JP2007257851A - Mass spectrometer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve work efficiency by reducing the number of times of repeating mass analyses according to an analysis target and improve detection sensitivity, and to enable to measure detailed two-dimensional substance distribution by a high spatial resolution, if necessary. <P>SOLUTION: A drive mechanism 13b to move in a z axis direction a test piece stage 13 to mount a test piece plate 14 mounting a test piece is provided, and the test piece stage 13 is moved by the control of an irradiation diameter control part 31, and by changing the distance between a laser condensing optical system 22 and the test piece 15, a laser irradiation diameter on the test piece 15 is changed. Furthermore, when the distribution of a target substance existing in the test piece 15 of which the location is not clear needs to be checked, firstly, the whole area of the test piece is scanned thoroughly at large scanning step width with a large laser irradiation diameter, and the location where the target substance exists is found out roughly. Thereafter, the laser irradiation diameter is reduced and only the area where the target substance is presumed to exist is scanned at a small step width thoroughly, and the detailed substance distribution is obtained. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a mass spectrometer equipped with an ion source that performs ionization by irradiating a sample with laser light. Specifically, a laser desorption ionization method (LDI = Laser Desorption / Ionization) or a matrix-assisted laser desorption ionization method ( The present invention relates to a mass spectrometer equipped with an ion source based on MALDI = Matrix Assisted Laser Desorption / Ionization, and more particularly to a mass spectrometer for performing mass analysis of a one-dimensional or two-dimensional region on a sample.

  In laser desorption ionization (LDI), a sample is irradiated with laser light, and ionization is performed by accelerating the movement of charges inside a substance that has absorbed the laser light. In addition, matrix-assisted laser desorption / ionization (MALDI) uses a material that easily absorbs laser light and ionizes in order to analyze a sample that is difficult to absorb laser light or a sample that is easily damaged by laser light such as protein. A sample is previously mixed as a matrix, and the sample is ionized by irradiating the sample with laser light. In particular, a mass spectrometer using MALDI can analyze a polymer compound having a large molecular weight without much cleavage, and is also suitable for microanalysis. Has been used. In this specification, mass spectrometers including an ion source based on LDI or MALDI are collectively referred to as LDI / MALDI-MS.

  In the above-mentioned LDI / MALDI-MS, by reducing the spot diameter of the irradiation laser beam and moving the irradiation position relatively on the sample, for example, the intensity distribution (two-dimensional) of ions having a certain mass number on the sample. An image representing the substance distribution) can be obtained. Such an apparatus is known as a mass spectroscopic microscope or a microscopic mass spectroscope, and is expected to be applied to obtain information on the distribution of proteins contained in cells in a living body, particularly in the biochemical field, the medical field, etc. (See Patent Document 1, Patent Document 1, etc.).

  In order to obtain useful knowledge about the sample in various fields as described above, it is desirable that the spatial resolution is high. Conventionally commercially available LDI / MALDI-MS has a laser beam focusing diameter of about several hundred μm. For example, in Non-Patent Document 2, the laser beam focusing diameter is reduced to about several tens μm for analysis. It is described to do. Further, Non-Patent Document 3 and the like include an example in which the condensing diameter of laser light is reduced to about 0.5 μm, and a substance distribution image is acquired in a cell having a size of about several tens of μm. However, there are the following problems when the spatial resolution is increased by reducing the condensing diameter of the laser beam.

  For example, when the target substance is localized only in a certain region in the sample, it is desired to obtain a detailed distribution image only of the region where the target substance exists. However, when the site where the target substance is localized is unknown, mass analysis must be performed sequentially over the entire sample to find the position where the target substance exists. If the spatial resolution is high, the range that can be analyzed by one mass analysis is quite narrow, and therefore, there is a high possibility that the mass analysis is repeated many times before reaching the position where the target substance exists. For this reason, there is a possibility that the time required to obtain the target distribution image becomes considerably long. In order to avoid such a problem, the interval between the positions where mass analysis is performed (that is, the step scanning step width) is increased to roughly analyze the entire sample, and based on the result, the region where the target substance exists is estimated and the step is performed. Another possible method is to analyze the region of interest by reducing the width. However, in this method, there is a possibility that the first rough analysis may be overlooked when the localization range of the target substance is narrow.

