JP2010085219A - Automatic position superimposing method of two-dimensional analysis image by mass spectrometry microscopy and two-dimensional visible image by optical photomicroscopy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a procedure capable of performing automatic and high-accuracy superimposition operation of each image position between a two-dimensional analysis image by mass spectrometry microscopy and a two-dimensional visible image by photomicroscopy, even when clear "characteristics of a concentration distribution shape of an object protein" which is an alignment key are difficult to be selected on the two-dimensional analysis image in mass spectrometry microscopy. <P>SOLUTION: The superimposition operation of each image position is performed between the two-dimensional analysis image by mass spectrometry microscopy and two-dimensional visible image by the photomicroscope photographing, by utilizing an artificial "marker for alignment", attached to the measurement domain of a histopathological slice. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for automatically superimposing the positions of two-dimensional images on a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image taken by an optical microscope, which are measured for a target region on the same section sample. .

  For example, in the histopathological diagnosis of a tumor, a light microscopic observation is performed on a histopathological section of a tumor excised from a patient. In histopathology, when observing a tissue slice of a lesion site excised from a patient, staining is usually performed prior to observation. For example, when observing a single cell in a tissue with a microscope, it is possible to distinguish cells in connective tissue and nuclei in cells based on the difference in morphology. However, for example, hematoxylin / eosin staining (HE staining) is performed, and cytoplasm is red to pink with eosin and nuclei are blue-purple with hematoxylin, thereby facilitating observation with an optical microscope. In a two-dimensional visible image obtained by staining a pathological tissue section and taking an optical microscope image, for example, in the cells in the tissue, the cytoplasm and nucleus are stained differently, and high resolution is obtained.

  In addition, in histopathological diagnosis, in order to detect the expression of a specific gene and the presence of a so-called “marker protein” due to the expression of the specific gene in the tissue of the lesion site in relation to the target disease. Immunostaining is also widely used. An antibody specific for the “marker protein” is labeled in advance, and the “marker protein” present in the tissue slice is detected using the labeled antibody. When various fluorescent dyes are bound to an antibody and fluorescently labeled, the presence of a “marker protein” reacted with the fluorescently labeled antibody can be detected by observing the fluorescence emitted by the fluorescent dye under photoexcitation. Immunostaining using a fluorescently labeled antibody is effective as a means for observing the localized site of the “marker protein” under a microscope in a tissue slice at a lesion site.

  In the two-dimensional fluorescence image obtained by immunostaining using fluorescent-labeled antibodies on the pathological tissue section and obtained by fluorescence microscopy, the cell outline (cell membrane) in the tissue and the nucleus in the cell are fully identified by their shadows. Is possible. Therefore, for the same region of the pathological tissue section, the image position between the two-dimensional visible image obtained by staining and immunostaining and the two-dimensional fluorescence image obtained by fluorescence microscopy, It is possible to automatically perform superposition with high accuracy.

  Although immunostaining is a useful tool for histopathological diagnosis, it has the essential limitation that it can only be applied to “marker proteins” when specific antibodies are available.

  The use of micromass spectrometry is expanding as a means for detecting a target protein or polypeptide present in a tissue slice without using a specific antibody. Usefulness of Peptide Mass Finger-print (PMF) method as a method to extract only mass spectrometry spectra derived from the target protein from mass spectrometry spectra derived from various proteins to be measured (For example, refer to Patent Document 1). Therefore, even when various proteins coexist, such as cells present in tissue slices, it is possible to extract only the mass spectrometry spectrum derived from the target known protein.

  The use of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) is expanding as a microscopic mass spectrometry method for proteins and polypeptides. Yes. In the MALDI-TOF MS method, desorption / ionization is performed by irradiating a matrix agent coated on the surface of a protein or polypeptide to be detected with a laser beam and utilizing light energy absorbed by the matrix agent. Is made. The UV-MALDI-TOF MS method using a laser beam in the ultraviolet region and the IR-MALDI-TOF MS method using a laser beam in the infrared region depending on the combination of the laser beam and the matrix agent used. .

  In micro-mass spectrometry for proteins and polypeptides, the UV-MALDI-TOF MS method is often used. In micro-mass spectrometry employing the UV-MALDI-TOF MS method, the laser beam that irradiates the matrix agent is collected, and proteins and polypeptides existing in the minute irradiation spot region are desorbed / ionized non-selectively. Accordingly, mass spectrometry spectra derived from various proteins and polypeptides existing in a minute irradiation spot region can be simultaneously measured. The intensity of the laser beam to be irradiated and the pulse irradiation time are fixed, and the minute irradiation spot position is moved in a two-dimensional matrix, and the mass spectrometry spectrum at each irradiation spot position is measured. Thereafter, a mass spectrometry spectrum derived from the protein to be detected is extracted from the mass spectrometry spectrum measured at each irradiation spot position. Furthermore, the two-dimensional analysis image of microscopic mass spectrometry is obtained by displaying the peak intensity: P (M / Z) caused by the ion species derived from the protein to be detected, corresponding to the irradiation spot position and two-dimensionally displaying it. Is obtained.

  When used for histopathological diagnosis of the tissue at the lesion site, for example, the same region of the histopathological section is obtained by microscopic mass spectrometry two-dimensional analysis image measured for “marker protein” and optical microscope photography. Comparison with the two-dimensional visible image to be made is made.

The two-dimensional analysis image of the microscopic mass analysis thus measured shows, for example, the concentration distribution of the “marker protein” quantitatively, and is used to identify the region where the “marker protein” is localized in the pathological tissue section. Is possible. Actually, the two-dimensional visible image obtained by optical microscopic imaging is aligned with the measured two-dimensional analysis image of microscopic mass spectrometry, for example, the concentration distribution of “marker protein” and the pathological tissue section. And the location of organelles such as nuclei in the cells.
JP 2007-10509 A

  When performing the above comparison, for example, when the “marker protein” has a feature localized in the nucleus, in consideration of the feature, on the two-dimensional analysis image of the micromass spectrometry measured for the “marker protein”, “ Assuming that the localization region of the “marker protein” is a nucleus, the image positions were visually overlapped so as to correspond to the positions of the nuclei on the two-dimensional visible image obtained by optical microscope photography.

  That is, on the measured two-dimensional analysis image of microscopic mass spectrometry, paying attention to the characteristics of the concentration distribution shape of the protein of interest, it is estimated that it corresponds to the characteristics of the concentration distribution shape, two-dimensional obtained by optical microscope photography The observer selected the shape on the visible image, and finally the image position was visually overlapped. In other words, when the “characteristic of the concentration distribution shape of the target protein”, which is a clue to alignment, is not clear, the image positions are superposed with great difficulty.

  In addition, even if the “characteristic of the concentration distribution shape of the target protein”, which is a clue to alignment, is clear, it is presumed that it corresponds to the feature of the concentration distribution shape, and is obtained by optical microscope photography. The alignment operation of selecting a shape on a three-dimensional visible image and superimposing the shapes is an operation that requires considerable skill.

  Therefore, it is desired to develop a method for automatically performing a superposition operation of image positions with high accuracy between a two-dimensional analysis image of microscopic mass analysis and a two-dimensional visible image taken by an optical microscope.

  The present invention solves the above problems. The first object of the present invention is to perform microscopic mass spectrometry even when it is difficult to select a clear “characteristic of the concentration distribution shape of the target protein” which is a clue to alignment on a two-dimensional analytical image of microscopic mass spectrometry. It is an object of the present invention to provide a method capable of automatically performing a superimposition operation of image positions between a two-dimensional analysis image of the above and a two-dimensional visible image taken by an optical microscope with high accuracy. In addition, the second object of the present invention, on the two-dimensional analysis image of the microscopic mass analysis, when a clear `` feature of the concentration distribution shape of the target protein '' that is a clue for alignment can be easily selected, It is an object of the present invention to provide a technique that makes it possible to automatically perform a superposition operation of image positions with high accuracy by utilizing the clear “characteristic of the concentration distribution shape of the target protein”.

  In order to find a means for solving the above-mentioned problems, the present inventor first automates the image position superposition operation between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image of optical microscope photography. We extracted technical problems in

  First, when used for histopathological diagnosis of tissue at a lesion site, a two-dimensional analysis image of microscopic mass analysis and a two-dimensional visible image obtained by optical microscope photography for the same region of a histopathological section are respectively Measured using another measuring device.

At that time, the two-dimensional analysis image data of the microscopic mass analysis is derived from the position information (x ^M _j , y ^M _j ) of each irradiation spot: j arranged two-dimensionally and the protein to be detected to be measured. peak intensity attributed to ionic species: P (M / Z: x M j, y M j) is composed of a combination of. Each irradiation spot: j arranged two-dimensionally needs to be arranged so that the irradiation spot areas do not overlap each other, and the positions (x ^M _j , y ^M _j ) of each irradiation spot have a predetermined interval: It is a discrete two-dimensional matrix having Δx ^M and Δy ^M. Of course, the predetermined intervals: Δx ^M and Δy ^M are set wider than the irradiation spot diameter of the focused laser beam: d _L.

On the other hand, a two-dimensional visible image obtained by optical microscope photography is essentially continuous information, but for example, it is digitized by detecting image information with a two-dimensional matrix photodetector. Yes. With this digitization process, the visible light intensity detected at each pixel constituting the two-dimensional matrix photodetector is, for example, primary color light component information in the “RBG” format: {P ^R , P ^B , P ^G } Is recorded. That is, the two-dimensional visible image data obtained by optical microscope photography is obtained by detecting the actual position: k position information (x ^O _k , y ^O _k ) and the detected visible light intensity: {P ^R (x ^O _k , y ^O _k ), P ^B (x ^O _k , y ^O _k ), P ^G (x ^O _k , y ^O _k )}. The actual position to be detected: k depends on the pixel size of the two-dimensional matrix photodetector, but the position (x ^O _k , y ^O _k ) has a predetermined minute interval: Δx ^O and Δy ^It is a discrete but dense two-dimensional matrix with ^O.

The resolution in the two-dimensional analysis image data of the microscopic mass analysis corresponds to a predetermined interval: Δx ^M and Δy ^M , and the resolution of the two-dimensional visible image data obtained by optical microscope photography is a predetermined minute interval: Δx ^O And Δy ^O. The predetermined interval: Δx ^M and Δy ^M are set in the range of 1 μm to 10 μm, for example, 3 μm to 5 μm, but the predetermined minute interval: Δx ^O and Δy ^O are in the range of 0.05 μm to 1.0 μm. For example, since it is set to 0.1 μm to 0.5 μm, there is usually a large gap between the resolutions of the two two-dimensional image data.

Of course, the same region of the pathological tissue section is measured, but in the two-dimensional analysis image data of the microscopic mass analysis, the position information (x ^M _j , y ^M _j ) of the irradiation spot: j (the origin of the coordinate axis) and In the two-dimensional visible image data obtained by optical microscope photography, the starting position (the origin of the coordinate axes) of the position information (x ^O _k , y ^O _k ) of the actual position: k is relatively shifted, and the shift ( The degree of δx, δy) is different for each pathological tissue section. The operation of aligning the deviations of the starting points of the position information of the two two-dimensional image data (the origin of the coordinate axes) corresponds to the image position overlapping operation.

Next, in the two-dimensional visible image obtained by optical microscope photography, for example, by performing hematoxylin and eosin staining (HE staining), the cytoplasm and nucleus of the cells in the tissue are stained differently. For example, the contour of the cell membrane and the contour of the nuclear membrane in the cell can be specified with high resolution based on the change in color tone over the entire measurement region of the tissue section. That is, the visible light intensity detected over the entire measurement region of the pathological tissue section: {P ^R (x ^O _k , y ^O _k ), P ^B (x ^O _k , y ^O _k ), P ^G (x ^O _k , y ^O _k )} is extracted, whereby the contour of the cell membrane and the contour of the nuclear membrane in the cell can be identified with high resolution.

On the other hand, in the two-dimensional analysis image of microscopic mass spectrometry, for example, the peak intensity P (M / Z: x ^M _j , y ^M _j ) due to the ion species derived from the “marker protein” to be detected is often It is measured only in a part of the measurement area of the pathological tissue section. Accordingly, the relative intensity change of the peak intensity: P (M / Z: x ^M _j , y ^M _j ) due to the ion species derived from the “marker protein” to be detected over the entire measurement region of the pathological tissue section. It is usually impossible to specify the contour of the cell membrane and the contour of the nuclear membrane in the cell with a considerable resolution.

  In particular, since the resolution in the two-dimensional analysis image data of the microscopic mass analysis is relatively low, the contour of the specified cell membrane, the contour of the nuclear membrane in the cell, and the optics in a part of the measurement region of the pathological tissue section When a two-dimensional visible image obtained by microscopic photography is overlaid with the contour of a cell membrane and the contour of a nuclear membrane in a cell that are specified with high resolution, this is a factor that lowers the accuracy of superposition.

That is, even when the “characteristic of the concentration distribution shape of the target protein” that is a clue to alignment is clear, the peak intensity due to the ion species derived from the “target protein”: P (M / Based on the relative intensity change of Z: x ^M _j , y ^M _j ), the region where “the characteristics of the concentration distribution shape of the target protein” can be extracted is usually limited to a part of the measurement region of the pathological tissue section. Yes. At that time, since the resolution is relatively low, the outline of the “characteristic of the concentration distribution shape of the target protein” and the outline of the cell membrane specified with high resolution on the two-dimensional visible image obtained by optical microscope photography When contrasting the contour of the nuclear membrane in the cell and superimposing the image positions, this is a factor that lowers the accuracy of superposition.

  In other words, the region that can extract “features of the concentration distribution shape of the target protein” that serves as a clue to alignment covers almost the entire measurement region of the pathological tissue section. It has been found that using the contour of “feature of density distribution shape”, it is necessary condition to improve the accuracy of superposition when superimposing image positions.

  On the other hand, if the "characteristic of the concentration distribution shape of the target protein", which is a clue to alignment, is not clear, an artificial "alignment marker" used for image position superimposition is used as a pathological tissue section. It was found that it is preferable to perform an image position superposition operation based on this “positioning marker”. Of course, this artificial "alignment marker" must be able to specify the contour shape of both the two-dimensional visible image obtained by optical microscope photography and the two-dimensional analysis image of microscopic mass spectrometry. It is.

  The inventors attach an artificial “alignment marker” that satisfies the above requirements to a measurement region of a pathological tissue section, and superimpose image positions based on this “alignment marker”. It was confirmed that adopting the operation mode has the following advantages.

  First, the planar shape of the artificial “positioning marker” provided can be selected as a predetermined planar shape. Further, the planar shape of the artificial “positioning marker” to be attached and the position to be attached to the measurement region of the pathological tissue section are set in advance. It can be automatically identified on a three-dimensional visible image without human judgment.

  On the other hand, the entire measurement region of the target pathological tissue section including the attachment site of the “alignment marker” is measured, and a two-dimensional visible image obtained by optical microscope photography, a two-dimensional analysis image of microscopic mass spectrometry , The attached portion of the “positioning marker” can be identified in both a two-dimensional visible image obtained by optical microscope photography and a two-dimensional analysis image of microscopic mass analysis.

  In particular, on the two-dimensional analysis image of micromass spectrometry, focusing on at least the peak intensity due to ion species derived from a specific protein, the interior of the planar shape of an artificial "alignment marker" and its surroundings It was found that it is possible to clearly distinguish the river basin.

  For example, in an irradiation spot that exists inside the planar shape of an artificial “alignment marker”, the peak intensity due to ionic species derived from a specific protein is clearly observed, but the “alignment marker” In the region surrounding the periphery of the planar shape, it was found that it is possible to create a situation in which the peak intensity due to the ionic species derived from a specific protein is not substantially observed. In that case, the peak intensity due to the ionic species derived from a specific protein is clearly observed. When attention is paid to the irradiation spot, the irradiation spot existing in the interior is caused by the ionic species derived from the specific protein. It was confirmed that “closed region” corresponding to the planar shape of “alignment marker” can be identified by selecting “closed region” in which peak intensity is clearly observed. In particular, it is possible to obtain the size of the planar shape of the “alignment marker” by using the outline of the planar shape of the identified “alignment marker” on the two-dimensional visible image obtained by optical microscope photography. Is possible. By comparing the size of the planar shape of the “alignment marker” and the size of the “closed region” specified on the two-dimensional analysis image of the microscopic mass analysis, the specified “closed region” is obtained. It was also confirmed that it can be verified that it corresponds to the planar shape of the “alignment marker”.

  Conversely, in the region surrounding the planar shape of the “alignment marker”, the peak intensity due to the ion species derived from a specific protein is clearly observed, but the artificial “alignment marker” It has been found that it is possible to create a situation in which the peak intensity due to an ionic species derived from a specific protein is not substantially measured in the irradiation spot existing inside the planar shape. In that case, the peak intensity due to the ionic species derived from the specific protein is not substantially observed. When attention is paid to the irradiation spot, the irradiation spot existing in the irradiation spot is attributed to the ionic species derived from the specific protein. It was confirmed that “closed regions” corresponding to the planar shape of the “alignment marker” can be identified by selecting “closed regions” in which the peak intensity is not substantially observed. In particular, it is possible to obtain the size of the planar shape of the “alignment marker” by using the outline of the planar shape of the identified “alignment marker” on the two-dimensional visible image obtained by optical microscope photography. Is possible. By comparing the size of the planar shape of the “alignment marker” and the size of the “closed region” specified on the two-dimensional analysis image of the microscopic mass analysis, the specified “closed region” is obtained. It was also confirmed that it can be verified that it corresponds to the planar shape of the “alignment marker”.

