JP4866098B2 - Mass spectrometer - Google Patents

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 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 LDI / MALDI-MS, by reducing the spot diameter of the irradiation laser light 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 (mass 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.).

  As described above, in order to move the laser irradiation position on the sample, the position of the sample is fixed and the laser irradiation unit is moved. The position of the laser irradiation unit is fixed and the position of the sample is moved. A method of moving the sample and the sample together can be considered, but in general, it is easiest to move the sample stage on which the sample is placed. Further, according to this, since the position of the ion introduction part for taking in ions generated from the vicinity of the laser irradiation position can be fixed, there is an advantage that the apparatus configuration does not have to be complicated. In this case, the spatial resolution of the two-dimensional mass distribution is determined by the irradiation area of the laser light and the adjacent laser irradiation position, that is, the distance between the adjacent measurement points (micro measurement areas). Therefore, if the spatial resolution is set to a certain value, the moving distance between the adjacent measurement points of the sample stage is determined accordingly.

  In the conventional apparatus, the sample stage is intermittently moved by the moving distance determined by the spatial resolution as described above for any measurement target region set on the sample, and the measurement point comes to the laser irradiation position by the movement. Each time the operation is stopped, the laser beam is irradiated, and mass analysis of the minute measurement region where the laser beam hits is executed. In the two-dimensional measurement target region, the measurement points are set, for example, in a lattice shape. Therefore, at the same spatial resolution, the number of measurement points increases as the measurement target region becomes wider, and the measurement time required for the two-dimensional mass distribution measurement of the entire measurement target region also increases.

For example, assuming that there are 100 measurement points in two orthogonal directions in a rectangular measurement target region, it is necessary to perform mass spectrometry repeatedly for each of the 10,000 measurement points in the entire measurement target region. . For example, if it takes 1 second for mass measurement and the movement time to move to the next measurement point for one measurement point, it will take nearly 3 hours to complete the measurement of the entire measurement target area. Become. For this reason, there is a problem that if a wide measurement target area is set or measurement is performed with high spatial resolution even when the measurement target area is relatively narrow, the measurement time becomes very long.

  Naturally, if the measurement time for one sample is long, the measurement efficiency is poor and the throughput is reduced. That is, the number of samples that can be measured within a fixed period such as one day or one week decreases, and the measurement cost for one sample increases accordingly. In addition, in a biological sample, the sample itself may be dried or denatured with the passage of time, and if the measurement time is long, the accuracy of measurement may become a problem due to such influence.

  The problems of the measurement time as described above are mainly due to the large number of measurement points and the time required to move the sample stage or the like from one measurement point to the next measurement point. For this reason, even if it is not the purpose of examining the exact two-dimensional mass distribution, for example, when it is desired to perform a rough measurement such as whether or not the target component exists in the measurement target region on the sample. It is difficult to shorten the measurement time in the conventional apparatus as described above.

  In addition, it has been reported that localization is observed in components contained in biological samples such as frozen sections of organs (for example, Non-Patent Document 2). Therefore, when measuring such a sample, it is desirable to measure a region where a localized component exists with a particularly high spatial resolution. However, if the spatial resolution is set to match the measurement of the part with the local component, the measurement is executed even with the high spatial resolution, that is, with a high density of measurement points even in the part without the local component, and the number of measurement points is very large. Increases the measurement time.

US Patent No. 5808300 Yasuhide Naito, “Mass Microscope for Biological Samples”, J. Mass Spectrom. Soc. Jpn., Vol. 53, No. 3, 2005, pp.125-132. Peter J Todd. And three others, “Organic Ion Imaging of Biological Tissues with Secondary Ion Mass Spectrometry and Matrix-Assisted Laser Desoap (Organic ion imaging of biological tissue with secondary ion mass spectrometry and matrix-assisted laser desorption / ionization) ", J. Mass Spectrom., 2001, 36: pp.355-369

  The present invention has been made to solve the above-mentioned problems, and its purpose is to improve measurement efficiency and throughput by shortening measurement time appropriately according to the purpose of two-dimensional mass distribution measurement. An object of the present invention is to provide a mass spectrometer that can be used.

  Another object of the present invention is to shorten the measurement time while measuring the target component with a sufficiently high spatial resolution when the target component is localized on the sample. An object of the present invention is to provide a mass spectrometer that can perform the above-described process.

A mass spectrometer according to the present invention made to solve the above problems is as follows.
a) laser irradiation means for irradiating a minute measurement region on the sample with laser light;
b) mass spectrometry means for separating and detecting ions generated around a minute measurement region corresponding to the laser light irradiation according to the mass number;
c) scanning means for changing the relative position of the sample and the laser irradiation means so that the minute measurement region on the sample changes discretely;
d) control means for controlling the laser irradiation means, the mass analysis means, and the scanning means, respectively, in order to obtain a two-dimensional mass distribution in the measurement target region set on the sample;
e) display means for displaying the two-dimensional mass distribution;
The control unit first scans the entire measurement target region to sequentially perform mass analysis of the minute measurement region at a predetermined interval, thereby displaying the coarse two-dimensional mass distribution of the measurement target region. Display on the means, and then scan the entire measurement target area or a part of the measurement target area to perform mass analysis of a micro measurement area different from the micro measurement area where mass analysis was performed during the previous scan. Thus, the spatial resolution of the two-dimensional mass distribution displayed on the display means is increased stepwise.