US Patent No. 5808300 Yasuhide Naito, “Mass Microscope for Biological Samples”, J. Mass Spectrom. Soc. Jpn., Vol. 53, No. 3, 2005, pp. 125-132. P. Chaurand and three others, “Profiling and imaging proteins in tissue sections by MS”, Analytical chemistry Chemistry), 2004, Vol.76, No.5, p.86A-93A B. Spengler and one other, "Scanning Microprobe Matrix-Assisted Laser Desorption Ionization (SMALDI) Mass Spectrometry: Instrumentation for Sub-Micrometer・ Resolved LDI ・ and ・ MALDI ・ Surface Analysis (Scanning Microprobe Matrix-Assisted Laser Desorption Ionization (SMALDI) Mass Spectrometry: Instrumentation for Sub-Micrometer Resolved LDI and MALDI Surface Analysis), Journal of American Society・ Mass Spectrometry (Jounal of American Society for Mass Spectrometry), 2002, Vol.13, No.6, p.735-748

  As described above, it is generally desirable that the spatial resolution is high. However, in some cases, it may be disadvantageous that the mass analysis range is narrow due to the high spatial resolution. The present invention has been made in view of these points, and its main purpose is to shorten the time required for analysis while achieving high spatial resolution when necessary according to the purpose of analysis and the like. An object of the present invention is to provide a mass spectrometer capable of performing analysis work efficiently.

A mass spectrometer according to the present invention made to solve the above problems is as follows.
a) laser irradiation means for irradiating the sample with laser light in order to ionize components contained in the sample;
b) scanning means for scanning one-dimensionally or two-dimensionally the laser irradiation position on the sample by the laser irradiation means;
c) irradiation range adjusting means for changing the laser irradiation area on the sample;
d) mass spectrometry means for mass-separating and detecting ions generated from the sample by the laser irradiation;
It is characterized by having.

  As an embodiment of the present invention, the irradiation range adjusting means may be configured to change the irradiation area by changing the position of the sample with respect to the laser irradiation means.

  In this configuration, the irradiation range adjusting unit is a unit that moves, for example, a sample stage on which the sample is placed or held so as to approach or move away from the laser irradiation unit. In general, in laser irradiation means, laser light is converged, so it is not a completely parallel light beam but a light that is focused in the traveling direction, and the light spreads in front of the focal point. Therefore, the laser irradiation area on the sample changes by changing the distance of the sample from the laser irradiation means.

  Considering only the movement closer to the laser irradiation means than the focal point now, the closer the sample is to the laser irradiation means, the larger the irradiation area and the lower the spatial resolution. Conversely, the farther the sample is from the laser irradiation means, the smaller the irradiation area and the higher the spatial resolution. Therefore, for example, when it is desired to know the position of the target substance localized in the sample, or when it is desired to examine the general distribution of the substance in the entire sample, if the irradiation area is increased, 2 over the entire area of the sample. Even if a dimensional analysis is performed, the number of repetitions of the analysis can be reduced. Thereby, the time required for analysis can be shortened. After the region where the target substance is localized is found, if the laser irradiation area is reduced, that is, the spatial resolution is increased, the limited region is scanned without omission and mass spectrometry is performed. Good.

  In addition, when the laser irradiation area is large, the number of molecules present in the area is large, and the amount of ions generated increases, thereby improving the S / N ratio of the mass spectrum and reducing the density of molecules of the target substance. However, detection with high sensitivity is possible. Therefore, for example, when it is desired to identify the target substance contained in the sample and the accuracy of the distribution of the substance is not very important, the mass spectrum can be increased with high sensitivity by increasing the laser irradiation area. It is also possible to make it easier to identify the target substance. Thus, an appropriate method can be selected according to the purpose of analysis.

  As another embodiment of the present invention, the laser irradiation means includes a laser light source that emits laser light and a convergence means that converges the laser light, and the irradiation range adjustment means changes the position of the convergence means relative to the sample. Thus, the irradiation area may be changed.

  Even with this configuration, on the side closer to the converging unit than the focal point of the laser beam converged by the converging unit, the irradiation area increases when the converging unit approaches the sample, and the irradiation area decreases when the converging unit is moved away from the sample. Therefore, it has the same action as the above embodiment.

  In addition, various forms can be considered as irradiation range adjusting means. That is, the irradiation range adjusting means may be a shielding means for shielding a part of the laser light, for example, an aperture having a variable light shielding area. The laser irradiation means includes a laser light source that emits laser light and a convergence means that converges the laser light, and the irradiation range adjustment means is a laser inserted in a space between the laser light source and the convergence means. An optical element that changes the cross-sectional area of light, for example, a lens with a variable convergence magnification, can also be used.

  Note that the total amount of laser light power on the sample is the same even if the irradiation area of the laser on the sample is changed, except when the irradiation area is changed by shielding part of the laser light as described above. Since they are the same, the laser light power (power density) per unit area increases when the irradiation area is reduced. For example, if the laser irradiation diameter is reduced to 1/10, the power density becomes 100 times. In general, in ionization by LDI / MALDI, a lower limit value of laser light intensity is determined by an ionization threshold. On the other hand, the upper limit value of the laser light intensity is determined by various factors depending on the apparatus configuration and the like, for example, the allowable degree of damage to the sample, the performance deterioration of the mass analyzer due to an excessive amount of ions, the detector range over, etc. . Therefore, as described above, if the power density of the laser beam becomes too large, the upper limit value may be exceeded.

