WO2019186965A1 - Procédé et programme de traitement de données dans une analyse de masse par imagerie - Google Patents

Procédé et programme de traitement de données dans une analyse de masse par imagerie Download PDF

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WO2019186965A1
WO2019186965A1 PCT/JP2018/013453 JP2018013453W WO2019186965A1 WO 2019186965 A1 WO2019186965 A1 WO 2019186965A1 JP 2018013453 W JP2018013453 W JP 2018013453W WO 2019186965 A1 WO2019186965 A1 WO 2019186965A1
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image
imaging
mass spectrometry
images
data processing
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PCT/JP2018/013453
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Japanese (ja)
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有里子 中木村
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株式会社島津製作所
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Priority to PCT/JP2018/013453 priority Critical patent/WO2019186965A1/fr
Priority to JP2020508761A priority patent/JP6927415B2/ja
Publication of WO2019186965A1 publication Critical patent/WO2019186965A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode

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  • the present invention relates to a method for processing data obtained by an imaging mass spectrometer capable of acquiring mass spectrum data for each of a large number of measurement points in a measurement region on a sample, and a method for performing the method on a computer.
  • the present invention relates to a data processing program for implementation.
  • mass spectrometry imaging In mass spectrometry imaging, the spatial distribution of a substance having a specific mass is obtained by performing mass analysis on each of a plurality of measurement points (micro-regions) in a two-dimensional measurement region on a sample such as a biological tissue section. It is a method to investigate, and its application to drug discovery, biomarker search, investigation of the cause of various diseases and diseases is being promoted.
  • a mass spectrometer for performing mass spectrometry imaging is generally called an imaging mass spectrometer (see Non-Patent Documents 1 and 2, etc.).
  • an imaging mass spectrometer obtains mass spectrum data (including MS n spectrum data where n is 2 or more) over a predetermined mass-to-charge ratio (m / z) range for each of a large number of measurement points on a sample. Then, when the user designates the m / z value of the ion derived from the compound to be observed, the ion intensity value at each measurement point at the designated m / z value is extracted, and the ion intensity value is extracted according to the gray scale or color scale.
  • a two-dimensional image (mass spectrometry imaging image, hereinafter referred to as “MS imaging image”) visualized and associated with the position of the measurement point is created and displayed on the screen of the display unit.
  • the imaging mass spectrometer uses a matrix-assisted laser desorption / ionization (MALDI) method to ionize components (compounds) in a sample.
  • MALDI matrix-assisted laser desorption / ionization
  • a matrix for MALDI is applied or sprayed on the surface of a sample such as a biological tissue section.
  • Various compounds are known as matrices, but the components to be ionized differ depending on the type of matrix.
  • Imaging mass spectrometry may be performed by applying different types of matrices to a plurality of section samples.
  • the above example is an example in which imaging mass spectrometry is performed for each of a plurality of samples collected from one biological tissue, specifically, for example, an organ such as a mouse or a human liver.
  • imaging mass spectrometry is performed on each sample cut from the same living tissue of a different individual.
  • the distribution of specific components can be compared, the relationship between the distributions of different components can be investigated, In order to grasp the three-dimensional distribution of components, it is necessary to superimpose a plurality of MS imaging images.
  • MS imaging image shows the intensity distribution of ions derived from components having a certain mass, that is, the distribution of abundance, and does not necessarily indicate the contour or boundary of a certain part or tissue structure. Since it is not limited, it has been difficult to accurately align MS imaging images.
  • a stained image obtained by staining the sample with, for example, HE (hematoxylin / eosin) staining is acquired, and the stained image and a specific MS imaging image are superimposed. Even in such a case, there is a problem similar to the case where the MS imaging images are superimposed.
  • the present invention has been made in view of the above problems, and the object of the present invention is to superimpose a plurality of MS imaging images obtained from different samples, or to superimpose stained images and the MS imaging images.
  • the data processing method which has been made to solve the above-mentioned problems, is an imaging mass spectrometry that processes mass spectrometry data respectively obtained at a plurality of measurement points in a measurement region on a sample.
