WO2021186577A1 - Dispositif de microdissection laser, procédé de microdissection laser et système d'analyse quantitative - Google Patents

Dispositif de microdissection laser, procédé de microdissection laser et système d'analyse quantitative Download PDF

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Publication number
WO2021186577A1
WO2021186577A1 PCT/JP2020/011793 JP2020011793W WO2021186577A1 WO 2021186577 A1 WO2021186577 A1 WO 2021186577A1 JP 2020011793 W JP2020011793 W JP 2020011793W WO 2021186577 A1 WO2021186577 A1 WO 2021186577A1
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sample
sample piece
unit
data
laser microdissection
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PCT/JP2020/011793
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English (en)
Japanese (ja)
Inventor
暎淳 洪
誠 澤田
健治 小野
弘美 鈴木
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国立大学法人東海国立大学機構
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Priority to PCT/JP2020/011793 priority Critical patent/WO2021186577A1/fr
Priority to JP2022508676A priority patent/JPWO2021186577A1/ja
Publication of WO2021186577A1 publication Critical patent/WO2021186577A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention relates to a laser microdissection (LMD) apparatus, a laser microdissection method, and a quantitative analysis system.
  • LMD laser microdissection
  • Mass spectrometric imaging is used to investigate the distribution of target compounds contained in samples such as biological tissue sections.
  • a plurality of measurement regions are set in a sample, and mass spectrometry is performed for each measurement region.
  • MALDI Matrix Assisted Laser Desorption / Ionization
  • Patent Document 1 Matrix Assisted Laser Desorption / Ionization
  • MALDI matrix-assisted laser desorption / ionization
  • the sample contains a large number of compounds such as biological tissue sections
  • the target compound is not isolated by the MALDI method, and contaminating components and matrix substances other than the target compound are also ionized at the same time as the target compound, so that the target compound is isolated.
  • the ionization efficiency may be lower than that in the case of the above, and the detection sensitivity may be deteriorated.
  • the abundance of contaminants and matrix substances is not uniform between the measurement regions, the quantitativeness of the target compound may be lacking.
  • thermoplastic resin sheet is irradiated with laser light from the support side in a state where the support made of a material (for example, acrylic resin or polycarbonate resin) is in contact with the support.
  • a material having a low melting point for example, 50 to 70 ° C.
  • the output of the laser beam and the irradiation time are adjusted so that the thermoplastic resin sheet does not melt and the sample is not denatured. ..
  • thermoplastic resin sheet melts in the measurement region irradiated with the laser beam, and then the molten thermoplastic resin sheet solidifies by stopping the irradiation of the laser beam, so that the sample is formed in the measurement region. Adhere to the thermoplastic resin sheet.
  • sample piece The portion obtained by peeling from the sample in this way is hereinafter referred to as a "sample piece".
  • sample pieces are collected from a large number of measurement areas.
  • a liquid sample for each measurement region can be obtained.
  • Data for each measurement region for imaging can be obtained by quantitatively analyzing the target component using the liquid chromatograph mass spectrometry for each of the liquid samples in each measurement region.
  • the problem to be solved by the present invention is a laser microdissection apparatus capable of reducing an error in data obtained by quantitative analysis caused by a difference in the amount of sample pieces obtained for each measurement region by the laser microdissection method. Is to provide.
  • the laser microdissection apparatus which has been made to solve the above problems, performs quantitative analysis for each sample piece obtained by the laser microdissection method from a plurality of measurement regions in the sample to be measured. It has a data correction unit that corrects the data obtained by doing so.
  • the data correction unit includes an area acquisition unit that obtains an area value of the sample piece for each sample piece, and a correction calculation unit that performs a calculation that corrects data for each sample piece based on the area value of the sample piece. ..
  • the calculation for correcting the data for each sample piece by the area value of the sample piece includes a calculation in which the data is divided by the area value, a calculation in which the data is further multiplied by a predetermined reference value after the calculation, or a predetermined value in the area value.
  • the calculation includes a calculation in which the correction parameter is obtained by multiplying and dividing the reference value of, and then the data is divided by the correction parameter.
  • the predetermined reference value the average value, the median value, or the like of the area values obtained from each of the plurality of sample pieces can be used.
