WO2004068131A1

WO2004068131A1 - Ionizer and fine area analyzer

Info

Publication number: WO2004068131A1
Application number: PCT/JP2004/000843
Authority: WO
Inventors: Katsutoshi Takahashi
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2003-01-31
Filing date: 2004-01-29
Publication date: 2004-08-12
Also published as: JP2004264043A

Abstract

(Issue): An ionizer, an ionizing method, a mass analyzer and a fine area analyzer for carrying out analysis in a fine area with a high resolution by using a mass analyzer. (Solutions): An ionizer, which is provided with a laser emitting means and a near-field light generating means for generating a near-field light on receiving a laser emitted from the laser emitting means, and which ionizes a sample mixed with a matrix by irradiating the matrix with a near-field light generated form the near-field light generating means, is used. Or, an ionizer, which is provided with a laser emitting means, an optical fiber for guiding a laser emitted from the laser emitting means, and a laser irradiating probe connected with the tip end of the optical fiber, and which ionizes a sample mixed with a matrix by irradiating the matrix with a laser emitted from laser irradiating probe, is used. In addition, a fine area analyzer is obtained by combining this ionizer with a mass analyzer.

Description

Description ^: Ionization device and micro area analyzer

The present invention relates to an apparatus and a method capable of performing analysis with high resolution in a minute area. More specifically, the present invention relates to an ionization apparatus and an ionization method for ionizing a sample by irradiating a sample-mixed matrix with near-field light or laser using a probe, and to a mass spectrometer and a micro-area analyzer equipped with the ionization apparatus . Background art

Matrix-assisted laser desorption / ionization (MALD I) mass spectrometers are highly useful in that they can mass-analyze molecules that are difficult to ionize, such as proteins. Mass spectrometers are usually used for the purpose of detecting a very small amount of sample with high sensitivity, but are not commonly used for observing microscopic areas like a microscope. As an example of observation of a micro area using a MALD I mass spectrometer, an example of imaging a brain cell section is known (see Non-Patent Documents 1 to 4). On the other hand, the technology using near-field light has been applied to a scanning near-field optical microscope (SNOM), and has realized an optical microscope with high resolution exceeding the diffraction limit of light.

As an example of applying near-field light to mass spectrometry, an ultraviolet laser is incident on a near-field light probe and irradiated with the generated near-field light to decompose the triazane compound. Is known by mass spectrometry (see Non-Patent Document 5).

(Non-Patent Document 1) Caprio1i, R.M.eta1., Ana1.Chem., 1997, 69, 4751-4760

(Non-Patent Document 2) Stoeckli, M. etal., J. Am. Soc. Mass Spectrom, 1999, 10, 67—71. (Non-Patent Document 3) Caur and, P. eta 1., An a 1. Chem., 1999, 71, 5263—5270

(Non-Patent Document 4) Stoeclkli, M.eta 1., NATURE MED I CINE, 2001, 7, 493

(Non-Patent Document 5) Stoec1e, R.eta1., Anal.Chem., 2001, Vol. 73, pl999—1402

However, in ordinary MALD I, the laser is focused by a lens and irradiates the matrix, so the diameter of the laser that can be focused is limited to 12 of the wavelength of one laser by the diffraction limit of light. Would. For this reason, it has not been possible to irradiate a laser to a region having a wavelength approximately equal to or less than 12 wavelengths to ionize the sample. In the measuring methods described in Non-Patent Documents 1 to 4, the resolution was as low as about 25 m.

As a method for improving these resolutions, it is conceivable to shorten the wavelength of the laser to be irradiated. However, ordinary MALD I uses an ultraviolet laser, and there is a limit to shortening the wavelength. In addition, ultraviolet lasers have a problem that they damage proteins and other samples due to their high energy.

On the other hand, when SNOM was used, it was practically impossible to measure what kind of substance was present in the micro area to be analyzed using only information obtained from an optical microscope.

In addition, the substance described in Non-Patent Document 5 cannot be directly used for mass analysis of proteins and the like because the analyte is a synthetic compound and decomposes the analyte into nitrogen molecules. .

Therefore, there has been a demand for a technique for performing mass spectrometry with high resolution without excessively damaging the sample in a minute area. Disclosure of the invention

An object of the present invention is to provide an apparatus and a method for performing analysis in a minute area with high resolution without excessively damaging a sample using a mass spectrometer. The present inventors have studied to solve the above problems, and as a result, have found that the above problems can be solved by applying laser irradiation using near-field light or a probe to MAL DI, thereby completing the present invention. Reached.