  • Data processing method that performs processing to superimpose a plurality of mass spectrometry imaging images created based on mass spectrometry data obtained for a plurality of samples having the same or similar parts to be observed.
  • a method a) an image selection step for allowing the user to select a plurality of mass spectrometry imaging images to be subjected to superposition processing; b) an optical microscopic image acquisition step for acquiring optical microscopic images for a plurality of samples respectively corresponding to the selected plurality of mass spectrometry imaging images; c) an image alignment step for aligning the images so that the same or similar parts coincide with each other while deforming one or a plurality of images among the plurality of obtained optical microscopic images; d) an image deformation information acquisition step for acquiring image deformation information for one or more images at the time of image alignment; e) an image deformation step of performing deformation processing of at least one image by applying the image deformation information acquired in the image deformation information acquisition step to a plurality of mass spectrometry imaging images that are targets of the superposition processing; f) an image superposition processing step for performing superposition processing of the plurality of mass spectrometry imaging images at least one of which has been deformed in the image deformation step;
  • a data processing program for performing a process of superimposing a plurality of mass spectrometry imaging images created based on mass spectrometry data respectively obtained for a plurality of samples comprising: a) an image selection function unit that allows a user to select a plurality of mass spectrometry imaging images to be subjected to superposition processing; b) an optical microscopic image acquisition function unit for acquiring optical microscopic images for a plurality of samples respectively corresponding to the selected plurality of mass spectrometry imaging images; c) an image alignment function unit that aligns images so that the same or similar parts match while deforming one or a plurality of images among a plurality of acquired optical microscopic images; d) an image deformation information acquisition function unit for acquiring image deformation information about one or more images at the time of
  • an imaging mass spectrometer (imaging mass microscope) disclosed in Non-Patent Document 1
  • an imaging mass spectrometer and an optical microscope are integrated, and sample morphology observation and mass spectrometry are performed by a single device. Yes.
  • an apparatus it is possible to acquire an optical microscopic image and a mass spectrometry imaging image in which the positions of the same parts on the sample are aligned.
  • the imaging mass spectrometer and the optical microscope are not integrated, they are usually prepared separately from the imaging mass spectrometer to determine the measurement range of the object to be subjected to imaging mass analysis on the sample.
  • An optical microscope image on the sample is taken with an optical microscope. In this case, the position of the same part on the sample should be aligned in the optical microscopic image and the mass spectrometry imaging image corresponding to the measurement range.
  • the specific region or tissue structure on the sample appears relatively clearly in the optical microscopic image. Therefore, in the data processing method realized by the data processing program according to the first aspect of the present invention, when a plurality of mass spectrometry imaging images to be superimposed are selected by the user in the image selection step, In the optical microscopic image acquisition step, optical microscopic images of a plurality of samples respectively corresponding to the selected plurality of mass spectrometry imaging images are acquired.
  • the same or mutually similar parts (tissue structure, etc.) between the optical microscopic images of the plurality of samples from which the plurality of mass spectrometry imaging images to be subjected to the overlay process are obtained.
  • image deformation information acquisition step image deformation information for one or a plurality of images is acquired in the image deformation information acquisition step.
  • deformation includes both linear deformation and non-linear deformation such as movement, rotation, enlargement / reduction, and the like of an image.
  • the deformation information obtained for the optical microscopic image is obtained by a plurality of masses. It can be used as it is for alignment between analytical imaging images. Therefore, in the deformation information image deformation step, the deformation information is applied to a plurality of mass spectrometry imaging images to be superimposed to deform at least one of the images. As a result, regardless of the pattern of the original mass spectrometry imaging image, the positions of the plurality of mass spectrometry imaging images to be superimposed are substantially aligned. In the image superimposition processing step, a plurality of mass spectrometry imaging images in such a state are superimposed, thereby creating a superimposed image that can compare, for example, distributions of predetermined components.
  • the data processing method which has been made to solve the above problems, is an imaging mass spectrometry for processing mass spectrometry data respectively obtained at a plurality of measurement points in a measurement region on a sample.