  The scanning unit may be either a unit that moves only the sample, a unit that moves only the laser irradiation unit, or a unit that moves both the sample and the laser irradiation unit. It is preferable to use a means for moving the sample holder that holds the sample.

  In the mass spectrometer according to the present invention, under the control of the control means, the scanning means moves the sample and the sample so that a predetermined minute measurement area within the measurement target area set on the sample comes to the laser irradiation position by the laser irradiation means. The relative position with respect to the laser irradiation means is determined, and at that time, the laser irradiation means irradiates the minute measurement region with laser light. The components constituting the sample are ionized by this laser light irradiation, and the mass analyzing means receives various ions emitted from the sample and separates and detects them. As a result, a mass analysis result of one minute measurement region can be obtained.

  When the mass analysis for one minute measurement region is completed, the scanning unit moves one or both of the sample and the laser irradiation unit so that the next minute measurement region to be analyzed in the measurement target region comes to the laser irradiation position. . Under the condition that the irradiation area of the laser beam is constant, the spatial resolution of the two-dimensional mass distribution is determined by the moving distance. Therefore, when the spatial resolution of the two-dimensional mass distribution is determined by the operator's input operation or by automatically setting a default value or the like, the adjacent minute measurement region interval necessary for achieving the spatial resolution, that is, the moving distance is determined, By sequentially performing mass analysis while changing the relative position between the sample and the laser irradiation means for each moving distance, a two-dimensional mass distribution having a set spatial resolution can be created.

  However, in the mass spectrometer according to the present invention, instead of scanning the plurality of minute measurement regions set in the measurement target region along the arrangement order, first, the movement distance determined by the set spatial resolution. The micromeasurement regions at positions separated from each other by a predetermined distance longer than that are sequentially scanned, and mass analysis is performed on each micromeasurement region. Then, the scan of the micro measurement area which is coarse (low density) compared to the micro measurement area interval corresponding to the set spatial resolution is completed for the entire measurement target area. In such a rough scan, since the number of times of mass analysis is reduced in the measurement target region compared to a more precise scan corresponding to the set spatial resolution, the entire measurement target region can be scanned in a relatively short time. Can be terminated. At the end of this scanning, the display means displays a coarse two-dimensional mass distribution having a spatial resolution lower than the set spatial resolution.

  After that, the scanning means sets a micro measurement area that has not been mass-analyzed at the time of the rough scan, that is, a micro measurement area that has been skipped in the previous scan, for the entire measurement target area or a part of the measurement target area. Perform mass spectrometry. At this time, since the mass analysis result is obtained so as to fill the portion where the information is missing in the coarse two-dimensional mass distribution obtained earlier, this result is added to the two-dimensional mass distribution obtained earlier, As information is enriched, the two-dimensional mass distribution gradually becomes more precise.

  As one aspect of the mass spectrometer according to the present invention, the apparatus further includes resolution setting means for setting the spatial resolution of the two-dimensional mass distribution, and the control means is a minute in which the interval is determined in accordance with the set spatial resolution. The measurement area is scanned so that one or more micro measurement areas are skipped, and mass analysis of the entire measurement target area is sequentially executed, and then a part or all of the micro measurement areas skipped earlier are selected. However, it can be set as the structure which controls so that mass spectrometry may be performed.

  First, the control means scans the micro measurement area determined by the distance between the micro measurement areas corresponding to the set spatial resolution so as to skip one or more micro measurement areas, and covers the entire measurement target area. Perform mass spectrometry. That is, as described above, a minute measurement region that performs mass spectrometry by driving the scanning unit while omitting a predetermined number of minute measurement regions set to achieve a desired spatial resolution, such as every other minute measurement region or every other minute measurement region. As for, a laser beam is irradiated by a laser irradiation means, and mass analysis is performed on ions generated from the vicinity of the laser irradiation position. In this way, a rough two-dimensional mass distribution can be obtained by performing skip scanning of the entire measurement target region, but naturally the spatial resolution is lower than the set one.

  When the skip-like scan is completed for the entire measurement target region, the control means then scans to select a part or all of the micro measurement region that has been skipped first, that is, the mass analysis was not performed. The mass analysis of the entire measurement target region or a part thereof is executed. As a result, the mass analysis result of the micro measurement region where the mass analysis was not performed first is obtained, so that at least a part of the blank between the micro measurement regions where the mass analysis was performed first is filled. Thus, the definition of the two-dimensional mass distribution image displayed by the display means gradually increases.

  As described above, according to the mass spectrometer according to the present invention, when a series of measurements is started, first, a coarse two-dimensional mass distribution having a spatial resolution lower than the desired spatial resolution is displayed for the entire measurement region, and the measurement is performed. As the process proceeds, the definition of the two-dimensional mass distribution increases, and a two-dimensional mass distribution having a desired spatial resolution is finally obtained.