  • the laser microdissection device since there is generally a correlation close to a proportional relationship between the amount of the sample piece and the area value, the data for each sample piece is obtained from the area value of the sample piece. By performing the correction calculation, it is possible to reduce the data error caused by the difference in the amount of each sample piece.
  • FIG. 6 is a schematic configuration diagram of an overall configuration of a quantitative analysis system including a laser microdissection device according to an embodiment of the present invention.
  • the schematic block diagram of the laser microdissection device which is the sampling part which the quantitative analysis system of this embodiment has.
  • the schematic block diagram of the microscope image acquisition part which the quantitative analysis system of this embodiment has.
  • the schematic block diagram of the liquid chromatograph part which the quantitative analysis system of this embodiment has.
  • the sampling section includes a slide glass with a sample attached to one surface and a support having a thermoplastic resin sheet attached to one surface. The figure which shows the state which held in.
  • the quantitative analysis system of the present embodiment is for explaining the operation of component separation in the LC section (upper figure), the operation of storing the eluate in the LC section (middle figure), and the operation of analysis in the MS section (lower figure). figure.
  • a micrograph of four sample pieces collected by the sampling unit in the quantitative analysis system of the present embodiment observed with a phase-contrast microscope. The figure which shows an example of the image which shows the data after correction at each position in a sample.
  • FIG. 1 shows the overall configuration of the quantitative analysis system 1 including the laser microdissection device 2 of the present embodiment.
  • the quantitative analysis system 1 includes a laser microdissection device 2, a liquid chromatograph mass spectrometer 30, a control unit 41, a data processing unit 42, an input unit 43, and a display unit 44.
  • the laser microdissection device 2 has a sampling unit 10 and a data correction unit 20. As will be described later, some of the components of the data correction unit 20 are incorporated in the data processing unit 42.
  • the sampling unit 10 includes an XY stage 11, a laser light source 12, and a thermoplastic film moving device 13.
  • the XY stage 11 is a table that supports the slide glass 92 on which the sample 91 is placed, and moves the supported slide glass 92 back and forth and left and right.
  • the laser light source 12 is provided so as to face the base of the XY stage 11, and emits a laser beam toward the XY stage 11.
  • the thermoplastic film moving device 13 is a slide in which a thermoplastic resin sheet (thermosoluble sheet) 94 supported by a support 93 made of a material that transmits a laser beam emitted by a laser light source 12 is supported by an XY stage 11.
  • thermoplastic film moving device 13 includes an adsorber 131 that sucks gas and adsorbs it on the upper surface of the support 93 (the surface opposite to the surface on which the thermoplastic resin sheet 94 is supported). It has an arm 132 that moves the adsorber 131 back and forth, left, right, up and down.
  • the data correction unit 20 includes a microscope image acquisition unit 21, an area acquisition unit 22, an area data storage unit 23, and a correction calculation unit 24.
  • the microscope image acquisition unit 21 includes a phase-contrast microscope 211 and a camera (CCD camera) 212 that captures an image observed by the phase-contrast microscope 211.
  • the phase-contrast microscope 211 used in this embodiment is a normal phase-contrast microscope having a light source 2111, a phase-contrast observation capacitor 2112, a sample stage 2113, and a phase-contrast observation objective lens 2114.
  • the electronic data of the microscope image taken by the camera 212 is transmitted to the area acquisition unit 22.
  • the area acquisition unit 22, the area data storage unit 23, and the correction calculation unit 24 are incorporated in the data processing unit 42, and will be described later together with other components of the data processing unit 42.
  • the liquid chromatograph mass spectrometer 30 has a pretreatment unit 31, an LC (liquid chromatograph) unit 32, a sample switching unit 33, and an MS (mass spectrometry) unit 34.
  • the pretreatment unit 31 prepares a liquid sample from each of a plurality of sample pieces collected by the sampling unit 10.
  • the LC unit 32 has n LC units 32a, 32b, 32c ... 32n having the same configuration (n is a natural number of 2 or more).
  • each LC unit 32k (k is any of a to n) is a mobile phase container 320 for storing the mobile phase, and a liquid feeding liquid that sucks and sends the mobile phase in the mobile phase container 320.
