi-stage differential exhaust system for increasing the vacuum degree can be adopted for each sub-chamber.

[0048] The data processing unit 400 includes a data storage unit 410 , an imaging image generation unit 411 , and an optical microscopic image generation unit 412 as functional blocks specific to the imaging mass analysis device 100 .

[0049] like Figure 3 As shown in FIG. 1 , the LC-MS device 300 includes: a liquid chromatography unit 310, a mass analysis unit 320, an analysis control unit 330, a data processing unit 400, a main control unit 500, an input unit 600, and a display unit 700. The data processing unit 400, the main control unit 500, the input unit 600, and the display unit 700 are respectively connected to Figure 1 and Figure 2 The data processing unit 400, main control unit 500, input unit 600 and display unit 700 shown are shared.

[0050] The liquid chromatography unit 310 includes: a mobile phase container 311, a liquid delivery pump 312, an automatic sampler 313, a syringe 314, and a column 315. The mass spectrometer 320 is an electrospray ionization (ESI) ion trap time-of-flight mass spectrometer, which has an ionization chamber 321 and a vacuum chamber 130. The ionization chamber 321, which is maintained at an atmosphere close to atmospheric pressure, is provided with an ESI probe 322. On the other hand, the structure inside the vacuum chamber 130 is similar to that of the conventional mass spectrometer 320. Figure 2 The configuration of the measuring unit 110 shown is exactly the same.

[0051] That is, the vacuum chamber 130 is made common, and the measuring section 110 of the imaging mass spectrometer 100 can be formed by installing the ionization chamber 120 for imaging mass analysis in the vacuum chamber 130, and the mass analysis section 320 of the LC-MS device 300 can also be formed by installing the ionization chamber 321 for atmospheric pressure ionization in the vacuum chamber 130. Of course, the imaging mass spectrometer 100 and the LC-MS device 300 can also be set as completely independent devices.

[0052] The data processing unit 400 includes a data storage unit 420 , a chromatogram generation unit 421 , a calibration line storage unit 422 , and a quantitative calculation unit 423 as functional blocks specific to the LC-MS apparatus 300 .

[0053] The LMD device 200 uses a sample collection method called thermal melt laser microdissection (see Patent Document 2, etc.), and includes a microscopic imaging unit 201 , a sample collection unit 202 , a sample pre-processing unit 203 , and the like.

[0054] In addition, in the system of this embodiment, the analysis control unit 140 and 330, the data processing unit 400, and the main control unit 500 are usually composed of a personal computer or a higher-performance workstation as the center, and the above-mentioned functional blocks can be realized by executing the dedicated control and processing software installed on the computer. In this case, the input unit 600 is a keyboard and a pointing device (mouse, etc.) attached to the computer, and the display unit 700 is a display monitor.

[0055] [General Operation of Each Device in the System of the Present Embodiment]

[0056] Next, the operations of the imaging mass spectrometer 100 , the LMD device 200 , and the LC-MS device 300 will be briefly described.

[0057] The measurement object of the imaging mass analyzer 100 is, for example, a slice sample obtained by cutting a biological tissue such as the brain or internal organs of an experimental animal into thin slices. The sample (slice sample) 122 is placed on a sample plate and mounted on a sample stage 121. The microscopic imaging unit 124 acquires the image moving to Figure 2 The optical microscopic image of the sample 122 on the sample stage 121 at the position 121' shown by the dotted line in FIG. 1 is displayed on the screen of the display unit 700 by the optical microscopic image generation unit 412. The user indicates the measurement area to be analyzed for imaging quality on the optical microscopic image. The analysis control unit 140 receives the instruction and controls the measurement unit 110 to perform quality analysis on the plurality of measurement points in the indicated measurement area in sequence. In addition, the matrix is ​​applied to the surface of the sample 122 at an appropriate time point before the measurement.

[0058] The measurement of one measurement point is performed as follows. That is, when the sample stage 121 is in Figure 2 , the laser irradiation unit 123 irradiates a measurement point in the measurement area with a pulsed laser. When irradiated with the laser, a part of the compound in the sample 122 is vaporized and ionized. The generated ions are sucked into the capillary 131 along with the airflow formed by the differential pressure at both ends of the capillary 131, and are sent to the vacuum chamber 130. The ions originating from the sample 122 are temporarily captured in the ion trap 133 via the ion guide 132. The captured ions are ejected from the inside of the ion trap 133 at the same time at a predetermined timing and are introduced into the time-of-flight mass separator 134.

[0059] During the flight of the flight time type mass separator 134, various ions with different mass-to-charge ratios are separated in space according to the mass-to-charge ratio m / z, and arrive at the ion detector 135 with a time difference. The ion detector 135 always outputs a signal corresponding to the amount of ions that have arrived. The data storage unit 410 converts the received signal into digital data, and stores the flight time with the ion emission time point as the starting point after converting it into a mass-to-charge ratio. Thus, it is possible to obtain mass spectrum data within a specified mass-to-charge ratio range for a certain measurement point in the measurement area on the sample 122.

