WO2021075254A1 - イメージング質量分析装置 - Google Patents

イメージング質量分析装置 Download PDF

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Publication number
WO2021075254A1
WO2021075254A1 PCT/JP2020/036855 JP2020036855W WO2021075254A1 WO 2021075254 A1 WO2021075254 A1 WO 2021075254A1 JP 2020036855 W JP2020036855 W JP 2020036855W WO 2021075254 A1 WO2021075254 A1 WO 2021075254A1
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Prior art keywords
unit
sample
mass spectrometer
irradiation
confirmation
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PCT/JP2020/036855
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English (en)
French (fr)
Japanese (ja)
Inventor
賢一 三嶋
建悟 竹下
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Shimadzu Corp
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Shimadzu Corp
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Priority to CN202080067563.6A priority Critical patent/CN114450587A/zh
Priority to US17/641,662 priority patent/US12154772B2/en
Priority to JP2021552300A priority patent/JP7215591B2/ja
Publication of WO2021075254A1 publication Critical patent/WO2021075254A1/ja
Anticipated expiration legal-status Critical
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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
    • G01N27/64—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 using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0004—Imaging particle spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • 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
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]

Definitions

  • the present invention relates to an imaging mass spectrometer.
  • Mass spectrometry imaging is a method for investigating the distribution of substances having a specific mass by performing mass spectrometry on multiple measurement points in a two-dimensional region of a sample such as a biotissue section. Applications such as biomarker search and investigation of the causes of various diseases are being promoted.
  • Mass Spectrometry A mass spectrometer for carrying out an imaging method is generally called an imaging mass spectrometer (see Patent Document 1, Non-Patent Document 1, etc.).
  • an analysis target region is determined based on the microscopic observation image, and imaging mass spectrometry is performed in that region, a microscopic mass spectrometer, a mass spectrometer, or the like is used. Although sometimes referred to, it is referred to herein as an "imaging mass spectrometer”.
  • an ion source by a laser desorption / ionization (LDI) method or a matrix-assisted laser desorption / ionization (MALDI) method is generally used.
  • the ion source by the LDI method / MALDI method the surface of the sample is irradiated with laser light focused by a condensing optical system including a lens and narrowed down to a small diameter, and the sample is included in the sample from the vicinity of the irradiation site of the laser light. Ions derived from the substance are generated.
  • the ions generated in this way are extracted from the vicinity of the sample surface by the action of an electric field, introduced into a mass spectrometer through an ion transport optical system, etc., and the ions are separated and detected according to the mass-to-charge ratio.
  • One of the measurement modes (usage) of the imaging mass spectrometer is an image showing the intensity distribution of ions having a certain mass-to-charge ratio on the sample by moving the irradiation position of the laser beam relatively on the sample.
  • the substance distribution image in the cell can be obtained by narrowing down the irradiation diameter of the laser beam to about 0.5 ⁇ m. That is, the diameter of the laser beam (irradiation diameter) irradiated on the sample is the spatial resolution of the imaging analyzer.
  • the irradiation diameter of the laser beam is, for example, the size of a mark (irradiation mark) formed by irradiating a sample in which a predetermined dye is uniformly applied to the surface of a slide glass with the laser light, and as a result, the dye is scattered by ablation. It is obtained by measuring the laser.
  • a special jig or measuring instrument such as the knife edge method is required to measure the diameter of the irradiation mark.
  • confirmation of whether or not the irradiation diameter of the laser beam is as set is performed at the time of inspection before shipment of the imaging mass spectrometer, maintenance inspection, repair, etc. performed at an appropriate timing after shipment. At that time, it was only done by the serviceman, and could not be done by the user.
  • the problem to be solved by the present invention is to enable the user of the imaging mass spectrometer to easily confirm the focused state of the laser beam.
  • the present invention which has been made to solve the above problems, is an imaging mass spectrometer that generates ions by irradiating a sample with laser light and mass-analyzes the ions.
  • a laser irradiation unit that emits laser light toward the sample
  • a condensing optical system that condenses the laser light emitted from the laser light source, which is arranged between the laser irradiation unit and the sample.