  • Pump 321, injector 322 that injects a liquid sample into the fed mobile phase
  • column 323 that separates various components contained in the liquid sample according to its holding time, and eluent that elutes from the outlet of column 323.
  • a sample storage unit 324 for storing is provided.
  • the injector 322 is held by a liquid sample container 3220 for accommodating a liquid sample, a needle 3221 and a sample holding unit 3222 for sucking and holding a predetermined amount of the liquid sample in the liquid sample container 3220, and a sample holding unit 3222. It includes a sample injection unit 3223 that injects a liquid sample into the mobile phase at a predetermined timing.
  • the sample storage unit 324 includes a plurality of storage containers 3241 and 3242, an inlet side flow path switching unit 3243 for selectively supplying the eluate from the outlet of the column 323 to one of the plurality of storage containers 3241 and 3242, and a storage container. Includes an outlet-side flow path switching unit 3244 that selectively supplies the eluate stored in 3241 and 3242 to the subsequent stage.
  • the eluate supplied from the storage containers 3241 and 3242 of the sample storage unit 324 will be referred to as an "eluent sample”.
  • the sample storage unit 324 is appropriately provided with a cleaning mechanism for cleaning the storage containers 3241 and 3242 and the flow path. In No. 4, the description of such a cleaning mechanism is omitted.
  • the sample switching unit 33 selects one of the eluate samples supplied in parallel from the sample storage unit 324 of each of the n LC units 32a to n and introduces the eluate sample into the MS unit 34. It switches the flow path.
  • the MS unit 34 is equipped with an ion source using an ionization method based on the Electrospray Ionization (ESI) method in the present embodiment, and is equipped with a Quadrupole-Time of Flight (Q-TOF) type mass.
  • the Q-TOF mass spectrometer ionizes the components of the eluent sample with an ion source and dissociates the ions by collision-induced dissociation (CID), resulting in a predetermined mass-to-charge ratio range for the product ions.
  • CID collision-induced dissociation
  • Mass spectrometry is performed over the period.
  • the Q-TOF mass spectrometer can acquire mass spectrum data for ions derived from sample components as well as product ion spectrum (MS / MS spectrum) data. Since the Q-TOF mass spectrometer is a known device, detailed description and illustration of the configuration will be omitted.
  • LC units 32a to 32n including all the components from the mobile phase container 320 to the sample storage unit 324 are arranged side by side, but these are in different samples. Any configuration may be used as long as the components can be separated in parallel. Therefore, although a plurality of columns 323 need to be arranged side by side, it is possible to have a configuration in which other components are shared. For example, the mobile phase container 320 can be shared. Further, the injector 322 shares each component except the sample injection unit 3223, and the flow path is switched so that the sample held in the common sample holding unit 3222 is selectively sent to one of the plurality of sample injection units 3223. It is also possible to provide a mechanism.
  • each column It is better to install a liquid feed pump in.
  • the control unit 41 controls the operations of the respective units described so far, as well as the data processing unit 42, the input unit 43, and the display unit 44 described below.
  • the data processing unit 42 has an area acquisition unit 22, an area data storage unit 23, and a correction calculation unit 24, which are a part of the data correction unit 20, as well as a measurement data storage unit 421 and a position information storage unit 422. It has a pre-correction data calculation unit 423, an imaging image creation unit 424, and a display processing unit 425. Details of each component of the data processing unit 42 will be described later together with a description of the operation of the quantitative analysis system 1 including the data correction unit 20.
  • the control unit 41 and the data processing unit 42 are configured such that the entity is a computer and their respective functions are realized by operating the dedicated control / processing software installed on the computer on the computer.
  • the quantitative analysis system 1 has an input unit 43 for inputting necessary information to the computer by the user, and a display unit (display) 44 for displaying a mass spectrometry imaging image or the like showing the analysis result.
  • FIGS. 5A to 5D the XY stage 11 and the thermoplastic film moving device 13 are not shown.
  • the user prepares one in which the sample 91 to be measured is attached to one surface of the slide glass 92 and one in which the thermoplastic resin sheet (film) 94 is attached to one surface of the support 93.
  • the sample 91 is placed on the XY stage 11 with the sample 91 facing upward, and in the latter, the thermoplastic resin sheet 94 is adsorbed on the adsorber 131 with the thermoplastic resin sheet 94 facing downward (FIG. 5A).