[0060] After the measurement is completed, the analysis control unit 140 immediately moves the sample stage 121 so that the next measurement point to be measured comes to the irradiation position of the laser of the laser irradiation unit 123. After the movement, the laser is irradiated to perform the mass analysis as described above. By sequentially performing this operation on multiple measurement points in the measurement area, that is, by repeatedly measuring while scanning the measurement points of the measurement object, mass spectrum data about all measurement points in the measurement area are obtained. In addition, the interval between adjacent measurement points is determined according to the desired spatial resolution.

[0061] When the user indicates the mass-to-charge ratio corresponding to the compound whose intensity distribution is to be confirmed from the input unit 600 at an appropriate time point, the imaging image generation unit 411 reads the signal intensity (ion intensity) of each measurement point in the indicated mass-to-charge ratio from the data storage unit 410, and generates an MS imaging image showing the two-dimensional distribution of the signal intensity. Then, the image is displayed on the screen of the display unit 700 through the main control unit 500. Thus, the MS imaging image reflecting the ion intensity distribution of the specified compound in the measurement area on the sample 122 can be provided to the user.

[0062] In addition, in the imaging mass spectrometer 100, for the ions captured in the ion trap 133, MS analysis can also be performed by selecting ions with a specific mass-to-charge ratio once or multiple times and performing collision-induced dissociation operations on the selected ions. nAnalysis (where n is an integer greater than or equal to 2). That is, it is also possible to generate and display an MS imaging image showing the ion intensity distribution of product ions derived from a predetermined compound.

[0063] The LMD device 200 collects a very small part of a given slice sample and prepares a sample solution containing the compound in the collected sample. As described above, the hot melt LMD method, which is one of the LMD methods, is used for sample collection. Figure 5 This is a schematic cross-sectional view for explaining the operation sequence of sample collection according to the hot melt LMD method. Figure 6 This is a perspective view for explaining sample collection and sample preprocessing in the LMD device 200 .

[0064] The user prepares a sample holding slide glass 10 having a slice sample 11, which is the object of LC / MS analysis (quantitative analysis), attached to one surface of the sample holding slide glass 10, and a sample collecting slide glass 12 having a thermosoluble film 13 attached to one surface of a sample collecting slide glass 12 different from the sample holding slide glass 10, and sets them at predetermined positions of the sample collecting section 202 (see Figure 5 (A)) The sample collecting section 202 holds the two slide glasses 10 and 12 in a state where the surface of the heat-soluble film 13 is in close contact with the slice sample 11 (see Figure 5 (B)). In this state, the surface of the sample collection slide glass 12 opposite to the surface on which the heat-soluble film 13 is attached is irradiated with a near-infrared laser 14 for a short time in a manner substantially perpendicular to the surface (see Figure 5 (C) The range of the irradiation with the laser beam 14 corresponds to the range of the portion to be analyzed by LC / MS on the slice sample 11 .

[0065] The irradiated laser light 14 passes through the sample collection slide glass 12 and heats the heat-soluble film 13. As a result, the heat-soluble film 13 near the range irradiated by the laser light 14 is dissolved and penetrates into the tissue of the slice sample 11. Then, the sample collection unit 202 separates the two slide glasses 10 and 12 to separate the heat-soluble film 13 from the slice sample 11. As a result, a portion 15 of the slice sample 11 is collected in a state where it adheres to the surface of the heat-soluble film 13 (see Figure 5 (D)).

[0066] The sample collecting unit 202 moves the sliding glasses 10 and 12 closer to each other toward the surface and repeats the same operation. Figure 6As shown, the sample pieces 15 near the plurality of measurement points 11b in the predetermined two-dimensional region 11a on the sliced ​​sample 11 are collected respectively on the thermosoluble film 13. At this time, although the intervals between the measurement points 11b on the sliced ​​sample 11 are narrow in accordance with the spatial resolution in the imaging mass analysis, the intervals between the sample pieces 15 on the thermosoluble film 13 can be expanded to, for example, several mm.

[0067] Then, the sample pre-processing unit 203 receives the sample collection slide glass 12 on which the sample pieces 15 are collected from the sample collection unit 202, and prepares a sample solution from each sample piece 15 collected on the thermosoluble film 13. Specifically, Figure 6 As shown, a microtiter plate (MTP) 16 having a plurality of wells 16a is used, and a predetermined extracting solution for extracting components in a sample piece 15 is preliminarily injected into each well 16a of the MTP 16. The slide glass 12 for sample collection is attached to the upper surface (opening surface) of the MTP 16 in such a manner that the sample piece 15 on the heat-soluble film 13 is located inside each well 16a. In this state, for example, by turning the MTP upside down as a whole, the sample piece 15 is immersed in the extracting solution in each well 16a, and a sample solution in which the components in the sample piece 15 are dissolved is prepared.