  For example, when it is desired to check whether or not a predetermined substance exists in the measurement target region on the sample, a two-dimensional mass distribution may be obtained by performing mass analysis focusing on the mass number corresponding to the substance, but it is rough. If it can be confirmed that the predetermined substance already exists at the stage where the two-dimensional mass distribution image is obtained (in whole or in part), the subsequent measurement, that is, the fine two-dimensional mass distribution image is obtained. Mass analysis of each minute measurement region to create is unnecessary. Therefore, the operator gives an instruction to stop the measurement at an appropriate time, and the control means stops the analysis in response to this. Thus, the measurement of this sample can be terminated before obtaining a two-dimensional mass distribution having a desired spatial resolution for the measurement target region, and for example, the measurement of the next sample can be started. Thus, according to the mass spectrometer of the present invention, the measurement efficiency can be improved and the throughput can be increased.

  Moreover, as one aspect of the mass spectrometer according to the present invention, the signal intensity of a specific mass number obtained for each minute measurement region is within a predetermined range when performing mass analysis while performing rough scanning on the entire measurement target region. Determination means for determining whether the signal intensity is within a predetermined range by the determination means or a peripheral region including the area, the mass spectrometry is performed during the previous rough scan. A configuration may be adopted in which scanning is performed to execute mass analysis of a micro measurement region different from the executed micro measurement region.

  According to this configuration, only a rough two-dimensional mass distribution is executed for a region that contains no or almost no target component on the sample, and a finer definition is given only for a region that contains a certain amount or more of the target component. A high two-dimensional mass distribution image can be obtained quickly.

  Further, in this case, the mass analysis may be performed with the same density of the minute measurement region as in the previous rough scanning, specifically, for example, with the same moving distance by the scanning means. When it is desired to obtain a two-dimensional mass distribution of a specific area with a spatial resolution, the control means scans in an area where the signal intensity is determined to be within a predetermined range by the determination means or a peripheral area including the area. When performing, it is good to set it as the structure which carries out mass spectrometry by making the density of a micro measurement area | region higher than the time of the rough scanning.

  According to this configuration, scanning is performed in the order of approaching minute measurement regions in which mass analysis has not been performed in a specific region, so that there is no waste in moving the scanning means, and a high-definition two-dimensional mass distribution image can be quickly obtained. Can get to.

  Further, only one measurement target region may be set on the sample, but a plurality of measurement target regions may be set. When there are multiple measurement target areas set on the sample, a signal of a specific mass number obtained for each micro measurement area when performing mass analysis while performing rough scanning for each measurement target area The control unit further includes a determination unit that determines whether or not the intensity is in a predetermined range, and the control unit performs mass analysis while sequentially performing a rough scan for each entire measurement target region for all the measurement target regions. At this time, only the measurement target region including the minute measurement region in which the signal intensity is determined to be within the predetermined range by the determination unit is subjected to mass analysis of the minute measurement region different from the minute measurement region in which the mass analysis was performed during the rough scan. A configuration may be adopted in which scanning is performed for execution.

  According to this configuration, since a schematic two-dimensional mass distribution image of a plurality of measurement target regions is first displayed on the display unit, it is only necessary to confirm that a predetermined component is included in any measurement target region. In some cases, such a check can be performed quickly and, for example, the next sample can be measured.

  However, there are cases where the target component cannot be detected well by mass spectrometry, or the measurement target region cannot be appropriately narrowed down even if the mass spectrometry result is used. Therefore, as one aspect of the mass spectrometer according to the present invention, the apparatus further comprises external information acquisition means for receiving two-dimensional distribution information on a sample different from the two-dimensional mass distribution, and the control means is a determination result by the determination means. And the two-dimensional distribution information obtained by the external information acquisition means may be configured to determine a region for performing mass analysis in a micro measurement region different from the micro measurement region in which mass analysis was performed during rough scanning. . Here, “two-dimensional distribution information on a sample different from two-dimensional mass distribution information” is, for example, two-dimensional distribution information obtained by labeling the distribution of a drug in a living cell with a fluorescent marker. It is.

  According to this configuration, for example, an area on a sample that is difficult to extract properly only by mass spectrometry can be selected or narrowed down using the results extracted by other methods. A useful two-dimensional mass distribution can be obtained in a short time.

  In the mass spectrometer according to the present invention, sample photographing means for obtaining an image of the whole or a part of the sample, and area setting means for setting an arbitrary measurement target area on the sample image obtained by the sample photographing means. It is good to set it as the structure further provided. According to this, the operator can arbitrarily set the range in which the two-dimensional mass distribution is to be examined by the region setting unit while visually confirming the sample image acquired by the sample photographing unit.

[First Example]
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. The sample 14 to be analyzed is placed on a sample stage 13 that can move with high positional accuracy in at least two directions of the x-axis and y-axis. 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.

  The stage drive unit 32 includes a drive source such as a stepping motor for moving the sample stage 13 in the x-axis and y-axis directions as described above, and is sufficiently higher than the minimum spatial resolution described later. The sample stage 13 is moved with accuracy. The laser beam for ionization emitted from the laser irradiation unit 20 is narrowed to a minute diameter (for example, about several μm) by the laser focusing optical system 22 and passes through an irradiation window 11 provided on the side surface of the vacuum chamber 10. Irradiated to a fixed position. Since this laser beam irradiation position is fixed, when the sample 14 moves in the xy plane by the movement of the sample stage 13, the position where the laser beam hits on the sample 14, that is, the target of mass analysis on the sample 14. The minute measurement area 15 is moved.