  • thermoplastic film moving device 13 brings the support 93 into contact with the slide glass 92 and holds it so that the surface of the thermoplastic resin sheet 94 is in close contact with the sample 91 (FIG. 5B).
  • a laser beam (near-infrared laser beam) 95 is irradiated for a short time from the surface of the support 93 opposite to the surface to which the thermoplastic resin sheet 94 is attached so as to be substantially orthogonal to the surface (near-infrared laser light) 95.
  • FIG. 5C The range of irradiating the laser beam 95 is a range corresponding to a portion (one measurement point) to be measured once on the sample 91.
  • the irradiated laser beam 95 passes through the support 93 and heats the thermoplastic resin sheet 94.
  • the thermoplastic resin sheet 94 near the area irradiated with the laser beam 95 melts and permeates into the structure of the sample 91.
  • the thermoplastic film moving device 13 pulls the support 93 away from the slide glass 92 so as to separate the thermoplastic resin sheet 94 from the sample 91.
  • the sample piece 911 which is a part of the sample 91, is collected on the surface of the thermoplastic resin sheet 94 (FIG. 5D).
  • the sample piece 911 having an inner diameter of 1 to several hundred ⁇ m does not affect the components contained in the collected biological tissue. Can be collected on the thermoplastic resin sheet 94.
  • the processing operation is an operation of collecting a small amount of sample piece 911 near a certain measurement point on the sample 91, and in the sampling unit 10, the position where the slide glass 91 and the support 93 are brought close to each other is set. The same operation is repeated while moving in the plane direction. As a result, as shown in FIG. 6, sample pieces 911 near a large number of measurement points 913 in a predetermined two-dimensional region 912 on the sample 91 are collected on the thermoplastic resin sheet 94, respectively.
  • the distance between the measurement points 913 on the sample 91 corresponds to the spatial resolution in the mass spectrometry imaging image, and is very narrow, for example, 1 ⁇ m, but the distance between the sample pieces 911 collected on the thermoplastic resin sheet 94 is
  • the sampling position is controlled so as to be as wide as several mm.
  • the two-dimensional positional relationship between the plurality of measurement points 913 on the sample 91 and the two-dimensional positional relationship of the sample piece 911 collected on the thermoplastic resin sheet 94 need not necessarily be maintained. As long as the correspondence between the position of the measurement point 913 on the sample 91 (the position defined by the address information in the X and Y directions) and the position of the sample piece 911 on the thermoplastic resin sheet 94 is determined. good.
  • FIG. 6 is a simplified drawing for illustration purposes only, and usually, the number of measurement points 913 on the sample 91 is much larger than that shown in the drawing, and a sample piece of several hundreds or more from one sample 91. 911 is obtained.
  • the phase contrast of the remaining part of the sample 91 (the part remaining without being collected as the sample piece 911) is possessed by the microscope image acquisition unit 21. It is placed on the sample stage 2113 of the microscope 211. The camera 212 of the microscope image acquisition unit 21 photographs the rest of the sample 91. The captured image data is transmitted to the area acquisition unit 22 via the control unit 41. The area acquisition unit 22 binarizes the obtained image data and analyzes it to obtain the position and area value of each portion of the remaining part of the sample 91 in which the sample piece 911 is collected and becomes a void. Ask for.
  • the position of each of these parts corresponds to the position of each measurement point 913, and the area value of each of these parts corresponds to the area value of each sample piece 911.
  • the contour becomes clearer than when a normal optical microscope or the like is used, so that the accuracy of the image analysis is improved. Binarizing the image data also contributes to clarifying the outline.
  • the data of the position of each of these cut parts, that is, the position of the measurement point 913 is stored in the position information storage unit 422, and the area value is associated with the position information data in the position information storage unit 422 and is associated with the area data storage unit. It is stored in 23.
  • thermoplastic resin sheet 94 Since the correspondence between the position of the measurement point 913 on the sample 91 and the position of the sample piece 911 on the thermoplastic resin sheet 94 is determined as described above, heat is obtained from the position of the measurement point 913 obtained by image analysis. The position of each sample piece 911 on the plastic resin sheet 94 can also be specified.