  In order to avoid such a situation, it is preferable that the mass spectrometer according to the present invention further includes an intensity adjusting unit that changes the intensity of the laser beam irradiated on the sample. The intensity adjusting means may be configured to adjust the laser intensity in conjunction with the size of the laser irradiation area, that is, in conjunction with the operation of the irradiation range adjusting means.

  Furthermore, in the mass spectrometer according to the present invention, the scanning unit can set any or a plurality of scanning steps, and the control for controlling the irradiation range adjusting unit to change the irradiation area of the laser beam according to the scanning step. A configuration including means may be employed.

  Specifically, the control means controls the irradiation range adjustment means so as to increase the irradiation area when the scanning step is large. Thus, for example, when it is desired to know roughly the distribution of the target substance within the predetermined range or the distribution of the target substance within a predetermined range, if the scanning step is set large, the irradiation area of the laser beam increases, and the entire sample or Analysis can be performed with high speed and high sensitivity without leaking within a predetermined range as much as possible.

  According to the mass spectrometer of the present invention, the laser light irradiation area is increased when it is desired to enlarge the region that can be observed by one mass analysis, and the laser is required when observation is performed with high mass resolution. A one-dimensional or two-dimensional substance distribution image on the sample can be obtained by appropriately switching depending on the purpose of analysis, such as reducing the light irradiation area. As a result, the time required to obtain the target substance distribution image can be shortened as compared with the prior art, and the analysis work can be advanced efficiently to improve the throughput. Then, a distribution image of the target substance can be obtained with high spatial resolution for a portion to be observed in detail.

  In addition, when spatial resolution is not so important, the accuracy of identification when performing identification based on the detection result can be improved by increasing the laser irradiation area and making it easy to detect substances with low existence density. .

[First embodiment]
Hereinafter, an LDI / MALDI-MS which is an embodiment (first embodiment) of a mass spectrometer according to the present invention will be described with reference to the drawings.

  FIG. 1 is an overall configuration diagram of LDI / MALDI-MS according to the present embodiment. A sample stage 13, an ion transport optical system 16, a mass analyzer 17, a detector 18, and the like are disposed inside a vacuum chamber 10 that is evacuated by a vacuum pump (not shown), and a laser is disposed outside the vacuum chamber 10. An irradiation unit 20, a laser focusing optical system 22, a CCD camera 23, an observation optical system 24, and the like are arranged. For example, an electrostatic electromagnetic lens, a multipolar high-frequency ion guide, or a combination thereof is used as the ion transport optical system 16. As the mass analyzer 17, for example, a quadrupole mass analyzer, a time-of-flight mass analyzer, a magnetic sector sector mass analyzer, or the like is used.

  A sample plate 14 on which a sample 15 to be analyzed is placed is placed on a sample stage 13, and this sample stage 13 is driven with high positional accuracy in two axial directions of the x axis and the y axis perpendicular to each other. The first drive mechanism 13a for movement and the second drive mechanism 13b for driving with high positional accuracy in the z-axis direction orthogonal to the xy plane are movable. The xy axis drive unit 33 drives a stepping motor and the like included in the first drive mechanism 13 a under the control of the scanning control unit 32 and the irradiation diameter control unit 31, and the z axis drive unit 34 is the irradiation diameter control unit 31. Under the control, the stepping motor and the like included in the second drive mechanism 13b are driven.

  The laser beam 21 for ionization emitted from the laser irradiation unit 20 is focused by the laser focusing optical system 22 and irradiated toward the sample 15 through the irradiation window 11 provided on the side surface of the vacuum chamber 10. Since the laser beam irradiation position is fixed when the sample stage 13 is at a certain position in the z-axis direction, when the sample stage 13 is moved in the xy plane by the first drive mechanism 13a, Thus, the position where the laser beam 21 hits, that is, the minute measurement region 15a to be subjected to mass analysis moves on the sample 15. Thereby, the position where mass analysis is performed on the sample 15 is scanned.

  On the other hand, the CCD camera 23 images a predetermined range on the sample plate 14 through the observation window 12 and the observation optical system 24 provided on the side surface of the vacuum chamber 10, and the image signal obtained here is subjected to image processing. A two-dimensional observation image is constructed by being sent to the unit 37. The central control unit 30 controls the overall operation of the apparatus. Specifically, the central control unit 30 moves and moves by the xy axis driving unit 33 via the scanning control unit 32 and the irradiation diameter control unit 31. The direction is controlled, and the amount of movement by the z-axis drive unit 34 is controlled via the irradiation diameter control unit 31. Further, the emission control unit 35 controls the emission / stop of the laser beam 21 in the laser irradiation unit 20 and the laser beam intensity. In FIG. 1, signal lines are not shown to avoid complexity, but the central control unit 30 also controls operations of the ion transport optical system 16, the mass analyzer 17, the detector 18, and the like.