  On the other hand, the CCD camera 23 images a predetermined range on the sample stage 13 through the observation window 12 and the observation optical system 24 provided on the side surface of the vacuum chamber 10, and the obtained image signal is subjected to image processing. A two-dimensional image is constructed by being sent to the unit 35. The central control unit 30 controls the overall operation of the apparatus. Specifically, the central control unit 30 controls the movement amount and movement direction of the stage driving unit 32 via the scanning control unit 31, and the irradiation control unit 33. The emission / stop of the laser beam at the laser irradiation unit 20 and the laser beam intensity are controlled. Although not shown in FIG. 1, 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 analysis data processing unit 34, where appropriate data processing is performed to create a two-dimensional mass distribution image as described later, for example. The central control unit 30 is connected to an operation unit 36 for setting analysis conditions such as a spatial resolution and a measurement target region by an operator's operation, and further displays a two-dimensional image, a two-dimensional mass distribution image, and the like of the sample. A display unit 37 is also connected.

  Next, a characteristic analysis operation by LDI / MALDI-MS of the present embodiment will be described with reference to FIGS. FIG. 2 is a plan view showing a microscopic observation image of the sample, FIG. 3 is an explanatory diagram of a scanning method of a minute measurement region (laser irradiation region) in the measurement target region, and FIG. 4 is a two-dimensional view at the time of scanning shown in FIG. It is a schematic diagram which shows the change of a mass distribution image.

  When the operator sets the sample 14 on the sample stage 13 and instructs the observation of the sample image by the operation unit 36, the image of the sample 14 is acquired by the CCD camera 23, and the screen of the display unit 37 shows the image shown in FIG. A two-dimensional sample image 40 on the surface of the sample 14 as shown is drawn. The operator instructs the area to be measured by the operation unit 36 while viewing the two-dimensional sample image 40. For example, as shown in FIG. 2A, a measurement target region instruction frame 41 is displayed on the two-dimensional sample image 40 by an operator's operation, and the size and shape of the instruction frame 41 is changed and its position is arbitrarily moved. By doing so, the range surrounded by the instruction frame 41 is set as the measurement target region. In this example, the measurement target region instruction frame 41 is rectangular, but it can be deformed into an arbitrary shape, and not only one certain location but also a plurality of locations can be set simultaneously on the sample 14. Also good.

  The operator also sets analysis conditions including the spatial resolution of the two-dimensional mass distribution from the operation unit 36. For example, when it is desired to examine a two-dimensional mass distribution for a component having a specific mass number M, this mass number M may be set together with the spatial resolution. Since the range of spatial resolution that can be set is determined by restrictions on the configuration of the apparatus such as the irradiation diameter of the laser beam, multiple levels of spatial resolution are usually prepared in advance. An appropriate spatial resolution may be selected from among them.

  Under the condition that the irradiation diameter of the laser beam is constant, the interval between the minute measurement regions is determined by the spatial resolution, and the higher the spatial resolution, the narrower the minute measurement region interval is, that is, the density of the minute measurement region is higher. Now, in FIG. 2A, a minute measurement region (laser irradiation region) is drawn with a small-diameter circle in the measurement target region instruction frame 41. This circle is not actually displayed on the image. Here, a total of 49 minute measurement regions 42 are provided in the measurement target region instruction frame 41, 7 in the x-axis direction and 7 in the y-axis direction. On the other hand, for example, when the spatial resolution is set to be twice as fine as that, as shown in FIG. 2 (b), it is doubled in each of the x-axis direction and the y-axis direction within the same measurement target region indicating frame 41. The minute measurement region 42 is set by the density, and 169 minute measurement regions 42 that are approximately four times as large are set. Conversely, if the spatial resolution is lowered (rough), the number of minute measurement regions set in the same measurement target region instruction frame 41 is reduced.

  When the operator instructs the start of analysis through the operation unit 36 after setting the measurement target region instruction frame 41, the scanning control unit 31 controls the determined measurement microregions on the sample 14 in order under the control of the central control unit 30. The stage drive unit 32 is controlled so as to move the sample stage 13 so as to come to the laser irradiation position. On the other hand, under the control of the central control unit 30, the irradiation control unit 33 has a predetermined intensity when the movement of the sample stage 13 is temporarily stopped when a predetermined minute measurement region on the sample 14 comes to the laser irradiation position. The laser irradiation unit 20 is controlled to irradiate the sample 14 with the laser beam.

  In the conventional apparatus, the sample stage is moved so that the plurality of minute measurement regions included in the measurement target region are moved to the laser irradiation position in the order of arrangement. On the other hand, in the LDI / MALDI-MS of this embodiment, the characteristic minute measurement region is scanned as follows.

  That is, as shown in FIG. 2 (a), for the 49 minute measurement regions 42 included in the measurement target region indication frame 41, first, as a first scan, mass analysis is performed in both the x-axis direction and the y-axis direction. The micro measurement area to be subjected to is made every two of the original micro measurement areas (that is, the density corresponding to the desired spatial resolution). Specifically, as shown in FIG. 3A, when the position of the minute measurement region to be subjected to mass spectrometry is expressed by an (X, Y) address, (0, 0) → (3, 0) → (6 , 0) → (6, 3) → (3, 3) →... → (6, 6). In FIG. 3A, the minute measurement region 421 where the mass analysis is performed is indicated by hatching.