That is, the present invention includes a laser emitting unit, and a near-field light generating unit that receives a laser emitted from the laser emitting unit and generates near-field light when the laser is emitted from the near-field light generating unit. The present invention relates to an ionization apparatus that irradiates a sample mixed with Matritas by irradiating a near-field light to a matrix.

Further, the present invention provides a laser emitting means, an optical fiber for guiding a laser emitted from the laser emitting means, and a laser irradiation probe connected to a tip of the optical fiber. The present invention relates to an ionization apparatus for irradiating a matrix with a laser emitted from the laser irradiation probe to ionize a sample mixed with the matrix.

Further, the present invention relates to an ionization apparatus and an ionization method for irradiating a sample mixed with a matrix by irradiating the matrix with near-field light.

Furthermore, the present invention relates to a mass spectrometer provided with the above-mentioned ionization device.

Further, the present invention provides the above ionization apparatus, information input means for inputting information on an area to be analyzed to be scanned,

Drive mechanism control means for controlling a drive mechanism based on the area information;

Mass spectrometry means;

Analysis result analysis means for analyzing the result obtained from the mass analysis means,

Storage means for storing position information transmitted from the drive mechanism control means and analysis results obtained from the analysis result analysis means;

Image means for performing imaging from the position information and the analysis result stored in the storage means,

Display means for displaying the imaged result;

The present invention relates to a micro-area analysis device provided with:

Further, the present invention provides a method for ionizing a sample using the above-mentioned ionization apparatus,

Place the near-field light generating means or laser irradiation probe at a predetermined distance from the object to be analyzed. Process, and

Irradiating the target with near-field light or laser emitted from a laser irradiation probe,

And a method for ionizing a sample.

ADVANTAGE OF THE INVENTION According to this invention, the analysis in a micro area | region can be performed with high resolution using a mass spectrometer, without excessively damaging a sample. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an enlarged view near the near-field optical probe of the ionization apparatus of the present invention.

FIG. 2 is a schematic configuration diagram of the ionization device and the mass spectrometer of the present invention.

FIG. 3 is an enlarged view near the near-field optical probe of the ionization apparatus of the present invention.

FIG. 4 is an enlarged view near the near-field optical probe of the ionization device of the present invention.

FIG. 5 is a schematic configuration diagram of the micro region analysis device of the present invention.

FIG. 6 is a view showing a laser irradiation probe used in the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, near-field light is light that is generated near the surface of an irradiation target when the irradiation target is irradiated with light and propagates only along the surface, and critical light from a medium having a high refractive index to a medium having a low refractive index. It includes both non-propagating light (so-called evanescent light) that penetrates into the low-refractive-index medium when light is introduced at an angle above the angle. Further, in the present invention, the minute region means a region having a diameter of 15 m or less. Hereinafter, the present invention will be described in more detail with reference to the drawings, but the present invention is not limited to these drawings.

1 and 2 show a first embodiment of the ionization apparatus of the present invention. 1 and 2, the ionizer has an ultraviolet laser oscillator 1, an optical fiber 13 connected to the ultraviolet laser oscillator 1, and a near-field optical probe 5 connected to the other end of the optical fiber 1.3. Further, a suction tube 12 is arranged near the near-field optical probe 5. The suction tube 12 is connected to a suction controller 30, and the suction controller 30 is connected to a mass spectrometer 50. The near-field optical probe 5 Below the sample, a protein sample 8 mixed with a matrix 9 is arranged. The ultraviolet laser oscillator 1, the near-field light probe 5, and the suction tube 12 constitute the laser emitting means, the near-field light generating means, and the sample transferring means of the present invention, respectively. The first embodiment of the present invention is configured as described above, and its operation will be described below. The laser light emitted from the ultraviolet laser oscillator 1 enters the near-field optical probe 5 through the optical fiber 3. When laser light is incident on the near-field optical probe 5, near-field light 7 is generated near the tip of the near-field optical probe 5. The generated near-field light 7 irradiates the matrix 9 mixed with the protein sample 8. When the matrix 9 irradiated with the near-field light 7 evaporates together with the protein sample 8, the protein sample 8 is ionized, and protein ions 10 are generated. The generated protein ions 10 are sucked into the suction tube 12 through the suction port 11. The protein ions 10 sucked into the suction tube 12 pass through the suction control section 30 and are sent to the mass spectrometry section 50. The mass spectrometer 50 performs mass spectrometry of the protein ions 10. As a result, it is possible to irradiate near-field light only to a small region smaller than the wavelength width of the ultraviolet laser, and mass spectrometry can be performed with high resolution.