  When calculating the simple average of the positions of all the pixels existing within the planar shape of the identified “alignment marker” on the two-dimensional visible image obtained by optical microscope photography, the simple average is calculated as follows: This corresponds to the barycentric position of the planar shape of the “alignment marker”. Further, when the simple average of the positions of all irradiation spot centers existing in the “closed region” specified on the two-dimensional analysis image of the micromass spectrometry is calculated, the simple average is calculated as “closed”. The barycentric position of the “region” is approximately shown. The relative deviation (δx, δy) between the origins of the position information (origin of the coordinate axes) between the two two-dimensional images can be approximately obtained as a deviation between the approximate values of the centroid positions. I also confirmed that. As will be described below, it was also confirmed that the accuracy of the approximation depends on the difference between the resolution of the two-dimensional analysis image of microscopic mass spectrometry and the resolution of the two-dimensional visible image obtained by optical microscope photography.

  In addition, the outline of the planar shape of the “alignment marker” to be identified on the two-dimensional visible image obtained by optical microscope photography and the “closed” specified on the two-dimensional analysis image of the microscopic mass analysis are identified. When overlapping the shape of the outer periphery of the “region”, a plurality of irradiation spots existing in the “closed region” are contained within the outline of the planar shape of the “alignment marker”. It was also confirmed that it was possible to perform alignment almost. At that time, compared with the resolution of the two-dimensional visible image obtained by optical microscope photography, the resolution in the two-dimensional analysis image of the microscopic mass analysis is relatively low, so that “uncertainty” of that resolution remains. I found out. As a technique to further reduce the degree of “uncertainty”, focus on the irradiation spot that is close to the spot diameter of the irradiation spot with respect to the outline of the planar shape of the “alignment marker” and position it on this outline. Then, it was also found that the relative position of the irradiation spot with respect to the contour can be estimated with high accuracy by using the peak intensity measured at the visible irradiation spot. By referring to the estimated relative position of the irradiation spot with respect to the contour, the contour of the planar shape of the “alignment marker” identified on the two-dimensional visible image obtained by optical microscope photography It was verified that the shape of the outer periphery of the “closed region” specified on the two-dimensional analysis image of microscopic mass spectrometry can be superimposed with higher accuracy.

  In addition, the technical principle of the present invention described above is that the contour of the planar shape of the “alignment marker” can be identified with extremely high accuracy on a two-dimensional visible image obtained by optical microscope photography. It has been found that a “closed region” corresponding to the planar shape of the “positioning marker” can be applied to a range that satisfies the requirement that it can be specified with high accuracy on a two-dimensional analysis image of mass spectrometry.

  For example, by performing hematoxylin and eosin staining (HE staining) on a two-dimensional visible image obtained by optical microscope photography, the cytoplasm and nucleus of the cells in the tissue are stained differently. For example, the contour of the cell membrane and the contour of the nuclear membrane in the cell can be specified with high resolution based on the change in color tone over the entire measurement region of the tissue section. Furthermore, in addition to the nucleus, there are various organelles that can specify the outline with high resolution and accuracy in the cell. Among these nuclei and various organelles that exist in the cell, the outline is composed of membrane tissue and the inside and outside are clearly separated. It has been found that when a plurality of irradiation spots can exist, the above requirement may be satisfied. For example, nuclei and mitochondria form a “closed space” that is divided by cytoplasm and membrane tissue. In the irradiation spot located inside this “closed space”, ionic species derived from specific proteins It was conceived that the resulting peak intensity is not substantially observed, or the peak intensity attributed to the ionic species derived from a specific protein may be clearly observed.

  Therefore, instead of artificially formed "positioning markers", cells that originally have cells and those that satisfy the above requirements among various organelles are used as predetermined selection criteria. Based on the selection, it was found that it can be used as an artificially set “positioning marker”. In addition, among the nuclei and various organelles inherently possessed by the cell, those that satisfy the above requirements, for example, even when selecting the nucleus, for the entire measurement area of the target pathological tissue section, When measurement is performed, the number of cells reaches a considerable number, and it is inefficient to use all the nuclei as artificially set “alignment markers”. In consideration of this, when a measurement is performed on the entire measurement area of the target pathological tissue section, a predetermined number of nuclei satisfying a certain size standard are automatically selected from a considerable number of identified nuclei. It was concluded that it was desirable to select and adopt a form that is used as an artificially set “positioning marker”.

  In particular, when selecting a “closed region” corresponding to the planar shape of the “alignment marker” on a two-dimensional analysis image of micromass spectrometry, the number of irradiation spots existing in the “closed region” is It was concluded that it would be desirable to automatically select a predetermined number of nuclei in the descending order and use them as artificially set “alignment markers”. Furthermore, with reference to the relative arrangement of a predetermined number of nuclei, the task of specifying a predetermined number of nuclei having a corresponding relative arrangement on a two-dimensional visible image obtained by optical microscope photography is also automated. It was also confirmed that it was easy. Of course, depending on the resolution of the two-dimensional analysis image of the microscopic mass analysis, the number of irradiation spots existing in “closed regions” corresponding to various organelles varies. Of course, other organelles satisfying the above requirements can be used instead of the nucleus in the range where the number of irradiation spots existing in the “closed region” satisfies a certain level. That is.

  That is, in the present invention, two two-dimensional images are measured between a two-dimensional analysis image of microscopic mass spectrometry measured on a pathological section of a subject and a two-dimensional visible image obtained by optical microscope photography of the pathological section. In order to automatically perform the alignment operation for displaying the images in an overlapping manner, an artificial “alignment marker” is used. On the two-dimensional visible image obtained by optical microscope photography, the outline of the planar shape of the “positioning marker” is specified based on the difference in “color tone”. On the other hand, on the two-dimensional analysis image of the microscopic mass analysis, attention is paid to the peak intensity caused by the ion species derived from the specific protein, and based on the significant difference in the peak intensity caused by the ion species, A “closed region” corresponding to the planar shape of the “marker” is specified. On the two-dimensional visible image obtained by optical microscope photography, the planar shape outline of the “positioning marker” to be identified and the “closed region” specified on the two-dimensional analysis image of microscopic mass analysis It was verified that it is possible to automatically align two two-dimensional images by superimposing the shape of the outer periphery of the two with high accuracy.

  At that time, by adopting the following six forms as artificial “alignment markers”, it is possible to improve the accuracy of alignment between two two-dimensional images when automatically performed. confirmed.

  (1) In the first embodiment, either a two-dimensional visible image obtained by optical microscope photography or a two-dimensional analysis image obtained by microscopic mass spectrometry so that the tissue slice is included in the target measurement region on the preparation on which the tissue slice is mounted. Are provided with “alignment markers” having a predetermined planar shape made of “marker substances” that can be identified.

  (2) In the second embodiment, the tissue slice can be identified in a two-dimensional visible image taken by an optical microscope so that the tissue slice is included in the target measurement region on the prepared slide. In the two-dimensional analysis image, a “positioning mask” having a predetermined planar shape that does not show an identifiable mass analysis peak intensity is provided.

  (3) In the third embodiment, a predetermined position on the tissue slice is provided as a “positioning marker” having a predetermined planar shape provided so as to be included in the target measurement region on the preparation on which the tissue slice is mounted. A “region where the matrix agent is not applied” having a predetermined planar shape provided in the region is used.

  (4) In the fourth embodiment, a predetermined position on the tissue slice is provided as a “positioning marker” having a predetermined planar shape provided to be included in the target measurement region on the preparation on which the tissue slice is mounted. A “region not subjected to protease cleavage treatment” having a predetermined planar shape provided at the site is used.

  (5) In the fifth embodiment, either a two-dimensional visible image obtained by optical microscope photography or a two-dimensional analysis image obtained by microscopic mass spectrometry so that the tissue slice is included in the target measurement region on the preparation on which the tissue slice is mounted. The “endogenous alignment marker” that can be discriminated in FIG.

  (6) In the sixth embodiment, a predetermined position on the tissue slice is provided as a “positioning marker” having a predetermined planar shape provided to be included in the target measurement region on the preparation on which the tissue slice is mounted. A “region that is not chemically modified to an amino acid residue” having a predetermined planar shape provided at the site is used.

  The present inventors have completed the present invention based on these findings.

The automatic position superposition method of the two-dimensional analysis image of microscopic mass spectrometry according to the first embodiment of the present invention and the two-dimensional visible image of optical microscope photography,
Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
It consists of a marker substance that can be discriminated in both a two-dimensional visible image obtained by optical microscope photography and a two-dimensional analysis image obtained by microscopic mass spectrometry so that it is included in the target measurement region on the preparation on which the tissue slice is mounted An automatic position superposition method characterized in that an alignment marker having a predetermined planar shape is provided.

In the first aspect of the present invention,
For example, the alignment marker having the predetermined planar shape is
It is possible to adopt an embodiment in which the alignment marker has a predetermined planar shape, which is composed of a dye-labeled protein and a dye-labeled polypeptide, with a dye label having a color distinguishable on a two-dimensional visible image taken by an optical microscope. it can.

For example, the alignment marker having the predetermined planar shape is
It is possible to adopt an embodiment in which the alignment marker has a predetermined planar shape made of a mixture in which a dye having a color identifiable on a two-dimensional visible image taken with an optical microscope is mixed with protein and polypeptide.

For example, the alignment marker having the predetermined planar shape is
Employing an embodiment of a positioning marker having a predetermined planar shape, which is made of a protein or a polypeptide, dyed with a dye having a color that can be identified on a two-dimensional visible image taken by an optical microscope. Can do.

  In addition, it is preferable to employ exogenous proteins and polypeptides that are essentially not present in tissue slices as the proteins and polypeptides used for the production of the alignment marker.

The automatic position superposition method of the two-dimensional analysis image of microscopic mass spectrometry and the two-dimensional visible image of optical microscope photography according to the second aspect of the present invention,
Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
Discriminated in the two-dimensional visible image of optical microscope photography and discriminated in the two-dimensional analytical image of microscopic mass spectrometry so that it is included in the target measurement area on the preparation on which the tissue slice is mounted An automatic position superposition method characterized by providing an alignment mask having a predetermined plane shape that does not show a strong mass analysis peak intensity.

In the second aspect of the present invention,
For example, the alignment marker having the predetermined planar shape is
It is possible to adopt a mode in which the coating film layer has a predetermined planar shape, which is made of a light masking material that does not exhibit optical transparency to laser light used in the MALDI-TOF MS method.

  For example, as a coating film layer made of a light masking substance that does not exhibit light transmittance with respect to the laser light, a light reflective coating film layer made of a metal material exhibiting high reflectance in the wavelength range of the irradiated laser light, or It is possible to use a light-absorbing coating film layer made of a material having a high extinction coefficient that exhibits strong light absorption with respect to laser light and substantially eliminates laser light absorption by the matrix agent.

For example, the alignment mask having the predetermined planar shape is
A coating film layer having a predetermined planar shape made of a physical masking substance that shows light permeability to laser light used in the MALDI-TOF MS method, but prevents desorption of ionic species derived from peptide fragments. Certain aspects can also be employed.

  For example, it is possible to use a polymer coating layer made of a polymer material exhibiting sufficiently high light transmittance in the wavelength range of the laser beam to be irradiated.

The automatic position superposition method of the two-dimensional analysis image of microscopic mass spectrometry according to the third aspect of the present invention and the two-dimensional visible image of optical microscope photography,
Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
An automatic position superposition method characterized in that a region having a predetermined planar shape and not coated with a matrix agent is provided at a predetermined site on a tissue slice.

The automatic position superposition method of the two-dimensional analysis image of microscopic mass analysis according to the fourth aspect of the present invention and the two-dimensional visible image of optical microscope photography,
Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
An automatic position superposition method characterized in that a region having a predetermined planar shape and not subjected to protease cleavage treatment is provided at a predetermined site on a tissue slice.

In the fourth aspect of the present invention,
In the region having the predetermined planar shape and not subjected to protease cleavage treatment, for example, a “coated protective film” having a predetermined planar shape is provided in advance on a predetermined site on the tissue slice, and then the entire surface of the tissue slice. It can be formed by subjecting to protease cleavage treatment.

The automatic position superposition method of the two-dimensional analysis image of the microscopic mass analysis according to the fifth aspect of the present invention and the two-dimensional visible image of the optical microscope,
Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
Intrinsic, identifiable in both two-dimensional visible images of optical microscopy and two-dimensional analytical images of microscopic mass spectrometry so that they are included in the target measurement area on the preparation on which the tissue slice is mounted An automatic position superimposing method characterized by setting an alignment marker.

In the fifth aspect of the present invention,
As an intrinsic alignment marker that can be identified in any of the two-dimensional visible image of the optical microscope and the two-dimensional analytical image of microscopic mass analysis,
On the surface of the tissue flakes, it is possible to adopt an embodiment in which a plurality of nuclei that can specify the position of the region can be adopted in both the two-dimensional visible image obtained by optical microscope photography and the two-dimensional analysis image obtained by microscopic mass spectrometry.

The automatic position superposition method of the two-dimensional analysis image of the microscopic mass analysis according to the sixth aspect of the present invention and the two-dimensional visible image of the optical microscope,
Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
In this automatic position superposition method, a region having a predetermined planar shape and not chemically modified to an amino acid residue is provided at a predetermined site on a tissue slice.

  For example, an S-alkylation treatment for a sulfanyl group (—SH) on the side chain of a Cys residue can be employed as the chemical modification treatment for the amino acid residue.

In the present invention, as the MALDI-TOF MS method,
It is preferable to use the UV-MALDI-TOF MS method.

  In the automatic position superimposing method according to the present invention, a two-dimensional analysis image of microscopic mass spectrometry and an optical microscope image using an artificial “positioning marker” attached to a measurement region of a pathological tissue section. Since the image position superimposing operation is performed between the two-dimensional visible image, the alignment can be automatically performed with high accuracy. In particular, even if the “characteristics of the concentration distribution shape of the target protein”, which is a clue to alignment on the two-dimensional analysis image of microscopic mass spectrometry, is not clear, the alignment is automatically performed with high accuracy. Can be done.

  Hereinafter, the automatic position superposition method of the two-dimensional analysis image of microscopic mass spectrometry and the two-dimensional visible image of optical microscope photography according to the present invention will be described in more detail.

  The present invention generalizes the technical significance based on the following idea.

In order to superimpose and display the analysis image of the microscopic mass analysis and the pathological slice image of the subject, the images are superimposed using information that can identify the position on the pathological slice image obtained by the micromass spectrometry. . Markers that can identify the position on the pathological slice image are marked with a “mark” that represents the position information on the image obtained by micromass analysis.
-In the case of adding a marker substance in advance on a preparation on which a pathological section is mounted,
・ When masking so that an image of a specific part falls out,
・ If the matrix agent is not partially applied or the protease treatment is not performed,
-As an internal standard for performing superposition, among the biological substances normally contained in pathological sections, substances present at positions showing image features, such as proteins localized in the nucleus, membranes that bind to cell membranes Using the protein, protein that exists only in the cytoplasm, etc., select the image that can display the specific part of the pathological image to be superimposed most characteristically, and use that image to identify the position of the overlay Is the case.

  Conventionally, in order to display an analysis image of micromass spectrometry and a pathological section image of a subject in a superimposed manner, the characteristics of the separately created images are visually determined or the photographing position is fixed in advance. Then, they were superposing. For this reason, it has been difficult to automatically perform accurate overlaying.

  By using the present invention, it is possible to automatically perform superimposition using spectral results of image processing and microscopic mass spectrometry, and at the same time, it is expected to improve the accuracy of superimposition.

  First, a two-dimensional analysis image of microscopic mass analysis and a two-dimensional visible image of optical microscope photography to which the automatic position superposition method according to the present invention is applied will be described.

(Two-dimensional visible image data taken with an optical microscope)
The present invention is directed to the technical field of histopathological diagnosis, and a two-dimensional visible image obtained by optical microscope is mainly used to prepare a tissue slice from a pathological tissue section excised from a patient, and in the tissue slice. The shape of the tissue, the cells constituting the tissue, the nucleus present in each cell, and various organelles are observed with an optical microscope. Alternatively, a tissue slice prepared from a tissue culture and used for various pathological studies and pharmacological studies, which is observed with an optical microscope, is also included.

  For example, a tissue slice prepared from a pathological tissue section is mounted on a preparation and then stained using various dyes. For example, hematoxylin and eosin staining (HE staining) is performed on individual cells constituting the tissue in order to clarify the contours of the cell membrane and the nuclear membrane in the cells.