[0068] The microscopic imaging unit 201 acquires an optical microscopic image of the slice sample as the sample collection target. This optical microscopic image is used to correct the difference in shape between the sample as the imaging mass analysis target and the sample collection target for LC / MS, etc., as described below.

[0069] In addition, the method of collecting the sample in the LMD apparatus 200 is not limited to the hot melt LMD method, and a general LMD method, that is, a method of cutting out a part of the sample using a laser, may also be used.

[0070] The autosampler 313 of the LC-MS device 300 is loaded with the plurality of sample solutions prepared in the LMD device 200 as described above. The LC-MS device 300 sequentially performs LC / MS analysis on the plurality of sample solutions.

[0071] That is, the liquid delivery pump 312 draws the mobile phase from the mobile phase container 311 and delivers it to the column 315 at a substantially constant flow rate. Under the control of the analysis control unit 330, the syringe 314 injects a sample solution selected in the automatic sampler 313 into the mobile phase at a predetermined timing. The injected sample solution is introduced into the column 315 along with the flow of the mobile phase, and while passing through the column 315, the components in the sample solution are separated in the time direction and eluted from the column 315.

[0072] The eluent from the column 315 is introduced into the ESI probe 322 and is electrostatically sprayed from the ESI probe 322 into the ionization chamber 321. As a result, the sample components contained in the eluent are ionized. The generated ions are sucked into the capillary 131 along with the airflow formed by the differential pressure at both ends of the capillary 131, and are transported into the vacuum chamber 130. Then, similarly to the imaging mass spectrometer 100, the ions derived from the sample components are temporarily captured inside the ion trap 133 and then introduced into the time-of-flight mass separator 134 for mass analysis. The accumulation of ions in the ion trap 133 and the mass analysis in the time-of-flight mass separator 134 and the ion detector 135 are repeated.

[0073] The ion detector 135 outputs a signal corresponding to the amount of ions that have arrived. The data storage unit 420 converts the received signal into digital data, converts the flight time starting from the ion ejection time point into a mass-to-charge ratio, and stores it. Therefore, as time passes from the sample injection time point of the injector 314, mass spectrum data within a specified mass-to-charge ratio range can be obtained successively. The mass-to-charge ratio corresponding to the compound to be quantified is pre-set, and when the LC / MS analysis of a sample solution is completed, the chromatogram generation unit 421 generates an extracted ion chromatogram (also commonly referred to as a mass chromatogram) based on the signal intensity in the mass-to-charge ratio of the quantification object. In the extracted ion chromatogram, peaks originating from the compound to be quantified appear.

[0074] The quantitative calculation unit 423 calculates the area of ​​the above-mentioned peak observed in the extracted ion chromatogram, and converts the peak area into a concentration with reference to the calibration line pre-stored in the calibration line storage unit 422. The calibration line is, for example, a calibration line obtained in advance by measuring a standard sample of a quantitative target compound with a known concentration using the LC-MS device 300. In this way, in the LC-MS device 300, based on the LC / MS analysis results of the prepared sample solution, the concentration values ​​can be obtained as quantitative values. Generally, in LC / MS analysis, since the influence of inclusions can be reduced by chromatography and ionization can be stably performed, the quantitative accuracy is much higher than that of the imaging mass spectrometer 100.

[0075] [Description of Characteristic Operations of the System of the Present Embodiment]

[0076] Next, in addition to the above figures, refer to Figure 7 , Fig. 9 An example of characteristic operation in the system of this embodiment will be described. Figure 7 This is a flowchart showing an example of a processing procedure for acquiring a density image in this system. Fig. 9 3 is a diagram showing the relationship between a small area on an MS imaging image and a collection site in an LMD device in the present system.

[0077] The user sets the target slice sample 122 in the imaging mass spectrometer 100, indicates the measurement area on the optical microscope image relative to the slice sample 122, and then indicates the execution of the analysis. After receiving the instruction, in the imaging mass spectrometer 100, as described above, mass analysis is performed on each of the plurality of measurement points in the measurement area (step S10). When the user indicates from the input unit 600 that he wants to confirm the mass-to-charge ratio of the two-dimensional intensity distribution, the imaging image generation unit 411 generates an MS imaging image showing the distribution of the signal intensity in the indicated mass-to-charge ratio based on the mass analysis result. The display processing unit 401 displays the generated MS imaging image on the screen of the display unit 700 through the main control unit 500 (step S11).

[0078] If an MS imaging image is generated, the acquisition site determination unit 403 determines one or more small areas with roughly the same signal intensity on the MS imaging image for each of the multiple different signal intensities. The small area is the area of ​​the quantitative analysis object (step S12). Fig. 9 In the example shown in (A), a total of three small areas are set for different signal intensities. The size and shape of the small area can be appropriately determined, and the sizes of multiple small areas can also be inconsistent. In addition, the small area can be determined not automatically based on the intensity distribution on the MS imaging image, but by the user visually confirming the intensity distribution, the range with roughly the same signal intensity is determined as a small area.