  • An imaging unit that acquires a focused state confirmation image, which is an optical microscopic image capable of confirming the focused state of the laser light emitted by the laser irradiation unit on the sample. It is provided with a display unit that displays a condensed state confirmation image acquired by the imaging unit on a display screen.
  • the user sees the focused state confirmation image displayed on the display screen, and whether the focused state of the laser light emitted by the laser irradiation unit is the desired state. Whether or not it can be easily confirmed.
  • the schematic block diagram of the imaging mass spectrometer which is one Embodiment of this invention.
  • FIG. 1 is a schematic configuration diagram of an imaging mass spectrometer of one embodiment.
  • This imaging mass spectrometer uses an atmospheric pressure matrix-assisted laser desorption / ionization (AP-MALDI) method or an atmospheric pressure laser desorption / ionization (AP-LDI) method as an ionization method, and ionization is maintained in a substantially atmospheric pressure atmosphere. It includes a chamber 10 and a vacuum chamber 20 that is evacuated by a vacuum pump 21.
  • AP-MALDI atmospheric pressure matrix-assisted laser desorption / ionization
  • AP-LDI atmospheric pressure laser desorption / ionization
  • a sample table 11 on which the sample 100 to be analyzed is placed is arranged.
  • the sample table 11 is configured to be movable in two axial directions, the X-axis and the Y-axis, which are orthogonal to each other by the driving force from the sample table driving unit 12 including the motor.
  • the sample table drive unit 12 corresponds to the irradiation position moving unit of the present invention.
  • the sample 100 is, for example, a tissue section cut out very thinly from a biological tissue, and is prepared as a sample for MALDI by applying or spraying an appropriate matrix on the sample 100.
  • a laser irradiation unit 30 and an imaging unit 40 are arranged outside the ionization chamber 10.
  • the laser irradiation unit 30 emits a laser beam 31 for ionizing a substance in the sample 100.
  • the laser beam 31 emitted from the laser irradiation unit 30 irradiates the surface of the sample 100 through the irradiation window 32 and the condensing optical system 33 provided on the side surface of the ionization chamber 10.
  • the condensing optical system 33 can be moved within a predetermined range in the optical axis direction of the laser beam 31 by the condensing optical system driving unit 34.
  • the imaging unit 40 is composed of, for example, a CCD camera, and photographs a predetermined range of the sample 100 placed on the sample table 11 via the photographing window 41 and the photographing optical system 42 provided on the side surface of the ionization chamber 10. To do.
  • the imaging signal obtained by the imaging unit 40 is sent to the data processing unit 50, and the image data processing unit 51 executes appropriate data processing to convert the data into optical microscopic image data.
  • the optical microscopic image data is stored in the image data storage unit 511 as needed.
  • the irradiation diameter confirmation screen creation unit 512 creates an irradiation diameter confirmation screen from the optical microscopic image data stored in the image data storage unit 511.
  • the data on the irradiation diameter confirmation screen is also stored in the image data storage unit 511. The irradiation diameter confirmation screen will be described later.
  • the inlet end of the ion transport pipe 22 that communicates the ionization chamber 10 and the vacuum chamber 20 is open.
  • an ion transport optical system 23 for transporting ions while converging them by the action of an electric field, a mass spectrometer that separates ions according to the mass-to-charge ratio, and detection that detects the separated ions.
  • An ion separation / detection unit 24 including a device is installed. The ionic strength signal obtained by the ion separation / detection unit 24 is input to the data processing unit 50, and the mass spectrometry data processing unit 52 included therein executes appropriate data processing to create, for example, a two-dimensional substance distribution image. Will be done.
  • the mass spectrometer in the ion separation / detection unit 24 includes a quadrupole mass filter, a linear ion trap, a three-dimensional quadrupole ion trap, a orthogonal acceleration type flight time mass spectrometer, and a Fourier transform ion cyclotron mass spectrometer. , A magnetic field sector mass spectrometer, etc. are used.
  • the control unit 60 includes an analysis control unit 61 and an irradiation diameter confirmation control unit 62.
  • An input unit 63 and a display unit 64 are connected to the control unit 60.
  • At least a part of the data processing unit 50 and the control unit 60 described above uses a personal computer (or a higher-performance workstation) including a CPU, RAM, ROM, etc. as a hardware resource, and has dedicated control installed on the computer. By operating the processing software on the computer, each function can be achieved.