  By eosin staining, the cytoplasm, cell membrane, and some extracellular structures are stained pink or red. Further, the nucleus is stained blue-purple or brown by hematoxylin staining. For hematoxylin staining, which is used for selective staining of nuclei, a mordant is required. By applying hematoxylin and eosin staining, which is a combination of hematoxylin staining and eosin staining, each with a different staining target and a different color tone after staining, the contours of the cell membrane for each cell constituting the tissue The outline of the nuclear envelope in the cell becomes clearer.

  The stained tissue slice mounted on the preparation is observed with a transmission microscope to obtain a two-dimensional visible image obtained by optical microscope photography.

  For example, the primary information of a two-dimensional visible image obtained by optical microscope photography can be a color micrograph taken using an optical microscope. The image information of the color micrograph is converted into digitized two-dimensional visible image data by a color digital scanner.

In addition, a two-dimensional visible image observed with an optical microscope is directly digitized by detecting image information using, for example, a two-dimensional matrix photodetector, and the digitized two-dimensional visible image data is obtained. It can also be obtained. With this digitization process, the visible light intensity detected at each pixel constituting the two-dimensional matrix photodetector is, for example, primary color light component information in the “RBG” format: {P ^R , P ^B , P ^G } Is recorded.

That is, the two-dimensional visible image data obtained by optical microscope photography includes the actual position on the tissue slice to be detected: position information (x ^O _k , y ^O _k ) and the detected visible light intensity: {P ^R (x ^O _k , y ^O _k ), P ^B (x ^O _k , y ^O _k ), P ^G (x ^O _k , y ^O _k )}. For example, depending on the resolution depending on the pixel size of the two-dimensional matrix photodetector or the resolution of the color digital scanner, the actual position detected: k is the position (x ^O _k , y ^O _k ) is a discrete but high-density two-dimensional matrix having predetermined small intervals: Δx ^O and Δy ^O.

The resolution of the two-dimensional visible image data obtained by optical microscope photography corresponds to the predetermined minute interval: Δx ^O and Δy ^O. The predetermined minute interval corresponding to the resolution: Δx ^O and Δy ^O depends on the magnification at the time of photographing with an optical microscope, but in the high magnification two-dimensional visible image data, the range is 0.05 μm to 1.0 μm. Preferably, it is selected in the range of 0.1 μm to 0.5 μm.

  For example, high-magnification two-dimensional visible image data is acquired for the entire tissue slice mounted on the preparation. At that time, depending on the size of the tissue slice, when observing with an optical microscope, the target measurement region may not fit within the frame size that can be photographed. In that case, the target measurement area is divided into a plurality of small areas, each small area is included in the center of the frame, and a plurality of high-magnification two-dimensional visible images having an overlap with adjacent small areas are acquired. . Then, based on the two-dimensional visible image data collected from each high-magnification two-dimensional visible image, two-dimensional visible image data that covers the entire target measurement area by connecting the two-dimensional visible image data of multiple frames. Build up.

  Since the two-dimensional visible image data of a plurality of frames are overlapped between adjacent frames and the resolution is equal, the two-dimensional visible image data can be obtained by applying an automatic method for automatically superimposing the image positions. Can be easily interconnected.

  The two-dimensional visible image data that covers the entire target measurement area and the two-dimensional visible image data of multiple frames that are the basis of the constructed two-dimensional visible image data are collectively displayed in the two-dimensional visible pathology image. The image data is stored in a data storage device attached to the image processing computer shown in FIG.

(Identification of characteristic structures found on “pathological visible images”)
The characteristic structures found on the “pathologically visible image” of the target measurement area are present in this measurement area, the contour of the cell membrane of each cell, the contour of the nuclear membrane in the cell, and the cell. The outline of various organelles, for example, the outline of the outer membrane of mitochondria, the outline of the outer membrane of the Golgi apparatus, etc. The outline (shape) of the entire tissue slice is eventually specified by specifying the outline of the cell membrane of each cell constituting the tissue.

  The size of the various organelles present in the cell is, for example, that the mitochondria are filamentous or granular with a width of about 0.5 μm, and the Golgi apparatus is a flat sac, small spherical vesicle (Golgi vesicle), Although it is a complex composed of Golgi vacuoles, its overall size depends on the cell. Therefore, the outline of the outer membrane of mitochondria and the outline of the outer membrane of the Golgi body are often difficult to discriminate by optical microscope photography. On the other hand, the contour of the cell membrane and the contour of the nuclear membrane in the cell can be easily distinguished.

  First, based on the two-dimensional visible image data obtained by optical microscope photography, the contour of the cell membrane and the contour of the nuclear membrane in the cell are specified for each cell constituting the tissue.

Specifically, based on the high-magnification two-dimensional visible image data, the change in the color tone existing between the cytoplasm and the nucleus is used, and the detected visible light intensity: {P ^R (x ^O _k , y ^O _k ), P ^B (x ^O _k , y ^O _k ), P ^G (x ^O _k , y ^O _k )}, paying attention to P ^B (x ^O _k , y ^O _k )}, its rate of change, ∂P _{^{B (x O k, y O}} k) / ∂x O k, ∂P B (x O k, y O k) is calculated by numerical differentiation of / ∂y ^O _k. The rate of _{^{change, ∂P B (x O k,}} y O k) / ∂x O k, ∂P B (x O k, y O k) / ∂y O k is the position shown remarkable values, the nuclear membrane It can be a contour. Position information of the contour of the nuclear membrane, the group of position indicating a closed _{^{curve: Gnuclear {(x O nuclear-}} k, y O nuclear-k)} constitutes the. Similarly, for example, cell membranes existing between adjacent cells can acquire positional information of the contours of the cell membranes by using a change in color tone existing between the cells. The position information of the contour of the cell membrane constitutes a group of positions indicating a closed curve: Gcell {(x ^O _cell-k , y ^O _cell-k )}.

The outline (shape) of the entire tissue slice is specified as the position information of the outer edge by specifying the outline of the cell membrane of each cell constituting the tissue. Position information of the tissue slices overall contour (shape), a group of contiguous _{^{positions: Gtissue {(x O tissue-}} k, y O tissue-k)} constitutes the.

(Two-dimensional analysis image data of micromass spectrometry)
The present invention is directed to the technical field of histopathological diagnosis. A two-dimensional visible image of microscopic mass spectrometry is mainly used to prepare a tissue slice from a pathological tissue section excised from a patient, and the tissue slice The peak intensity due to the ion species derived from the protein or polypeptide detected by microscopic mass spectrometry: P (M / Z: x ^M _j , y ^M _j ) It corresponds to what is displayed in a dimension. Alternatively, proteins and polypeptides that are used in various pathological and pharmacological studies, prepared from tissue cultures, present in tissue slices, and detected in mass spectrometry, are detected in mass spectrometry. peak intensity attributed to ionic species derived from the peptide: P (M / Z: x M j, y M j) also include those displaying the two-dimensionally.

  As a mass spectrometry method for proteins and polypeptides present in tissue slices, a MALDI-TOF MS method, particularly a UV-MALDI-TOF MS method using laser light in the ultraviolet region is used.

  In micro-mass spectrometry employing the UV-MALDI-TOF MS method, a matrix agent is applied to the upper surface of the tissue slice, and the laser beam irradiated on the upper surface of the tissue slice coated with the matrix agent is condensed to form a minute irradiation spot region. Non-selectively desorbs / ionizes proteins and polypeptides. Accordingly, mass spectrometry spectra derived from various proteins and polypeptides existing in a minute irradiation spot region can be simultaneously measured. The intensity of the laser beam to be irradiated and the pulse irradiation time are fixed, and the minute irradiation spot position is moved in a two-dimensional matrix, and the mass spectrometry spectrum at each irradiation spot position is measured. Thereafter, a mass spectrometry spectrum derived from the protein to be detected is extracted from the mass spectrometry spectrum measured at each irradiation spot position.

The extracted mass spectrometry spectrum derived from the protein to be detected includes a plurality of peaks (peak positions: M / Z) due to ionic species derived from the protein to be detected. Position information (x ^M _j , y ^M _j ) of each irradiation spot: j and peak intensity due to ion species derived from the protein to be detected measured at the irradiation spot: j: P (M / Z: x ^M _j , y ^M _j ) are combined to construct two-dimensional analysis image data for microscopic mass spectrometry.

Further, the peak intensity P (M / Z: x ^M _j , y ^M _j ) caused by the ion species derived from the protein to be detected is made to correspond to the irradiation spot position (x ^M _j , y ^M _j ), By displaying in two dimensions, a two-dimensional analysis image of microscopic mass analysis for each ion species (peak position: M / Z) can be obtained.

  For example, with respect to the whole tissue slice mounted on the preparation, the minute irradiation spot position is moved in a two-dimensional matrix, and the mass spectrometry spectrum at each irradiation spot position is measured. The two-dimensional analysis image data of the microscopic mass analysis configured based on the measurement result becomes the two-dimensional analysis image data of the microscopic mass analysis covering the entire target measurement region.

  The mass spectrometry spectrum measured at each irradiation spot position includes mass spectrometry spectra derived from various proteins and polypeptides present in the minute irradiation spot region, which are measured simultaneously.

  As a technique for extracting mass spectrometry spectra derived from individual proteins and polypeptides from mass spectrometry spectra measured at each irradiation spot position, in the present invention, peptide mass fingerprint (PMF: Peptide Mass Finger-print) is used. ) Law is applied.

Specifically, a protease having specificity for a specific partial amino acid sequence is allowed to act on a protein to be detected, and a peptide chain (P ₀ : molecular weight M ₀ ) constituting the protein is cleaved to produce a plurality of types. Peptide fragment {(P _fi : molecular weight M _fi )} is prepared. When a plurality of types of peptide fragments {(P _fi : molecular weight M _fi )} obtained by performing this protease cleavage treatment are subjected to mass spectrometry using the UV-MALDI-TOF MS method, they are derived from a plurality of types of peptide fragments. Multiple types of peaks due to ionic species are measured.

  From the mass spectrometry spectrum measured at each irradiation spot position, multiple types of peaks derived from ionic species derived from multiple types of peptide fragments prepared from the target protein are derived from the target protein. As a mass spectrometry spectrum to be extracted.

The amount of the plurality of types of peptide fragments {(P _fi : molecular weight M _fi )} obtained by performing the protease cleavage treatment described above is essentially equal. The UV-MALDI-TOF MS method, as the main ion species derived from each peptide fragment, protons (H ⁺⁾ cation species are added with ^{_{(M fi + H + / Z}} ), protons (H ⁺⁾ is disconnected Anion species (M _fi -H ⁺ / Z) are generated. The production ratio of the cationic species and the anionic species depends on the amino acid sequence of each peptide fragment: (P _fi : molecular weight M _fi ). Furthermore, the ionization efficiency of each peptide fragment: (P _fi : molecular weight M _fi ) depends on the molecular weight and the amino acid sequence of the peptide fragment.

In the MALDI-TOF MS method, mass spectrometry spectra in two measurement modes, that is, a mode for detecting only a cation species (positive mode) and a mode for detecting only an anion species (negative mode) are individually measured. Can be measured. For example, in a mass spectrometry spectrum measured in a positive mode for detecting only cationic species (cation mode derived from multiple types of peptide fragments (M _fi + H ⁺ / Z) prepared from the protein to be detected) The peak intensity: P (M _fi + H ⁺ / Z) depends on the molecular weight of each peptide fragment and its amino acid sequence. Therefore, the peak position (M _fi + H ⁺ / Z) of a cationic species derived from a plurality of types of peptide fragments prepared from the protein to be detected and its peak intensity: P (M _fi + H ⁺ / Z) The relative ratio: P _rel (M _fi + H ⁺ / Z) depends on the amino acid sequence of the protein to be detected.

Therefore, there is a difference in the entire amino acid sequence between the two proteins, but it is possible that a plurality of types of peptide fragments {(P _fi : molecular weight M _fi )} obtained by subjecting to protease cleavage treatment are completely identical. Sexuality is not completely absent, but the possibility is extremely low. In the present invention, the above feature is used in the step of applying the PMF method and extracting the mass spectrometry spectrum derived from the protein to be detected.

For example, from the mass spectrometry spectrum measured in the positive mode for detecting only the cation species, the peak positions of the cation species derived from multiple types of peptide fragments (M _fi + H ⁺ / Z) and its peak intensity: P (M _fi + H ⁺ / Z) relative ratio: P _rel (M _fi + H ⁺ / Z), a group of peaks derived from the protein to be detected Extracted as a mass spectrum.

  At that time, if the number of peptide fragments obtained by subjecting the protein to be detected to protease cleavage treatment is small, the probability that another protein that gives the same multiple types of peptide fragments happens to exist by chance. , Relatively high. Therefore, it is preferable to perform the cleavage treatment using a protease such that the number of the plurality of types of peptide fragments obtained by the protease cleavage treatment is usually 4 or more.

  Conversely, if the number of peptide fragments obtained by subjecting the protein to be detected to protease cleavage is too large, there is another protein that happens to give the same multiple types of peptide fragments by chance. The probability is relatively high. Therefore, it is preferable to perform the cleavage treatment using a protease such that the number of the plurality of types of peptide fragments obtained by the protease cleavage treatment is usually 15 or less, more preferably 10 or less.

  Among proteases having specificity for specific partial amino acid sequences, those that meet the above conditions are proteases that selectively cleave peptide bonds on the N-terminal side or C-terminal side of specific amino acids. is there. As a protease that selectively cleaves the N-terminal peptide bond of a specific amino acid or the C-terminal peptide bond, trypsin that cleaves the C-terminal peptide bond of lysine or arginine residue, C-terminal of glutamic acid residue A widely used protease for peptide fragmentation treatment, such as the V8 enzyme that cleaves the side peptide bond, can be used.

  Even if there are four or more sites that can be cleaved by the protease in the peptide chain constituting the protein to be detected, the actual protein has a three-dimensional structure, There is a high possibility that the number of severable parts exposed on the surface is reduced. Considering this point, trypsin that cleaves the C-terminal peptide bond of lysine or arginine residue is preferably used for protease cleavage treatment. If the number of peptide fragments generated by protease cleavage treatment using one type of protease does not reach the above level, select a form to perform protease cleavage treatment using two types of protease. It is also possible.

  In the present invention, the tissue slice mounted on the preparation is subjected to the protease cleavage treatment described above, and then the treatment for removing the utilized protease is not performed, but the treatment for evaporating moisture is performed. After the removal of moisture, a matrix agent coating layer is formed on the upper surface of the tissue slice. In the UV-MALDI-TOF MS method, when a nitrogen gas laser is used as the laser beam to be irradiated, examples of the matrix agent include 3HPA (3-hydroxy-picolinic acid), α-CHCA (α-cyano-4- Hydroxycinnamic acid) can be used.

  As a result, a plurality of types of peptide fragments derived from various proteins prepared in the protease cleavage treatment step are held on the surface of the tissue slice mounted on the preparation. Furthermore, the protease used for the protease cleavage treatment and its self-digested fragment also remain on the surface of the tissue slice. A matrix agent coating layer surrounds a plurality of types of peptide fragments derived from various proteins, proteases, and self-digested fragments, which are held on the surface of these tissue slices. Of course, when the protease cleavage treatment is applied to the entire surface of the tissue slice, the protease and its self-digested fragment remain on the entire surface of the tissue slice.

For example, with respect to the entire tissue slice mounted on the preparation, the laser beam focused on the matrix agent coating layer formed at each irradiation spot position is moved by moving the minute irradiation spot position in a two-dimensional matrix. Irradiate. At that time, for example, the irradiation spots: j arranged in a two-dimensional manner are arranged so that the irradiation spot areas do not overlap. In that case, the position (x ^M _j , y ^M _j ) of each irradiation spot is a discrete two-dimensional matrix having a predetermined interval: Δx ^M and Δy ^M. Of course, the predetermined intervals: Δx ^M and Δy ^M are set wider than the irradiation spot diameter of the focused laser beam: d _L.

When the laser beam was focused on, the irradiation spot diameter of the focused laser beam: d _L will generally not be less than the spread caused by the spherical aberration of a lens system using the condenser. Considering this point, for example, when using a nitrogen gas laser with an oscillation wavelength λ = 337.1 nm and a pulse width Δt: several ns, the irradiation spot diameter of the focused laser beam: d _L is usually It is selected in the range of 1 μm to 10 μm, preferably in the range of 1 μm to 5 μm, desirably in the range of 1 μm to 3 μm.

A predetermined interval provided between the center positions of adjacent irradiation spots: Δx ^M and Δy ^M is at least Δx ^M and Δy ^M > d _L with reference to the irradiation spot diameter of the condensed laser beam: d _L. satisfies the condition, _{typically, 4/3 · d L ~3} · d L range, preferably, it is desirable to select the range of _{3/2 · d L ~2 ·} d L. Therefore, it is desirable that the predetermined intervals: Δx ^M and Δy ^M are generally selected in the range of 2 μm to 15 μm, preferably in the range of 2 μm to 10 μm, for example, in the range of 3 μm to 5 μm. The resolution in the two-dimensional analysis image data of the microscopic mass analysis corresponds to the predetermined interval: Δx ^M and Δy ^M.