[0079] The user takes out the target slice sample from the imaging mass spectrometer 100 and sets it at a predetermined position of the LMD device 200. The microscopic imaging unit 201 acquires an optical microscopic image of the set slice sample. The acquisition position determination unit 403 that receives the image associates the optical microscopic image with the optical microscopic image and the MS imaging image acquired in the imaging mass spectrometer 100, thereby identifying a range corresponding to the previously determined small area in the slice sample set in the LMD device 200. In addition, within the range, the position of the sample acquisition unit 202 is determined to be acquired (step S13).

[0080] As a method for determining the sample collection position, any of the following methods can be used: Fig. 9 (C) As shown on the right, a method for setting the sample collection site in a manner that avoids the measurement point of the imaging mass analysis; Fig. 9 (C) The left side shows a method of setting the sample collection site in a manner that includes the measurement point of the imaging mass analysis and ensures the largest possible area. In the former, the sample collection site is determined in a manner that does not overlap with the adjacent measurement points. In the latter, the sample collection site of a predetermined diameter is determined in a manner that does not overlap with the adjacent sample collection site with the measurement point as the center.

[0081] Fig.10 The actual MS imaging image (A) and an example of the sample collection position set for the area with different signal intensities on the image (B). Fig.10 The area surrounded by a rectangular frame in (B) is a small area, and a plurality of sample collection sites are determined in one small area.

[0082] When the sample collection sites are determined, the sample collection unit 202 collects sample pieces from the sample collection sites on the sliced ​​sample as described above. Then, the sample preprocessing unit 203 prepares sample solutions for LC / MS analysis corresponding to the collected sample pieces (step S14).

[0083] As described above, the LC-MS device 300 performs LC / MS analysis on each sample solution (step S15). Then, the chromatogram generation unit 421 and the quantitative operation unit 423 obtain the concentration of the target compound in each sample solution based on the analysis results. Further, the concentration conversion information generation unit 404 calculates the average value of multiple concentration values ​​corresponding to small areas of the same signal intensity on the MS imaging image, and obtains the concentration value (average value) relative to a certain signal intensity on the MS imaging image (step S16).

[0084] Next, the concentration conversion information generating unit 404 calculates concentration conversion information showing the relationship between the signal intensity of the target compound in the imaging mass analysis and the concentration value based on the LC / MS analysis result based on the concentration values ​​(average values) respectively corresponding to the plurality of small areas of different signal intensities on the MS imaging image (step S17). The concentration conversion information is a calibration curve.

[0085] The concentration image generation unit 405 generates a concentration image by converting the signal intensities of the entire MS imaging image into concentration values ​​using the above-mentioned concentration conversion information. The display processing unit 401 displays the concentration image on the screen of the display unit 700 through the main control unit 500 (step S18). Thus, a high-precision concentration image corresponding to the MS imaging image for a specific compound obtained by the imaging mass spectrometer 100 can be provided to the user.

[0086] Of course, if the user wants to observe only a concentration image of a part of the measurement area rather than the concentration image of the entire measurement area, the user specifies the area to be observed on the MS imaging image or the optical microscope image, and the concentration image generating unit 405 generates and displays a concentration image corresponding only to the specified area. In addition, if the user wants to know the correct concentration value of a specific part on the MS imaging image more accurately, the concentration image generating unit 405 calculates and displays the concentration value corresponding to the position indicated by the user.

[0087] [Other Examples of Characteristic Operations in the System of the Present Embodiment]

[0088] In the above example, the slice sample used in mass spectrometry imaging is also used in LC / MS analysis. However, when the spatial resolution is improved in imaging mass spectrometry or the laser power of the irradiated laser is increased, a sufficient amount of the target compound may not be obtained from the slice sample after the imaging mass spectrometry. In this case, as the slice sample for collecting the sample for LC / MS analysis, instead of the target slice sample used in mass spectrometry imaging, a slice sample adjacent to or close to the target slice sample in the thickness direction when the biological tissue is sliced ​​(hereinafter referred to as a "continuous slice sample") may be used.

[0089] In addition to the above figures, see Figure 8 An example of characteristic operation in the system of this embodiment in this case will be described. Figure 8 FIG. 4 is a flowchart showing an example of a processing procedure for acquiring a density image in this case. Figure 8 In the above, each process of steps S20 to S21, S25 to S29 is the same as that of the above-described Figure 7 The processes of steps S10 to S12 and S14 to S18 in FIG. 1 are substantially the same, and therefore, detailed description of these processes will be omitted.

