JP2009168673A - Ionization method and apparatus - Google Patents

Abstract

[Objective] To improve spatial resolution and ionization efficiency.
The first and second laser beams L1 and L2 are directed toward the sample SA from different directions so that the spots S1 and S2 of the two laser beams partially overlap on the sample surface. , Condensing and irradiating, detaching the fine particles from the sample portion where these two laser beams overlap, and irradiating with the third laser beam L3 toward the space where the detached fine particles exist and vaporizing Let The vaporized sample ions are guided to the mass spectrometer 42 through the capillary 41.
[Selection] Figure 2

Description

  The present invention relates to an ionization method and apparatus, and more particularly to a method and apparatus for ionizing a sample (biological tissue, cell, organic material, etc.) for mass spectrometry.

MALDI (Matrix Assisted Laser Desorption / Ionization), which is a typical ionization method for biomolecules, is a phenomenon in which ultraviolet laser light is absorbed into a solid matrix and ablation (the material that absorbed the beam energy explodes as a fragment with large energy). ) To desorb and ionize biopolymers. In recent years, attempts have been made to apply this technique to two-dimensional imaging.
Koichi Tanaka, Hiroaki Waki, Yutaka Ido, Satoshi Akita, Yoshikazu Yoshida, Tamio Yoshida, T. Matsuo “Protein and polymer analyzes up to m / z 100 000 by laser ionization time-of-flight mass spectrometry” Rapid Communications in Mass Spectrometry, Volume 2, Issue 8, August 1988, pp151-153 Richard M. Caprioli, Terry B. Farmer, and Jocelyn Gile “Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOF MS” Analytical Chemistry, Vol.69, No.23, December 1, 1997, pp4751-4760

  However, since the spatial resolution of MALDI is determined by the diffraction limit of the light used, the wavelength order is the limit. In addition, MALDI has the problem that the sample cannot be completely vaporized, so that the ionization efficiency is low.

  The present invention provides a method and apparatus capable of generating fine particles of a sample in a range finer than the wavelength order and ionizing them with high efficiency.

  The ionization method according to the present invention is such that the first and second laser beams are directed to the sample from different directions, and at least one of the two laser beams overlaps the sample surface in part. Condensing and irradiating the beam, detaching the fine particles from the sample portion where these two laser beams overlap, and irradiating the third laser beam toward the space where the desorbed fine particles exist It vaporizes fine particles.

  An ionization apparatus according to the present invention includes a laser optical system that generates first, second, and third laser beams, and separately collects and projects these three laser beams. The laser beam was directed to the sample from different directions, and at least one of the laser beams was focused and projected so that the two laser beams overlapped on the sample surface in part. The direction, intensity, beam diameter, and the like are adjusted so that the fine particles are detached from the sample in the region, and the third laser beam is a space where the fine particles detached from the sample portion where the laser beam overlaps the second laser beam exists. (Direction, intensity, beam diameter, etc.) are adjusted so as to be focused and projected toward the center.

  According to the present invention, the first and second laser beams are condensed (to near the diffraction limit of light if necessary), and the irradiation ranges of these two laser beams are partially overlapped on the sample surface. . In the irradiation range (region) where the two laser beams overlap, the laser intensity is adjusted so that the laser intensity is such that fine particles of the sample are detached. Fine particles are detached from the sample only in the region where the laser beams overlap. The laser beam spot cannot be made smaller than the range defined by the diffraction limit, but the area where the two laser beams overlap can be made smaller than the range defined by the diffraction limit. . Since desorption from the sample is performed only in the region where the two laser spots overlap, a spatial resolution exceeding the diffraction limit of light can be obtained.

  The sample fine particles detached from the fine region in this way are then irradiated by the third laser beam, and are almost completely vaporized and ionized.

  The ionized sample molecules are guided to a mass spectrometer and subjected to mass analysis.

  As described above, since the spatial resolution is increased, a two-dimensional molecular image image can be obtained by scanning a place where two laser light spots overlap.

  Two or three laser beams can be generated by beam splitting the laser beams emitted from one or two laser devices.

  The ionization method according to the present invention can be performed under atmospheric pressure or in vacuum.

  FIG. 1 shows a schematic configuration of an ionization apparatus capable of forming a molecular imaging image according to an embodiment of the present invention.

  The ionizer can operate at atmospheric pressure. The sample ions desorbed and ionized from the sample by the ionizer are guided to the mass spectrometer 42 through the capillary 41, for example. An example of a mass spectrometer is an orthogonal time-of-flight mass spectrometer, but the present invention is also applicable to a mass spectrometer such as a (linear) ion trap apparatus.

  A sample stage 10 is provided in place in the ionizer. A substrate 12 coated with a sample is placed on the sample stage 10 and fixed by an appropriate fixture (not shown). For convenience, the upper surface of the sample stage 10 is the XY plane, and the direction perpendicular thereto is the Z direction.

  An XYZ manipulator 11 is provided in the ionizer, and the XYZ manipulator 11 holds the sample stage 10 so as to be independently movable in the X, Y, and Z directions. The drive source for displacement in the X direction, Y direction and Z direction can give mechanical vibration such as a drive device using a piezo element or a stepping motor, and in the X, Y, Z direction with a resolution of nm order. It is desirable to be able to control the displacement of each separately. It is only necessary to be able to displace in at least one of the X and Y directions, and movement in the Z direction is not necessarily required.

