Abstract:
An imaging mass spectrometer, an image of a sample is generated, and a region in the image is selected in accordance with predetermined criteria. Then, a mass analysis of the region is performed while scanning the sample in the selected region with a laser beam spot. By computing the total or average of the results in the region, a high precision analytical value in the region can be obtained. In a biological sample, by preliminarily performing a staining process on the biological sample using a certain dye, only the objective tissues can be analyzed. Also, a fluorescence microscope can be used.

Description:
BACKGROUND OF THE INVENTION AND RELATED ART STATEMENT  
       [0001]     The present invention relates to an imaging mass spectrometer wherein a sample is moved and stopped repeatedly, and while the sample is stopped, a laser beam is irradiated to ionize the sample; and individual sections of the sample are analyzed and images of the sample analyzed are generated. Specifically, the invention relates to an imaging mass spectrometer equipped with an ion source by means of laser desorption ionization (LDI) or matrix-assisted laser desorption ionization (MALDI). One of the typical applications of such an apparatuses is a microscope mass spectrometer or a mass microscope.  
         [0002]     Using LDI, samples are ionized by irradiating a laser beam thereon to enhance the movements of electric charges in the substances that adsorbs the laser beam. In MALDI, in order to analyze the samples not readily laser beam absorbent, or samples susceptible to laser damage such as proteins, such samples are laser irradiated and ionized after being mixed with a matrix made of a laser beam absorbent material.  
         [0003]     MALDI mass spectrometry apparatuses, in particular, are capable of analyzing high molecular compounds with minimum degradation of such compounds and well suited for trace analysis, and thus, in recent years, have been widely utilized in the life science and other fields. A mass spectrometry apparatus comprising an LDI or MALDI ion source is referred to generally as an LDI/MALDI-MS herein.  
         [0004]     Employing an LDI/MALDI-MS while reducing the laser beam spot diameter and moving the laser irradiated positions on a sample (typically, the sample is moved into position) produces an image showing the distribution of mass analysis measurements. This is referred to as an imaging mass spectrometry apparatus. It is often used as a microscope (mass spectrometry microscope) by focusing the laser beam with spot diameter from several hundreds to several microns (μm) (non-patent reference 1 and patent reference 1).  
         [0005]      FIG. 1  shows one example of a conventional mass spectrometry microscope construction. An operator observes the sample  12  through a CCD or an ocular lens in the viewing system  11  and determines the region to be analyzed based on the image observed. When the operator performs the start operation, the irradiating system  13  irradiates a laser beam onto the sample  12  while the stage actuator  14  effects the two-dimensional movements of the sample stage  16  on which the sample  12  is placed.  
         [0006]     The sample  12  is ionized in the locations where the laser beam is directed, and ions  17  generated enter the mass analyzer  18 . The ions are separated according to the mass numbers (mass-to-charge ratio) and detected by the detector  19 .  
         [0007]     The signals from the detector  19  are sent to the measuring and control system  20  (a PC with dedicated software installed therein is often used). At the measuring and control system  20 , the mass analysis data is correlated with the locations on the sample  12  (i.e., the positions on the sample  12  where the laser beam is irradiated) to generate an image. The image generated is displayed and/or printed out.  
         [0008]     Patent reference 1: U.S. Pat. No. 5,808,300  
         [0009]     Non-patent reference 1: Yasuhide Naito, “Mass Spectrometry Microscope Suited for Biological Samples,” Journal of Mass Spectrometry Society of Japan, Vol. 53, No. 3, 2005, pp. 125-132.  
         [0010]     One of the major objectives of an imaging mass spectrometry or microscope mass spectrometry is analysis of the composition of biological tissues and cells. The need to analyze proteins and saccharides in biological samples is particularly considerable. Certain proteins and saccharides, however, are sparsely present in a biological sample, which makes it difficult for mass spectrometry to obtain a sufficient level of target signal intensity for reliable results.  
         [0011]     It is therefore an object of the present invention to solve these problems and provide an imaging mass spectrometry apparatus capable of performing a highly reliable analysis of even a sparsely present component.  
         [0012]     Further objects and advantages of the invention are apparent from the following description of the invention.  