  • the area value has a different value for each sample piece 911. This is when the support 93 is removed from the upper surface of the sample 91 when the adhesion of the sample 91 to the thermoplastic resin sheet 94 is partially insufficient due to the presence of irregularities on the surface of the sample 91 or the like. Since a part of the sample 91 remains on the slide glass 91 side without adhering to the thermoplastic resin sheet 94, for example, as shown in FIG. 7, each sample piece 911 is collected in a different planar shape. However, in general, since there is a closely proportional correlation between the amount of the sample piece 911 and the area value, the area value for each sample piece 911 is based on the data obtained by the quantitative analysis as described later. It can be used for correction.
  • the pretreatment unit 31 receives the support 93 from which the sample piece 911 is collected from the sample collection unit 10, and the individual sample pieces collected on the thermoplastic resin sheet 94. Prepare a liquid sample from 911. Specifically, as shown in FIG. 6, a microtiter plate (MTP) 3200 having a large number of wells 320 is used, and a predetermined value for extracting components in the sample piece 911 in advance in each well 320 of the MTP 3200 is used. Inject the extract.
  • MTP microtiter plate
  • the support 93 is attached to the upper surface of the MTP3200 (the surface on the side where each well 320 is open) so that the sample piece 911 on the thermoplastic resin sheet 94 is located inside each well 320.
  • the sample piece 911 is immersed in the extract in each well 320 to prepare a liquid sample in which the components in the sample piece 911 are dissolved. If the correspondence between the position of the measurement point 913 on the sample 91 and the position of the sample piece 911 on the thermoplastic resin sheet 94 is determined as described above, each prepared liquid sample and the measurement point 913 on the sample 91 are determined. The relationship with the position of is also uniquely determined.
  • FIG. 6 is a simplified drawing for explanation, and the number of wells 320 is much larger than that shown in the figure.
  • FIG. 9 is a diagram for explaining the operations of the LC unit 32 and the MS unit 34, in that order from the top, the LC unit component separation operation (upper figure), the LC unit eluate storage operation (middle figure), and the MS unit analysis operation. ( Figure below) is shown.
  • the case where the number of LC units 32a to 32n is 100 will be described as an example, but the present invention is not limited to this example.
  • the 100 LC units 32a to 32n perform a process of separating the components in the given liquid sample in parallel (upper figure of FIG. 9). That is, in the injector 322 of the LC unit 32, the sample holding unit 3222 sucks and holds a predetermined amount of the liquid sample in the well (liquid sample container) 3220 through the needle 3221.
  • the mobile phase is fed to the column 323 at a constant flow rate by the liquid feed pump 321 via the sample injection unit 3223, and the sample injection unit 3223 is held in the sample holding unit 322 in response to an instruction from the control unit 41. Inject the sample into the mobile phase.
  • the injected liquid sample is introduced into the column 323 along with the flow of the mobile phase, the components in the liquid sample are separated according to the retention time while passing through the column 323, and the eluate containing the separated components is the column. Elute from the outlet of 323.
  • the inlet side flow path switching unit 3243 switches the supply destination of the eluate every t1 (in this example, every 10 seconds) for a predetermined time. As shown by an arrow in FIG. 4, when the eluate is supplied to one storage container 3241, the outlet side flow path switching unit 3244 closes the eluate outflow path from the storage container 3241, and the storage container The eluate is stored in 3241.
  • the inlet side flow path switching unit 3243 switches the supply destination of the eluate to the storage container 3242.
  • the outlet-side flow path switching unit 3244 closes the eluate outflow path from the storage container 3242, and this time, the eluate is stored in the storage container 3242.
  • the eluate sample which is the stored eluate in a predetermined amount, contains the components in the eluate eluted from the outlet of the column 323 within a predetermined time t1, and the plurality of components once separated in the column 323 are stored. It may remix when stored in container 3241 or 3242.
  • the LC unit 32 it can be considered that the liquid sample after the components are separated by the column 323 is fractionated and stored every t1 for a predetermined time.
  • the outlet side flow path switching unit 3244 stores the eluent.
  • the eluent discharge path on the container 3241 side is opened, and the eluent sample stored in the storage container 3241 is flowed to the sample switching unit 33.