  When the minute measurement region to be analyzed is at the laser irradiation position, the laser beam emitted from the laser irradiation unit 20 and condensed by the laser focusing optical system 22 is irradiated to the minute measurement region on the sample 14. When the laser beam 21 is irradiated, various substances contained in the sample 14 are ionized in the irradiation range, and ions are mainly emitted in a direction substantially orthogonal to the surface of the sample 14, that is, substantially right above. The ions are converged by the ion transport optical system 16 and introduced into the mass analyzer 17. Under the control of the central control unit 30, the mass analyzer 17 is set to select only ions having the mass number M, and the detector 18 outputs a detection signal corresponding to the amount of the ions. The mass analysis data processing unit 34 obtains a relative intensity value with respect to the mass number M based on this detection signal, and stores it in the memory together with information (for example, the above (X, Y) address) indicating the position of the measurement target region.

  Since the mass analysis as described above is executed every time the micro measurement region 421 moves in accordance with the movement of the sample stage 13, a relative intensity value of the mass number M is obtained for each micro measurement region 421, as described above. The information is stored in the memory in association with the position information of the minute measurement region 421. A two-dimensional mass analysis image is created based on the stored contents of the memory. Here, as a two-dimensional mass distribution image, as shown in FIG. 4, a rectangular divided region obtained by dividing the display frame 50 corresponding to the measurement target region in a one-to-one correspondence with the minute measurement region is considered. The magnitude of the intensity value is expressed by, for example, the darkness or luminance of the display color in each divided area.

  According to such an expression method, when the mass analysis results in the nine minute measurement regions 421 are obtained at the stage where the first scan shown in FIG. As shown in FIG. 4A, a two-dimensional mass distribution image in which the display indicating the relative intensity value is made in nine divided regions 511 is drawn. In FIG. 4, all the divided areas 511 where the relative intensity values are displayed are indicated by the same diagonal lines, but actually the display color density, luminance, and the like are changed according to the relative intensity values. Since the divided region 512 corresponding to the minute measurement region skipped in the scanning as described above is blank where nothing is displayed, the display of the two-dimensional mass distribution image is sparse, that is, rough drawing.

  For example, when it is desired to check only whether or not a substance having a mass number M exists in the measurement target region, the substance having the mass number M is displayed in the two-dimensional mass distribution image displayed corresponding to the first scan as described above. If the existence can be confirmed, there is no need to continue the measurement. In this case, the operator instructs to stop the measurement halfway through the operation unit 36, and the central control unit 30 immediately stops the analysis. Therefore, the measurement can be finished before starting the measurement of the next sample, for example, before the two-dimensional mass distribution of the entire measurement target region can be acquired with the set spatial resolution. Thus, it is possible to perform efficient measurement without taking unnecessary measurement time. In addition, when the sample is a biological sample, it is desirable to reduce the number of laser irradiations as much as possible to reduce damage to the sample surface. However, if the measurement is interrupted as described above, it is given to a valuable biological sample. Damage can be suppressed.

  When the entire area to be measured is scanned by the first scan as described above, the next minute scan is performed by sequentially scanning every second minute measurement area skipped by the first scan. Mass spectrometry is performed on the measurement region 422. Specifically, as shown in FIG. 3B, when the position of the minute measurement region to be subjected to mass spectrometry is expressed by an (X, Y) address, (1, 1) → (4, 1) → (4 , 4) → (1, 4), scanning in order while skipping. Since the relative intensity value of the mass number M for the four minute measurement regions 422 is obtained during the scanning in the same manner as in the first scanning, as shown in FIG. In addition to the nine divided areas 511, a display indicating the relative intensity value is added to the four divided areas 511 as well. This slightly improves the spatial resolution of the two-dimensional mass distribution image.

  If the measurement is continued without ending, then, as the third scan, the micro measurement areas skipped in the first and second scans are scanned every two in turn, and the mass is measured for each micro measurement area 423. Perform analysis. Specifically, as shown in FIG. 3C, when the position of the minute measurement region to be subjected to mass spectrometry is expressed by an (X, Y) address, (2, 2) → (5, 2) → (5 , 5) → (2, 5), scanning sequentially. Since the relative intensity values of the mass number M for the four minute measurement regions 423 are obtained during the scanning in the same manner as in the first and second scanning, as shown in FIG. In addition to the previous 13 divided areas 511, a display indicating the relative intensity value is also added to the four divided areas 511. As a result, the spatial resolution of the two-dimensional mass distribution image is further improved.

  By repeating the scan while avoiding the minute measurement region in which the mass spectrometry is performed once as described above, the blank divided region 512 is gradually formed in the display frame 50 as shown in FIGS. As shown in FIG. 4F, all the divided areas in the display frame 50 become divided areas 511 on which display indicating relative intensity values is performed. At this time, the two-dimensional mass distribution image has the initially set spatial resolution. Of course, the measurement can be aborted at any point before such a final fine two-dimensional mass distribution image is displayed.

[Second Embodiment]
Next, LDI / MALDI-MS of another embodiment (second embodiment) according to the present invention will be described. FIG. 5 is an overall configuration diagram of the LDI / MALDI-MS according to this embodiment. Constituent elements that are the same as or correspond to those of the apparatus of the first embodiment shown in FIG.