       SUMMARY OF THE INVENTION  
       [0013]     The imaging mass spectrometry apparatus or imaging mass spectrometer according to the present invention devised to solve the aforementioned problems comprises a sample image generator for generating an image of a sample, a region selector for selecting a predetermined region from the sample image, a laser irradiator for irradiating a spot-shaped laser beam against the sample, a scanning portion for changing the position of the sample relative to the laser beam spot within the selected region, and amass analyzer for analyzing the ions generated from the laser irradiated locations of the sample.  
         [0014]     In the imaging MS apparatus according to the present invention, mass analysis is performed with scanning, with a laser beam spot, on a predetermined region, which is selected beforehand based on the image extracted information of the sample generated. The analysis of the target region, therefore, can be accomplished in a short period of time. In many samples, a region is defined by the color or the brightness, which contains substantially the same or similar components, so the analysis can be expedited. Moreover, by computing the sum (or the average) of the results in the region, the analysis of the components present in the region can be effected at a high level of sensitivity (S/N ratio). By computing complex statistical values, such as variances, more in-depth information related to the presence of the components in the sample can be obtained.  
         [0015]     For example, performing a staining process on a biological sample using a certain dye or the like enables the coloring of specific tissues. The use of this apparatus of the present invention, therefore, enables the analysis of the components that are present in the tissue quickly and with a high level of accuracy.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]      FIG. 1  is a schematic diagram showing a construction of a microscope mass spectrometer conventionally used, and also used in the present invention.  
         [0017]      FIG. 2  is a block diagram showing the functions of the measuring and control system in the microscope mass spectrometer of the invention.  
         [0018]      FIG. 3  is a flow chart showing the operation of the measuring and control system in the microscope mass spectrometer in the example.  
         [0019]      FIG. 4 ( a ) shows a CCD image of a sample, and  FIG. 4 ( b ) shows an image of a selected region.  
         [0020]      FIG. 5  is an explanatory diagram showing the scanning condition of the selected region.  
         [0021]      FIG. 6  is a graph showing the relationship between the overall brightness and individual colors red, green and blue in the sample image. 
     
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS  
       [0022]     A microscope mass spectrometer, which is one example of the present invention, will be explained below. The hardware composition of the microscope mass spectrometry apparatus is essentially the same as the aforementioned conventional microscope. In other words, the main body, as shown in  FIG. 1 , comprises a sample chamber  15  and a mass analyzer  18 , an image generating system  11  for viewing and generating the image of a sample through a window provided therein, and a laser irradiating system  13  for irradiating a laser beam, which is narrowed to form a fine spot, on the sample  12  through a laser irradiation window. The main body is connected to a personal computer with dedicated software programs installed thereon for the control and measurements performed by the microscope mass spectrometer, as well as for data processing, which constitute the measuring and control system  20 .  
         [0023]     In executing the aforementioned dedicated programs, the measuring and control system  20  operates as a system having the functional blocks shown in  FIG. 2 .  
         [0024]     The operation of the microscope mass spectrometer constructed as described in the above example will be explained with reference to the flow chart shown in  FIG. 3 . A sample  12  is set on the sample stage  16  within the sample chamber (Step S 11 ). At this time, the sample chamber  15  wherein the sample stage  16  is located is completely sealed from the mass analyzer  18  so as not to reduce the degree of vacuum in the mass analyzer  18 . After the sample  12  is set, the door to the sample chamber  15  is tightly sealed and air in the sample chamber  15  is exhausted until the degree of vacuum reaches a predetermined level. Then, the small opening located between the sample chamber  15  and the mass analyzer  18  is opened to allow ions to pass through. Ionization may be performed without creating a vacuum in the sample chamber  15 , and thus performed at atmospheric pressure, on occasion.  
         [0025]     In the image generating system  11 , the image of the sample  12  is generated by a CCD color camera via a window. The image data is sent from the CCD camera to the image generator  202  of the measuring and control system  20 . The measuring and control system  20  displays the sample image at a predetermined region (window) on the display device (S 12 ).  FIG. 4 ( a ) shows an example of such an image.  
         [0026]     In analyzing a biological sample using this microscope mass spectrometer, for example, a user often is able to roughly identify, by the color, tissues based on the user&#39;s empirical knowledge when viewing the color image. The user, while viewing the color image on the screen, designates the locations to be analyzed using an input device  22 , such as a mouse (S 13 ). The region selector  203  then selects the regions falling within the range of colors designated by the user (S 14 ,  FIG. 4 ( b )). A region maybe selected by setting a range of colors or, as shown in  FIG. 6 , a range of brightness levels based on the brightness data prepared using color specific data (or data for specific colors). When a fluorescence microscope is used as an image generating system  11 , the range is set by using only brightness values. In either way, it is desirable to have the region selector  203  arranged so as to allow the user to freely set the range of colors or brightness to be used when selecting the region of interest. By setting a desired range, the user can determine which of the selected regions whose sizes vary in accordance with the set range would be appropriate for analysis.  