  A detection signal from the detector 18, that is, an ion intensity signal, is input to the mass spectrometry data processing unit 36, where appropriate data processing is executed to create, for example, a two-dimensional substance distribution image as described later. The central control unit 30 is connected to an operation unit 38 for setting analysis conditions such as a spatial resolution and a measurement target region by an operator's operation. Further, a two-dimensional observation image and a two-dimensional substance distribution image of the sample 15 are displayed. A display unit 39 for displaying is also connected.

A basic operation for acquiring a two-dimensional substance distribution image of a target substance having a known mass number using the LDI / MALDI-MS of the present embodiment is as follows.
First, the operator determines which part on the sample 15 is to be analyzed. For this purpose, the CCD camera 23 acquires a two-dimensional observation image of the sample 15 through the observation window 12 and the observation optical system 24 provided on the side surface of the vacuum chamber 10 and displays them on the screen of the display unit 39. The operator determines the range for mass analysis, sets the range by the operation unit 38, sets the mass number of the target substance, and instructs the start of analysis.

  When the analysis is started, the laser beam 21 emitted from the laser irradiation unit 20 is collected by the laser focusing optical system 22 and irradiated onto the sample 15 through the irradiation window 11 provided on the side surface of the vacuum chamber 10. . When the laser beam 21 is irradiated, various substances existing in the vicinity of the minute measurement region 15a on the sample 15 are ionized, and ions are mainly emitted in a direction substantially orthogonal to the surface of the sample 15, that is, directly above. The ions are converged by the ion transport optical system 16 and introduced into the mass analyzer 17, and only the ions having the mass number of the target substance are separated by the mass analyzer 17 and reach the detector 18. The detector 18 outputs a current corresponding to the number of reached ions as a detection signal. The data processing unit 36 receives this detection signal and obtains the relative intensity of the target substance corresponding to the laser irradiation position (micro measurement region 15a) on the sample 15.

  The scanning control unit 32 moves the first driving mechanism 13a via the xy axis driving unit 33 so that the minute measurement region 15a irradiated with the laser light 21 sequentially moves within the analysis range set on the sample 15. And the sample stage 13 is moved stepwise. Each time the sample stage 13 moves and stops for a minute distance, the laser beam 21 is irradiated as described above to determine the relative intensity of the target substance corresponding to the minute measurement region 15a. In this way, mass analysis is performed over the entire analysis range set first, and a map of the relative intensity of the target substance is created and displayed on the screen of the display unit 39.

  Next, characteristic operations in the LDI / MALDI-MS of the present embodiment will be described. As described above, the laser beam 21 is focused by the laser condensing optical system 22 and hits the sample 15. However, by moving the sample stage 13 in the z-axis direction, the laser irradiation area on the sample 15 (that is, a minute measurement region). 15a) can be changed. That is, as shown in the schematic diagram of FIG. 2, the laser focusing optical system 22 has a focal point at a position separated by a distance D, but when the surface of the sample 15 is at that position, the area of the micro measurement region 15a is minimized. (FIG. 2B), the area of the minute measurement region 15a increases as the sample 15 approaches the laser focusing optical system 22 from that position (FIG. 2A). It is possible to change the size of the range to be subjected to mass spectrometry and the spatial resolution.

  However, when the laser beam 21 is irradiated from directly above the sample 15 as shown in FIG. 2, even if the sample 15 is moved in the z-axis direction, the position of the center point of the minute measurement region 15a does not change. As shown in FIG. 1, when the sample 15 is irradiated with the laser beam 21 from an oblique direction, the position of the center point of the minute measurement region 15a is shifted when the sample 15 is moved in the z-axis direction. Since the incident angle of the laser beam 21 is determined, the amount of displacement of the center point of the minute measurement region 15a with respect to the amount of movement in the z-axis direction can be easily obtained by calculation. Therefore, when the laser irradiation area is changed by moving the sample stage 13 in the z-axis direction, xy is taken into account that the center point of the minute measurement region 15a is displaced according to the amount of movement. The axial position is also controlled. Thereby, the area (laser irradiation diameter) can be changed while maintaining the position of the center point of the minute measurement region 15a on the sample 15.

  Further, when the area of the minute measurement region 15a is changed by moving the sample stage 13 in the z-axis direction in the configuration of FIG. 1, the relative position or distance between the observation optical system 24 or the ion transport optical system 16 and the sample 15 is changed. However, for example, when the depth of focus of the observation optical system 24 is shallow or the field of view is narrow, the sample image is captured when the sample 15 moves in the z-axis direction. Therefore, it is necessary to move the observation optical system 24 in a direction perpendicular to the optical axis in conjunction with the movement of the sample stage 13. Further, when the mass analyzer 17 is a time-of-flight type, it may be necessary to appropriately adjust the ion acceleration voltage according to the movement of the sample stage 13 or to correct the time axis for the obtained signal. is there. Of course, when the amount of movement of the sample stage 13 for changing the laser irradiation area is such that it does not significantly affect the observation position and spatial resolution of the sample 15, or the signal intensity, mass accuracy, and mass resolution in the analysis. The above considerations are not necessary.