  • the analysis control unit 61 operates the sample table drive unit 12, the irradiation control unit 37, the condensing optical system drive unit 34, the ion transport optical system 23, the ion separation / detection unit 24, and the like in response to an instruction from the input unit 63. Is controlled to perform mass spectrometry on sample 100. Specifically, the analysis control unit 61 emits the laser beam 31 from the laser irradiation unit 30 toward the sample 100 placed on the sample table 11 via the irradiation control unit 37. As a result, the components existing on the sample 100 at the site (measurement point) irradiated with the laser beam 31 are ionized.
  • the ionized components are transported into the vacuum chamber 20 via the ion transport tube 22 where mass spectrometry is performed. Further, the analysis control unit 61 moves the sample table 11 in the XY plane via the sample table drive unit 12. As a result, the position where the laser beam 31 is irradiated on the sample 100 moves, and the laser beam irradiation position is scanned on the sample 100. As a result, mass spectrometry is performed on a plurality of measurement points in the two-dimensional region on the sample 100. As a result of executing the mass spectrometry, the obtained detection signal is sent to the data processing unit 50, and the mass spectrometry data processing unit 52 performs predetermined data processing. The result of the data processing performed by the mass spectrometry data processing unit 52 is input to the control unit 60 and output to the display unit 64.
  • the irradiation diameter confirmation control unit 62 controls the operations of the sample stand drive unit 12, the irradiation control unit 37, and the condensing optical system drive unit 34 in response to an instruction from the input unit 63, and laser light for the sample 100. The operation of confirming the irradiation diameter of is executed.
  • the laser light 31 emitted from the laser irradiation unit 30 is focused by the condensing optical system 33 and then irradiated on the surface of the sample 100.
  • the condensing optical system 33 is arranged so that the diameter (laser irradiation diameter) of the laser beam 31 irradiated on the surface of the sample 100 becomes a predetermined size.
  • the condensing optical system 33 is arranged so that the surface of the sample comes to the position where the laser beam 31 is most focused, that is, the position where the laser irradiation diameter is minimized, but the present invention is not limited to this.
  • the position of the condensing optical system 33 when the laser irradiation diameter becomes a desired size is determined by the focal length of the condensing optical system 33. Therefore, originally, the distance from the surface of the sample 100 to the condensing optical system 33 is adjusted so that the laser irradiation diameter becomes a predetermined size, but due to disturbance or the like, the sample table 11 and the condensing optical system 33 The placement may shift. The deviation of the arrangement between the sample table 11 and the condensing optical system 33 affects the size of the laser irradiation diameter even if the deviation is slight.
  • the laser beam 31 is irradiated to a predetermined region on the sample, and the optical microscopic image of the confirmation region at that time is displayed on the display unit 64. It has become like.
  • the optical microscopic image displayed in the confirmation area corresponds to the condensed state confirmation image of the present invention.
  • the user In displaying the optical microscopic image of the confirmation area on the display unit 64, the user prepares a sample in which a predetermined dye is uniformly applied to the surface of the slide glass, and sets this on the sample table 11. Subsequently, when the user of the imaging mass spectrometer gives an instruction to execute the irradiation diameter confirmation operation from the input unit 63, the irradiation diameter confirmation control unit 62 designates one or a plurality of confirmation regions on the sample 100. The irradiation diameter confirmation control unit 62 stores the size and position of the confirmation area according to the number of the designated confirmation areas, and the position of the condensing optical system 33 when irradiating each confirmation area with the laser beam.
  • the irradiation diameter confirmation control unit 62 moves the sample table 11 through the sample table drive unit 12 so that the irradiation position of the laser beam moves with a step width corresponding to the size and position of the confirmation area. Further, the condensing optical system 33 is moved through the condensing optical system driving unit 34 so that the condensing optical system 33 is at the position set for the confirmation region. Then, the laser irradiation unit 30 is driven via the irradiation control unit 37 to irradiate the laser beam in a pulsed manner. When the surface of the sample is irradiated with the laser beam, the dye is scattered by ablation and a mark (irradiation mark) of the laser beam is formed.