         [0027]     The region selector  203  may also be arranged so as to allow the user to set multiple ranges of colors or luminous intensity values, such as concurrently selecting both red and violet regions, or two brightness levels ranging 0 (black)-0.2 and 0.8-1 (white), for example.  
         [0028]     After determining that the selected region is appropriate, the user operates the input device  22  to effect the command to begin the analysis. The scanning controller  204  transmits a control signal to the stage actuator  14  to move the sample stage  16  to position an edge (dot A in  FIG. 5 ) of the selected region to the laser beam irradiation location. When the dot A reaches that location, the scanning controller  204  sends a command to the stage actuator  14  to allow for the scanning of the region with a laser beam spot. In response to the command, the stage actuator  14  operates the sample stage  16  to repeat the following: move the sample stage in the direction X by a short predetermined distance Δx and pause for a short predetermined duration at each location ( FIG. 5 ). The laser controller  205  transmits a command signal to the laser irradiating system  13  to emit a laser beam to that location while the sample  12  is at a stop. This generates ions from the sample, and the ions generated are drawn into the mass separator  182  due to the pressure difference between the sample chamber  15  and the mass analyzer  18  as well as the electrical field created by the ion guide  181 . The ions are separated in accordance with the mass numbers (mass-to-charge ratio) in the mass separator  182 . The separated ions are detected by the detector  19 .  
         [0029]     When the location irradiated by the laser beam reaches the other edge of the selected region, the stage actuator  14  moves the sample in the direction Y by a predetermined distance to perform the scanning of the next row. If the region consists of multiple islands, the sample is moved between the spaces between the islands at high speed.  
         [0030]     During the scanning process, the detector  19  of the mass analyzer  18  transmits the signals based on the ions separated and detected for each mass number at each location to the detected data processor  206  of the measuring and control system  20 . The detected data processor  206  computes the intensity per mass number based on the signals transmitted from the detector  19 , and transmits the data (detected data) to the central processing unit  201 . Based on the control signals transmitted from the scanning controller  204  (or the stage position signals transmitted from the stage actuator  14 ), the central processing unit  201  correlates the information for each measured location of the sample  12  with the detected data to be stored in a predetermined memory region (S 15 ).  
         [0031]     When the entire region selected is scanned, the central processing unit  201  computes the sum or the average, as well as statistical values, such as variances and standard deviations as needed, based on the detected data for the entire region (S 17 ). When the sum or the average statistical value is obtained in this manner, the value represents the sum or the average of the analyzed values of the region of the same color of the sample. Accordingly, in a biological sample where there is strong correlation between colors and tissue composition, for example, a high sensitivity (high S/N ratio) mass spectrum of a tissue section represented by a particular color can be obtained.  
         [0032]     When a staining process is applied to a biological sample, the colored section can be selectively analyzed, which enables high sensitivity analysis of the biological composition of the stained region. Since biological samples can be colored with tissue specific dyes, this technique can provide a useful effect in analyzing biological samples.  
         [0033]     By installing an excitation light source, such as an ultraviolet light, in the image generating system  11  so as to generate the fluorescent image of a sample, even more information about the biological sample can be obtained.  
         [0034]     Instead of computing the statistical values from the aggregated detected data for the entire region (all measured locations) as described above, the detected data for each location may be superimposed on the sample image  4 ( a ) (or the image of a selected region  4 ( b )). In this case, if a color is set beforehand, instead of designating a representative location to be analyzed, in the step S 13  described above, the region selector  203  automatically selects and extracts a colored or fluorescing section. This eliminates the need for the user to individually match the colored or fluorescing section to the laser ionized region, and thus provides the benefit of simplifying the mass analysis of the colored or fluorescing section.  
         [0035]     The disclosure of Japanese Patent Application No. 2005-319495 filed on Nov. 2, 2005 is incorporated as a reference.  
         [0036]     While the invention has been explained with reference to the specific embodiments of the invention, the explanation is illustrative and the invention is limited only by the appended claims.