In some cases, when performing two-dimensional mass spectrometry, the irradiation spot moving pitches: Δx ^M _pitch and Δy ^M _pitch , Δx ^M _pitch , Δy with respect to the irradiation spot diameter: d _{L of} the focused laser beam. ^M _pitch ≈d _L ~ 2 / 3d _L may be selected and continuous measurement may be performed. That is, the measurement may be performed in a state where there is an overlap at the bottom of the spot diameter of the adjacent irradiation spot. In that case, the interval between the center positions of the irradiation spots becomes 2Δx ^M _pitch and 2Δy ^M _pitch , and the second proximity irradiation spot with no overlapping of the irradiation spot diameter is selected, and the two-dimensional analysis image data of the microscopic mass analysis is constructed. To do. Note that the measurement results of all the measured irradiation spots are stored as the original measurement results based on the construction of the two-dimensional analysis image data of the microscopic mass analysis.

In each irradiation spot: j, the light intensity of the laser beam: P _Excitation (W), the pulse width Δt: proportional to the irradiation amount: Δt · P _Excitation (W · s) due to the irradiation of the ultraviolet laser beam of several ns. Thus, desorption / ionization of protein molecules occurs. Irradiation spot diameter present in the tissue slice surface in d _L, various proteins, polypeptides, ionizing conflict. When the surface density of each protein: P _p existing on the tissue slice surface is C (P _p ) and its ionization efficiency is f _ion (P _p ), the ionization amount of each protein: P _p is approximately f _It is proportional to _ion (P _p ) · C (P _p ) / Σ {f _ion (P _p ) · C (P _p )}. The total ionization amount of each protein: P _p : Σ {f _ion (P _p ) · C (P _p )} is proportional to the laser beam irradiation amount: Δt · P _Excitation (W · s).

Usually, for each irradiation spot: j, the surface density: C (P _p : x ^M _j , y ^M _j ) of each existing protein: P _P is different. Accordingly, the total ionization amount of each protein: P _p : Σ {f _ion (P _p ) · C (P _p : x ^M _j , y ^M _j )} is different for each irradiation spot: j. Even if the detection target protein: P _{i has} the same surface density: C (P _i : x ^M _j , y ^M _j ), due to the difference in the surface density of other competing proteins, protein to be detected: ionization of P _{i is, f ion (P i) ·} C (P i: x M j, y M j) / Σ {f ion (P p) · C (P p: x M j , Y ^M _j )}.

On the other hand, the relative ratio of the ionization amount of each peptide fragment: (P _fi : molecular weight M _fi ) derived from the detection target protein: P _i is the surface density of each peptide fragment at the irradiation spot: j: C (P _fi : X ^M _j , y ^M _j ) are essentially equal, and only depend on the ionization efficiency of each peptide fragment: f _ion (P _fi ).

For example, from the mass spectrometry spectrum measured in the positive mode for detecting only the cation species, the peak positions of the cation species derived from multiple types of peptide fragments (M _fi + H ⁺ / Z) and the relative ratio of its peak intensity: P (M _fi + H ⁺ / Z): P _rel (M _fi + H ⁺ / Z) shows essentially variations in any irradiation spot: j Absent. In the present invention, the above feature is utilized in the step of applying the PMF method and extracting the mass spectrometry spectrum derived from the protein to be detected for each irradiation spot: j.

In some irradiation spots, peptides derived from other proteins at one of the peak positions (M _fi + H ⁺ / Z) of cationic species derived from multiple types of peptide fragments derived from the protein to be detected The peak positions of cationic species derived from the fragments may overlap by chance. At that time, the peak intensity of cationic species derived from a plurality of types of peptide fragments measured at other irradiation spots: relative ratio of P (M _fi + H ⁺ / Z): P _rel (M _fi + H ⁺ / With reference to Z), the peak intensity of the cationic species derived from the remaining peptide fragments at the irradiation spot: from P (M _fi + H ⁺ / Z), the peak of the cationic species derived from another peptide fragment by chance. It is also possible to reasonably estimate the peak intensity that overlaps the position.

If necessary, after correcting the peak intensity: P (M _fi + H ⁺ / Z) at the irradiation spot where the above-mentioned incidental peak overlap occurs, position information (x ^M _j , y ^M _j ) and the irradiation spot: the peak position (M _fi + H ⁺ / Z) of a cationic species derived from a plurality of types of peptide fragments derived from the protein to be detected, measured at j Two-dimensional analysis image data of micromass spectrometry for a plurality of types of peptide fragments derived from the protein to be detected by combining the peak intensities: P (M _fi + H ⁺ / Z: x ^M _j , y ^M _j ) Build up.

  Furthermore, two-dimensional analysis image data of microscopic mass analysis is similarly constructed for a plurality of types of peptide fragments derived from other proteins that are simultaneously observed at each irradiation spot: j.

The above-mentioned method for detecting the accidental peak overlap can detect, for example, the presence of a modified protein or a mutant protein in addition to the protein to be detected. It is said. Plural types of peptide fragments derived from modified proteins and mutant proteins, except for peptide fragments containing modified sites and mutation sites, and other peptide fragments derived from multiple types of proteins derived from the protein to be detected. It is the same as the peptide fragment. Accordingly, the relative ratio of the peak intensities P (M _fi + H ⁺ / Z: x ^M _j , y ^M _j ) of cationic species derived from a plurality of types of peptide fragments derived from the protein to be detected: P _rel (M Focusing on _fi + H ⁺ / Z), it seems that the relative ratio of the peptide fragments containing the site to be modified appears to decrease at first glance. When applying the criteria in the above method for detecting the accidental peak overlap, the peak positions (M _fi + H ⁺ / Z) are all determined to cause an accidental peak overlap. That is, it can be reasonably concluded that a modified protein or mutant protein having essentially the same amino acid sequence as the protein to be detected coexists.

  Multiple peptide fragments derived from various proteins that cover the entire target measurement area and are constructed after correcting the peak intensity at the irradiation spot where the above-mentioned incidental peak overlap occurs In contrast, two-dimensional analysis image data of microscopic mass analysis is stored in a data storage device attached to the image processing computer shown in FIG. In addition, the two-dimensional analysis image data of the micromass spectrometry for the plurality of types of peptide fragments derived from various proteins not subjected to the correction of the peak intensity is also the uncorrected two-dimensional analysis image data of the micromass analysis. In addition, the data is stored in a data storage device attached to the image processing computer shown in FIG.

Of course, the data of the mass spectrometry spectrum measured at each irradiation spot: j is combined with the positional information (x ^M _j , y ^M _j ) of each irradiation spot: j, and the “data of the mass spectrometry spectrum is the basis. As a set, it is stored in a data storage device attached to the image processing computer shown in FIG.

(Automatic positioning method between two types of two-dimensional images)
Next, regarding the automatic position superposition method of the two-dimensional analysis image of microscopic mass analysis and the two-dimensional visible image of optical microscope photography according to the present invention, the configuration of each form and the technical principle thereof are specifically described. Further details will be described with reference to these embodiments.

Of course, the two-dimensional analysis image of the microscopic mass analysis and the two-dimensional visible image of the optical microscope photographed by the above method are based on the measurement result obtained by measuring the same region of the pathological tissue section. In addition, efforts have been made to match the starting point (the origin of the coordinate axes) where each position information is described as much as possible, but as the measurement is performed using different measuring devices, both position information is described. The starting point (the origin of the coordinate axes) is slightly shifted. That is, in the two-dimensional analysis image data of the microscopic mass analysis, in the two-dimensional visible image data obtained by optical microscope photography, the starting point (the origin of the coordinate axes) of the position information (x ^M _j , y ^M _j ) of the irradiation spot: j , actual position: position information of _{^{k (x O k, y O}} k) origin (the origin of the coordinate axes) of relatively has _{^{deviated, (x M j, y M}} j) = (x O k + δx, y ^O _k + δy). Further, the degree of the deviation (δx, δy) differs in each pathological tissue section. The operation of aligning the deviations of the starting points of the position information of the two two-dimensional image data (the origin of the coordinate axes) corresponds to the image position overlapping operation.

Furthermore, the resolution of the two-dimensional analysis image of the microscopic mass analysis and the resolution of the two-dimensional visible image of the optical microscope photography are essentially different, and the position information of the two-dimensional visible image of the optical microscope photography having a high resolution ( The displacement (δx, δy) indicated by the positional information (x ^M _j , y ^M _j ) in the two-dimensional analysis image of the microscopic mass spectrometry with inferior resolution with reference to x ^O _k , y ^O _k ) The operation of automatically estimating corresponds to the image position overlapping operation.

In the automatic position superposition method according to the present invention, since the relative deviation (δx, δy) between the starting points of the position information (the origin of the coordinate axes) is automatically estimated, the first to fifth embodiments Then, each of the following means is adopted, that is, by adopting the following five forms as an artificial “alignment marker”, when performing automatically, the two two-dimensional images The alignment accuracy is improved.

  (1) In the first embodiment, either a two-dimensional visible image obtained by optical microscope photography or a two-dimensional analysis image obtained by microscopic mass spectrometry so that the tissue slice is included in the target measurement region on the preparation on which the tissue slice is mounted. Are provided with “alignment markers” having a predetermined planar shape made of “marker substances” that can be identified.

  (2) In the second embodiment, the tissue slice can be identified in a two-dimensional visible image taken by an optical microscope so that the tissue slice is included in the target measurement region on the prepared slide. In the two-dimensional analysis image, a “positioning mask” having a predetermined planar shape that does not show an identifiable mass analysis peak intensity is provided.

  (3) In the third embodiment, a predetermined position on the tissue slice is provided as a “positioning marker” having a predetermined planar shape provided so as to be included in the target measurement region on the preparation on which the tissue slice is mounted. A “region where the matrix agent is not applied” having a predetermined planar shape provided in the region is used.

  (4) In the fourth embodiment, a predetermined position on the tissue slice is provided as a “positioning marker” having a predetermined planar shape provided to be included in the target measurement region on the preparation on which the tissue slice is mounted. A “region not subjected to protease cleavage treatment” having a predetermined planar shape provided at the site is used.

  (5) In the fifth embodiment, either a two-dimensional visible image obtained by optical microscope photography or a two-dimensional analysis image obtained by microscopic mass spectrometry so that the tissue slice is included in the target measurement region on the preparation on which the tissue slice is mounted. The “endogenous alignment marker” that can be discriminated in FIG.

(First form)
In the first form of the automatic position superimposing method according to the present invention, a two-dimensional visible image of an optical microscope, a microscopic mass so that a tissue slice is included in a target measurement region on a prepared slide. A “positioning marker” having a predetermined planar shape, which is made of a “marker substance” that can be identified in any two-dimensional analysis image of analysis, is provided. Thereafter, the entire target measurement region including the “alignment marker” is measured to obtain two-dimensional visible image data captured by an optical microscope and two-dimensional analysis image data of microscopic mass analysis.

On the two-dimensional analysis image of the micromass analysis displayed based on the two-dimensional analysis image data of the micromass analysis, the “point S characterizing the planar shape” of the “alignment _marker ” is selected, and the micromass analysis based on the two-dimensional analysis image data, with respect to the "S _marker points characterizing the planar shape", on the two-dimensional analysis image of imaging mass spectrometry, the position information (x ^M _marker of the "S _marker points characterizing the planar shape", y ^M _marker ). Next, positional information (x ^O _marker , y ^O _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional visible image taken by the optical microscope, which is displayed based on the two-dimensional visible image data taken by the optical microscope. Image data of “alignment marker” having a predetermined planar shape on a two-dimensional analysis image of microscopic mass spectrometry and “alignment marker having a predetermined planar shape on a two-dimensional visible image taken by an optical microscope” Is estimated based on the image data.

Position information (x ^M _marker , y ^M _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis, and the estimated two-dimensional visible image of the optical microscope Position information satisfying (x ^M _marker , y ^M _marker ) = (x ^O _marker + δx, y ^O _marker + δy) from the position information (x ^O _marker , y ^O _marker ) of “point S _marker characterizing the planar shape” The relative deviation (δx, δy) between the starting points (the origin of the coordinate axes) is calculated.

Based on the calculated relative deviation (δx, δy), the position (x ^M _j , y ^M _j ) of each irradiation spot: j on the two-dimensional analysis image of the microscopic mass analysis is represented by the two-dimensional image of the optical microscope. Automatic position superposition is performed so as to match the position (x ^O _j , y ^O _j ) ≡ (x ^M _j −δx, y ^M _j −δy) on the visible image.

  The “alignment marker” having a predetermined planar shape made of a “marker substance” that can be identified in both a two-dimensional visible image taken by an optical microscope and a two-dimensional analytical image obtained by microscopic mass spectrometry is, for example, the following form: can do.

  First, in order to enable identification on a two-dimensional analysis image of microscopic mass spectrometry, the “marker substance” must be capable of mass spectrometry at the same time under the conditions for performing mass analysis of the protein to be detected. is there. Therefore, when protease analysis is performed, when mass analysis is performed simultaneously under the conditions for mass analysis of multiple types of peptide fragments derived from the protein to be detected, within the M / Z measurement range of the ionic species, It is necessary to apply the PMF method to give an ion species peak that can identify the presence of the “marker substance”. Specifically, it is preferable to employ a protein or polypeptide as the “marker substance”. More preferably, an exogenous protein or polypeptide that is essentially not present in the tissue slice is employed rather than an endogenous protein or polypeptide of the tissue cell that is present in the tissue slice.

  Proteins and polypeptides themselves generally do not have a color that can be identified on a two-dimensional visible image taken with an optical microscope. Therefore, “dye-labeled protein, dye-labeled polypeptide” with a dye label having a color distinguishable on a two-dimensional visible image taken with an optical microscope is used as a “marker substance”. Alternatively, a mixture of a protein and a polypeptide mixed with a dye is used as a “marker substance”. By applying this “marker substance” in a predetermined planar shape so as to have a predetermined surface density, a “positioning marker” is produced.

  Alternatively, a mode of non-specifically staining proteins and polypeptides used as “marker substances” using Coomassie blue (Coomassie blue or Coomassie brilliant blue, CBB) can also be used.

The “alignment marker” having a predetermined planar shape is found on the above-described two-dimensional visible image taken by the optical microscope. In the “characteristic structure identification” operation step, a “closed region” indicating a specific color tone is displayed. ". A group of positions indicating a closed curve, which outlines a “closed region” indicating a specific color tone, corresponding to this “alignment marker”: G ^O _{Marker-contour} {(x ^O _Marker-ck , y ^O _Marker-ck )} Is specified. Next, the inside of the outline of the “closed region” indicating this specific color tone, specified as a group of positions indicating a closed curve: G ^O _{Marker-contour} {(x ^O _Marker-ck , y ^O _Marker-ck )} The total number of pixels present in N: N ^O _Matker-size is specified. Size of the "alignment marker" having a predetermined planar shape: S ^O _Marker, the spacing between pixels: [Delta] x ^O and [Delta] y ^O, and the total number of pixels: using _{^{N O Matker-size, S O}} Marker = ( It is estimated that Δx ^O × Δy ^O ) · (N ^O _Matker-size +1).

At that time, on a two-dimensional analysis image of imaging mass spectrometry, the total number of radiation spots existing inside the "alignment marker" having the predetermined planar shape: N ^M _Marker-spot, the distance between the irradiation spots: [Delta] x Considering ^M and Δy ^M , it is estimated to be around S ^O _Marker / (Δx ^M × Δy ^M ).

On the other hand, an “alignment marker” having a predetermined planar shape is used as a “marker substance” on a two-dimensional analysis image of microscopic mass spectrometry, for example, a peptide fragment derived from an exogenous protein or polypeptide. It can be identified as a “closed region” in which the resulting ion species is specifically observed. Next, the total number of irradiation spots: N ^M _Marker-spot existing in this “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis is specified. Confirm that the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is around the previously estimated value: S ^O _Marker / (Δx ^M × Δy ^M ) Thus, it is verified that the “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis actually corresponds to the “positioning marker” having a predetermined planar shape. Specifically, the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is, for example, _3/2 · S ^O _Marker / (Δx ^M × Δy ^M ) to 1 / 2. When the criterion of S ^O _Marker / (Δx ^M × Δy ^M ) is satisfied, the identified “closed region” actually corresponds to the “alignment marker” having a predetermined planar shape That is verified.

At that time, on the two-dimensional analysis image of the microscopic mass analysis, the size of the “alignment marker” having a predetermined planar shape: S ^M _Marker is the interval between the irradiation spots: Δx ^M and Δy ^M , and the irradiation spot It is inferred that N ^M _Marker-spot can be used to approximate S ^M _Marker = (Δx ^M × Δy ^M ) · (N ^M _Matker-size + 1). The size “S ^M _Marker ” of the “alignment marker” having the estimated predetermined planar shape is also about the previously estimated value: S ^O _Marker / (Δx ^M × Δy ^M ). Specifically, the size of the “alignment marker” having a predetermined predetermined planar shape: a criterion that S ^M _Marker is, for example, a range of _3/2 · S ^O _Marker to 1/2 · S ^O _Marker If it satisfies, it is verified that the identified “closed region” actually corresponds to the “alignment marker” having a predetermined planar shape.