  Next, various analysis methods using the above characteristic operation in the LDI / MALDI-MS of the present embodiment will be described.

(1) When narrowing down the analysis location A situation is assumed in which the target substance has been found to be localized only in a certain region in the sample 15, but its position is unknown. In this case, it takes a lot of time to search for the position of the target substance by repeating mass spectrometry while scanning the entire area of the sample 15 uniformly with high spatial resolution, and a large step width is set and high spatial resolution is required. If sparse mass spectrometry is executed, the target substance may be missed. Therefore, in the apparatus of this embodiment, the analysis is executed in the following procedure.

  First, the sample stage 13 is moved in the z-axis direction so as to approach the laser condensing optical system 22, and the irradiation diameter of the laser light on the sample 15 is made relatively large. In this state, as shown in FIG. 3 (a) or (b), the laser beam is irradiated while scanning stepwise so as to cover the entire area of the sample 15 (or the entire analysis range set by the operator). The data processing unit 36 obtains the strength of the target substance by performing mass analysis of each minute measurement region 151 that has been performed. And based on the intensity | strength in each location, the presence or absence of a target substance is confirmed, respectively. Note that FIG. 3B is an example in which the laser irradiation area is further increased as compared with FIG. 3A, and the region that does not enter the analysis target on the sample 15 can be made smaller even with the same step width.

  Now, it is assumed that the target substance is detected only in the minute measurement region 151a in the above analysis. Next, the sample stage 13 is moved in the z-axis direction so as to be away from the laser condensing optical system 22, and the irradiation diameter of the laser beam on the sample 15 is narrowed down. Then, as shown in FIG. 3C, the region 40 near the minute measurement region 151a in which the presence of the target substance has been confirmed is narrowed down, and scanning is performed stepwise with a small movement interval so as to cover the region 40. The data processing unit 36 obtains the intensity of the target substance by performing mass analysis of each minute measurement region 152 irradiated with the laser. As a result, for example, the presence of the target substance is confirmed only in the minute measurement region 152a. As a result, the portion where the target substance exists can be determined in more detail, that is, with high spatial resolution.

  When scanning is initially performed with a large laser irradiation area, since the absolute number of molecules present in the minute measurement region 151 is large, the S / N ratio of the mass spectrum is improved. Therefore, even if the density of the molecule of the target substance is low, ions derived from this molecule can be detected with high sensitivity.

(2) When examining the general distribution of the target substance For example, when observing and measuring a biological sample such as a biological section, the sample may be divided into a plurality of regions morphologically (for example, color). Sometimes it is clear. In such a case, it is not necessary to know the location of the target substance in detail, and it is often sufficient to know in which region the target substance is present in the form. As an example, let us consider a case where the sample 41 can be clearly divided into four regions Ua, Ub, Uc and Ud as shown in FIG. At this time, as shown in FIG. 4 (c), mass spectrometry is performed so as to cover the entire sample 41 while scanning with a relatively small laser beam irradiation diameter and high spatial resolution and a small step width. Is also possible. As a result, the minute measurement region 152a in which the target substance exists is known, and it is found that the target substance exists in the region Ua. However, the efficiency is poor because the number of repetitions of mass spectrometry becomes very large.

  Therefore, the sample stage 13 is moved in the z-axis direction so as to approach the laser condensing optical system 22, and the irradiation diameter of the laser beam on the sample 15 is intentionally increased. Then, as shown in FIG. 4B, mass analysis is performed on each minute measurement region 151 irradiated with the laser light while scanning in a stepped manner so as to cover the entire region of the sample 15, and the result is Determine the presence or absence of the target substance. Thereby, it is possible to detect that the target substance is present in the region Ua in a short time by reducing the number of repetitions of mass spectrometry. Also in this case, since the scanning is performed with a large laser irradiation area, the absolute number of molecules existing in the minute measurement region 151 is large, and detection can be performed with high sensitivity even when the molecular density of the target substance is low.

(3) When switching the magnification of the microscopic observation When, for example, it is desired to examine the two-dimensional material distribution of the target substance while performing microscopic observation of the sample with the CCD camera 23 (or a microscope provided separately), for example, there is a certain observation. It is assumed that the two-dimensional observation image shown in FIG. 5A is obtained with the magnification. Then, it is assumed that the image shown in FIG. 5B is observed by increasing the observation magnification in order to finely observe the specific region 42 in the image. In the configuration of the above embodiment, for example, the observation optical system 24 is controlled in order to switch the observation magnification. At this time, the central control unit 30 causes the irradiation diameter control unit 31 to reduce the laser irradiation diameter as the observation magnification is increased. Is given a directive. Accordingly, when observation is performed at a high observation magnification as shown in FIG. 5B, the laser irradiation diameter on the sample is small, and a more detailed two-dimensional distribution is obtained as shown in FIG. 5D. It is possible. On the other hand, when observation is performed at a low observation magnification as shown in FIG. 5A, the laser irradiation diameter on the sample is large, and a rough two-dimensional substance distribution image is obtained as shown in FIG. It is possible to obtain. Thereby, even if the observation magnification is switched, the number of repetitions of the mass analysis necessary for obtaining the two-dimensional substance distribution can be made the same, so that it can be avoided that the analysis takes a long time.