  • a data processing method in which a mass spectrometric imaging image created based on mass spectrometric data obtained for one of a plurality of samples whose parts to be observed are the same or similar to each other;
  • Image alignment is performed between the acquired optical microscopic image and the reference image of the same sample so that one or a plurality of images are deformed and the same or similar parts are matched.
  • An image alignment step d) an image deformation information acquisition step for acquiring image deformation information for one or more images at the time of image alignment; e) an image deformation step of applying the acquired image deformation information to the mass spectrometry imaging image that is the object of the superimposition process to perform an image deformation process; f) an image superposition processing step for performing superposition processing of the mass spectrometry imaging image deformed in the image deformation step and the reference image; It is characterized by having.
  • the data processing program according to the second aspect of the present invention which has been made to solve the above-mentioned problems, processes on the computer the mass spectrometry data respectively obtained at a plurality of measurement points in the measurement region on the sample.
  • a data processing program for obtaining a mass spectrometry imaging image based on mass spectrometry data obtained for one of a plurality of samples and a measurement or observation technique other than mass spectrometry for the other one A data processing program for performing a process of superimposing a reference image on a computer, a) an image selection function unit that allows the user to select one mass spectrometry imaging image that is the target of the overlay process; b) an optical microscopic image acquisition function unit for acquiring an optical microscopic image corresponding to the selected mass spectrometry imaging image; c) Image alignment is performed between the acquired optical microscopic image and the reference image of the same sample so that one or a plurality of images are deformed and the same or similar parts are matched.
  • An image alignment function d) an image deformation information acquisition function unit for acquiring image deformation information about one or more images at the time of image alignment; e) an image deformation function unit that applies image deformation information acquired by the image deformation information acquisition function unit to a mass spectrometry imaging image that is an object of the superposition process, and performs image deformation processing; f) an image superimposition processing function unit that performs superposition processing of the mass spectrometry imaging image deformed by the image deformation function unit and the reference image; It is characterized by operating.
  • the reference image can be obtained by various methods as long as the shape such as the contour or pattern of the site such as the tissue structure on the sample can be observed to some extent clearly.
  • image deformation information is obtained from an optical microscopic image of one sample and a reference image of another sample. If the mass spectrometry imaging image corresponding to the optical microscopic image is deformed by using, the mass spectrometry imaging image and the reference image can be aligned.
  • a general optical microscopic image can be obtained together with the imaging mass spectrometer. . Therefore, images are aligned using the optical microscopic image obtained at the time of Raman microscopic imaging measurement or infrared microscopic imaging measurement and the optical microscopic image obtained at the time of mass spectrometry imaging.
  • the deformation information can be used for alignment between the mass spectrometry imaging image and the Raman microscopic imaging image or the infrared microscopic imaging image.
  • the data processing method is a data processing method in imaging mass spectrometry that processes mass spectrometry data respectively obtained at a plurality of measurement points in a measurement region on a sample, and is an observation target.
  • a mass spectrometry imaging image created based on mass spectrometry data obtained for one of a plurality of samples having the same or similar site, and an imaging measurement other than imaging mass spectrometry for the other Other than mass spectrometry imaging images and imaging mass spectrometry, which is performed by superimposing the second imaging image obtained by the technique, or created based on the mass spectrometry data obtained for the same observation target part of one sample
  • Data for processing to superimpose the second imaging image obtained by the imaging measurement method of A management method a) an image selection step that allows the user to select one mass spectrometry imaging image and a second imaging image that are to be superposed; b) an optical microscopic image acquisition step of acquiring optical microscopic images respectively corresponding to the selected mass spectrometry imaging image and
  • the data processing program is a data processing program for processing, on a computer, mass spectrometry data respectively obtained at a plurality of measurement points in a measurement region on a sample.