Further, on the two-dimensional visible image obtained by optical microscope photography, the position information of all the pixels existing in the outline of the planar shape of the “alignment marker” to be identified: (x ^O _Marker-k , y ^O _Marker-k ) is calculated as a simple average. This simple average: (x ^O _marker-k-av. , Y ^O _marker-k-av .) Can be regarded as position information of the center of gravity of the planar shape of the identified “alignment marker”. . Calculates the simple average of the position information (x ^M _Marker-j , y ^M _Marker-j ) of all irradiation spots that exist inside the “closed region” identified on the two-dimensional analysis image of microscopic mass spectrometry To do. This simple average: (x ^M _Marker-j-av. , Y ^M _Marker-j-av. ) Approximately represents the position of the center of gravity of the “closed region” to be identified. That is, using the relative deviation (δx, δy) between the origins of the position information (the origin of the coordinate axes), approximately (x ^M _Marker-j-av. , Y ^M _Marker-j-av. ) ≈ (X ^O _Marker-k-av. + Δx, y ^O _Marker-k-av. + Δy).

Is sufficiently high resolution two-dimensional analysis image itself imaging mass spectrometry, the total number of radiation spots existing inside the "closed region": if N ^M is sufficiently large _Marker-spot, accuracy of the approximation high It has become. The degree of approximation depends on the resolution of the two-dimensional analysis image itself of microscopic mass analysis, and includes the following “uncertainty” (approximation error).

The resolution of the two-dimensional analysis image itself of the microscopic mass analysis corresponds to the interval between the irradiation spots: Δx ^M and Δy ^M , and the outline of the identified “closed region” is “unsatisfied due to the resolution. Certainty ”. Specifically, the apparent contour of the identified "closed region", the group of radiation spots existing inside the "closed _{^{region": G M Marker-spot {(}} x M Marker-j, y M Marker _-j )} is displayed as the outer periphery. However, having a predetermined planar shape contour of "alignment marker", the group of radiation spots existing inside the "closed region": surrounding "outer periphery" of the G ^M _Marker-spot, from a "marker substance" It is determined that it is located between the irradiation spot where the peak intensity of the ion species to be measured is not substantially measured and the irradiation spot located on the outer periphery of the “closed region”. In other words, with respect to the apparent contour of the “closed region” identified, the contour of the “alignment marker” having a predetermined planar shape has a resolution, that is, an interval between the irradiation spots: Δx ^M and Δy ^M As a result, in some cases, “Δx ^M −d _L” to (Δx ^M −1 / 2d _L ), (Δy ^M −d _L ) to (Δy ^M −1 / 2d _L ) Sex ".

  However, “uncertainty” can be reduced by selecting the planar shape of the “alignment marker” as follows, for example.

For example, when the planar shape of the “alignment marker” is a rectangle, the shape is selected as (a × Δx ^M ) × (b × Δy ^M ). As an example, consider the case of selecting (3 × Δx ^M ) × (3 × Δy ^M ).

When the planar shape of the “alignment marker” is a rectangle of (3 × Δx ^M ) × (3 × Δy ^M ), the center of the irradiation spot matches the contour of the planar shape of the “alignment marker”. And, the area of the irradiation spot: ½ of 1/4 (π · d _L ² ) exists inside the “alignment marker”, but the other half is outside. At this time, the peak intensity of the ionic species derived from the “marker substance” observed at this irradiation spot is proportional to the area existing inside the “alignment marker”. Relative position of “irradiation spot center” located on the outer periphery of “closed region” with respect to the planar outline (boundary in the X-axis direction) of “alignment marker”: δx _relative and the area of the irradiation spot Among them, the relationship of the ratios included in the “alignment marker” shows the correspondence as follows.

As shown in Table 1-1, the relative position of the “irradiation spot center” located on the outer periphery of the “closed area” with respect to the planar outline of the “alignment marker” is −1 / 2 · d. when present in the range of _{L ~1} / 2 · d _L, the change in peak intensity in the irradiation spot 1,4 at both ends is observed. In other words, based on the change in peak intensity at the irradiation spots 1 and 4 at both ends, the “positioning marker” is positioned on the outer periphery of the “closed region” with respect to the planar contour (boundary in the X-axis direction). It is possible to estimate the relative position δx _{relative of the} “center of the irradiation spot”. Similarly, it is possible to estimate the relative position δy _relative of the “irradiation spot center” located on the outer periphery of the “closed region” with respect to the planar contour (boundary in the Y-axis direction) of the “alignment marker” It is.

When the planar shape of the “alignment marker” is a rectangle of (3 × Δx ^M ) × (3 × Δy ^M ), an “irradiation spot” positioned on the outer periphery of the “closed region” with respect to the outline of the rectangle It is estimated that the position of the “center of” is relatively biased by (δx _relative , δy _relative ).

The position information (x ^M _marker , y ^M _marker ) of the irradiation spot: j that is closest to the apex of the “alignment marker” having a rectangular shape on the two-dimensional analysis image of the microscopic mass analysis is an optical microscope image. Based on the position information (x ^O _{marker-corner} , y ^O _{marker-corner} ) of the “alignment marker” having the corresponding rectangular shape found on the two-dimensional visible image, (x ^O _{marker-corner} + δx _relative) , Y ^O _{marker-corner} + δy _relative ).

Therefore, the position information (x ^M _marker , y ^M _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis and the estimated two-dimensional visible image of the optical microscope From the positional information (x ^O _marker , y ^O _marker ) ≡ (x ^O _{marker-corner} + δx _relative , y ^O _{marker-corner} + δy _relative ) of the “point S _marker characterizing the planar shape”, (x ^M _marker , y ^M The relative deviation (δx, δy) between the origins of the position information (the origin of the coordinate axes) that satisfies _marker ) = (x ^O _marker + δx, y ^O _marker + δy) can be accumulated with high accuracy.

When generalized, based on the outline of the planar shape of the “alignment marker” specified on the two-dimensional visible image taken by the optical microscope, the interval between irradiation spots used for the two-dimensional analysis of the microscopic mass analysis: Δx ^{M Assuming} that the “ideal position” of the irradiation spot: (x ^O _marker-k-hyp , y ^O _marker-k-hyp ) located on the outer periphery of the planar shape of the “alignment marker” is Ions derived from the “marker substance” actually measured at the irradiation spot with reference to the “ideal position” (x ^O _marker-k-hyp , y ^O _marker-k-hyp ) of the irradiation spot Estimate _relative position variations (δx _relative , δy _relative ) that reproduce the peak intensity of the species with high accuracy. As a result, regarding the irradiation spot located on the outer periphery of the planar shape of the “alignment marker”, a reasonable estimated position on the two-dimensional visible image of the optical microscope is (x ^O _marker-k , y ^O _{marker- k} ) ≡ (x ^O _marker-k-hyp + δx _relative , y ^O _marker-k-hyp + δy _relative ) and is estimated with high accuracy.

Position information (x ^M _marker-j , y ^M _marker-j ) located on the outer periphery of the planar shape of the “alignment marker” on the two-dimensional analysis image of micromass spectrometry is reasonably estimated (X ^O _marker-k , y ^O _marker-k ) ≡ (x ^O _marker-k-hyp + δx _relative , y ^O _marker-k-hyp + Δy _relative ). After that, the relative deviation between the origins of the position information (the origin of the coordinate axes) satisfying (x ^M _marker-j , y ^M _marker-j ) = (x ^O _marker-k + δx, y ^O _marker-k + δy) (Δx, δy) can be accumulated with high accuracy.

However, the relative positional variation: (δx _relative , δy _relative ) shows a slight variation depending on a slight variation in the peak intensity measured at the irradiation spot. In order to eliminate the slight variation, the average of the _relative position variation: (δx _relative , δy _relative ) is calculated to be a representative value of the _relative position variation: (δx _relative , δy _relative ). Is also possible.

In the above description, when the planar shape of the “alignment marker” is a rectangle, the shape is selected as (a × Δx ^M ) × (b × Δy ^M ), for example, (3 × Δx ^M ) × The technical principle of the present invention is described with reference to the case of selecting (3 × Δy ^M ).

When estimating the relative position δx _relative of the “irradiation spot center” located on the outer periphery of the “closed region” with respect to the planar shape outline (boundary in the X-axis direction) of the “alignment marker”, the “alignment marker” The width in the X-axis direction: (a × Δx ^M ) is used in the planar shape. Furthermore, by providing a plurality of levels as the width in the X-axis direction, specifically, two levels (a × Δx ^M ) and (a × Δx ^M ) + d _L , “vernier” "Function can be added.

For example, when the width in the X-axis direction is (a × Δx ^M ) + d _L , the “irradiation spot of the irradiation spot” located on the outer periphery of the “closed region” with respect to the planar outline (boundary in the X-axis direction) When the arrangement is such that the “center” is a distance of 1/2 d _L , the peak intensity changes in the irradiation spots 1 and 4 at both ends shown in Table 1-2. When Table 1-2 is combined with Table 1-1, the “irradiation spot center” located on the outer periphery of the “closed region” with respect to the planar shape contour (boundary in the X-axis direction) of the “alignment marker” The accuracy in estimating the relative position δx _relative is improved.

In particular, the width in the X-axis direction includes a plurality of levels, specifically, (a × Δx ^M ), (a × Δx ^M ) + d _L , (a × Δx ^M ) +1/2. and doing, it is possible to add a ^{1/2 · Δx M> δx} relative> -1/2 · Δx M covers a whole range "vernier (vernier)" function.

Of course, the same applies when estimating the relative position δy _relative of the “irradiation spot center” located on the outer periphery of the “closed region” with respect to the planar contour (boundary in the Y-axis direction) of the “alignment marker”. In addition, it is desirable to provide a plurality of levels as the width in the Y-axis direction.

(Second form)
In the second form of the automatic position superposition method according to the present invention, the identification is made in the two-dimensional visible image of the optical microscope so that the tissue slice is included in the target measurement region on the prepared slide. In a two-dimensional analysis image of microscopic mass analysis, a “positioning mask” having a predetermined planar shape that does not show an identifiable mass analysis peak intensity is provided. Thereafter, the entire target measurement region including the “positioning mask” is measured to obtain two-dimensional visible image data obtained by optical microscope photography and two-dimensional analysis image data obtained by microscopic mass analysis.

On the 2D analysis image of the micromass analysis displayed based on the 2D analysis image data of the micromass analysis, the planar shape of the “alignment mask” is defined as an area that does not show the identifiable mass analysis peak intensity. Identify. Select "S _mask that characterize the planar shape" as specified, on the basis of two-dimensional analysis image data of imaging mass spectrometry, with respect to the "S _mask that characterize the planar shape", on the two-dimensional analysis image of imaging mass spectrometry , Position information (x ^M _mask , y ^M _mask ) of the “point S _mask characterizing the planar shape” is specified. Next, position information (x ^O _mask , y ^O _mask ) of the “point S _mask characterizing the planar shape” on the two-dimensional visible image taken by the optical microscope, which is displayed based on the two-dimensional visible image data taken by the optical microscope. Image data of a “positioning mask” having a predetermined planar shape on a two-dimensional analysis image of microscopic mass spectrometry and “positioning having a predetermined planar shape on a two-dimensional visible image taken by an optical microscope” Is estimated based on the image data of the “mask”.

Position information (x ^M _mask , y ^M _mask ) of the “point S _mask characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis, and the estimated two-dimensional visible image of the optical microscope Position information satisfying (x ^M _mask , y ^M _mask ) = (x ^O _mask + δx, y ^O _mask + δy) from the position information (x ^O _mask , y ^O _mask ) of the “point S _mask characterizing the planar shape” The relative deviation (δx, δy) between the starting points (the origin of the coordinate axes) is calculated.

Based on the calculated relative deviation (δx, δy), the position (x ^M _j , y ^M _j ) of each irradiation spot: j on the two-dimensional analysis image of the microscopic mass analysis is represented by the two-dimensional image of the optical microscope. Automatic position superposition is performed so as to match the position (x ^O _j , y ^O _j ) ≡ (x ^M _j −δx, y ^M _j −δy) on the visible image.

  A two-dimensional visible image taken with an optical microscope is identifiable, and in a two-dimensional analytical image obtained by microscopic mass spectrometry, an “alignment mask” having a predetermined planar shape and showing no identifiable mass analysis peak intensity is For example, it can be set as the following form.

  First, on the two-dimensional analysis image of the microscopic mass analysis, it is preferable that the following two situations indicate that no identifiable mass analysis peak intensity is shown.

  One of the situations is that, when performing mass spectrometry, the laser beam irradiated to perform non-selective desorption / ionization of peptide fragments derived from proteins or polypeptides does not reach the matrix agent. It is.

  For example, as a “positioning mask” having a predetermined planar shape, a method of providing a “coating film layer” made of a “light masking substance” that does not exhibit optical transparency to laser light can be used. Specifically, the “light-reflective coating film layer” made of a metal material exhibiting a high reflectance in the wavelength range of the laser beam to be irradiated is “positioned” having a predetermined planar shape in the second mode. It can be used as a “mask”. Alternatively, a “light-absorbing coating film layer” made of a material having a high extinction coefficient, which exhibits strong light absorption with respect to laser light and substantially eliminates absorption of laser light by the matrix agent, in the second embodiment It can be used as a “positioning mask” having a predetermined planar shape.

  The other is a situation in which, even when the irradiated laser light reaches the matrix agent, non-selective desorption / ionization of peptide fragments derived from proteins or polypeptides is not substantially achieved.

  For example, as a “positioning mask” having a predetermined planar shape, a “coating” made of a “physical masking substance” that shows optical transparency to laser light but prevents desorption of ionic species derived from peptide fragments. A method of providing a “film layer” can be used. Specifically, in the wavelength range of the irradiated laser beam, a “polymer coating film layer” made of a polymer material exhibiting sufficiently high light transmittance is formed in a predetermined planar shape in the second mode. It can also be used as a “positioning mask”.

  In the “coating film layer” consisting of “light masking material” that does not show optical transparency to laser light, use the “light masking material” that has a wavelength range where the light transmittance is reduced in the visible light region. Then, the “coating film layer” made of the “photomasking substance” can be identified in a two-dimensional visible image taken with an optical microscope.

  In addition, in the “coating film layer” made of “physical masking material” that exhibits optical transparency to laser light, as a “physical masking material”, there is a wavelength range in which the light transmittance decreases in the visible light region. When the existing one is used, the “coating film layer” made of the “physical masking substance” can be identified in a two-dimensional visible image taken by an optical microscope.

  The “coating film layer” composed of the “light masking substance” or the “coating film layer” composed of the “physical masking substance” is applied to the protease cutting treatment step and the matrix agent coating on the preparation on which the tissue slice is mounted. After finishing the process, it is formed in a predetermined region.

  Before the protease cleavage treatment step and the matrix agent coating step, and after the formation of the “coating film layer” composed of the “photomasking substance” or the “coating film layer” composed of the “physical masking substance”, Two-dimensional visible images taken by two types of optical microscopes are acquired.

  Except for the part where a “positioning mask” having a predetermined planar shape is provided, in the two-dimensional visible images taken by the two types of optical microscopes before and after, in the visible light region, there is a matrix agent coating film that exhibits light transmittance. There is only a slight change in the derived color tone. Therefore, the superposition of the positions of the two-dimensional visible images taken by the two types of front and rear optical microscopes can be performed very easily.

  The position of the “alignment mask” having a predetermined planar shape is based on a clear difference between its surroundings and color tone on a two-dimensional visible image taken by an optical microscope obtained after the step of forming the “alignment mask”. Specified.

Further, the “alignment marker” having a predetermined planar shape is found on the above-described two-dimensional visible image taken by the optical microscope, and shows a difference in specific color tone in the step of “identification of characteristic structure” operation. Identified as “closed area”. A group of positions indicating a closed curve, which forms an outline of a “closed region” indicating a difference in specific color tone, corresponding to this “alignment marker”: G ^O _{Marker-contour} {(x ^O _Marker-ck , y ^O _{Marker -ck} )} is specified. Next, the inside of the outline of the “closed region” indicating this specific color tone, specified as a group of positions indicating a closed curve: G ^O _{Marker-contour} {(x ^O _Marker-ck , y ^O _Marker-ck )} The total number of pixels present in N: N ^O _Matker-size is specified. Size of the "alignment marker" having a predetermined planar shape: S ^O _Marker, the spacing between pixels: [Delta] x ^O and [Delta] y ^O, and the total number of pixels: using _{^{N O Matker-size, S O}} Marker = ( It is estimated that Δx ^O × Δy ^O ) · (N ^O _Matker-size +1).

At that time, on a two-dimensional analysis image of imaging mass spectrometry, the total number of radiation spots existing inside the "alignment marker" having the predetermined planar shape: N ^M _Marker-spot, the distance between the irradiation spots: [Delta] x Considering ^M and Δy ^M , it is estimated to be around S ^O _Marker / (Δx ^M × Δy ^M ).