FIG. 3 shows a second embodiment of the ionization device of the present invention. In the embodiment of FIGS. 1 and 2, an ultraviolet laser oscillator is provided, whereas in the present embodiment, an infrared laser oscillator (not shown) is provided. In FIG. 3, the ionizer has a hollow optical fiber 130 connected to an infrared laser oscillator (not shown), and a near-field optical probe 5 connected to the other end of the hollow optical fiber 130. Further, a suction tube 12 is disposed near the near-field optical probe 5. The suction tube 12 is connected to a mass spectrometer (not shown). A cell section 100 is arranged below the near-field optical probe 5, and the cell section 100 is fixed to a sample stage 150. The sample stage 150 is connected to a stage drive mechanism (not shown), and the stage drive mechanism is connected to a drive mechanism controller. The infrared laser oscillator, near-field light probe 5, suction tube 12, and cell section 100 constitute the laser emitting means, near-field light generating means, sample transfer means, and analysis object of the present invention, respectively. I do. The second embodiment of the present invention is configured as described above, and its operation will be described below. Laser light emitted from the infrared laser oscillator passes through the hollow optical fiber 130 and enters the near-field optical probe 5. When the laser light is incident, near-field light 7 is generated near the tip of the near-field light probe 5. The generated near-field light 7 is applied to a matrix existing in the cell section 100. The matrix is mixed with a protein sample present in the cell section 100, and when the matrix is irradiated with near-field light 7 and vaporized together with the protein sample, the protein sample is ionized and the protein ion 1 0 is generated. The generated protein ions 10 are sucked into the suction tube 12 through the suction port 11. The protein ions 10 sucked into the suction tube 12 are sent to the mass spectrometer. The mass spectrometer performs mass spectrometry of protein ions 10. Further, the cell section 100 is fixed to a sample stage 150, and the sample stage 150 can be slid in a horizontal direction by a stage driving mechanism. The stage and the drive mechanism are controlled by the drive mechanism control unit, and slide the sample stage 150 back and forth, left and right. As a result, the near-field light probe 5 can scan the surface of the cell slice 100. Then, the mass spectrometry of the protein sample existing at each position of the cell section can be performed, and it is possible to examine what portion of the cell section contains what kind of protein.

FIG. 4 shows a third embodiment of the ionization device of the present invention. Although not shown, an infrared laser oscillator is also provided in this embodiment. In FIG. 4, the ionizer is provided with a mirror 210 for receiving a laser from an infrared laser oscillator and an atomic force microscope (AFM) probe 230. The AFM probe 230 has a pinhole 235 open. A skimmer 250 is arranged above the AFM probe 230, and a mass analyzer (not shown) is arranged ahead of the skimmer 250. A cell sample 280 is arranged below the pinhole 235, and the cell sample 280 is fixed to a sample stage 270. The sample stage 270 is connected to a stage drive mechanism (not shown), and the stage drive mechanism is connected to a drive mechanism control unit. The infrared laser oscillator, the AFM probe 230, the skimmer 250, and the cell sample 280 correspond to the laser emitting means of the present invention. The means for generating the in-field light, the means for transferring the sample, and the object to be analyzed are configured.

The third embodiment of the present invention is configured as described above, and its operation will be described below. The laser light emitted from the infrared laser oscillator is reflected by the mirror 210 and enters the AFM probe 230. Near-field light 7 is generated from the pinhole 2 35 of the AFM probe 230 into which the laser beam has entered. The generated proximity light 7 is applied to the matrix existing inside or on the surface of the cell membrane 290 of the cell sample 280. The matrix is mixed with the membrane protein sample 300, and when the near-field light 7 is applied to the matrix and evaporates together with the membrane protein sample 300, the membrane protein sample 300 is ionized and the membrane is sampled. Protein ions 310 are produced. The generated membrane protein ion 310 is sucked by the skimmer 250, and the membrane protein ion 310 sucked by the skimmer 250 is sent to the mass spectrometer. In the mass spectrometry section, mass spectrometry of the membrane protein ion 310 is performed. In addition, the cell sample 280 is fixed to a sample stage 270, and the sample stage 270 can be slid in a horizontal direction by a stage driving mechanism. The stage driving mechanism is controlled by a driving mechanism control unit, and slides the sample stage 270 forward and backward and left and right. Thus, the AFM probe 230 can scan the surface of the cell membrane 290. Then, mass spectrometry of the membrane protein sample present at each position of the cell membrane can be performed, and it is possible to examine what part of the cell membrane contains what kind of membrane protein.