  In addition, in any of the analysis methods as described in (1) to (3) above, since it is necessary to scan the entire sample or within a predetermined range on the sample as much as possible, there is no difference between the laser irradiation diameter and the scanning step width. It is more convenient to be linked. Therefore, the central control unit 30 may be configured to perform control such that the laser irradiation diameter is automatically changed according to, for example, a two-dimensional scanning step width of mass spectrometry.

(4) When detection with a particularly high sensitivity is required, such as identification of a target substance. Now, for example, a two-dimensional substance distribution is measured with a predetermined spatial resolution for a sample 43, and the result shown in FIG. Assume that the following results are obtained. Here, consider a case where it is found that an unknown target substance is distributed as shown in the minute measurement region 43 and it is desired to identify the substance. For identification purposes, there is a method of cleaving ions derived from a substance and examining the mass number of the ions produced thereby to estimate the structure of the original substance, and the internal energy of the ions is used as the cleavage method. There are known methods (Post Source Decay) and methods (Collision-Induced Dissociation) in which ions are once trapped in an ion trap and collide with a gas. In any case, if the ion of interest can be detected with a high S / N ratio, the identification is easy and the identification accuracy is improved accordingly.

  Therefore, in the range where it was confirmed that the unknown substance exists in FIG. 6A, as shown in FIG. 6B, the laser irradiation diameter 45 is not as small as that in the previous two-dimensional substance distribution measurement, As shown in FIG. 6C, the laser irradiation diameter 46 is deliberately increased, and the irradiation position is set so that the range in which the unknown substance is present occupies the majority. According to this, since the number of molecules of the target substance to be subjected to one mass analysis increases, not only the ions derived from it are directly detected, but also, for example, ions generated by cleavage as described above are detected. Even in this case, the ionic strength becomes relatively large, and the identification becomes easy.

  In general MALDI-TOFMS, a sample is dissolved in a solvent and further mixed with a matrix, then dropped onto a sample plate, and laser beam is irradiated to the sample plate for analysis. In this case, since the laser beam is sequentially irradiated to the two-dimensionally and discretely arranged samples, the spatial resolution is not questioned at all. In such a case, it is better to increase the laser irradiation diameter in order to improve sensitivity.

  Next, mass spectrometers according to some other embodiments, which are different from the first embodiment in the method of changing the laser irradiation diameter, will be described.

[Second Embodiment]
FIG. 7 is a schematic view of an optical path between the laser irradiation unit and the sample in the second embodiment. As described with reference to FIG. 2, the laser irradiation diameter (area) on the sample 15 changes by changing the distance between the laser focusing optical system 22 and the surface of the sample 15. That is, even if the position of the sample stage 13 is fixed and the position of the laser focusing optical system 22 is changed along the optical axis of the laser light 21, the same thing can be said. As shown in FIG. 7B, when the laser condensing optical system 22 is at a position away from the surface of the sample 15 by a distance D, the area of the minute measurement region 15a on the sample 15 is minimized, and the laser beam is collected from that position. When the optical optical system 22 moves so as to approach the sample 15, the area of the micro measurement region 15a in the sample 15 increases.

  In the case of this configuration, the sample 15 does not need to be moved in the z-axis direction. Therefore, unlike the above embodiment, when the laser irradiation diameter is changed, elements other than the laser focusing optical system 22 (for example, the observation optical system 24) ) Need not be considered. Therefore, the configuration is simple. The laser focusing optical system 22 may be a single lens or a combination lens or mirror. In FIG. 7, the incident laser light to the laser condensing optical system 22 is drawn as a parallel light beam, but it is not necessarily a parallel light beam.

[Third embodiment]
Theoretically, laser light is known to have a smaller condensing diameter as it is condensed with a large numerical aperture. Specifically, according to the Gaussian beam theory, the relationship between the numerical aperture (NA) and the condensing diameter (2w ₀ ) is as follows regarding the condensing of the laser light.
w ₀ = λ / (π · sin ⁻¹ NA)
As an example, FIG. 8 shows the result of calculating the relationship between the numerical aperture and the focused diameter when the wavelength (λ) of the laser beam is 355 nm. Therefore, the laser condensing diameter on the sample can be reduced by changing the numerical aperture for laser condensing to be large.