  • Mass spectrometric imaging image created based on mass spectrometric data obtained for one of multiple samples of the same or similar target site, and imaging other than imaging mass spectrometric analysis for the other A mass spectrometric imaging image and an imaging mass spectrometric process that are performed by superimposing the second imaging image obtained by the measurement technique or that are created based on the mass spectrometric data obtained for the same observation target part of one sample
  • the computer a) an image selection function unit that allows a user to select one mass spectrometry imaging image and a second imaging image that are to be superposed; b) an optical microscopic image acquisition function unit for acquiring optical microscopic images respectively corresponding to the selected mass spectrometry imaging image and the second imaging image
  • the second imaging image is an imaging image obtained by a technique other than mass spectrometry, such as a Raman microscopic imaging image or an infrared microscopic imaging image.
  • a technique other than mass spectrometry such as a Raman microscopic imaging image or an infrared microscopic imaging image.
  • an image obtained by accurately superimposing a mass spectrometry imaging image, a Raman microscopic imaging image, an infrared microscopic imaging image, and the like can be created. Can be provided to the user.
  • the plurality of samples from which a plurality of mass spectrometry imaging images to be superimposed can be obtained from different individuals. If the parts to be observed are not the same or similar to each other, it is meaningless to perform the overlay process substantially.
  • the plurality of samples may be slice samples continuously cut out from one living tissue of one individual.
  • a plurality of mass spectrometry imaging images respectively obtained from different samples, a superposition of a stained image and the like and a mass spectrometry imaging image Or, when superimposing a mass spectrometry imaging image with a Raman microscopic imaging image, an infrared microscopic imaging image, or the like, the same part or tissue structure on different samples or one sample is as much as possible in the same position or shape.
  • highly accurate positioning can be performed easily, that is, while omitting complicated manual work by the user. Thereby, the images can be efficiently superimposed, and the burden on the user for such work is reduced.
  • FIG. 6 is a flowchart for creating a characteristic superimposed image in the imaging mass spectrometer of the present embodiment.
  • the conceptual diagram for demonstrating the superimposition image creation process shown in FIG. The conceptual diagram of the measurement area
  • FIG. 1 is a schematic configuration diagram of an embodiment of an imaging mass spectrometer for carrying out a data processing method according to the present invention.
  • the imaging mass spectrometer of the present embodiment includes an imaging mass spectrometer 1, an optical microscope observation unit 2, a data processing unit 3, an operation unit 4, and a display unit 5.
  • the operation unit 4 and the display unit 5 are user interfaces.
  • the imaging mass spectrometer 1 includes, for example, a matrix-assisted laser desorption ionization ion trap time-of-flight mass spectrometer (MALDI-IT-TOFMS), and a two-dimensional measurement region on a sample 6 such as a biological tissue section.
  • the mass spectrum data of each of a large number of measurement points (small regions) can be acquired.
  • This mass spectrum data also includes MS n spectrum data in which n is 2 or more.
  • the optical microscope observation unit 2 acquires an optical microscope image in a range including at least a measurement region on the sample 6.
  • the data processing unit 3 receives the mass spectrum data at each measurement point collected by the imaging mass spectrometry unit 1 and the optical microscopic image data obtained by imaging by the optical microscopic observation unit 2, and performs a predetermined process.
  • Optical image data storage unit 30, MS imaging data storage unit 31, optical image creation unit 32, MS imaging image creation unit 33, image alignment processing unit 34, image deformation information storage unit 35, MS imaging image adjustment unit 36, image superposition Functional blocks such as an alignment processing unit 37 and an image display processing unit 38 are provided.
  • the substance of the data processing unit 3 is a personal computer (or a higher-performance workstation).
  • dedicated software that is, a computer program
  • the imaging mass spectrometer 1 and the optical microscope observation section 2 are integrated, that is, a sample set at a predetermined position of the apparatus.
  • 6 is an apparatus that moves between the measurement position by the imaging mass spectrometry unit 1 and the imaging position by the optical microscope observation unit 2 automatically or in response to a manual operation.
  • the imaging mass spectrometer 1 and the optical microscope observation unit 2 may not be integrated.