FIG. 5 is a diagram showing an embodiment of the micro region analysis device of the present invention. In FIG. 5, the control unit 500 is configured by a computer including a drive mechanism control unit 520, an analysis result analysis unit 540, an imaging unit 550, and a storage unit 560. The control section 500 is connected to the drive mechanism 510 via the drive mechanism control section 5200, and the drive mechanism 5100 is connected to the sample stage 150. Further, the control section 500 is connected to the mass analysis section 50 via the analysis result analysis section 540. Further, the control unit 500 is connected to the keyboard 530 via the drive mechanism control unit and to the display 570 via the imaging unit 550, respectively. Other configurations are the same as in FIG. The keyboard 530, drive mechanism controller 520, mass spectrometer 50, analysis result analyzer 540, storage 560, imager 550, and display 570 Departure The information input means, the drive mechanism control means, the mass analysis means, the analysis result analysis means, the storage means, the imaging means, and the display means are constituted respectively.

The microregion analyzer of the present invention is configured as described above, and its operation will be described below. The area information of the cell section 100 to be scanned is input to the keyboard 5300, and the input area information is transmitted to the drive mechanism control section 5200. Here, the region means a scanning range of the cell section 100, and the region is specified by, for example, the coordinates of the sample stage 150 on which the cell section 100 is fixed. The drive mechanism control section 520 slides the sample stage 150 forward, backward, left, and right by the drive mechanism 510 based on the input area information. At the same time, the position information of the sample stage 150, that is, the position information indicating which part of the cell slice 100 is irradiated with the near-field light 7 is stored from the drive mechanism control unit 520. Transmitted to part 560. Analysis result analysis unit 5

40 analyzes the result obtained from the mass spectrometry unit 50 and transmits the analysis result to the storage unit 560. The storage unit 560 stores an analysis result corresponding to the position information. The imaging unit 550 uses the position information and the analysis result stored in the storage unit 560 to determine which substance is present at which position in the cell slice 100, for example, for each substance. Create an image as a color-coded plot. The imaged result is displayed on the display

Displayed at 570. As a result, it becomes possible to easily understand what kind of substance is present at which position in a minute area such as a cell slice.

The near-field light generating means used in the present invention is not particularly limited as long as it can generate near-field light. For example, a near-field light probe for SNOM, an AFM probe, or the like can be used. . Examples of the material of the probe include a material in which glass or a transparent plastic is coated in a single layer or a multilayer with aluminum, magnesium fluoride, silver, or the like.

As the structure of the probe, for example, a probe for SNOM can be a cantilever type, a straight fiber type, or a small scatterer type. Thus, for example, even if the wavelength of the incident laser is 0.1 to 15 m, it exceeds the diffraction limit of the laser beam and is close to the minute area of about 0.01 to L wm on the analysis target. Field light can be applied. The irradiation distance of near-field light in the case of the SNOM probe is about 1/2 of the wavelength of the laser, and the distance between the SNOM probe and the analysis target is small. The distance is also within this irradiation distance.

In the case of an AFM probe, a pinhole is formed as shown in FIG. 4, and the diameter of the pinhole can be, for example, about 0.1 μm. Thus, for example, even if the wavelength of the incident laser beam is 3 to L0 m, the near-field light exceeds the diffraction limit of the laser beam and reaches a minute area with a diameter of about 0.1 to 1111 on the analysis target. Can be irradiated.

When an infrared laser is used as the laser, a hollow fiber can be used as shown in FIGS. 3 and 5, in addition to the solid optical fiber and the fiber probe. As a result, laser scattering in the fiber is suppressed, and the sample can be efficiently irradiated with near-field light with little energy.