[0090] In step S21, after the MS imaging image is generated, the microscopic imaging unit 201 of the LMD device 200 obtains an optical microscopic image of a continuous slice sample different from the target slice sample 122. Since the continuous slice sample is adjacent to or close to the target slice sample in the thickness direction in the original biological tissue, although the shape and material distribution of the tissue in the cross section of the two are very similar, they are not completely the same. For example, when the blood vessels in the biological tissue exist obliquely relative to the cross section, even in the continuous slice sample, the difference in the position of the blood vessels is obvious. Therefore, in order to reduce the influence of the difference in position and shape of the same part between the target slice sample and the continuous slice sample or the distortion of the shape, a technology called image registration is used as used in patent documents 3, 4, etc. That is, the following processing is performed: deforming either the MS imaging image of the target slice sample 122 or the optical microscopic image of the continuous slice sample, and identifying the position of the area corresponding to the small area set on the MS imaging image on the continuous slice sample. Fig.11 This is a diagram showing an example of applying image registration to two MS imaging images with different shapes so as to deform the images so that the shapes become consistent.

[0091] Specifically, the image deformation processing unit 402 uses image registration to match the optical microscopic image of the continuous section sample to deform the MS imaging image for the target section sample 122 (step S22). Afterwards, the collection site determination unit 403 determines one or more small areas with substantially the same signal intensity for multiple different signal intensities on the deformed MS imaging image (step S23). Then, on the continuous section sample, the sample collection site in the area corresponding to the above-mentioned small area is determined as the range of the actual sample cut out (step S24). If the sample collection site is determined in this way, the sample slice can be collected from the specified sample collection site using the LMD device 200 as described above to prepare the sample for LC / MS analysis.

[0092] In this case, on serial section specimens, e.g. Fig. 9 As shown in (C), the shape of the sample collection portion is circular, so there is an advantage that the sample can be easily cut out in the LMD device 200.

[0093] In addition, the following sequence may be adopted instead of the above sequence. That is, after determining the small area on the undeformed MS imaging image, the MS imaging image is deformed to match the optical microscope image of the continuous section sample. Since the shape and position of the small area also change with the deformation, on the continuous section sample, the sample collection site of the size and position corresponding to the deformed small area is determined as the range of the actual sample cut out. In this case, for example, Fig. 9 As shown in (A), even if the small area is set to be a rectangle on the MS imaging image, the shape of the small area after deformation is likely not to be a rectangle. Fig. 9 The shape of the sample collection portion shown in (C) is also likely not to be circular, so it is necessary to cut out (collect) a sample corresponding to such a special shape.

[0094] In addition, as described above, when a small area is determined on the deformed MS imaging image, an area corresponding to the small area may be shown on the MS imaging image before deformation, and the area may be set as a region of interest (ROI) for MS imaging data analysis. The region of interest may be used for the following purposes: for example, calculating the average of mass analysis obtained at all measurement points included in the region of interest to obtain an average mass spectrum or performing comparative analysis and difference analysis between different regions of interest.

[0095] As described above, according to the imaging mass analysis system of the present embodiment, the user can confirm not only the intensity distribution of ions originating from a specific substance but also the correct concentration of the substance at a specific position therein and the correct concentration distribution of the substance.

[0096] [Modification of the system of this embodiment]

[0097] In the system of the above embodiment, LC / MS analysis is used for quantitative analysis, but any analysis method other than LC / MS analysis can be used as long as it can obtain higher quantitative accuracy than general imaging mass analysis. For example, any of liquid chromatography analysis using a photodiode array detector and an ultraviolet-visible light detector as a detector, gas chromatography analysis, gas chromatography mass spectrometry, Raman spectrometry, infrared spectrometry, fluorescence analysis, and staining quantitative analysis can be used.

[0098] In addition, in a mass spectrometer using the MALDI method as an ionization method, a sample is repeatedly measured multiple times in a device that performs mass analysis on a sample prepared by mixing a sample and a matrix solution in advance, dropping the mixture into a well of a sample plate, and then drying and solidifying the mixture, and a mass spectrum is generated by integrating the data obtained from each measurement. Therefore, in such a mass spectrometer method, the quantitative performance can be improved compared to imaging mass spectrometry, and thus this mass spectrometer method can also be used for quantitative analysis.

[0099] Furthermore, the imaging mass spectrometer 100 is not limited to the MALDI method used as the ionization method, and may also use a laser dissociation ionization method, a surface-assisted laser dissociation ionization method, or the like.

[0100] In the system of the above embodiment, the transfer of the slice sample from the imaging mass spectrometer 100 to the LMD device 200 and the transfer of the sample solution from the LMD device 200 to the LC-MS device 300 may be performed automatically without human intervention.

[0101] The above-described embodiment and modified examples are merely examples of the present invention, and any appropriate changes, corrections, additions, etc. made within the scope of the gist of the present invention are naturally also included in the scope of the claims of the present invention.

[0102] [Various options]

[0103] Those skilled in the art may understand that the above exemplary embodiments are specific examples of the following schemes.