  Although the basic configuration of this apparatus is the same as that of the first embodiment, in the apparatus of the second embodiment, the central control unit 30 includes an intensity value determination unit 301 and a scanning region determination unit 302 as functional blocks. The intensity value determination unit 301 receives the relative intensity value in each minute measurement region obtained as a result of mass spectrometry from the mass analysis data processing unit 34, and determines whether or not the intensity value is within a predetermined range. Further, the scanning region determination unit 302 sets a region to be scanned for mass analysis on the sample according to the determination result of the intensity value determination unit 301.

  The operation will be described with a specific example. FIG. 6A is a microscopic observation image of a sample, and FIGS. 6B and 6C are explanatory diagrams of a scanning method of a minute measurement region in a measurement target region. When the operator sets the measurement target area instruction frame 41 and instructs to start measurement on the image as shown in FIG. 6A displayed on the display unit 37, the measurement target area instruction is displayed as shown in FIG. 6B. Within the range specified by the frame 41, mass analysis is sequentially performed on every other minute measurement region arranged at intervals determined by the set spatial resolution, that is, on the minute measurement region 421 indicated by hatching. . This corresponds to the first scan of the first embodiment.

  When the relative intensity value at the mass number M of each minute measurement region 421 is obtained by mass spectrometry, the intensity value determination unit 301 determines whether the relative intensity value is equal to or greater than a predetermined value, and the relative intensity value equal to or greater than the predetermined value. Is provided to the scanning region determination unit 302. The predetermined value for determination may be set freely by the operator or may be set automatically. Based on the position information of the given micro measurement area, the scanning area determination unit 302 determines the second scanning area so as to include it. For example, as shown in FIG. 6B, when the entire measurement object area indication frame 41 is scanned, if the relative intensity value shows a predetermined value or more only in the center six minute measurement areas 424, scanning is performed. The region determination unit 302 determines the scanning region range 43 so as to include these minute measurement regions 424.

  When it is desired to observe the range including the component of the mass number M on the sample with a particularly high spatial resolution, the relative intensity value at the mass number M is not less than a predetermined value at the site including the component of the mass number M. There is a high possibility that a component of mass number M is included in the determined scanning region range 43. Therefore, in the second scan, only the minute measurement region 425 that exists in the scan region range 43, but not skipped in the first scan, instead of the entire measurement target region indication frame 41 is selectively and sequentially scanned. Perform analysis. At this time, instead of every other scan, the movement distance of the sample stage 13 is set at the time of the first scan so as to scan all the minute measurement regions 425 other than the minute measurement region 424 subjected to the mass analysis in the first scan. Shorter than that. This is to quickly create a two-dimensional mass distribution image with a predetermined spatial resolution within the scanning region range 43.

  As described above, in the apparatus of the second embodiment, a two-dimensional mass distribution image with a high spatial resolution is obtained in a range where a specific component exists on a sample (or a range where the concentration of the specific component is high) or in the vicinity thereof. And a coarse two-dimensional mass distribution image can be obtained in other regions. Thereby, useless measurement time can be shortened.

  In the above description, the moving distance of the sample stage 13 is made shorter in the second scanning than in the first scanning, but stepwise scanning is repeated in the scanning region range 43 as in the first embodiment. Alternatively, the mass analysis may be performed so that the spatial resolution of the two-dimensional mass distribution image in the range 43 gradually increases.

  In the above description of the first and second embodiments, only one measurement target region is set on the sample. However, even when a plurality of measurement target regions are set, a two-dimensional mass distribution image in each measurement target region is created by the same method. can do. When a plurality of measurement target areas are set on the sample, various modes can be considered using the relative intensity values as in the second embodiment.

  For example, consider a case where three measurement target region instruction frames 41a, 41b, and 41c are set on a sample as shown in FIG. In the first scan, scanning is performed while skipping appropriately, such as every other minute measurement area arranged in each measurement target area instruction frame 41a, 41b, 41c corresponding to the set spatial resolution. By doing so, coarse two-dimensional mass distribution images of the measurement target region instruction frames 41a, 41b, and 41c are created. In parallel, the intensity value determination unit 301 determines whether or not the relative intensity value of the mass number M in each minute measurement region is equal to or greater than a predetermined value, and includes a minute measurement region that indicates a relative intensity value greater than or equal to the predetermined value. Find the measurement target area indication frame. For example, assuming that a minute measurement region showing a relative intensity value greater than or equal to a predetermined value exists only in the measurement target region indication frame 41b, the second scan is executed only for the measurement target region indication frame 41b. As a result, a fine two-dimensional mass distribution is obtained for the measurement target region designation frame 41b, and a coarse two-dimensional mass distribution is obtained for the other two measurement target region designation frames 41a and 41c.

  Furthermore, instead of performing the second scan for the entire measurement target region instruction frame 41b, a minute measurement region having a relative intensity value equal to or larger than a predetermined value is also detected in the measurement target region specification frame 41b as in the second embodiment. The second scan may be executed by narrowing the range to be included.