  • FIG. 2 shows a plurality of confirmation regions 110 designated on the sample 100.
  • FIG. 2 shows an example in which 11 rectangular confirmation regions 110 are designated on the sample 100, but the shape, number, size, etc. of the confirmation regions 110 are not limited to the example shown in FIG.
  • the laser irradiation unit 30 may irradiate each confirmation region 110 with the laser beam only once, or may irradiate each of a plurality of different locations in each confirmation region 110 with the laser beam once. That is, the laser beam is irradiated multiple times in each confirmation area).
  • the irradiation diameter confirmation control unit 62 causes the imaging unit 40 to acquire an optical microscopic image of the surface of the sample 100 including the confirmation region 110.
  • the optical micro image acquired by the imaging unit 40 is sent to the image data processing unit 51 of the data processing unit 50, where appropriate data processing is performed to create optical micro image data.
  • the created optical microscopic image data is stored in the image data storage unit 511 in association with the position information of the condensing optical system 33 set in the confirmation area 110.
  • the irradiation diameter confirmation control unit 62 is subjected to the image data storage unit.
  • the optical microscopic image data of each confirmation area 110 is read from 511, an irradiation diameter confirmation screen is created, and the screen is output to the display unit 64.
  • FIG. 3 shows an example of the irradiation diameter confirmation screen 642 displayed on the display screen 641 of the display unit 64.
  • the irradiation diameter confirmation screen 642 displays the optical microscopic images 643 of the sample surface including 11 confirmation regions side by side. The user looks at these optical microscopic images 643, and determines a confirmation region in which the irradiation mark of the laser beam is in a desired state as a determination confirmation region.
  • the determination confirmation area can be determined, for example, by moving the cursor on the display screen 641 to the vicinity of the desired confirmation area using a mouse and performing a click operation.
  • the mouse is the selection operation unit.
  • the irradiation diameter confirmation control unit 62 reads out the position of the condensing optical system 33 when the laser beam 31 is irradiated to the determination confirmation area from the image data storage unit 511, and the position setting unit 621.
  • the condensing optical system 33 set in the position setting unit 621 can be the position of the condensing optical system 33 at the time of the next mass spectrometry.
  • the laser beam irradiation position on the sample is moved by moving the sample table (that is, the sample table drive unit 12 is used as the irradiation position drive unit), but the laser irradiation unit 30 is moved.
  • the laser irradiation position on the sample may be moved by moving the laser irradiation unit 30 or changing the posture (direction) of the laser irradiation unit 30 (that is, the laser irradiation unit drive unit that changes the position or orientation of the laser irradiation unit 30). May be provided).
  • the irradiation position driving unit may be configured to move both the sample table and the laser beam.
  • the position of the condensing optical system 33 corresponding to the confirmation area was configured to be automatically set to the position for the next mass spectrometry, but the user who saw the optical microscopic image 643 displayed in the confirmation area was allowed to manually set the position of the condensing optical system. You may.
  • the image data processing unit 51 performs, for example, binarization processing of the optical microscopic image acquired by the imaging unit 40 to extract the contour of the laser beam irradiation mark in the confirmation region 110, and the diameter of the irradiation mark (from the contour).
  • the irradiation diameter) may be calculated.
  • the irradiation diameter data of each confirmation area 110 calculated by the image data processing unit 51 is stored in the image data storage unit 511.
  • the irradiation diameter data stored in the image data storage unit 511 is read out together with the optical microscopic image data of each confirmation area 110 and displayed on the irradiation diameter confirmation screen.
  • the image data processing unit 51 corresponds to the irradiation diameter calculation unit.
  • a plurality of optical microscopic images are collectively displayed on one display screen, but a plurality of optical microscopic images may be displayed one by one on the display screen in order.
  • the imaging mass spectrometer is A device that generates ions by irradiating a sample with laser light and mass spectrometrically analyzes the ions.
  • a laser irradiation unit that emits laser light toward the sample
  • a condensing optical system that is arranged between the laser irradiation unit and the sample and that collects the laser light emitted from the laser irradiation unit.
  • An imaging unit that acquires a focused state confirmation image, which is an optical microscopic image capable of confirming the focused state of the laser light emitted by the laser irradiation unit on the sample. It is provided with a display unit that displays a condensing state confirmation image acquired by the imaging unit on a display screen.