  • FIG. 5 different types of matrixes are applied to a plurality of slice samples continuously cut out from one living tissue, and each is subjected to imaging mass spectrometry.
  • MS imaging images are superimposed.
  • the size and shape of the biological tissue appearing in each sample are substantially the same.
  • the user sets a sample 6 placed on a dedicated plate and not coated with a matrix at a predetermined position of the apparatus, and performs a predetermined operation with the operation unit 4. Then, the optical microscope observation unit 2 takes an optical microscope image on the sample 6 and displays the image on the screen of the display unit 5. The user confirms this image, determines a measurement region to be subjected to imaging mass spectrometry on the sample 6, and designates the measurement region by setting a frame surrounding the measurement region on the screen by the operation unit 4, for example. Thereby, for example, as shown in FIG. 4, a measurement region 60 is set on a sample 6 derived from a living body such as a mouse liver slice. At this time, the optical microscopic image data obtained by photographing the sample 6 is stored in the optical image data storage unit 30 together with information for specifying the position of the measurement region 60.
  • the imaging mass spectrometer 1 performs mass analysis (or MS n analysis) on each of a large number of measurement points 61 within the range of the measurement region 60 set as described above. Acquire mass spectral data over the m / z range. As a result, a set of mass spectrum data corresponding to the number of measurement points 61 in the measurement region 60 (hereinafter referred to as “MS imaging data”) is obtained, and this data is transferred from the imaging mass analyzer 1 to the data processor 3. The data is input and stored in the MS imaging data storage unit 31.
  • optical microscopic image data and MS imaging data for one sample 6 are stored in association with the optical image data storage unit 30 and the MS imaging data storage unit 31. Then, as described above, measurement is similarly performed on the plurality of samples 6 cut out from one living tissue, and optical microscopic image data and MS imaging data are collected.
  • optical microscopic observation may be performed after applying the matrix on the sample 6, but in general, when the matrix is applied, the color or pattern of the tissue on the sample 6 becomes difficult to see. Therefore, generally, a clear optical microscopic image on the sample 6 is acquired prior to the matrix coating, and the measurement region 60 is determined using the image.
  • FIG. 2 is a flowchart showing the procedure of the data processing
  • FIG. 3 is a conceptual diagram for explaining the data processing.
  • the user designates a plurality of MS imaging images to be subjected to the overlay process by the operation unit 4 (step S1).
  • one MS imaging image can be designated by information (for example, serial numbers assigned to a plurality of samples) and m / values that specify the sample.
  • information for example, serial numbers assigned to a plurality of samples
  • m / values that specify the sample.
  • the optical image creating unit 32 is an optical microscopic image in the measurement region corresponding to each of the designated MS imaging images, that is, an optical microscopic image in the almost same measurement region of the sample A and the sample B.
  • the optical microscopic image data constituting the image is read out from the optical image data storage unit 30 (step S2).
  • the optical image creation unit 32 may create an optical microscopic image from the read optical microscopic image data and display it on the screen of the display unit 5.
  • the image alignment processing unit 34 executes alignment processing according to a predetermined algorithm so that the position, size, and shape of the same part are aligned between the two optical microscopic images.
  • a predetermined algorithm for example, an image registration technique widely used in the medical field can be used.
  • Image registration technology can be broadly divided into linear registration that performs alignment by translation, rotation, and enlargement / reduction, and two-dimensional lattice points on the image, and the lattice points can be moved freely.
  • Nonlinear registration for example, FFD (Free Form Deformation) method, etc.
  • the method of alignment processing is not limited to this.
  • image deformation information for translating, rotating, enlarging / reducing, non-linear deformation, etc. of an image when one image is used as a reference and the other image is matched with the reference image is provided. can get. Therefore, the image deformation information is stored in the image deformation information storage unit 35 as image deformation information for alignment between the sample A and the sample B (step S3).
  • the MS imaging image creation unit 33 reads out MS imaging data constituting the plurality of MS imaging images specified in Step S1 from the MS imaging data storage unit 31 (Step S4).