  • the sample switching unit 33 sequentially selects the eluate samples supplied from the sample storage units 324a to 324n in the 100 LC units 32a to 32n and sends them to the MS unit 34. Switch the flow path to supply.
  • the time for supplying the eluate sample supplied from the sample storage unit 324 in one LC unit 32 to the MS unit 34 is about 100 msec. Therefore, within a predetermined time t1 of 10 seconds, the eluate samples supplied from all the sample storage units 324a to 324n of 100 LC units 32a to 32n can be selected once and supplied to the MS unit 34. Is.
  • the MS unit 34 ionizes various components contained in the eluate sample supplied from the sample storage unit 324 in one LC unit 32 during the period of 100 msec, and mass spectrometrically analyzes the generated ions. Alternatively, an ion having a specific mass-to-charge ratio is selected from the generated ions and then dissociated by CID, and the product ion generated by the dissociation is mass-analyzed.
  • the MS unit 34 executes such mass spectrometry each time the eluate supplied by the sample switching unit 33 is switched (lower figure of FIG. 9). That is, mass spectrometry or MS / MS analysis is performed once every 100 msec period.
  • the spectral data obtained for each liquid sample in this way is stored in the measurement data storage unit 421.
  • the measurement data storage unit 421 the spectrum data of each liquid sample stored therein and the position information of the sample piece 911 (or measurement point 913) corresponding to each liquid sample stored in the position information storage unit 422. Data is associated.
  • the pre-correction data calculation unit 423 acquires information on the range of the mass-to-charge ratio and the holding time corresponding to the component from the memory, and stores the measurement data for each liquid sample. From the spectrum data stored in the part 421, the spectrum data at the mass-to-charge ratio within the holding time range is extracted, and the integrated value obtained by integrating the mass chromatogram within the range is obtained.
  • the integrated value obtained here corresponds to the amount of the component to be analyzed that each liquid sample has, but also depends on the amount of each sample piece 911 that is the source of each liquid sample. Therefore, the correction calculation unit 24 acquires the area value of the sample piece 911 from the area data storage unit 23 for each liquid sample, and divides the integrated value of the mass chromatogram calculated by the pre-correction data calculation unit 423 by the area value. By doing so, the data error caused by the difference in the amount of 911 for each sample piece is corrected.
  • Table 1 shows the area values of the sample pieces and the integrated values of the mass chromatograms before and after the correction for a certain component for the four sample pieces 9111 to 9114 shown in FIG. show.
  • FIG. 10 is an image obtained by observing the sample pieces 9111 to 9114 with a phase contrast microscope 211. By observing with a phase contrast microscope 211, the contrast between the sample pieces 9111 to 9114 and their surroundings becomes clear.
  • Table 1 also shows the standard deviation, the average value, and the CV value obtained by dividing the standard deviation of the four sample pieces 9111 to 9114 by the average value for each of the mass chromatograms before and after the correction. bottom.
  • the CV value is an index showing the variation of the data with respect to the average value, and is smaller after the correction than before the correction. This means that the data variability that was larger than it actually was before the correction was corrected by the correction.
  • one corrected data (integral value) can be obtained for one sample piece 911.
  • the imaging image creation unit 424 is based on the corrected data of each sample piece 911 and the position information of each sample piece 911 stored in the position information storage unit 422, and the hue and saturation of the color are displayed on the two-dimensional map. And any one or more differences in brightness create an image representing the corrected data at each position in the sample 91.
  • FIG. 11 shows an example of an image created by the imaging image creation unit 424.
  • the created image is converted into a signal for display on the display unit 44 by the display processing unit 425, and the signal is transmitted to the display unit 44.
  • the display unit 44 receives the signal and displays the created image.
  • the data is corrected by the area value of each sample piece 911, so that the error of the data caused by the difference in the amount of each sample piece 911 is reduced. be able to.
  • a microscope image is acquired using a phase-contrast microscope 211 and then image analysis is performed.
  • a normal optical microscope is used.
  • a laser microscope or the like may be used.
  • the sample piece may be photographed with a camera without using a microscope.
  • the area of the sample piece 911 may be obtained by a method other than image analysis.