[0104] (Item 1) An embodiment of an imaging mass analysis system of the present invention comprises:

[0105] An imaging mass analysis unit collects data by performing mass analysis on a plurality of micro-areas within a measurement area set on a target sample, and acquires an image showing a distribution of signal intensity with respect to a specific mass-to-charge ratio or a range of mass-to-charge ratios based on the data;

[0106] a quantitative analysis unit for performing analysis by a predetermined analysis method showing higher quantitativeness than the mass analysis performed by the imaging mass analysis unit on a sample collected as described below, wherein the sample is a sample collected from a predetermined portion within the measurement area or a substantially corresponding measurement area for the target sample or a similar sample different from the target sample but having a substance distribution considered to be substantially the same as that of the target sample, and obtaining a quantitative value using the analysis result;

[0107] The processing unit calculates the relationship between the signal intensity obtained by the imaging mass analysis unit and the quantitative value obtained by the quantitative analysis unit based on the quantitative value of the sample relative to the specified part obtained in the quantitative analysis unit and the signal intensity at the position corresponding to the specified part in the signal intensity distribution obtained by the imaging mass analysis unit, and uses the relationship to infer the quantitative value of any position in the signal intensity distribution.

[0108] (Item 8) In addition, an analysis method using the imaging mass analysis system of the present invention comprises:

[0109] A first analysis execution step of performing imaging quality analysis on a measurement area on a target sample to obtain an image showing a distribution of signal intensity with respect to a specific mass-to-charge ratio or a mass-to-charge ratio range;

[0110] a second analysis execution step of executing analysis by a predetermined analysis method showing higher quantitativeness than the analysis performed in the first analysis execution step on a sample collected as described below, wherein the sample is a sample collected from a predetermined portion within the measurement area or a substantially corresponding measurement area with respect to the target sample or a similar sample different from the target sample but having a substance distribution considered to be substantially the same as that of the target sample, and obtaining a quantitative value using the analysis result;

[0111] A processing step, based on the quantitative value of the sample relative to the specified part obtained in the second analysis execution step and the signal intensity at the position corresponding to the specified part in the signal intensity distribution obtained in the first analysis execution step, calculates the relationship between the signal intensity obtained by the imaging mass analysis and the quantitative value obtained by the analysis in a specified analysis method, and uses the relationship to infer the quantitative value of any position in the signal intensity distribution.

[0112] According to the system described in item 1 and the analysis method described in item 8, compared with quantitative analysis performed by the in-tissue method, it is possible to obtain highly accurate quantitative results of a specific substance in a predetermined portion of an MS imaging image while reducing cumbersome, complicated, and labor-intensive operations. In addition, it is also possible to obtain an image showing a highly accurate concentration (existence amount) distribution of a predetermined substance corresponding to the entire MS imaging image corresponding to the measurement area on the target sample or a portion of the image.

[0113] In the system described in item 1 and the analysis method described in item 8, various methods generally used for quantitative analysis can be used as the predetermined analysis method.

[0114] (Item 2, Item 9) That is, in the system described in Item 1 or the analysis method described in Item 8, the specified analysis method can be any one of liquid chromatography analysis, gas chromatography analysis, liquid chromatography mass analysis, gas chromatography mass analysis, matrix-assisted laser desorption ionization mass analysis, Raman spectroscopy analysis, infrared spectroscopy analysis, ultraviolet-visible spectroscopy analysis, fluorescence analysis, or staining quantification.

[0115] (Item 3) The system described in Item 1 or Item 2 may further include a quantitative site determination unit, which determines the specified site of the sample to be collected as an analysis object of the quantitative analysis unit by using an image obtained by the imaging mass analysis unit showing the distribution of signal intensity in one or more specific mass-to-charge ratios.

[0116] (Item 10) In addition, in the analysis method described in Item 8 or Item 9, there can be further a quantitative site determination step, which uses the image showing the distribution of signal intensity in one or more specific mass-to-charge ratios obtained in the first analysis execution step to determine the specified site of the sample to be collected as the object of quantitative analysis in the second analysis execution step.

[0117] In the system described in item 3, the quantitative site determination unit determines a small area with substantially the same intensity as the specified site for each of a plurality of signal intensities different from each other on the mass analysis imaging image, for example, in a signal intensity distribution image at a specific mass-to-charge ratio. Thus, according to the imaging mass analysis system described in item 3 and the analysis method described in item 10, the relationship between the signal intensity obtained by the imaging mass analysis unit and the quantitative value obtained by the quantitative analysis unit can be obtained with high accuracy. Specifically, even when the relationship between the signal intensity and the quantitative value is nonlinear, the relationship can be accurately obtained and the concentration value and concentration distribution can be calculated with high accuracy.

[0118] (Item 4) In the system described in Item 3, the target sample is one of the slice samples cut out in the form of thin slices from the block sample, and the analysis object in the quantitative analysis unit is the similar sample, which can be set as other slice samples adjacent to or close to the target sample.