  The first and second laser devices 21 and 22 and their condensing optical systems (lenses, etc.) 31 and 32 (including the laser device and the condensing optical system) are obliquely above the sample stage 10. ) Is provided. These laser optical systems can adjust the focal point position, laser projection direction, and the like, and can also control the laser beam diameter and laser intensity. Further, on the side of the sample stage 10, a third laser device 23 and its condensing optical system (lens or the like) 33 (referred to as a laser optical system including the laser device and the condensing optical system) are arranged. This laser optical system can also adjust the focal point position, laser projection direction, beam diameter, and laser intensity. The laser beams emitted from the laser devices 21, 22, and 23 are denoted as L1, L2, and L3, respectively. It is also possible to divide the light emitted from one laser device into two (or three) and use these as laser beams L1, L2 (and L3).

  The tip of the ion sampling capillary 41 is arranged above the vicinity of the place on the sample stage 10 where the sample is placed. The capillary 41 is for guiding the sample ions generated here to the mass spectrometer 42. It is desirable that the capillary 41 is also held so that its angle and position can be adjusted.

  A suitable matrix suitable for the sample to be analyzed (peptide, protein, synthetic polymer, pigment, DNA, etc.) is selected (the matrix is not necessarily required, depends on the sample to be analyzed) and mixed with the sample. The sample mixed with the matrix is dissolved or ground in a suitable solvent and applied onto the glass substrate 12. The target sample may be placed or coated on the substrate 12 as it is, or an appropriate matrix may be thinly coated on the sample. In any case, the sample SA includes both those that include the matrix and those that do not. The glass substrate 12 is fixed on the sample stage 10.

  FIG. 2 shows the irradiation direction and irradiation range of the three laser beams L1, L2, and L3 in an enlarged manner, and FIG. 3 shows the overlap of the laser spots of the two laser beams L1 and L2. Laser spots formed on the surface of the sample SA by the lasers L1 and L2 are denoted by S1 and S2.

  The focal positions and projection directions (and the laser beam diameter if necessary) of the first and second laser beams L1 and L2 by the first and second laser optical systems are adjusted, and the laser beams L1 and L2 are used. The laser spots S1 and S2 are partially overlapped on the surface of the sample SA. The laser beams L1 and L2 have a laser beam intensity enough to prevent the sample from being desorbed by the laser beam L1 or the laser beam L2 alone, and the fine particles are desorbed from the sample only in the region where the laser spots S1 and S2 overlap. The laser intensity is adjusted. The laser beams L1 and L2 may be continuous light or pulsed light. However, in the case of pulsed light, the laser beams L1 and L2 are simultaneously irradiated with the sample synchronously.

  Each of the laser beams L1 and L2 cannot be focused below the spot diameter defined by the light diffraction limit, but the region where the two laser beams L1 and L2 overlap as described above exceeds the light diffraction limit. It is possible to achieve a very narrow spatial resolution.

  When the sample SA includes a matrix, it is preferable to use an ultraviolet laser as the second laser beams L1 and L2, but an infrared laser may be used to desorb and ionize the aqueous solution system.

  FIGS. 1 to 3 show the superposition of two laser beams focused close to the diffraction limit. For example, the second laser beam L2 is guided near the sample SA by an optical fiber to generate near-field light. , And this may be superimposed on the first laser beam L1 (condensed).

The third laser beam L3 is condensed and projected toward a space where fine particles desorbed from the sample SA (before being sucked by the capillary 41) are present. The third laser beam L3 is preferably pulsed or continuous wave infrared light (for example, CO ₂ laser light). Fine particles containing droplets of various sizes are almost completely vaporized by irradiation with the third laser beam L3 and introduced into the mass spectrometer 42 through the capillary 41 (since the mass analyzer 42 is a vacuum). , Sample ions are aspirated).

  As another embodiment, a discharge electrode is provided between the sample stage 10 and the ion intake port (tip of the capillary 41) to fragment sample ions. This makes it possible to analyze proteins. A housing including the sample stage 10, the XYZ manipulator 11 and the like may be provided, and the inside of the housing may be evacuated. The laser devices 21, 22, and 23 are disposed outside the housing, and can irradiate the laser beam through a window transparent to the laser beam.

1 shows a schematic configuration of an ionization apparatus. The focal positions and directions of the three laser beams are shown enlarged. 2 shows the overlap of two laser beam spots.

Explanation of symbols

10 Sample stage
12 Glass substrate
21, 22, 23 Laser equipment
31, 32, 33 Condensing optical system
41 capillary
42 Mass spectrometer L1, L2, L3 Laser beam SA Sample S1, S2 Laser spot

Claims (4)

The first and second laser beams are directed to the sample from different directions, and at least one laser beam is focused so that the two laser beams partially overlap the sample surface. , Irradiate and remove the fine particles from the sample part where these two laser beams overlap,
The third laser beam is irradiated toward the space where the detached fine particles exist to vaporize the fine particles.
Ionization method. Lead the vaporized sample ions to the mass spectrometer,
The ionization method according to claim 1. A laser optical system for generating first, second and third laser beams and separately focusing and projecting the three laser beams;
The first and second laser beams are focused and projected from different directions toward the sample, and the two laser beams partially overlap on the sample surface. Adjusted so that
The third laser beam is adjusted so as to be focused and projected toward the space where the fine particles detached from the sample portion where the two laser beams overlap are present.
Ionizer.   The ionization according to claim 3, further comprising means for desorbing the sample ions by irradiation with the first and second laser beams and guiding the sample ions vaporized by the third laser beam irradiation to a mass spectrometer. apparatus.

Images