  • the image analysis is performed after binarizing the data of the microscope image, but the image analysis may be performed without binarizing.
  • a microscope image of the rest of the sample 91 that remains without being collected as the sample piece 911 is acquired, and the area value of the sample piece 911 is based on the image of each part where the sample piece 911 is collected and becomes a void.
  • a microscopic image of the surface of the thermoplastic resin sheet 94 to which the collected sample piece 911 is attached is acquired, and the sample piece 911 is taken from the image of the sample piece 911 included in the microscopic image. The area value of may be obtained.
  • the MS unit 34 is not limited to the above-mentioned Q-TOF type mass spectrometer, and may be appropriately used as a single type quadrupole mass spectrometer, a triple quadrupole mass spectrometer, an ion trap-time-of-flight mass spectrometer, or the like.
  • the device of the above method can be used.
  • the ESI method it is equipped with an ion source that uses other ionization methods such as the atmospheric pressure chemical ionization (APCI) method and the atmospheric pressure photoionization (APPI) method. It may be a mass analyzer.
  • APCI atmospheric pressure chemical ionization
  • APPI atmospheric pressure photoionization
  • a plurality of sample pieces are analyzed in parallel using a liquid chromatograph mass spectrometer provided with a plurality of LC units 32a to 32n, but a liquid chromatograph mass spectrometer provided with only one LC unit. May be used to analyze the sample pieces one by one.
  • the laser microdissection device is data for correcting data obtained by performing quantitative analysis for each sample piece obtained by the laser microdissection method from a plurality of measurement regions in the sample to be measured.
  • the data correction unit includes an area acquisition unit that obtains an area value of the sample piece for each sample piece, and a correction calculation unit that calculates data for each sample piece by the area value of the sample piece. Be prepared.
  • the data for each sample piece is used to obtain the area value of the sample piece.
  • the calculation corrected by it is possible to reduce the data error caused by the difference in the amount of each sample piece.
  • Data obtained by performing quantitative analysis for each sample piece include, for example, the peak area and peak top value of the chromatogram or mass spectrum obtained by the liquid chromatograph mass spectrometry.
  • Calculations to be corrected by the area value of a sample piece include a calculation in which the data obtained by performing a quantitative analysis on the sample piece is divided by the area value, and a calculation in which a predetermined reference value is further multiplied after the calculation.
  • a calculation or the like in which the correction parameter is obtained by multiplying the area value by a predetermined reference value and then the data is divided by the correction parameter is included. By performing the operation of multiplying and dividing the reference value in this way, the data can be brought closer to a more accurate value.
  • the reference value the average value, the median value, or the like of the area values obtained from each of the plurality of sample pieces can be used.
  • the calculation is performed by dividing the data by those values.
  • the value after the calculation is a numerical value that does not represent the original data (peak area or peak top value), but such a numerical value is used. It is possible to determine the relative difference between the sample pieces (measurement areas in the sample).
  • the laser microdissection device according to the second item is the laser microdissection device according to the first item. Also has a microscope, The area acquisition unit obtains an area value for each sample piece by performing image analysis on a microscope image acquired for each sample piece by the microscope.
  • the data can be easily corrected by obtaining the area value of each sample piece by using a known image analysis method.
  • the microscope is a phase contrast microscope in the laser microdissection device according to the second item.
  • the laser microdissection apparatus by using a phase contrast microscope, even if the sample piece is almost transparent, the light transmitted through the sample piece and the light passing around the sample piece can be obtained. Since the image of the sample piece can be clearly obtained by the phase difference of the above, the area value can be obtained more accurately based on the image analysis in the area acquisition unit. As a result, the data can be corrected more accurately.
  • the laser microdissection device according to the fourth item is the laser microdissection device according to the second or third item, in which the area acquisition unit binarizes the microscope image and then performs the image analysis. Is.
  • the laser microdissection device by performing image analysis on the microscope image acquired for each sample piece, the outline of the sample piece becomes clearer, so that the area acquisition unit performs image analysis.
  • the area value can be obtained more accurately based on. As a result, the data can be corrected more accurately.
  • the laser microdissection method is A sample piece acquisition process in which a sample piece is obtained from a plurality of measurement regions in the sample to be measured by the laser microdissection method, respectively.