  • the user sees the focused state confirmation image displayed on the display screen of the display unit and confirms the focused state of the laser light emitted by the laser irradiation unit on the sample.
  • the condensed state confirmation image includes an image in which the irradiation diameter at the irradiation position of the laser light can be confirmed (for example, an image in which the outline of the irradiation mark of the laser light is clearly shown), and the laser light at the irradiation position of the laser light.
  • An image in which the light intensity per unit area of the above can be confirmed for example, an image in which the light intensity is represented by shading) is included.
  • the display unit should display the scale together with the condensing state confirmation image on the display screen.
  • the imaging mass spectrometer of the second aspect is further described in the imaging mass spectrometer of the first aspect.
  • An irradiation diameter calculation unit for calculating the irradiation diameter, which is the diameter of the irradiation mark of the laser beam on the sample, from the focused state confirmation image acquired by the imaging unit is provided.
  • the display unit may display the irradiation diameter on the display screen together with the condensing state confirmation image.
  • the user can confirm the focused state of the laser beam using the irradiation diameter displayed on the display screen as an index.
  • the sample used for acquiring an image in which the irradiation diameter of the laser beam can be confirmed is, for example, a slide glass in which a dye is uniformly applied to the surface of the slide glass, and the laser beam is used. It is possible to use a sample in which the dye in the region is scattered by ablation and a mark (irradiation mark) of being irradiated with the laser beam is formed when the light is irradiated. Further, for example, a sample in which the matrix used for preparing the sample for MALDI is applied to the surface of the slide glass can be used as a sample for confirming the irradiation diameter.
  • a sample for MALDI (that is, a sample in which a matrix is applied on a tissue section cut out from a living tissue) can also serve as a sample for confirming the irradiation diameter.
  • the region of the MALDI sample that is out of the analysis target region is used to acquire an image in which the irradiation diameter can be confirmed.
  • the imaging mass spectrometer of the third aspect is the imaging mass spectrometer of the second aspect.
  • the imaging unit It is an image of the non-analytical target area acquired by.
  • a condensing optical system drive unit that moves the condensing optical system so that the condensing state of the laser light on the sample changes. It is provided with an irradiation position moving portion for moving the irradiation position of the laser beam on the sample.
  • the display unit displays a condensing state confirmation image of each confirmation region when the laser beam is irradiated at different positions of the condensing optical system with respect to the plurality of confirmation regions on the sample. It may be displayed in.
  • the irradiation position moving unit may move the laser beam or the sample table.
  • Examples of the method of moving the laser beam include moving the emitting laser irradiation unit and changing the posture (direction) of the laser irradiation unit so that the emission direction of the laser beam changes.
  • the user sees the focused state confirmation images of a plurality of confirmation areas displayed on the display screen, and the focused optics when the focused state of the laser light is in a desired state. The position of the system can be confirmed.
  • the position of the condensing optical system corresponding to the confirmation region where the irradiation diameter of the laser beam is the minimum is determined by the predetermined laser irradiation diameter in relation to the sample and the measurement purpose.
  • the position of the condensing optical system corresponding to the confirmation region in the state closest to the laser irradiation diameter can be set as the position of the condensing optical system at the time of measurement.
  • the imaging mass spectrometer of the fifth aspect is the imaging mass spectrometer of the fourth aspect.
  • the display unit may display the condensed state confirmation images of the plurality of confirmation areas side by side on the display screen.
  • the imaging mass spectrometer of the fifth aspect it becomes easy to compare the focused state of the laser light irradiated to each of the plurality of confirmation areas displayed on the display screen.
  • the user can easily adjust the position of the condensing optical system.

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PCT/JP2020/036855 2019-10-16 2020-09-29 イメージング質量分析装置 Ceased WO2021075254A1 (ja)

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CN202080067563.6A CN114450587A (zh) 2019-10-16 2020-09-29 成像质量分析装置
US17/641,662 US12154772B2 (en) 2019-10-16 2020-09-29 Imaging mass spectrometer
JP2021552300A JP7215591B2 (ja) 2019-10-16 2020-09-29 イメージング質量分析装置

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