FIG. 6 is a diagram showing an example of the structure of the laser irradiation probe 700 used in the present invention. In FIG. 6, the distal end portion 705 of the laser irradiation probe 700 is thinner and open than the main body portion 702. The diameter of the opening 707 is not particularly limited, and can be appropriately determined according to the area of the region to be irradiated with the laser beam. From the viewpoint of irradiating the laser beam to the minute region, the diameter of the opening 707 is preferably 15 m or less, more preferably 5 or less, further preferably l ^ m or less, and most preferably 0.1 m or less. There is no particular lower limit on the diameter of the opening 707, but it is preferably at least 0.01 m from the viewpoint of ease of processing. Further, from another viewpoint, it is preferable that the diameter of the opening is smaller than the wavelength of the laser incident on the probe.

In FIGS. 1 to 3 and 5, another embodiment of the present invention is shown by using a laser irradiation probe 700 instead of the near-field light probe 5. In this case, for example, if the wavelength of the incident laser is 0.1 to 15 m, the laser can be applied to a minute area of 0.1 to 15 m. By using the laser irradiation probe 700, the irradiation distance can be increased and the energy of the laser to be irradiated can be increased as compared with the case where the near-field optical probe 5 is used. It becomes possible.

The subject to be scanned in the present invention is not particularly limited. A cell membrane is a preferred example. As shown in Figs. 3 to 5 above, scanning can be performed by sliding the sample stage on which the object to be analyzed is fixed and scanning, or by using a near-field optical probe or the like with the sample stage fixed. It is also possible to scan by moving the field light generating means or the laser irradiation probe. Further, both the sample stage, the near-field light generating means, or the laser irradiation probe may be moved. There is no particular limitation on the sample stage as long as it can fix the analysis target.However, when scanning cell sections or cell membranes, from the viewpoint of maintaining the shape of cells at a very low temperature, for example, 4 Those that can maintain the temperature around K are preferred.

Next, a method for controlling the distance between the near-field light generating means or the laser irradiation probe used in the present invention and the object to be analyzed will be described. There are no particular restrictions on the method of controlling the distance between the near-field light generating means or the laser irradiation probe and the object to be analyzed, and any method can be used as long as it can be placed within the distance that the near-field light or laser is irradiated. Is also good. For example, the thickness of the object to be analyzed is measured in advance, and the near-field light generating means or the laser irradiation probe is fixed within the range where the near-field light or the laser is irradiated according to the thickness, and the predetermined distance is set. Can be placed at a distance. Further, an atomic force acting between the near-field light generating means or the laser irradiation probe and the object to be analyzed is detected, and the near-field light generating means or the laser irradiation probe is set so that the atomic force becomes a predetermined value. A method of keeping the distance between the object and the analysis target may be used. That is, by applying the principle of the atomic force microscope (AFM), the near-field light generating means or the laser By detecting the distance between the irradiation probe and the object to be analyzed, the distance can be set to a predetermined value. Furthermore, the near-field light generating means or the laser irradiation probe is once brought into contact with the object to be analyzed, and the near-field light generating means or the laser irradiation probe is separated from the contacted state by a predetermined distance, so that the near-field light can be obtained. The generating means or the laser or laser irradiation probe and the object to be analyzed can be placed at a predetermined distance. In this case, for example, the deflection of the near-field light generating means or the laser irradiation probe is detected using an optical lever, or a near-field light generating means or a pressure sensor such as a tuning fork attached to the laser irradiation probe is used. By detecting the presence or absence of contact with a sensor, it is possible to detect that the object to be analyzed has come into contact with the near-field light generating means or the laser irradiation probe.

In any of the embodiments of the present invention, the laser emitting means is not particularly limited as long as it can emit a laser. For example, when analyzing proteins from cell sections or cell membranes, an infrared laser is preferred from the viewpoint of not damaging protein samples.

The matrix used in the present invention is not particularly limited as long as it can be vaporized together with the sample by near-field light or laser to ionize the sample. For example, glycerol derivatives such as those used in ordinary MAL DI, such as cinnamic acid derivatives, can be used.In addition, when analyzing proteins from cell sections or cell membranes, glycerol is used because it is difficult to alter cells. Is preferably used.

The sample transfer means used in the present invention is not particularly limited as long as the sample can be sent to the mass spectrometry means. For example, as shown in the above-described embodiment, a suction suction means is used. Examples include tubing and electrically skimmed skimmers. The sample may be directly introduced into the mass spectrometer without using the sample moving means.