On the other hand, the “alignment marker” having a predetermined planar shape is, for example, a peak intensity of ionic species derived from a peptide fragment derived from a protein or polypeptide to be detected on a two-dimensional analysis image of microscopic mass spectrometry. Can be identified as a “closed region” where substantially no is observed. Next, the total number of irradiation spots: N ^M _Marker-spot existing in this “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis is specified. Confirm that the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is around the previously estimated value: S ^O _Marker / (Δx ^M × Δy ^M ) Thus, it is verified that the “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis actually corresponds to the “positioning marker” having a predetermined planar shape. Specifically, the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is, for example, _3/2 · S ^O _Marker / (Δx ^M × Δy ^M ) to 1 / 2. When the criterion of S ^O _Marker / (Δx ^M × Δy ^M ) is satisfied, the identified “closed region” actually corresponds to the “alignment marker” having a predetermined planar shape That is verified.

The resolution of the two-dimensional analysis image itself of the microscopic mass analysis corresponds to the interval between the irradiation spots: Δx ^M and Δy ^M , and the outline of the identified “closed region” is “unsatisfied due to the resolution. Certainty ”. Specifically, the apparent contour of the identified "closed region", the group of radiation spots existing inside the "closed _{^{region": G M Marker-spot {(}} x M Marker-j, y O Marker _-j )} is displayed as the outer periphery. However, the outline of the "alignment marker" having a predetermined planar shape, the group of radiation spots existing inside the "closed region": surrounding "outer periphery" of the G ^M _Marker-spot, from any peptide fragment It is determined that it is located between the irradiation spot where the peak intensity of the ion species is measured and the irradiation spot located on the outer periphery of the “closed region”. In other words, with respect to the apparent contour of the “closed region” identified, the contour of the “alignment marker” having a predetermined planar shape has a resolution, that is, an interval between irradiation spots: Δx ^M and Δy ^M Due to the maximum in some cases. “Uncertainty” of (Δx ^M −d _L ) to (Δx ^M −1 / 2d _L ) and (Δy ^M −d _L ) to (Δy ^M −1 / 2d _L ) is shown.

  However, “uncertainty” can be reduced by selecting the planar shape of the “alignment marker” as follows, for example.

For example, when the planar shape of the “alignment marker” is a rectangle, the shape is selected as (a × Δx ^M ) × (b × Δy ^M ). As an example, consider the case of selecting (3 × Δx ^M ) × (3 × Δy ^M ).

When the planar shape of the “alignment marker” is a rectangle of (3 × Δx ^M ) × (3 × Δy ^M ), the center of the irradiation spot matches the contour of the planar shape of the “alignment marker”. And, the area of the irradiation spot: ½ of 1/4 (π · d _L ² ) exists inside the “alignment marker”, but the other half is outside. At this time, the peak intensity of the ionic species derived from the peptide fragment observed in this irradiation spot is proportional to the area existing inside the “alignment marker”. Relative position of “irradiation spot center” located on the outer periphery of “closed region” with respect to the planar outline (boundary in the X-axis direction) of “alignment marker”: δx _relative and the area of the irradiation spot Among them, the relationship of the ratios included in the “alignment marker” shows the correspondence as follows.

As shown in Table 2-1, the relative position of the “center of the irradiation spot” located on the outer periphery of the “closed region” with respect to the planar outline of the “alignment marker” is −1 / 2 · d. when present in the range of _{L ~1} / 2 · d _L, the change in peak intensity in the irradiation spot 1,4 at both ends is observed. In other words, based on the change in peak intensity at the irradiation spots 1 and 4 at both ends, the “positioning marker” is positioned on the outer periphery of the “closed region” with respect to the planar contour (boundary in the X-axis direction). It is possible to estimate the relative position δx _{relative of the} “center of the irradiation spot”. Similarly, it is possible to estimate the relative position δy _relative of the “irradiation spot center” located on the outer periphery of the “closed region” with respect to the planar contour (boundary in the Y-axis direction) of the “alignment marker” It is.

When the planar shape of the “alignment marker” is a rectangle of (3 × Δx ^M ) × (3 × Δy ^M ), an “irradiation spot” positioned on the outer periphery of the “closed region” with respect to the outline of the rectangle It is estimated that the position of the “center of” is relatively biased by (δx _relative , δy _relative ).

The position information (x ^M _marker , y ^M _marker ) of the irradiation spot: j that is closest to the apex of the “alignment marker” having a rectangular shape on the two-dimensional analysis image of the microscopic mass analysis is an optical microscope image. Based on the position information (x ^O _{marker-corner} , y ^O _{marker-corner} ) of the “alignment marker” having the corresponding rectangular shape found on the two-dimensional visible image, (x ^O _{marker-corner} + δx _relative) , Y ^O _{marker-corner} + δy _relative ).

Therefore, the position information (x ^M _marker , y ^M _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis and the estimated two-dimensional visible image of the optical microscope From the positional information (x ^O _marker , y ^O _marker ) ≡ (x ^O _{marker-corner} + δx _relative , y ^O _{marker-corner} + δy _relative ) of the “point S _marker characterizing the planar shape”, (x ^M _marker , y ^M The relative deviation (δx, δy) between the origins of the position information (the origin of the coordinate axes) that satisfies _marker ) = (x ^O _marker + δx, y ^O _marker + δy) can be accumulated with high accuracy.

When generalized, based on the outline of the planar shape of the “alignment marker” specified on the two-dimensional visible image taken by the optical microscope, the interval between irradiation spots used for the two-dimensional analysis of the microscopic mass analysis: Δx ^{M Assuming} that the “ideal position” of the irradiation spot: (x ^O _marker-k-hyp , y ^O _marker-k-hyp ) located on the outer periphery of the planar shape of the “alignment marker” is Ions derived from the “marker substance” actually measured at the irradiation spot with reference to the “ideal position” (x ^O _marker-k-hyp , y ^O _marker-k-hyp ) of the irradiation spot Estimate _relative position variations (δx _relative , δy _relative ) that reproduce the peak intensity of the species with high accuracy. As a result, regarding the irradiation spot located on the outer periphery of the planar shape of the “alignment marker”, a reasonable estimated position on the two-dimensional visible image of the optical microscope is (x ^O _marker-k , y ^O _{marker- k} ) ≡ (x ^O _marker-k-hyp + δx _relative , y ^O _marker-k-hyp + δy _relative ) and is estimated with high accuracy.

Position information (x ^M _marker-j , y ^M _marker-j ) located on the outer periphery of the planar shape of the “alignment marker” on the two-dimensional analysis image of micromass spectrometry is reasonably estimated (X ^O _marker-k , y ^O _marker-k ) ≡ (x ^O _marker-k-hyp + δx _relative , y ^O _marker-k-hyp + Δy _relative ). After that, the relative deviation between the origins of the position information (the origin of the coordinate axes) satisfying (x ^M _marker-j , y ^M _marker-j ) = (x ^O _marker-k + δx, y ^O _marker-k + δy) (Δx, δy) can be accumulated with high accuracy.

However, the relative positional variation: (δx _relative , δy _relative ) shows a slight variation depending on a slight variation in the peak intensity measured at the irradiation spot. In order to eliminate the slight variation, the average of the _relative position variation: (δx _relative , δy _relative ) is calculated to be a representative value of the _relative position variation: (δx _relative , δy _relative ). Is also possible.

(Third form)
In a third form of the automatic positioning method according to the present invention, a “positioning marker having a predetermined planar shape provided to be included in a target measurement region on a preparation on which a tissue slice is mounted. The “region where the matrix agent is not applied” having a predetermined planar shape provided at a predetermined site on the tissue slice is used.

  Since the matrix agent is not applied to this “predetermined planar shape,” the matrix agent is not applied, so that desorption / ionization is not performed even when laser light irradiation is performed. On the analysis image, it is specified as a region that does not show an identifiable mass analysis peak intensity.

  In addition, the boundary between the “matrix agent non-application region” and the “matrix agent application region”, that is, the planar shape of the “matrix agent non-application region”, is a two-dimensional visible image taken with an optical microscope, measured after the matrix agent application. Can be identified.

  The entire measurement area including the “matrix agent non-application region” is measured before and after the matrix agent application, and two-dimensional visible image data of an optical microscope before the matrix agent application is measured. And the two-dimensional visible image data of the optical microscope photography after matrix agent application | coating are acquired.

  The image positions of the “two-dimensional visible image taken by an optical microscope before applying the matrix agent” and the “two-dimensional visible image taken by the optical microscope after applying the matrix agent” can be completely matched. At this time, when “color tone change” due to “matrix agent application” is extracted, “matrix agent non-application region” is specified as a region where “color tone change” does not substantially exist.

Is displayed on the basis of two-dimensional analysis image data of imaging mass spectrometry, on a two-dimensional analysis image of imaging mass spectrometry, the "matrix agent-coated area" type "alignment markers" of "S points characterizing the planar shape _marker The "point S _marker characterizing the planar shape" on the two-dimensional analytical image of the microscopic mass analysis with respect to the "point S _marker characterizing the planar shape" based on the two-dimensional analytical image data of the microscopic mass analysis. Position information (x ^M _marker , y ^M _marker ) is specified. Next, positional information (x ^O _marker , y ^O _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional visible image taken by the optical microscope, which is displayed based on the two-dimensional visible image data taken by the optical microscope. Image data of “alignment marker” having a predetermined planar shape on a two-dimensional analysis image of microscopic mass spectrometry and “alignment marker having a predetermined planar shape on a two-dimensional visible image taken by an optical microscope” Is estimated based on the image data.

Position information (x ^M _marker , y ^M _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis, and the estimated two-dimensional visible image of the optical microscope Position information satisfying (x ^M _marker , y ^M _marker ) = (x ^O _marker + δx, y ^O _marker + δy) from the position information (x ^O _marker , y ^O _marker ) of “point S _marker characterizing the planar shape” The relative deviation (δx, δy) between the starting points (the origin of the coordinate axes) is calculated.

Based on the calculated relative deviation (δx, δy), the position (x ^M _j , y ^M _j ) of each irradiation spot: j on the two-dimensional analysis image of the microscopic mass analysis is represented by the two-dimensional image of the optical microscope. Automatic position superposition is performed so as to match the position (x ^O _j , y ^O _j ) ≡ (x ^M _j −δx, y ^M _j −δy) on the visible image.

  The position of the “alignment mask” having a predetermined planar shape is compared with the “two-dimensional visible image taken by optical microscope before applying the matrix agent” and “two-dimensional visible image taken by optical microscope after applying the matrix agent”. Then, when “color tone change” due to “matrix agent application” is extracted, “matrix agent non-application region” is specified as a region where “color tone change” does not substantially exist.

Further, the “alignment marker” having a predetermined planar shape is found on the above-described two-dimensional visible image taken by the optical microscope, and is caused by “matrix agent application” in the process of “identification of characteristic structure” operation. It is identified as a “closed area” that does not show “color change”. A group of positions showing a closed curve, which forms an outline of a “closed region” that does not show a “color tone change” caused by “matrix agent application”, corresponding to this “positioning marker”: G ^O _{Marker-contour} {( x ^O _Marker-ck , y ^O _Marker-ck )} is specified. Next, a “color change” caused by this “matrix agent application” specified as a group of positions indicating a closed curve: G ^O _{Marker-contour} {(x ^O _Marker-ck , y ^O _Marker-ck )} The total number of pixels existing in the outline of the “closed area” not shown: N ^O _Matker-size is specified. Size of the "alignment marker" having a predetermined planar shape: S ^O _Marker, the spacing between pixels: [Delta] x ^O and [Delta] y ^O, and the total number of pixels: using _{^{N O Matker-size, S O}} Marker = ( It is estimated that Δx ^O × Δy ^O ) · (N ^O _Matker-size +1).

At that time, on a two-dimensional analysis image of imaging mass spectrometry, the total number of radiation spots existing inside the "alignment marker" having the predetermined planar shape: N ^M _Marker-spot, the distance between the irradiation spots: [Delta] x Considering ^M and Δy ^M , it is estimated to be around S ^O _Marker / (Δx ^M × Δy ^M ).

On the other hand, the “alignment marker” having a predetermined planar shape is, for example, a peak intensity of ionic species derived from a peptide fragment derived from a protein or polypeptide to be detected on a two-dimensional analysis image of microscopic mass spectrometry. Can be identified as a “closed region” where substantially no is observed. Next, the total number of irradiation spots: N ^M _Marker-spot existing in this “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis is specified. Confirm that the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is around the previously estimated value: S ^O _Marker / (Δx ^M × Δy ^M ) Thus, it is verified that the “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis actually corresponds to the “positioning marker” having a predetermined planar shape. Specifically, the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is, for example, _3/2 · S ^O _Marker / (Δx ^M × Δy ^M ) to 1 / 2. When the criterion of S ^O _Marker / (Δx ^M × Δy ^M ) is satisfied, the identified “closed region” actually corresponds to the “alignment marker” having a predetermined planar shape That is verified.

  A series of operations for estimating the relative deviation (δx, δy) between the starting points of the subsequent position information (the origin of the coordinate axes) is substantially the same as in the second embodiment.

By applying this series of operations, the position information (x ^M _marker-j , y ^M _{marker) of the} irradiation spot located on the outer periphery of the planar shape of the “alignment marker” on the two-dimensional analysis image of microscopic mass spectrometry _-j ) and position information (x ^O _marker-k , y ^O _marker-k ) ≡ (x ^O _marker-k-hyp ) on a reasonably estimated two-dimensional visible image taken by an optical microscope + Δx _relative , y ^O _marker-k-hyp + δy _relative ). After that, the relative deviation between the origins of the position information (the origin of the coordinate axes) satisfying (x ^M _marker-j , y ^M _marker-j ) = (x ^O _marker-k + δx, y ^O _marker-k + δy) (Δx, δy) can be accumulated with high accuracy.

(Fourth form)
In a fourth form of the automatic position superimposing method according to the present invention, a “positioning marker having a predetermined planar shape provided to be included in a target measurement region on a preparation on which a tissue slice is mounted. As “,” a “region not subjected to protease cleavage treatment” having a predetermined planar shape provided at a predetermined site on the tissue slice is used.

  In this “predetermined planar shape, protease cleavage untreated region”, since the protease cleavage treatment is not performed, the ionization efficiency of the protein having a large molecular weight itself is low, and the peak position (M / Z) exceeds the molecular weight range of a normal measurement object, and is usually specified as a region that does not show a substantially distinguishable mass analysis peak intensity on a two-dimensional analysis image of microscopic mass spectrometry.

  For example, the "protease cleavage treatment unexecuted region" is a "coating protective film" having a predetermined planar shape in advance at a predetermined site on the tissue slice, and then subjecting the entire surface of the tissue slice to protease cleavage treatment, Form. In the region covered with the “coating protective film”, the protease cannot act on the protein present in the tissue slice, and as a result, the region is not subjected to protease cleavage treatment.

  Prior to the protease cleavage treatment, the entire target measurement area is measured before the formation of the “coating protective film” and after the formation of the “coating protective film”. Two-dimensional visible image data of photographing and two-dimensional visible image data of photographing with an optical microscope after forming the “coating protective film” are acquired.

  In addition, the formation part of “coating protective film”: “protease cutting treatment unexecuted region” and the “coating protective film” non-forming part: “protease cutting processed region”, ie, the planar shape of “coating protective film” Can be identified in a two-dimensional visible image taken by light microscopy, measured prior to protease cleavage.

  The image positions of the “two-dimensional visible image taken by the optical microscope before forming the protective coating” and the “two-dimensional visible image taken by the optical microscope after forming the protective coating” can be completely matched. . At that time, when “color change” caused by “coating protective film” is extracted, “coating protective film” unformed part: “protease cleaved region” is an area where “color change” is not substantially present. As specified. As a result, the planar shape of the formation part of the “coating protective film”: “protease cleavage treatment unexecuted region” is also specified.

On the two-dimensional analysis image of the micromass analysis displayed based on the two-dimensional analysis image data of the micromass analysis, the “point S characterizing the planar shape” of the “alignment marker” of the “protease cleavage treatment unexecuted region” type _marker "is selected, based on the two-dimensional analysis image data of imaging mass spectrometry, with respect to the" S _marker points characterizing the planar shape ", on the two-dimensional analysis image of imaging mass spectrometry, S _marker that characterizes the" planar shape Position information (x ^M _marker , y ^M _marker ). Next, positional information (x ^O _marker , y ^O _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional visible image taken by the optical microscope, which is displayed based on the two-dimensional visible image data taken by the optical microscope. Image data of “alignment marker” having a predetermined planar shape on a two-dimensional analysis image of microscopic mass spectrometry and “alignment marker having a predetermined planar shape on a two-dimensional visible image taken by an optical microscope” Is estimated based on the image data.

Position information (x ^M _marker , y ^M _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis, and the estimated two-dimensional visible image of the optical microscope Position information satisfying (x ^M _marker , y ^M _marker ) = (x ^O _marker + δx, y ^O _marker + δy) from the position information (x ^O _marker , y ^O _marker ) of “point S _marker characterizing the planar shape” The relative deviation (δx, δy) between the starting points (the origin of the coordinate axes) is calculated.