  Various configurations are possible to change the numerical aperture for laser focusing. For example, in FIG. 9, an aperture member 50 in which the diameter of the opening 51 is variable (or switchable) is inserted between the laser irradiation unit 20 and the laser focusing optical system 22. In FIG. 10, an aperture member 50 having the same configuration is inserted between the laser focusing optical system 22 and the sample 15. The diameter (that is, the area) of the opening 51 of the aperture member 50 can be controlled by the central control unit 30. When the opening 51 is made small as shown in FIGS. The laser irradiation diameter on the surface 15 (area of the minute measurement region 15) is increased, and when the opening 51 is enlarged as shown in FIGS. 9B and 10B, the laser irradiation on the sample 15 is performed. The diameter becomes smaller.

[Modification of Third Embodiment]
In FIG. 11, a zoom lens 52 is inserted between the laser irradiation unit 20 and the laser focusing optical system 22 instead of the aperture member. The zoom lens 52 is a beam expander with a variable magnification. If the light beam of the laser light 21 is expanded by this, the laser irradiation diameter on the sample 15 becomes smaller.

  In FIG. 12, the aperture member 50 is provided in the immediate vicinity of the laser irradiation unit 20 between the laser irradiation unit 20 and the laser focusing optical system 22. The optical system having this configuration functions as a reduction imaging type optical system using the opening 51 of the aperture member 50 as a light source. That is, the reduction imaging system uses a reduction ratio of a ratio between the distance D1 between the sample 15 and the laser focusing optical system 22 and the distance D2 between the laser focusing optical system 22 and the aperture member 50. In order to reduce the laser irradiation diameter, it is necessary to make the distance D2 larger than the distance D1, and the aperture member 50 is disposed in the immediate vicinity of the laser irradiation unit 20 in order to realize this.

  As shown in FIGS. 12A and 12B, when the opening 51 of the aperture member 50 is reduced from a large state, laser irradiation on the sample 15 is performed according to the same principle as the configuration shown in FIG. The diameter increases. However, if the opening 51 is further reduced from a state in which the opening 51 is reduced to some extent, the opening 51 of the aperture member 50 behaves as a minute light source and functions as a reduction optical system. For this reason, as shown in FIG. 12C, the laser beam irradiation diameter becomes smaller as the opening 51 is made smaller.

  As an example, in accordance with the theory of Gaussian beam, the wavelength of the laser beam is 355 nm, the focal length of the laser focusing optical system 22 is 100 mm, and the distance between the laser irradiation unit 20 and the laser focusing optical system 22 is 1000 mm. FIG. 13 shows the result of calculating the relationship between the aperture opening diameter and the laser irradiation diameter when the beam waist is positioned at the opening 51 by placing the aperture member 50 at the emitting portion of the irradiation section 20. The optical system having the configuration shown in FIG. 12 uses the region P in the drawing, and the optical system having the configuration shown in FIG. 9 uses the region on the right side of the region.

  In FIG. 14, a spatial filter 55 including a condenser lens 56 and a pinhole 57 is inserted between the laser irradiation unit 20 and the laser condensing optical system 22. In this configuration, the laser irradiation diameter on the sample 15 can be changed by changing the hole diameter of the pinhole 57. Also in this case, the hole of the pinhole 57 functions as a reduced image forming system considering the light source as in the above example.

  In the third embodiment and its modifications, the theoretical laser focusing diameter is changed by changing the cross-sectional area of the propagating laser beam. All of the above explanations are based on the Gaussian beam theory, but qualitatively, the same configuration can be used for a non-Gaussian beam such as a multimode oscillation gas laser.

  The great advantage of the configuration of the third embodiment and its modification is that it is only necessary to add the aperture member 50 and its switching control circuit to the existing apparatus except for FIG. It is. Further, when the zoom lens 52 is used, it is more expensive than the aperture member 50, but there is no loss of laser power, and it is only necessary to insert the zoom lens 52 in one of the laser light propagation paths. The degree of freedom in layout is great when designing. Further, the configuration of the third embodiment has an advantage that, as in the second embodiment, it is not necessary to move other elements such as the observation optical system 24 in accordance with the change of the laser irradiation diameter.

  As described above, in the configuration of the first embodiment, the intensity (laser power) of the laser beam 21 emitted from the laser irradiation unit 20 can be adjusted under the control of the irradiation control unit 35. Therefore, in mass spectrometry, the laser emission light is adjusted to a laser light intensity suitable for analysis. In general, in order to perform appropriate ionization, there is an appropriate range of laser light intensity, the lower limit is mainly determined by the ionization threshold, and the upper limit is determined by various constraints depending on the apparatus configuration and the like. Specifically, the degree of damage to the sample, the performance degradation of the mass analyzer due to an excessive amount of ions entering, the range over of the detector, etc. are the limiting conditions.