  • the laser microdissection method by performing a calculation in which the data for each sample piece is corrected by the area value of the sample piece, the error of the data caused by the difference in the amount for each sample piece can be obtained. It can be made smaller.
  • the quantitative analysis system according to item 6 is The laser microdissection device according to any one of items 1 to 4 and It is provided with a liquid chromatograph mass spectrometer that performs quantitative analysis for each sample piece.
  • the quantitative analysis system by using the laser microdissection device according to any one of the first to fourth items, it is possible to perform a quantitative analysis with a small error caused by a difference in the amount of each sample piece. can.
  • Quantitative analysis system 10 ... Sampling unit 11 ... XY stage 12 ... Laser light source 13 ... Thermoplastic film moving device 131 ... Adsorber 132 ... Arm 2 ... Laser microdissection device 20 ... Data correction unit 21 ... Microscopic image acquisition unit 211 ... Phase difference microscope 2111 ... Light source 2112 ... Phase difference observation capacitor 2113 ... Sample stage 2114 ... Phase difference observation objective lens 212 ... Camera 22 ... Area acquisition unit 23 ... Area data storage unit 24 ... Correction calculation unit 30 ... Liquid chromatography Graph mass analyzer 31 ... Pretreatment unit 32, 32a to 32n ... LC (liquid chromatograph) unit 320 ... Mobile phase container 3200 ... MTP 321 ...

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Abstract

L'invention concerne un dispositif de microdissection laser (2) comportant une unité de correction de données (20), qui corrige des données obtenues en mettant en oeuvre une analyse quantitative sur des échantillons (911) obtenus par un procédé de microdissection laser à partir d'une pluralité de régions de mesure d'un échantillon (91) à mesurer. L'unité de correction de données (20) comprend : une unité d'acquisition de zone (22) qui, pour chaque échantillon (911), obtient une valeur de zone dudit échantillon (911) ; et une unité de calcul de correction (23), qui effectue un calcul pour corriger des données relatives à chacun des échantillons (911) à l'aide de la valeur de zone dudit échantillon (911).
PCT/JP2020/011793 2020-03-17 2020-03-17 Dispositif de microdissection laser, procédé de microdissection laser et système d'analyse quantitative WO2021186577A1 (fr)

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JP2022508676A JPWO2021186577A1 (fr) 2020-03-17 2020-03-17

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013245998A (ja) * 2012-05-24 2013-12-09 Olympus Corp 組織分布計算方法
WO2015053039A1 (fr) * 2013-10-07 2015-04-16 国立大学法人名古屋大学 Dispositif de microdissection laser, dispositif d'analyse contenant un dispositif de microdissection laser, et procédé pour produire une micropuce
US20160126073A1 (en) * 2013-06-07 2016-05-05 Vanderbilt University Pathology interface system for mass spectrometry
WO2016163385A1 (fr) * 2015-04-06 2016-10-13 国立大学法人名古屋大学 Appareil de microdissection par laser, appareil d'analyse comprenant un dispositif de microdissection par laser, procédé de prélèvement d'échantillon, et dispositif utilisé dans un appareil de microdissection par laser
WO2018037491A1 (fr) * 2016-08-24 2018-03-01 株式会社島津製作所 Dispositif d'imagerie par spectrométrie de masse

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013245998A (ja) * 2012-05-24 2013-12-09 Olympus Corp 組織分布計算方法
US20160126073A1 (en) * 2013-06-07 2016-05-05 Vanderbilt University Pathology interface system for mass spectrometry
WO2015053039A1 (fr) * 2013-10-07 2015-04-16 国立大学法人名古屋大学 Dispositif de microdissection laser, dispositif d'analyse contenant un dispositif de microdissection laser, et procédé pour produire une micropuce
WO2016163385A1 (fr) * 2015-04-06 2016-10-13 国立大学法人名古屋大学 Appareil de microdissection par laser, appareil d'analyse comprenant un dispositif de microdissection par laser, procédé de prélèvement d'échantillon, et dispositif utilisé dans un appareil de microdissection par laser
WO2018037491A1 (fr) * 2016-08-24 2018-03-01 株式会社島津製作所 Dispositif d'imagerie par spectrométrie de masse

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