Based on the calculated relative deviation (δx, δy), the position (x ^M _j , y ^M _j ) of each irradiation spot: j on the two-dimensional analysis image of the microscopic mass analysis is represented by the two-dimensional image of the optical microscope. Automatic position superposition is performed so as to match the position (x ^O _j , y ^O _j ) ≡ (x ^M _j −δx, y ^M _j −δy) on the visible image.

  The position of the “positioning mask” having a predetermined planar shape corresponds to the position where the “coating protective film” is formed. In addition, the formation part of “coating protective film”: “protease cutting treatment unexecuted region” and the “coating protective film” non-forming part: “protease cutting processed region”, ie, the planar shape of “coating protective film” Can be identified in a two-dimensional visible image taken by light microscopy, measured prior to protease cleavage.

  The image positions of the “two-dimensional visible image taken by the optical microscope before forming the protective coating” and the “two-dimensional visible image taken by the optical microscope after forming the protective coating” can be completely matched. . At that time, when “color change” caused by “coating protective film” is extracted, “coating protective film” unformed part: “protease cleaved region” is an area where “color change” is not substantially present. As specified. As a result, the planar shape of the formation part of the “coating protective film”: “protease cleavage treatment unexecuted region” is also specified.

Furthermore, the “alignment marker” having a predetermined planar shape is found on the above-described two-dimensional visible image taken by the optical microscope, and is caused by the “coating protective film” in the process of “identification of characteristic structure” operation. Is identified as a “closed area” indicating some “color change”. A group of positions indicating a closed curve, which forms an outline of a “closed region” indicating some “change in color tone” caused by the “coating protective film”, corresponding to this “positioning marker”: G ^O _{Marker- The contour} {(x ^O _Marker-ck , y ^O _Marker-ck )} is specified. Then, the group of position indicating a closed _{^{curve: G O Marker-contour {(}} x O Marker-ck, y O Marker-ck)} are identified as due to the "covering layer", a change in some "color The total number of pixels existing in the outline of the “closed area” indicating “N ^O _{Matker-size”} is specified. Size of the "alignment marker" having a predetermined planar shape: S ^O _Marker, the spacing between pixels: [Delta] x ^O and [Delta] y ^O, and the total number of pixels: using _{^{N O Matker-size, S O}} Marker = ( It is estimated that Δx ^O × Δy ^O ) · (N ^O _Matker-size +1).

At that time, on a two-dimensional analysis image of imaging mass spectrometry, the total number of radiation spots existing inside the "alignment marker" having the predetermined planar shape: N ^M _Marker-spot, the distance between the irradiation spots: [Delta] x Considering ^M and Δy ^M , it is estimated to be around S ^O _Marker / (Δx ^M × Δy ^M ).

On the other hand, the “alignment marker” having a predetermined planar shape is, for example, a peak intensity of ionic species derived from a peptide fragment derived from a protein or polypeptide to be detected on a two-dimensional analysis image of microscopic mass spectrometry. Can be identified as a “closed region” where substantially no is observed. Next, the total number of irradiation spots: N ^M _Marker-spot existing in this “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis is specified. Confirm that the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is around the previously estimated value: S ^O _Marker / (Δx ^M × Δy ^M ) Thus, it is verified that the “closed region” identified on the two-dimensional analysis image of the microscopic mass analysis actually corresponds to the “positioning marker” having a predetermined planar shape. Specifically, the total number of irradiation spots existing in the identified “closed region”: N ^M _Marker-spot is, for example, _3/2 · S ^O _Marker / (Δx ^M × Δy ^M ) to 1 / 2. When the criterion of S ^O _Marker / (Δx ^M × Δy ^M ) is satisfied, the identified “closed region” actually corresponds to the “alignment marker” having a predetermined planar shape That is verified.

  A series of operations for estimating the relative deviation (δx, δy) between the starting points of the subsequent position information (the origin of the coordinate axes) is substantially the same as in the second embodiment.

By applying this series of operations, the position information (x ^M _marker-j , y ^M _{marker) of the} irradiation spot located on the outer periphery of the planar shape of the “alignment marker” on the two-dimensional analysis image of microscopic mass spectrometry _-j ) and position information (x ^O _marker-k , y ^O _marker-k ) ≡ (x ^O _marker-k-hyp ) on a reasonably estimated two-dimensional visible image taken by an optical microscope + Δx _relative , y ^O _marker-k-hyp + δy _relative ). After that, the relative deviation between the origins of the position information (the origin of the coordinate axes) satisfying (x ^M _marker-j , y ^M _marker-j ) = (x ^O _marker-k + δx, y ^O _marker-k + δy) (Δx, δy) can be accumulated with high accuracy.

(Fifth form)
In the fifth form of the automatic position superposition method according to the present invention, a two-dimensional visible image obtained by optical microscopy, a microscopic mass so that the tissue slice is included in the target measurement region on the prepared slide. An “endogenous alignment marker” that can be identified in any two-dimensional analysis image of the analysis is set. Thereafter, measurement is performed on the entire target measurement region including the set “endogenous alignment marker”, and two-dimensional visible image data of optical microscope photography, two-dimensional analysis image data of microscopic mass analysis, and To get.

As the "endogenous alignment marker" to be set, it is commonly found in cells found on a two-dimensional visible image obtained by light microscopy on the surface of a tissue slice, and the above-mentioned "characteristic structure identification" Adopt a nucleus that can be located by operation. Specifically, on the surface of the tissue slice, a plurality of nuclei satisfying the following selection criteria are selected from the nuclei of the cell group found on the two-dimensional visible image taken by optical microscope, and the plurality of the nuclei are selected. Is set as the “endogenous alignment marker”.

  On the other hand, on the two-dimensional analysis image of the microscopic mass analysis, as a means for identifying a plurality of nuclei (indicator proteins) set as “endogenous alignment markers”, a large number of commonly existing cells Among the endogenous proteins, a cytoplasmic protein that is abundant and is present in principle in the cytoplasm but not in the nucleus is selected. That is, a plurality of nuclei set as “endogenous alignment markers” are “closed” in which the peak intensity of ionic species derived from “index protein” is not substantially measured in the cells divided by each cell membrane. Can be identified.

First, a “closed region” in which the peak intensity of the ionic species derived from the “index protein” is not substantially measured is specified in a cell classified by each cell membrane on a two-dimensional analysis image of microscopic mass spectrometry. In this step, it is confirmed that there is a region where the peak intensity of the ionic species derived from the “index protein” is significantly measured so as to surround the identified “closed region”. Next, the size of the approximate "closed region" specified: calculating the S ^M _N.

One of the methods is the following operation. First, the total number of irradiation spots: N ^M _N-spot that is present in each identified “closed region” and in which the peak intensity of the ionic species derived from the “index protein” is not substantially measured is identified. . Using the approximate size of identified “closed region”: S ^M _N , the spacing between irradiated spots: Δx ^M and Δy ^M , and the total number of identified irradiated spots: N ^M _{N-spot Let} ^M _N = (Δx ^M × Δy ^M ) · (N ^M _N-spot +1).

On the other hand, the “position information of the contour of the nuclear membrane” identified by the “identification of characteristic structure” operation found on the above-described two-dimensional visible image of the optical microscope image, that is, a group of positions indicating a closed curve: Gnuclear _{^{{(x O nuclear-k,}} y O nuclear-k)} "closed region" surrounded by, i.e., the average size of the nucleus to calculate the S ^O _N.

One of the methods is the following operation. First, an average total number of pixels: N ^O _N-size existing in the “closed region” specified as “the contour of the nuclear envelope” is specified. Using the average size of the nuclei: S ^O _N , the spacing between pixels: Δx ^O and Δy ^O , and the average total number of pixels: N ^O _N-size , S ^O _N = (Δx ^O × Δy ^O ) · (N ^O _N-size +1).

On the two-dimensional analysis image of the microscopic mass analysis, those that satisfy the following criteria are selected from the group of “closed regions” that are identified as having a high probability of being “nuclei”. Calculation has been the size of the approximate identified "closed region": S ^M _N is to screen those in the range of _{^{S O N> S M N>}} 1/2 · S O N. It is determined that the identified “closed region” selected by the secondary criterion also corresponds to the average nucleus size.

Calculation has been average nuclear size: Considering S ^O _N, on a two-dimensional analysis image of imaging mass spectrometry, the maximum value of the total number of radiation spots present in the nucleus: _N ^M _N-spot-MAX is _{^{N M N-spot-MAX =}} MOD (S O N / (Δx M × Δy M)) and estimated. Therefore, among the irradiation spots existing inside the nucleus, the total number of irradiation spots in which the peak intensity of the ion species derived from the “index protein” is not substantially measured: N ^M _N-spot is N ^M _N-spot-MAX Below, the probability of being in the range of 1/2 · N ^M _N-spot-MAX or higher is extremely high.

Among the group of identified “closed regions” selected by the above secondary criteria, the total number of irradiation spots: the upper 4 to 8 in descending order of N ^M _N-spot , A plurality of “nuclei” set as “markers” are selected.

  A plurality of “nuclei” selected as “endogenous alignment markers” on a two-dimensional analysis image of microscopic mass spectrometry, a plurality of corresponding “ Identify "nuclear".

  The planar shape of the “nucleus” can be generally regarded as “circular”. Accordingly, when the planar shape of the “nucleus” is approximated to “circular”, the “center” of the nucleus can be regarded as the position of the center of the “circular”.

“Position information of the contour of the nuclear membrane” identified on the two-dimensional visible image taken with an optical microscope, that is, a group of positions indicating a closed curve: G _{nuclear-contour} {(x ^O _nuclear-ck , y ^O _nuclear-ck ) }, For a “closed region” surrounded by}, the center when the shape is approximated as “circular” is the “center” of the “core”. Alternatively, “position information of the contour of the nuclear membrane”, that is, a group of positions indicating a closed curve: G _{nuclear-contour} {(x ^O _nuclear-ck , y ^O _nuclear-ck )} describes the position of “circumference” Then assuming, this _{group: G nuclear-contour {(x} O nuclear-ck, y O nuclear-ck)} simple average of _{^{(x O nuclear-ck-av}} , y O nuclear-ck-av) and the estimated It is also possible to set the center position.

On a two-dimensional visual image of the optical microscope imaging, the present within the "outline of the nuclear membrane", the position information of all the pixels _{^{(x O nuclear-k, y}} O nuclear-k) simple average of _(x ^O nuclear- _k-av , y ^O _nuclear-k-av ) corresponds to the position of the center of gravity of the “closed region” surrounded by the “outer membrane contour”. When the shape is approximated to “circular”, the center of gravity coincides with the center of the circle. Thus, the simple average _{^{(x O nuclear-k-av}} , y O nuclear-k-av) , and it is also possible to position of the center to be estimated.

Based on the outer shape of the “closed region” that was inferred as “nucleus” on the two-dimensional analysis image of micromass spectrometry, the center when the outer shape is approximated to “circular” is the “nucleus”. It is also possible to be the “center”. However, when the resolution determined by the interval between the irradiation spots: Δx ^M and Δy ^M is not high, it is easy to specify the outer shape of the “closed region” of the total number of irradiation spots: N ^M _N-spot with considerable accuracy. Not. The total number of radiation spots: the center of the "closed region" of _N ^M _N-spot, the total number: simple average of the position of the irradiation spot of the _{^{N M N-spot (x M}} nuclear-j, y M nuclear-j) ( x ^M _nuclear-j-av , y ^M _nuclear-j-av ) can be set as the estimated center position.

The position of the “center” of a plurality of “nuclei” selected as the “endogenous alignment marker” on the two-dimensional analysis image of micromass spectrometry, for example, (x ^M _nuclear-j-av , y ^M _{nuclear -j-av} ) is used to identify the relative relationship between the “center” positions of multiple “nuclei”. For example, as a polygon whose apex is the position of the “center” of a plurality of “nuclei” selected as the “alignment marker”, for example, (x ^M _nuclear-j-av , y ^M _nuclear-j-av ) The relative relationship can be expressed.

The position of the “center” of the “nucleus”, for example, (x ^O _nuclear-k-av , y ^O _nuclear-k-av ) To identify the relative relationship between the “center” positions of a plurality of “nuclei”. For example, the position of the “center” of a plurality of “nuclei” selected as the “endogenous alignment marker”, for example, (x ^O _nuclear-k-av , y ^O _nuclear-k-av ) is the vertex. The relative relationship can be expressed as a polygon.

With regard to the “nucleus” specified on the two-dimensional visible image taken by the optical microscope, the above (x ^M _nuclear-j-av , y ^M _nuclear-j-av ) is the vertex at the center of the “nucleus”. One vertex of the square is arranged, and it is determined whether or not a “nucleus” exists at the position of the other vertex. A plurality of “nuclei” satisfying this criterion is identified as a corresponding plurality of “nuclei” on the two-dimensional visible image taken by the optical microscope.

The position of the “center” of a plurality of corresponding “nuclei” on the two-dimensional visible image taken by the optical microscope identified by the above method, for example, (x ^O _nuclear-k-av , y ^O _nuclear-k and polygonal shape whose vertices _-av), verify that the criteria _{^{(x M nuclear-j-av}} , the polygonal shape with the y ^M _{nuclear-j-av)} vertices substantially equal to To do.

The actual position of the “center” of a plurality of “nuclei” selected as “endogenous alignment markers” on the two-dimensional analysis image of micromass spectrometry: (x ^M _{nuclear-j-center} , y ^M _nuclear for _-j-center), the approximation error of the approximate location _{^{(x M nuclear-j-av}} , y M nuclear-j-av) are usually the spacing between irradiation spots: not exceeding about [Delta] x ^M and [Delta] y ^M . In consideration of this approximation error, for example, the shape of a polygon having (x ^O _nuclear-k-av , y ^O _nuclear-k-av ) as a vertex and the above-mentioned criterion (x ^M _nuclear-j-av , Verify that the polygonal shapes with y ^M _nuclear-j-av ) as the vertex are substantially equal.

On the 2D analysis image of the micromass analysis displayed based on the 2D analysis image data of the micromass analysis, the “point S _marker characterizing the planar shape” of the “endogenous alignment _marker ” is selected and based on the two-dimensional analysis image data of mass spectrometry, with respect to the "S _marker points characterizing the planar shape", on the two-dimensional analysis image of imaging mass spectrometry, the position information (x ^M of the "S _marker points characterizing the planar shape" _marker , y ^M _marker ). Next, positional information (x ^O _marker , y ^O _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional visible image taken by the optical microscope, which is displayed based on the two-dimensional visible image data taken by the optical microscope. The image data of the “endogenous alignment marker” having a known planar shape on the two-dimensional analysis image of the microscopic mass analysis and the predetermined planar shape on the two-dimensional visible image of the optical microscope image “ It is estimated based on the image data of the “alignment marker”.

Position information (x ^M _marker , y ^M _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis, and the estimated two-dimensional visible image of the optical microscope Position information satisfying (x ^M _marker , y ^M _marker ) = (x ^O _marker + δx, y ^O _marker + δy) from the position information (x ^O _marker , y ^O _marker ) of “point S _marker characterizing the planar shape” The relative deviation (δx, δy) between the starting points (the origin of the coordinate axes) is calculated.

Based on the calculated relative deviation (δx, δy), the position (x ^M _j , y ^M _j ) of each irradiation spot: j on the two-dimensional analysis image of the microscopic mass analysis is represented by the two-dimensional image of the optical microscope. Automatic position superposition is performed so as to match the position (x ^O _j , y ^O _j ) ≡ (x ^M _j −δx, y ^M _j −δy) on the visible image.

  A plurality of “nuclei” selected as “endogenous alignment markers” on the two-dimensional analysis image of the microscopic mass analysis by the selection procedure described above are substantially in the two-dimensional visible image of the optical microscope. Is identifiable, and corresponds to a “positioning marker” having a known planar shape and showing no identifiable mass analysis peak intensity in a two-dimensional analysis image of microscopic mass spectrometry.

Therefore, by applying the technical principle described in the second embodiment, it is positioned on the outer periphery of the “closed region” with respect to the planar outline (boundary in the X-axis direction) of the “alignment marker”. It is possible to estimate the relative position δx _{relative of} “the center of the irradiation spot”. Similarly, it is possible to estimate the relative position δy _relative of the “irradiation spot center” located on the outer periphery of the “closed region” with respect to the planar contour (boundary in the Y-axis direction) of the “alignment marker” It is.

Since the planar shape of the “endogenous alignment marker” is specified in the two-dimensional visible image taken by the optical microscope, the “irradiation spot of the irradiation spot” located on the outer periphery of the “closed region” with respect to the known contour. It is estimated that the position of the “center” is relatively biased by (δx _relative , δy _relative ).

The position information (x ^M _marker , y ^M _marker ) of the irradiation spot: j closest to the contour of the “endogenous alignment marker” having a known shape on the two-dimensional analysis image of the microscopic mass analysis is With reference to position information (x ^O _{marker-corner} , y ^O _{marker-corner} ) on the contour of the “alignment marker” having the corresponding contour shape found on the two-dimensional visible image taken by the optical microscope, (x ^O _{marker-corner} + δx _relative , y ^O _{marker-corner} + δy _relative ).