  As described above, when the laser beam intensity is appropriately adjusted in a state where the laser irradiation area on the sample 15 is large, the laser beam density on the sample 15 increases as the laser irradiation area decreases (however, (Except when an aperture member is used in the third embodiment). For example, if the laser irradiation diameter is reduced to 1/10, the laser beam density is increased 100 times. For this reason, unless the intensity of the emitted laser beam from the laser irradiation unit 20 is adjusted, the laser beam intensity at the time of hitting the sample 15 is likely to deviate from the appropriate range as described above. Therefore, when the central control unit 30 adjusts the irradiation area of the laser beam, it is preferable to control the irradiation control unit 35 so as to adjust the laser beam intensity in conjunction therewith. The adjustment of the laser light intensity can be realized by various methods. For example, a stepless attenuator may be used.

  The above-described embodiment is an example of the present invention, and it is a matter of course that changes, modifications, and additions within the scope of the present invention are included in the scope of the claims of the present application.

The whole LDI / MALDI-MS block diagram by 1st Example of this invention. The schematic of the optical path from the laser irradiation part of LDI / MALDI-MS by a 1st example to a sample. Explanatory drawing of an example of the analysis method in the case of narrowing down an analysis part. Explanatory drawing of an example of the analysis method when it wants to investigate rough distribution of the target substance. Explanatory drawing of an example of the analysis method in the case of switching the magnification of microscopic observation. Explanatory drawing of an example of the analysis method in case detection with especially high sensitivity is required. Schematic of the optical path from the laser irradiation part of LDI / MALDI-MS by a 2nd Example to a sample. The figure which shows the result of having calculated the relationship between a numerical aperture and a condensing diameter when the wavelength of a laser beam is 355 nm. Schematic of the optical path from the laser irradiation part of LDI / MALDI-MS by a 3rd Example to a sample. Schematic of the optical path from the laser irradiation part of LDI / MALDI-MS by a 3rd Example to a sample. The schematic of the optical path from the laser irradiation part of LDI / MALDI-MS by the deformation | transformation of 3rd Example to a sample. The schematic of the optical path from the laser irradiation part of LDI / MALDI-MS by the deformation | transformation of 3rd Example to a sample. The figure which shows the result of having calculated the relationship between an aperture opening diameter when a beam waist is located in the emission part of a laser irradiation part, and a laser irradiation diameter. The schematic of the optical path from the laser irradiation part of LDI / MALDI-MS by the deformation | transformation of 3rd Example to a sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Vacuum chamber 11 ... Irradiation window 12 ... Observation window 13 ... Sample stage 13a ... 1st drive mechanism 13b ... 2nd drive mechanism 14 ... Sample plate 15 ... Sample 15a, 151, 151a, 152, 152a ... Minute measurement area DESCRIPTION OF SYMBOLS 16 ... Ion transport optical system 17 ... Mass analyzer 18 ... Detector 20 ... Laser irradiation part 21 ... Laser beam 22 ... Laser condensing optical system 23 ... CCD camera 24 ... Observation optical system 30 ... Central control part 31 ... Irradiation diameter Control unit 32 ... Scanning control unit 33 ... y-axis drive unit 34 ... z-axis drive unit 35 ... irradiation control unit 36 ... mass analysis data processing unit 37 ... image processing unit 38 ... operation unit 39 ... display unit 50 ... aperture member 51 ... Opening 52 ... Zoom lens 55 ... Spatial filter 56 ... Condensing lens 57 ... Pinhole

Claims (7)

a) laser irradiation means for irradiating the sample with laser light in order to ionize components contained in the sample;
b) scanning means for scanning one-dimensionally or two-dimensionally the laser irradiation position on the sample by the laser irradiation means;
c) irradiation range adjusting means for changing the laser irradiation area on the sample;
d) mass spectrometry means for mass-separating and detecting ions generated from the sample by the laser irradiation;
A mass spectrometer comprising:   The mass spectrometer according to claim 1, wherein the irradiation range adjusting unit changes an irradiation area by changing a position of the sample with respect to the laser irradiation unit.   The laser irradiation means includes a laser light source that emits laser light and a convergence means that converges the laser light, and the irradiation range adjustment means changes an irradiation area by changing a position of the convergence means with respect to the sample. The mass spectrometer according to claim 1, wherein:   The mass spectrometer according to claim 1, wherein the irradiation range adjusting means is a shielding means for shielding a part of the laser beam.   The laser irradiation means includes a laser light source that emits laser light and a convergence means that converges the laser light, and the irradiation range adjustment means is inserted in a space between the laser light source and the convergence means The mass spectrometer according to claim 1, wherein the mass spectrometer is an optical element that changes a cross-sectional area of the laser beam.   The mass spectrometer according to any one of claims 1 to 5, further comprising intensity adjusting means for changing the intensity of laser light applied to the sample. The scanning means can set arbitrary or a plurality of scanning steps, and includes a control means for controlling the irradiation range adjusting means so as to change the irradiation area of the laser beam in accordance with the scanning steps. The mass spectrometer in any one of 1-6.

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