  • the MS imaging image adjustment unit 36 reads image deformation information for alignment between the sample A and the sample B from the image deformation information storage unit 35, and uses the image deformation information to read the plurality of MS imaging images. Image processing for deforming one of these is performed (step S5). That is, here, regardless of the pattern of the MS imaging image, the image is appropriately moved, rotated, enlarged / reduced, or nonlinearly deformed based on the given image deformation information.
  • step S3 If the accuracy of the alignment performed in step S3 is high and the positional deviation between the optical microscopic image and the MS imaging image is negligible in each sample, the same parts of the MS imaging images after image deformation are located at substantially the same position. , Become size and shape. That is, alignment of a plurality of MS imaging images is realized with high accuracy by image deformation.
  • the imaging mass spectrometer of the present embodiment performs high-precision alignment between the MS imaging image of the sample A at an arbitrary m / z value and the MS imaging image of the sample B at an arbitrary m / z value.
  • a superimposed image can be created.
  • the above embodiment is an overlay of two MS imaging images, but the same may be applied to an overlay of three or more MS imaging images. That is, image deformation information of another optical microscopic image based on one optical microscopic image may be obtained, and a plurality of MS imaging images may be processed using the image deformation information.
  • MS imaging derived from a continuous section sample cut from one living tissue is not an image, but MS imaging derived from a sample cut from the same living tissue of another individual (for example, another mouse). The same process can be applied when images are superimposed.
  • imaging mass spectrometry is performed on each of a large number of section samples continuously cut out from the living tissue, and as shown in FIG. 7, the three-dimensional MS imaging image at a specific m / z value is obtained.
  • a three-dimensional distribution image of a specific compound can be created.
  • the image deformation information is obtained by aligning the optical microscopic images in order to superimpose the MS imaging images, but obtained by a technique other than the MS imaging image and mass spectrometry.
  • a similar method can be used when superimposing other images.
  • FIG. 6 is a conceptual diagram for explaining processing in the case of superimposing one stained image and one MS imaging image.
  • a stained image is observed in such a manner that a specific tissue, a specific substance, or the like is stained and can be distinguished from others. However, the shape of other parts can be observed sufficiently clearly.
  • an apparatus capable of imaging measurement such as a Raman microscopic imaging apparatus or an infrared microscopic imaging apparatus, can acquire an optical microscopic image of a site on a sample to be imaged, similar to an imaging mass spectrometer. Therefore, processing according to the conceptual diagram shown in FIG. 8 obtained by modifying FIG. 3 may be performed.

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Abstract

Selon la présente invention, lorsqu'un utilisateur désigne une pluralité d'images d'imagerie par SM provenant d'échantillons contenant différents objets à chevaucher, une unité de traitement de mise en correspondance de position d'image (34) lit, à partir d'une unité de stockage de données d'image optique (30), des données d'image microscopique optique sur une pluralité d'échantillons qui correspondent aux images désignées et exécute une mise en correspondance de position au moyen d'une technologie d'enregistrement d'image connue pour obtenir des informations de modification d'image. Étant donné que la forme d'un échantillon peut être observée distinctement dans l'image microscopique optique, la mise en correspondance de position peut être effectuée avec une plus grande précision. Une unité de réglage d'image d'imagerie par SM (36) utilise les informations de modification d'image obtenues et modifie une pluralité d'images d'imagerie par SM. L'unité de traitement de chevauchement d'image (37) amène les images modifiées à se chevaucher. Même lorsque la forme d'une partie sur l'échantillon ne peut pas être observée dans l'image d'imagerie par SM, il est possible d'effectuer un chevauchement dans lequel les emplacements, les amplitudes et les formes de la même partie sont mis en correspondance. Il est ainsi possible d'obtenir un chevauchement précis d'images d'imagerie par SM dérivées d'une pluralité d'échantillons de tranche qui sont successivement coupés à partir d'un morceau de tissu biologique.
PCT/JP2018/013453 2018-03-29 2018-03-29 Procédé et programme de traitement de données dans une analyse de masse par imagerie WO2019186965A1 (fr)

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