Therefore, the position information (x ^M _marker , y ^M _marker ) of the “point S _marker characterizing the planar shape” on the two-dimensional analysis image of the microscopic mass analysis and the estimated two-dimensional visible image of the optical microscope From the positional information (x ^O _marker , y ^O _marker ) ≡ (x ^O _{marker-corner} + δx _relative , y ^O _{marker-corner} + δy _relative ) of the “point S _marker characterizing the planar shape”, (x ^M _marker , y ^M The relative deviation (δx, δy) between the origins of the position information (the origin of the coordinate axes) that satisfies _marker ) = (x ^O _marker + δx, y ^O _marker + δy) can be accumulated with high accuracy.

When generalized, based on the outline of the planar shape of the “alignment marker” specified on the two-dimensional visible image taken by the optical microscope, the interval between irradiation spots used for the two-dimensional analysis of the microscopic mass analysis: Δx ^M and based on [Delta] y ^M, located on the outer periphery of the planar shape of "endogenous alignment marker", "ideal position" of radiation spots: a _{^{(x O marker-k-hyp}} , y O marker-k-hyp) Assume. Based on the “ideal position” of the irradiation spot (x ^O _marker-k-hyp , y ^O _marker-k-hyp ), ions derived from “labeled protein” actually measured at the irradiation spot Estimate _relative position variations (δx _relative , δy _relative ) that reproduce the peak intensity of the species with high accuracy. As a result, regarding the irradiation spot located on the outer periphery of the planar shape of the “endogenous alignment marker”, a reasonable estimated position on the two-dimensional visible image of the optical microscope is (x ^O _marker-k , y ^O _marker-k ) ≡ (x ^O _marker-k-hyp + δx _relative , y ^O _marker-k-hyp + δy _relative ).

Position information (x ^M _marker-j , y ^M _marker-j ) on the outer periphery of the planar shape of the “endogenous alignment marker” on the two-dimensional analysis image of micromass spectrometry and rational (X ^O _marker-k , y ^O _marker-k ) ≡ (x ^O _marker-k-hyp + δx _relative , y ^O _{marker- k-hyp} + δy _relative ). After that, the relative deviation between the origins of the position information (the origin of the coordinate axes) satisfying (x ^M _marker-j , y ^M _marker-j ) = (x ^O _marker-k + δx, y ^O _marker-k + δy) (Δx, δy) can be accumulated with high accuracy.

However, the relative positional variation: (δx _relative , δy _relative ) shows a slight variation depending on a slight variation in the peak intensity measured at the irradiation spot. In order to eliminate the slight variation, the average of the _relative position variation: (δx _relative , δy _relative ) is calculated to be a representative value of the _relative position variation: (δx _relative , δy _relative ). Is also possible.

  In the above description, an example of using a “labeled protein” selected from an endogenous protein that is commonly present in cells as an identification means when specifying an “endogenous alignment marker” is used as an example. For

  In the case of an endogenous biological substance that shows a peak at a specific M / Z position on the mass spectrometry spectrum measured by the MALDI-TOF-MS method, instead of the “labeled protein” selected from the endogenous protein , And can be used as a means for identifying the “endogenous alignment marker”.

  When applying the MALDI-TOF-MS method, for example, an endogenous biological substance that is firmly immobilized on a cell membrane needs to be desorbed and desorbed / ionized, and is generally ionized. The efficiency is quite low. Considering this point, when using an endogenous biological substance that is firmly immobilized on the cell membrane, it is preferable to eliminate the immobilization in advance to eliminate the desorption / ionization failure.

  A membrane-bound endogenous protein, for example, a transmembrane-type endogenous protein, is usually subjected to protease cleavage treatment to produce a plurality of peptide fragments that do not normally show a membrane-bound fragment. Some are included. Therefore, a membrane-bound endogenous protein, for example, a transmembrane endogenous protein, utilizes a protease cleavage process, and the peptide fragment itself is desorbed / desorbed when the MALDI-TOF-MS method is applied. In terms of ionization, there is no obstacle.

  On the other hand, phospholipids and the like contained in the constituent elements of the cell membrane itself are in a state of low ionization efficiency when the treatment for detaching from the cell membrane tissue is not performed in advance. If solubilization treatment is performed on the entire cell membrane tissue, it becomes difficult to specify the shape of the cell, so that the entire cell membrane tissue is not solubilized, and phospholipids contained in the components of the cell membrane itself can be used. It is desirable to employ a solubilizing process. Of course, phospholipids that have not been taken up by membrane tissues such as cell membranes are in a state of no obstacle in terms of desorption / ionization when the MALDI-TOF-MS method is applied.

  In addition to the first to fifth embodiments described above, the same effect can be achieved by adopting the following embodiments.

(Sixth form)
In a sixth form of the automatic positioning method according to the present invention, a “positioning marker having a predetermined planar shape provided to be included in a target measurement region on a preparation on which a tissue slice is mounted. As “,” a “region not chemically modified to amino residues” having a predetermined planar shape provided at a predetermined site on a tissue slice is used.

  In the fourth embodiment of the automatic position superposition method according to the present invention described above, “protease cutting” having a predetermined planar shape provided at a predetermined site on a tissue slice as a “positioning marker” having a predetermined planar shape. "Unprocessed area" is used.

When a cysteine residue (Cys) is contained in a peptide fragment generated by protease cleavage treatment, a Cys-Cys bond between chains (SS bond) between two peptide fragments
Formation may occur.

  In order to prevent the linking of two peptide fragments via the Cys-Cys bond (SS bond) between the chains, a sulfanyl group (-SH) on the side chain of the Cys residue was previously added to carboxymethylation or pyridyl. Ethylation or the like is performed and the protection is performed in advance. Formation of a Cys-Cys bond (SS bond) is inhibited by a chemical modification corresponding to the S-alkylation treatment on the sulfanyl group (-SH) on the side chain of the Cys residue. On the other hand, since the chemical modification corresponding to the S-alkylation treatment is not normally subjected to dissociation in the desorption / ionization process in the MALDI-TOF-MS method, the ions that retain the chemical modification corresponding to the S-alkylation treatment are retained. Species are measured.

  For example, if the chemical modification treatment for the sulfanyl group (-SH) on the side chain of the Cys residue is not performed only in a predetermined region, the peptide fragment in which the chemical modification to the original Cys residue is not performed in the region. Is not generated. As a result, the region is a region where the peak intensity of the ionic species derived from the peptide fragment without chemical modification to the original Cys residue is not substantially measured on the two-dimensional analysis image of microscopic mass spectrometry. As identified.

  That is, instead of the “region not subjected to protease cleavage treatment” having a predetermined planar shape provided at a predetermined site on the tissue slice, which is used in the fourth form of the automatic position superimposing method according to the present invention described above. Then, use a “region not chemically modified to Cys residue” having a predetermined planar shape provided at a predetermined site on the tissue slice as a “positioning marker” having a predetermined planar shape. Is also possible.

In addition to “chemical modification treatment to Cys residue”, chemical modification treatment specific to amino acid residue, for example, phenolic hydroxyl group (—OH) on the side chain of tyrosine residue is sulfated (− It is also possible to use a chemical modification treatment (O—SO ₃ H).

  That is, in the “protease cleavage treatment” and the “desorption / ionization process in the MALDI-TOF-MS method”, “chemical modification to amino acid residues” that does not undergo dissociation is adopted and provided at a predetermined site on the tissue slice. It is also possible to provide a “region that is not chemically modified to an amino residue” having a predetermined planar shape and use it as a “positioning marker” having a predetermined planar shape.

  The method of providing a “region not chemically modified to amino residues” having a predetermined planar shape at a predetermined site on the tissue slice is the first of the above-described automatic position superposition methods according to the present invention. This is in accordance with the method described in the fourth embodiment in which a “region not subjected to protease cleavage treatment” having a predetermined planar shape is provided at a predetermined site on a tissue slice.

  The method described in the fourth embodiment of the automatic position superimposing method according to the present invention described above can also be applied to other processes.

  The automatic position superposition method of the two-dimensional analysis image of the microscopic mass analysis and the two-dimensional visible image of the optical microscope according to the present invention measures, for example, “marker protein” for the same region of the pathological tissue section. When performing histopathological diagnosis of the tissue at the lesion site based on the comparison between the two-dimensional analysis image of the microscopic mass analysis and the two-dimensional visible image obtained by optical microscope photography, the two-dimensional analysis image of the micromass analysis And two-dimensional visible image obtained by optical microscope photography can be used to improve the efficiency of image alignment in two-dimensional image analysis.

FIG. 1 is a diagram schematically showing an apparatus configuration that can be used when performing the automatic position superposition method according to the present invention. FIG. 2 is a diagram schematically showing a process flow showing an execution order of a series of steps and an outline of operations in each step in the first embodiment of the automatic position superposition method according to the present invention. FIG. 3 is a diagram schematically showing a process flow showing an execution order of a series of steps and an outline of operations in each step in the second embodiment of the automatic position superposition method according to the present invention. FIG. 4 is a diagram schematically showing a process flow showing an execution order of a series of steps and an outline of operations in each step in the third embodiment of the automatic position superposition method according to the present invention. FIG. 5 is a diagram schematically showing a process flow showing an execution order of a series of steps and an outline of operations in each step in the third embodiment of the automatic position superposition method according to the present invention.

Claims (19)

Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
It consists of a marker substance that can be discriminated in both a two-dimensional visible image obtained by optical microscope photography and a two-dimensional analysis image obtained by microscopic mass spectrometry so that it is included in the target measurement region on the preparation on which the tissue slice is mounted. An automatic position superimposing method comprising providing an alignment marker having a predetermined planar shape. The alignment marker having the predetermined planar shape is
An alignment marker having a predetermined planar shape, comprising a dye-labeled protein and a dye-labeled polypeptide, to which a dye label having a color distinguishable on a two-dimensional visible image taken by an optical microscope is attached. Item 2. The automatic position overlay method according to Item 1. The alignment marker having the predetermined planar shape is
2. The alignment marker having a predetermined planar shape, wherein the pigment having a color distinguishable on a two-dimensional visible image taken with an optical microscope is a mixture of protein and polypeptide. The automatic positioning method described. The alignment marker having the predetermined planar shape is
It is an alignment marker having a predetermined planar shape, which is made of a protein or a polypeptide, dyed with a dye having a color that can be identified on a two-dimensional visible image taken by an optical microscope. The automatic position superposition method according to claim 1. The exogenous protein or polypeptide that is essentially not present in the tissue slice is adopted as the protein or polypeptide used for the production of the alignment marker. The automatic position overlay method according to claim 1. Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
Discriminated in the two-dimensional visible image of optical microscope photography and discriminated in the two-dimensional analytical image of microscopic mass analysis so that it is included in the target measurement area on the preparation on which the tissue slice is mounted An automatic position superimposing method comprising providing an alignment mask having a predetermined plane shape that does not show a strong mass analysis peak intensity. The alignment marker having the predetermined planar shape is
7. The automatic positioning layer according to claim 6, wherein the coating layer is a coating film layer having a predetermined planar shape, which is made of an optical masking material that does not exhibit optical transparency to laser light used in the MALDI-TOF MS method. How to match. As a coating film layer made of a light masking material that does not show light transmittance to the laser light,
8. The automatic position superposition method according to claim 7, wherein a light-reflective coating film layer made of a metal material exhibiting a high reflectance is used in the wavelength range of the irradiated laser beam. As a coating film layer made of a light masking material that does not show light transmittance to the laser light,
8. A light-absorbing coating film layer made of a material having a high extinction coefficient that exhibits strong light absorption with respect to laser light and substantially eliminates laser light absorption by the matrix agent. Automatic position overlay method. The alignment mask having the predetermined planar shape,
A coating film layer having a predetermined planar shape made of a physical masking substance that shows light permeability to laser light used in the MALDI-TOF MS method, but prevents desorption of ionic species derived from peptide fragments. 7. The automatic position superimposing method according to claim 6, wherein: As a coating film layer consisting of a physical masking substance that shows light permeability to the laser light used in the MALDI-TOF MS method, but prevents desorption of ionic species derived from peptide fragments,
11. The automatic position overlay method according to claim 10, wherein a polymer coating film layer made of a polymer material exhibiting sufficiently high light transmittance is used in the wavelength range of the irradiated laser beam. Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
An automatic position superimposing method comprising providing a region having a predetermined planar shape and not coated with a matrix agent at a predetermined site on a tissue slice. Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
An automatic position superposition method comprising providing a region having a predetermined planar shape and not subjected to protease cleavage treatment at a predetermined site on a tissue slice. The region having the predetermined planar shape and not subjected to protease cleavage treatment is
14. The automatic coating according to claim 13, wherein a coating protective film having a predetermined planar shape is formed in advance by providing a predetermined site on the tissue slice, and then subjecting the entire surface of the tissue slice to protease cleavage treatment. Method of superimposing positions. Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
Intrinsic, identifiable in both two-dimensional visible images of optical microscopy and two-dimensional analytical images of micromass spectrometry so that the tissue slice is included in the target measurement area on the prepared preparation. An automatic positioning method characterized by setting an alignment marker. As an intrinsic alignment marker that can be identified in any of the two-dimensional visible image of the optical microscope and the two-dimensional analytical image of microscopic mass analysis,
A plurality of nuclei that can specify the position of the region of the surface of the tissue flake can be specified in either a two-dimensional visible image obtained by optical microscope photography or a two-dimensional analysis image obtained by microscopic mass spectrometry. Item 16. The automatic position overlay method according to Item 15. Two two-dimensional images are superimposed and displayed between a two-dimensional analysis image of microscopic mass spectrometry measured for a pathological section of a subject and a two-dimensional visible image obtained by optical microscopic imaging of the pathological section. A method for automatically performing an alignment operation for
The two-dimensional visible image obtained by the optical microscope photography is
A two-dimensional visible image obtained by mounting a tissue slice prepared from the pathological section on a preparation and photographing the cells contained in the tissue slice, as well as at least the nucleus and cytoplasm present in the cells, with an optical microscope. And
Image information constituting a two-dimensional visible image obtained by the optical microscope photographing is composed of digitized color information of pixels of a predetermined size having a two-dimensional matrix arrangement and position information of the pixels. , Digitized two-dimensional visible image data;
The two-dimensional analysis image of the microscopic mass analysis is a two-dimensional microscopic mass analysis of the surface of the tissue slice using the MALDI-TOF MS method in the measurement region of the two-dimensional visible image obtained by the optical microscope photography. It is an image obtained by two-dimensionally displaying the distribution of the peak intensity of the ionic species specified on the mass spectrometry spectrum,
The image information constituting the two-dimensional analysis image of the microscopic mass analysis is arranged in a two-dimensional matrix at a predetermined interval, and is specified on a mass spectrometry spectrum measured at the irradiation spot having a predetermined irradiation spot diameter. Digitized two-dimensional analysis image data composed of peak intensity of each ion species and position information of the irradiation spot;
Automatic alignment operation to display two two-dimensional images superimposed between a two-dimensional analysis image of microscopic mass spectrometry and a two-dimensional visible image obtained by optical microscope photography of the pathological section. The steps performed in
Provided in advance a positioning marker having a predetermined planar shape at a predetermined position included in the measurement region of the two-dimensional visible image obtained by the optical microscope photography,
The digital data processing described in (i) to (iv) below includes a process that is automatically performed.
(I) a step of identifying the alignment marker on the two-dimensional visible image obtained by the optical microscope imaging and specifying the contour of the planar shape of the identified alignment marker;
(ii) identifying a closed region corresponding to a planar shape of the alignment marker on the two-dimensional analysis image of the microscopic mass analysis;
(iii) The position information of the contour of the planar shape of the alignment marker specified on the two-dimensional visible image, and the alignment marker specified on the two-dimensional analysis image of the microscopic mass analysis Using the position information of the shape of the outer periphery of the closed region corresponding to the planar shape,
Calculating a relative shift between the positional information of the two two-dimensional image data by superimposing the planar contour of the alignment marker and the position of the outer peripheral shape of the closed region;
(iv) Based on the calculated value of the relative deviation between the positional information of the two two-dimensional image data, the relative positional deviation between the two two-dimensional images is corrected, and the two-dimensional analysis image of the micromass spectrometry And a step of superimposing and displaying a two-dimensional image of a two-dimensional visible image obtained by optical microscope photography;
As an alignment marker having the predetermined planar shape,
An automatic position superposition method comprising: providing a region having a predetermined planar shape and not chemically modified to an amino acid residue at a predetermined site on a tissue slice. As a chemical modification treatment to the amino acid residue,
18. The automatic position superposition method according to claim 17, wherein an S-alkylation treatment is adopted for a sulfanyl group (-SH) on a side chain of a Cys residue. As MALDI-TOF MS method,
The automatic position overlay method according to any one of claims 1 to 18, wherein a UV-MALDI-TOF MS method is used.

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