[0119] (Item 11) In addition, similarly, in the analysis method described in Item 10, the target sample is one of the slice samples cut out in the form of thin slices from the block sample, and the analysis object of the second analysis execution step is the similar sample, which can be set as other slice samples adjacent to or close to the target sample.

[0120] In the ionization method using laser such as MALDI method and LDI method, there is a case where sample components are depleted in the part irradiated with laser, and if the measurement points are densely arranged on the target sample in order to improve the spatial resolution of imaging mass analysis, it may not be possible to extract a sufficient amount of components from the target sample for quantitative analysis. In contrast, according to the system described in item 4 and the analysis method described in item 11, since quantitative analysis can be performed using a similar sample different from the target sample, it is easy to ensure a sufficient amount of sample components and improve quantitative accuracy.

[0121] (Item 5) In the system described in Item 4, there is further provided an image deformation processing unit, which performs image deformation based on image registration on the mass analysis imaging image or observation image of the target sample and the observation image before sample collection in the similar sample, and the quantitative site determination unit can use the deformed image to determine the specified site of the sample to be collected as the object of quantitative analysis.

[0122] (Item 6) More specifically, in the system described in Item 5, the image deformation processing unit deforms the mass analysis imaging image of the target sample to match it with an observation image of the similar sample before sample collection, and the quantitative site determination unit is able to associate a set area on the deformed mass analysis imaging image with an area on the observation image of the similar sample before sample collection, thereby determining the specified site of the sample to be collected as the object of quantitative analysis.

[0123] (Item 12) In addition, the analysis method described in Item 11 further comprises an image deformation step, in which image deformation based on image registration is performed on the mass analysis imaging image or observation image of the target sample and the observation image before sample collection in the similar sample, and in the quantitative site determination step, the deformed image can be used to determine the specified site of the sample to be collected as the object of quantitative analysis.

[0124] (Item 13) More specifically, in the analysis method described in Item 12, in the image deformation step, the mass analysis imaging image of the target sample is deformed to match the observation image of the similar sample before the sample is collected, and in the quantitative site determination step, the area set on the deformed mass analysis imaging image can be associated with the area on the observation image of the similar sample before the sample is collected, so as to determine the specified site of the sample to be collected as the object of quantitative analysis.

[0125] According to the systems described in items 5 and 6 and the analysis methods described in items 12 and 13, even when a similar sample different from the target sample is used for quantitative analysis, the effects of positional deviations and differences in size and shape of the same tissue can be reduced, thereby achieving high quantification.

[0126] (Item 7) In the system described in Item 5 or 6, it is possible to determine a region set on the mass analysis imaging image deformed by the image deformation processing unit as a region of interest when analyzing data collected by the imaging mass analysis unit.

[0127] (Item 14) In the analysis method described in Item 12 or Item 13, the area set on the deformed mass analysis imaging image in the image deformation step can be determined as the area of ​​interest when analyzing the data collected in the first analysis execution step.

[0128] According to the system described in item 7 and the analysis method described in item 14, the area on the target sample corresponding to the area for quantitative analysis can be analyzed in detail as the area of ​​interest for imaging mass analysis.

[0129] Description of Reference Numerals

[0130] 100 Imaging Quality Analysis Device

[0131] 110 Measurement Department

[0132] 120 Ionization Chamber

[0133] 121 Sample table

[0134] 122 Slice specimen

[0135] 123 Laser irradiation unit

[0136] 124 Microscopic Photography Department

[0137] 130 Vacuum Chamber

[0138] 131 Capillary

[0139] 132 Ion Guide

[0140] 133 Ion Trap

[0141] 134 Time-of-Flight Mass Separator

[0142] 135 Ion Detector

[0143] 140 Analysis and Control Department

[0144] 200 Laser Microdissection Device

[0145] 201 Microscope Photography Department

[0146] 202 Sample Collection Department

[0147] 203 Sample pre-treatment department

[0148] 300 Liquid Chromatography Mass Analyzer

[0149] 310 Liquid Chromatography Department

[0150] 311 Mobile phase container

[0151] 312 Liquid delivery pump

[0152] 313 Autosampler

[0153] 314 Syringe

[0154] 315 columns

[0155] 320 Quality Analysis Department

[0156] 321 Ionization Chamber

[0157] 322 ESI probe

[0158] 330 Analysis and Control Department

[0159] 400 Data Processing Department

[0160] 401 Display Processing Unit

[0161] 402 Image deformation processing unit

[0162] 403 Collection Site Determination Department

[0163] 404 Concentration conversion information generation unit

[0164] 405 Concentration image generation unit

[0165] 410 Data Storage Department

[0166] 411 Imaging Image Generation Unit

[0167] 412 Optical Microscopic Image Generation Unit

[0168] 420 Data Storage Department

[0169] 421 Chromatogram Generation Unit

[0170] 422 Calibration line storage unit

[0171] 423 Quantitative Calculation Department

[0172] 500 Main control unit

[0173] 600 Input

[0174] 700 Display unit.