  In addition, instead of performing the first scan sequentially for a plurality of measurement target region instruction frames, for example, the first scan is executed for the measurement target region instruction frame 41a, and the relative intensity value is a predetermined value or more. If the area is included, the second scanning is executed for the whole or a part of the instruction frame 41a to obtain a fine two-dimensional mass distribution, and then the first scanning is performed for the second measurement target area instruction frame 41b. The processing may be advanced in order, for example. Which procedure is appropriate for proceeding depends on the purpose of measurement, for example, the degree of importance of obtaining a rough two-dimensional mass distribution, so that the operator can select the procedure. You may leave it.

  Moreover, in the said Example, although the determination of the area | region which performs the scan after the 2nd time used the mass spectrometry result, you may utilize the result by other measuring devices other than mass spectrometry. For example, when examining the distribution of a drug in an organ of an animal that has been administered a certain drug in animal experiments, etc., a fluorescent marker that binds to the target drug is administered simultaneously, and then the fluorescence intensity distribution of the collected section sample Can be used to understand the distribution of the target drug. Therefore, if you want to examine the two-dimensional mass distribution with the drug as the target component, use the fluorescence intensity distribution, and only perform a rough two-dimensional mass distribution measurement in the region where the fluorescence intensity is smaller than the predetermined value on the sample. In a region where the fluorescence intensity is greater than or equal to a predetermined value, a fine two-dimensional mass distribution measurement having a desired spatial resolution can be performed. In addition, the region to be scanned for the second time may be determined by combining the result of mass spectrometry obtained by executing the first scan and the result of other measurements such as the fluorescence intensity distribution.

  In the above-described embodiments, the arrangement of the optical system for irradiating the sample with the laser and capturing the sample image can be modified as appropriate. Such modifications are shown in FIGS. In FIG. 8 and FIG. 9, the same or corresponding components as those in FIG. 1 and FIG.

  In the apparatus having the configuration shown in FIG. 8, an observation / laser condensing optical system 25 with a hole and a mirror 26 with a hole are disposed above the sample stage 13, and a wavelength selection mirror 27 is provided outside the observation window 12. Has been placed. An image of the sample 14 is bent at a substantially right angle by a mirror 26 with a hole through an observation / laser condensing optical system 25 with a hole, and is captured by the CCD camera 23 through the observation window 12 and the wavelength selection mirror 27. On the other hand, the laser beam 21 emitted from the laser irradiation unit 20 is bent at a substantially right angle by the wavelength selection mirror 27, passes through the observation window 12, is reflected downward by the mirror 26 with a hole, and has observation / laser condensing with a hole. The sample 14 is condensed by the dual-purpose optical system 25 and irradiated onto the sample 14. Ions generated from the sample 14 by this laser irradiation reach the ion transport optical system 16 through the holes of the holed observation / laser converging optical system 25 and the holed mirror 26. Even in such a configuration, since the irradiation position of the laser beam is fixed, the laser irradiation position can be moved on the sample 14 by moving the sample stage 13.

  In the apparatus having the configuration shown in FIG. 9, the sample stage 13 can be largely slid by the stage driving mechanism 28 particularly in the x-axis direction. That is, in FIG. 9, the position indicated by the solid line of the sample stage 13 is the mass analysis position, and the position indicated by the dotted line is the observation position. When the sample 14 is at the analysis position, the laser light 21 emitted from the laser irradiation unit 20 is condensed by the laser condensing optical system 22 arranged close to the sample 14 and hits a predetermined position of the sample 14. The ion transport optical system 16, the mass analyzer 17, and the detector 18 for performing mass analysis are arranged along the axis C above the sample 14 at the analysis position. On the other hand, the CCD camera 23 is arranged so as to shoot substantially vertically downward, and when the sample 14 is present at the observation position due to the movement of the sample stage 13, the upper surface of the sample 14 is passed through the observation window 12 and the observation optical system 24. A predetermined range of images is taken. Even with such a configuration, the same measurement as described above is possible.

  In any of the above configurations, the sample stage 13 moves in the x-axis and y-axis directions so that the laser beam is irradiated to an appropriate position of the sample 14 placed on the sample stage 13. However, if it is necessary to move the sample 14 in the thickness direction, the sample stage 13 may be moved along the z-axis orthogonal to the x-axis and the y-axis. Further, the sample 14 can be moved by other moving methods. FIG. 10 is a schematic view showing another embodiment of the sample stage.

  In this embodiment, a disc-shaped sample stage 61 capable of rotating motion is disposed on a pedestal portion 60 capable of linear reciprocating motion, and the sample 14 is placed on the sample stage 61. An arbitrary position on the sample 14 comes to the laser beam irradiation position by a combination of the reciprocating motion of the pedestal 60 and the rotational motion of the sample stage 61. Further, an inlet of an ion transport tube 62 that sucks ions generated from the sample 14 and transports them to a mass analyzer (not shown) is provided above the laser beam irradiation position. According to the sample moving method having such a structure, when moving from one position on the sample to another position, it is possible to move at high speed by appropriately using linear motion and rotational motion. Contributes to shortening. In general, the shape of the range in which the target component exists on the sample is indefinite, but there is an advantage that positioning near the range in which the target component exists is easy and positioning can be performed with high accuracy.

  Of course, any of the above-described embodiments and modifications is merely an example of the present invention, and changes, modifications, and additions as appropriate within the scope of the present invention are included in the scope of the claims of the present application. It is natural.