The atmospheric pressure for ionization in the present invention is not particularly limited, and it can be performed under vacuum, as in ordinary MALDI, or under normal pressure or low pressure. When analyzing cell sections or cell membranes, it is preferable to perform the analysis under normal pressure or low pressure, from the viewpoint that damage to cells and proteins is small and generated ions are not easily broken. Note that the low pressure is not particularly limited as long as it is a pressure at which a sample such as a cell is not easily damaged, and examples thereof include a pressure of 0.1 to 100 Pa. The mass spectrometer used in the present invention is not particularly limited as long as it can perform mass spectrometry, and examples thereof include a time-of-flight mass spectrometer, a quadrupole mass spectrometer, and a tandem mass spectrometer. It can be appropriately selected and used depending on the purpose.

Claims

The scope of the claims

1. Near-field light generated by the near-field light generating means, comprising: a laser-emitting means; and near-field light generating means for receiving a laser emitted from the laser-emitting means to generate near-field light. An ionization device that irradiates the matrix with ions to ionize the sample mixed with the matrix.

2. The ionizer according to claim 1, wherein the near-field light generating means is a near-field light probe.

3. The ionization device according to claim 1, wherein the laser is an infrared laser.

4. The ionization apparatus according to any one of claims 1 to 3, further comprising scanning means for causing the near-field light to scan an object to be analyzed.

5. An ionizer that irradiates the sample mixed with the matrix by irradiating the matrix with near-field light.

6. The ionization apparatus according to claim 5, wherein the near-field light is irradiated by a near-field light probe.

7. The ionization apparatus according to claim 5, wherein the near-field light is near-field light obtained by causing an infrared laser to be incident on a near-field light probe.

8. The ionization apparatus according to any one of claims 5 to 7, wherein the near-field light scans an object to be analyzed.

9. A laser emitting means, an optical fiber for guiding a laser emitted from the laser emitting means, and a laser irradiation probe connected to a tip of the optical fiber. An ionizer that irradiates the matrix with the emitted laser to ionize the sample mixed with the matrix.

10. The ionization apparatus according to claim 9, wherein the laser irradiation probe has an opening having a diameter of 0 to 15 m.

11. The ionization apparatus according to claim 9, wherein the laser irradiation probe has an opening having a diameter smaller than the wavelength of the laser.

12. The method according to claim 9, wherein the laser is an infrared laser. The ionization device according to any one of the above.

13. The ionization apparatus according to any one of claims 9 to 12, further comprising scanning means for causing the laser irradiation probe to scan an object to be analyzed.

14. The ionization device according to claim 4, wherein the analysis target is a cell membrane or a cell slice.

15. The ionization device according to any one of claims 1 to 14, wherein the ionization is performed under normal pressure or low pressure.

16. The ionization apparatus according to any one of claims 1 to 15, further comprising a sample transfer unit that sends the ionized sample to the mass analysis unit.

17. A mass spectrometer comprising the ionization device according to any one of claims 1 to 16.

18. An ionization apparatus according to any one of claims 1 to 16, and information input means for inputting information on an area to be analyzed to be scanned.

Mass spectrometry means;

Imaging means for performing imaging from the position information and the analysis result stored in the storage means,

Display means for displaying the imaged result;

Micro-area analyzer equipped with

19. A method for ionizing a sample using the ionization device according to any one of claims 1 to 16,

Placing the near-field light generating means or laser irradiation probe at a predetermined distance from the object to be analyzed, and

A method for ionizing a sample, comprising:

20. The step of placing at the predetermined distance,

A step of detecting an atomic force acting between a near-field light generating means or a laser irradiation probe and an object to be analyzed, and

A step of setting a distance between the near-field light generating means or the laser-irradiation probe and the analysis target so that the detected atomic force becomes a predetermined value,

10. The method according to claim 19, comprising:

2 1. The step of placing at the predetermined distance,

Bringing the near-field light generating means or the laser irradiation probe into contact with the object to be analyzed, and

A step of separating the near-field light generating means or the laser irradiation probe and the object to be analyzed from the contact state by a predetermined distance,

10. The method according to claim 19, comprising:

2 2. A method in which the sample mixed with the matrix is ionized by irradiating the matrix with near-field light.