Claims

1. An imaging quality analysis system, characterized in that: have: An imaging mass analysis unit collects data by performing mass analysis on a plurality of micro-areas within a measurement area set on a target sample, and acquires an image showing a distribution of signal intensity with respect to a specific mass-to-charge ratio or a range of mass-to-charge ratios based on the data; a quantitative analysis unit for performing analysis by a predetermined analysis method showing higher quantitativeness than the mass analysis performed by the imaging mass analysis unit on a sample collected as described below, wherein the sample is a sample collected from a predetermined portion within the measurement area or a substantially corresponding measurement area for the target sample or a similar sample different from the target sample but having a substance distribution considered to be substantially the same as that of the target sample, and obtaining a quantitative value using the analysis result; The processing unit calculates the relationship between the signal intensity obtained by the imaging mass analysis unit and the quantitative value obtained by the quantitative analysis unit based on the quantitative value of the sample relative to the specified part obtained in the quantitative analysis unit and the signal intensity at the position corresponding to the specified part in the signal intensity distribution obtained by the imaging mass analysis unit, and uses the relationship to infer the quantitative value of any position in the signal intensity distribution.

2. The imaging quality analysis system according to claim 1, characterized in that: The specified analysis method is any one of liquid chromatography analysis, gas chromatography analysis, liquid chromatography mass analysis, gas chromatography mass analysis, matrix-assisted laser desorption ionization mass analysis, Raman spectroscopy analysis, infrared spectroscopy analysis, ultraviolet-visible spectroscopy analysis, fluorescence analysis or staining quantification.

3. The imaging quality analysis system according to claim 1 or 2, characterized in that: The method further comprises a quantitative analysis unit for determining the predetermined site for sampling the sample to be analyzed by the quantitative analysis unit, using the mass analysis imaging image obtained by the imaging mass analysis unit and showing the distribution of signal intensity at one or more specific mass-to-charge ratios.

4. The imaging quality analysis system according to claim 3, characterized in that: The target sample is one of the slice samples cut out in a thin sheet form from the bulk sample, and the analysis object in the quantitative analysis unit is the similar sample, which is another slice sample adjacent to or close to the target sample.

5. The imaging quality analysis system according to claim 4, characterized in that: It further includes an image deformation processing unit, which performs image deformation based on image registration on the mass analysis imaging image or observation image of the target sample and the observation image before sample collection in the similar sample, and the quantitative site determination unit uses the deformed image to determine the specified site of the sample to be collected as the object of quantitative analysis.

6. The imaging quality analysis system according to claim 5, characterized in that: The image deformation processing unit deforms the mass analysis imaging image of the target sample to match it with the observation image of the similar sample before the sample is collected, and the quantitative site determination unit associates the area set on the deformed mass analysis imaging image with the area on the observation image of the similar sample before the sample is collected, thereby determining the specified site for collecting the sample as the object of quantitative analysis.

7. The imaging quality analysis system according to claim 5 or 6, characterized in that: A region set on the mass analysis imaging image deformed by the image deformation processing unit is determined as a region of interest when analyzing data collected by the imaging mass analysis unit.

8. An analysis method using imaging quality analysis, characterized in that: have: A first analysis execution step of performing imaging quality analysis on a measurement area on a target sample to obtain an image showing a distribution of signal intensity with respect to a specific mass-to-charge ratio or a mass-to-charge ratio range; a second analysis execution step of executing analysis by a predetermined analysis method showing higher quantitativeness than the analysis performed in the first analysis execution step on a sample collected as described below, wherein the sample is a sample collected from a predetermined portion within the measurement area or a substantially corresponding measurement area with respect to the target sample or a similar sample different from the target sample but having a substance distribution considered to be substantially the same as that of the target sample, and obtaining a quantitative value using the analysis result; A processing step, based on the quantitative value of the sample relative to the specified part obtained in the second analysis execution step and the signal intensity at the position corresponding to the specified part in the signal intensity distribution obtained in the first analysis execution step, obtains the relationship between the signal intensity obtained by the imaging mass analysis and the quantitative value obtained by the analysis in a specified analysis method, and uses the relationship to infer the quantitative value at any position in the signal intensity distribution.

9. The analysis method using imaging quality analysis according to claim 8, characterized in that: The specified analysis method is any one of liquid chromatography analysis, gas chromatography analysis, liquid chromatography mass analysis, gas chromatography mass analysis, matrix-assisted laser desorption ionization mass analysis, Raman spectroscopy analysis, infrared spectroscopy analysis, ultraviolet-visible spectroscopy analysis, fluorescence analysis or staining quantification.

10. The analysis method using imaging mass analysis according to claim 8 or 9, characterized in that: There is further a quantitative site determination step for determining the specified site of the sample to be collected as the object of quantitative analysis in the second analysis execution step, using the image showing the distribution of signal intensity in one or more specific mass-to-charge ratios obtained in the first analysis execution step.

Images