The whole LDI / MALDI-MS block diagram by 1st Example of this invention. The top view which shows the microscopic observation image of the sample in LDI / MALDI-MS of 1st Example. Explanatory drawing of the scanning method of the micro measurement area | region (laser irradiation area | region) in the measurement object area | region in LDI / MALDI-MS of 1st Example. FIG. 4 is a schematic diagram showing a change in a two-dimensional mass distribution image at the time of scanning shown in FIG. 3. The whole LDI / MALDI-MS block diagram by 2nd Example of this invention. The microscopic observation image (a) of the sample in LDI / MALDI-MS of 2nd Example, and explanatory drawing (b), (c) of the scanning method of the micro measurement area | region in a measurement object area | region. Explanatory drawing of the scanning method of the micro measurement area | region by the other form in LDI / MALDI-MS of 2nd Example. The block diagram which shows the modification of optical system arrangement | positioning for the laser irradiation to a sample, and the imaging of a sample image. The block diagram which shows the modification of optical system arrangement | positioning for the laser irradiation to a sample, and the imaging of a sample image. The block diagram which shows the modification of a sample stage.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Vacuum chamber 11 ... Irradiation window 12 ... Observation window 13 ... Sample stage 14 ... Sample 15 ... Micro measurement area | region 16 ... Ion transport optical system 17 ... Mass analyzer 18 ... Detector 20 ... Laser irradiation part 21 ... Laser beam DESCRIPTION OF SYMBOLS 22 ... Laser condensing optical system 23 ... CCD camera 24 ... Observation optical system 25 ... Observation / laser condensing optical system 26 ... Mirror with hole 27 ... Wavelength selection mirror 28 ... Stage drive mechanism 30 ... Central control part 301 ... Intensity Value determination unit 302 ... scanning region determination unit 31 ... scanning control unit 32 ... stage drive unit 33 ... irradiation control unit 34 ... mass spectrometry data processing unit 35 ... image processing unit 36 ... operation unit 37 ... display unit 40 ... two-dimensional sample image 41, 41a, 41b, 41c ... measurement target area indication frames 42, 421, 422, 423, 424, 425 ... minute measurement area 43 ... scanning area range 50 ... display frames 511, 51 ... divided region 60 ... base portion 61 ... sample stage 62 ... ion transport tube

Claims (6)

a) laser irradiation means for irradiating a minute measurement region on the sample with laser light;
b) mass spectrometry means for separating and detecting ions generated around a minute measurement region corresponding to the laser light irradiation according to the mass number;
c) scanning means for changing the relative position of the sample and the laser irradiation means so that the minute measurement region on the sample changes discretely;
d) control means for controlling the laser irradiation means, the mass analysis means, and the scanning means, respectively, in order to obtain a two-dimensional mass distribution in the measurement target region set on the sample;
e) resolution setting means for setting the spatial resolution of the two-dimensional mass distribution;
f) display means for displaying the two-dimensional mass distribution;
The control means sequentially executes mass analysis of the entire measurement target region while scanning so as to skip one or a plurality of minute measurement regions for a minute measurement region whose interval is determined in accordance with the set spatial resolution. by, the measurement to display the two-dimensional mass distribution rough target area on said display means, then performing a scan while being mass analyzed to select some or all of the microscopic measurement region skipped previously According to the mass spectrometer, the spatial resolution of the two-dimensional mass distribution displayed on the display means is increased stepwise. A determination means for determining whether or not the signal intensity of the specific mass number obtained for each minute measurement region when performing mass analysis while performing rough scanning for the entire measurement target region is a predetermined range;
The control means has a mass in a micro measurement area that is different from the micro measurement area in which mass analysis was performed at the time of rough scanning in an area where the signal intensity is determined to be within a predetermined range by the determination means or a peripheral area including the area. The mass spectrometer according to claim 1 , wherein scanning is performed to perform analysis. The control means increases the scanning density of the micro measurement area when scanning in the area where the signal intensity is determined to be within the predetermined range by the determination means or in the peripheral area including the area. The mass spectrometer according to claim 2 , wherein mass spectrometry is performed. There are a plurality of measurement target areas set on the sample, and the signal intensity of a specific mass number obtained for each micro measurement area when performing mass analysis while performing a coarse scan for each measurement target area. A determination means for determining whether or not the predetermined range,
The control means performs mass analysis while sequentially performing a rough scan for all the measurement target areas in order for all the measurement target areas, and at that time, the signal intensity is determined to be within a predetermined range by the determination means. for measurement target region including the measurement region only, to claim 1, characterized in that to perform the scan in order to execute a mass spectrometric analysis of microscopic measurement region that performed the mass spectrometry during earlier coarse scan different microscopic measurement region The mass spectrometer as described. It further comprises external information acquisition means for receiving two-dimensional distribution information on a sample different from the two-dimensional mass distribution information, and the control means includes a determination result by the determination means and two-dimensional distribution information obtained by the external information acquisition means. The mass spectrometer according to claim 2, further comprising: determining a region for performing mass analysis of a micro measurement region different from the micro measurement region for which mass analysis was performed during the previous rough scanning. Claim wherein the sample capturing means for acquiring all or part of the image of the sample, further comprising an area setting means for setting an arbitrary measurement target region on the acquired sample image by the sample taking means 1 The mass spectrometer in any one of -5 .

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