JP3959744B2 - Trace element analyzer in living tissue - Google Patents

Description

  The present invention relates to a trace element analyzer in a biological tissue for analyzing trace metal elements in a biological tissue using an X-ray microanalyzer, a sample stage, and an analysis method using the same.

  In some diseases such as Wilson's disease, metal elements bind to biomolecules such as proteins and accumulate abnormally. In such diseases, there is a traditional method in which a site where metal is accumulated by a special staining method is expressed as a difference in color and confirmed pathologically. However, this method has a problem in reliability because the result greatly depends on the instability of the dye preparation conditions, the temperature during dyeing, the dyeing time, the fatigue level of the dyeing liquid, and the like.

  In addition, when analyzing fine particles taken up into the alveoli, most of the fine particles are inorganic compounds such as oxides, and thus cannot be dyed. It was.

  Incidentally, an apparatus called an X-ray microanalyzer (hereinafter referred to as EPMA) is known. This is an apparatus for analyzing the type and amount of an element by irradiating a sample with an electron beam and detecting X-rays (characteristic X-rays) specific to the element generated therefrom. Therefore, if the elemental analysis using this device can be applied to the analysis of metallic elements in living tissues, such as the analysis of diseases such as Wilson's disease and fine particles in the alveoli, the above problems will be solved and the reliability of pathological diagnosis will be improved. It is expected that

  However, since the biological tissue contains a very small amount of the metal element to be analyzed, the characteristic X-ray of the element cannot be distinguished from the background noise, and the detection sensitivity is increased by increasing the characteristic X-ray intensity. In order to increase the intensity of the electron beam, it is impossible to analyze the metal element in the living tissue by EPMA because the living tissue section which is weak against heat is broken or deformed by the electron beam.

Patent Document 1 discloses a biological sample stage for EPMA in which a conductive thin film such as carbon is coated on a plate-like body made of silicon or quartz. According to this sample stage, the charge of electrons is prevented by the conductive thin film, and the metal element in the living tissue can be analyzed by EPMA.
JP 2003-177108 A

  However, when the sample stage disclosed in Patent Document 1 is used, even though the charge of electrons can be prevented by the conductive thin film formed on the sample stage, the background noise is large and the trace element The detection sensitivity was limited. That is, the above sample stage is obtained by coating a plate-like body made of silicon, quartz or the like with a conductive thin film such as carbon having a thickness of 10 to 100 mm, and an electron beam of EPMA is transmitted through the conductive thin film. Thus, there is a problem that a large amount of continuous X-rays are generated from a plate-like body made of silicon, quartz or the like, and this continuous X-ray becomes background noise, resulting in a decrease in detection sensitivity of trace elements.

  Even if trace metal elements contained in living tissue can be analyzed with high sensitivity using an X-ray microanalyzer (EPMA), analysis of trace metal elements with high sensitivity is possible. The resolution needs to be increased and it takes a long time. Therefore, in order to perform analysis efficiently and in a short time, it is necessary to focus on the target area.

  By the way, the analysis area in a living tissue is often instructed by a doctor based on a photograph of a section of a stained living tissue or the like for the purpose of diagnosing a disease. However, the biological tissue to be analyzed by the X-ray microanalyzer is not stained, and the analyst accurately searches and analyzes only the analysis area designated by the doctor from the biological tissue placed on the sample stage. It is difficult to do.

  Furthermore, the optimal analysis conditions such as electron beam intensity and scanning speed differ depending on the type and concentration of the metal element, but the type of trace metal element to be analyzed differs depending on the disease, as well as the trace metal element contained in the living tissue. Since it is difficult to predict the type and concentration of the metal, there is a problem that skill is required to determine the metal element to be analyzed and to determine the optimal analysis conditions.

  In view of the above problems, the present invention can easily analyze only a target analysis area when analyzing a trace metal element contained in a biological tissue using an X-ray microanalyzer (EPMA). A trace element analysis device in a biological tissue, a sample stage, and the like, which are capable of determining optimum analysis conditions, and capable of analyzing trace metal elements contained in the biological tissue with high sensitivity. The purpose is to provide the analysis method used.

According to a first aspect of the present invention, there is provided an apparatus for analyzing a trace element in a biological tissue, a sample stage on which the biological tissue is placed, and a trace metal element that is irradiated with an electron beam on the biological tissue placed on the sample stage. an X-ray microanalyzer analyzes, the analytical area determination means for determining the analysis area of the living tissue to be analyzed by the X-ray microanalyzer, the X-ray microanalyzer in the analysis area of a biological tissue as determined by the analysis area determination means Analysis condition determination means for determining the analysis conditions of the biological tissue, and the analysis condition determination means causes the X-ray microanalyzer to perform a preliminary quantitative analysis of trace elements in the analysis area of the biological tissue determined by the analysis area determination means. The preliminary quantitative analysis means for obtaining the preliminary quantitative analysis value, and the X-ray microanalyzer based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means. Analytical element determination means for determining the element to be analyzed by the riser. This analytical element determination means compares the element accumulated in the database with the information on the minimum concentration for analyzing the element and the preliminary quantitative analysis value. And determining the element to be analyzed, and determining to measure the nitrogen distribution .

Trace element analyzer in a biological tissue according to claim 2 of the present invention, Oite to claim 1, and stained image acquiring means for acquiring a stained image of the living tissue, the living tissue obtained by the stained image acquisition unit And an image synthesis means for synthesizing the trace element distribution image obtained by the X-ray microanalyzer.

The trace element analyzer in biological tissue according to claim 3 of the present invention is the surface of the biological tissue according to claim 1 or 2 , wherein the sample stage is formed of ultra-high purity glassy carbon. The average roughness (Ra) is 1 to 5 nm.

The trace element analysis device in biological tissue according to claim 4 of the present invention is the specimen image of biological tissue placed on the sample stage in any one of claims 1 to 3. Obtained by the sample image obtaining means for obtaining the analysis area information obtaining means for obtaining the analysis area information of the living tissue, the sample image of the living tissue obtained by the sample image obtaining means and the analysis area information obtaining means. Image correspondence means for associating the analysis area information of the biological tissue, and stage coordinate determination for determining the stage coordinates of the biological tissue placed on the sample table based on the sample image and the analysis area information associated with the image correspondence means Means.

Trace element analyzer in a biological tissue according to claim 5 of the present invention, according to claim 1 in any one of 4, the X-ray microanalyzer on the basis of the preliminary quantitative analysis values obtained in the previous SL preliminary quantitative analysis means The electron beam intensity determining means for determining the intensity of the electron beam irradiated by the above and the analysis time determining means for determining the analysis time by the X-ray microanalyzer based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means. It is characterized by that .

According to the trace element analyzing apparatus for living tissue according to claim 1 of the present invention, the analysis area determining means for determining the analyzing area of the living tissue is provided, so that the target analyzing area can be efficiently narrowed down in a short time. In addition, the person in charge of the analysis can accurately search and analyze only the analysis area designated by the doctor from the biological tissue placed on the sample stage. In addition, by providing an analysis condition determining means for determining the analysis conditions in the analysis area, the optimum type of metal element to be analyzed, electron beam intensity, scanning speed, etc., depending on the type and concentration of the metal element in the analysis area. Analysis conditions can be easily determined. Furthermore, by providing the analysis element determination means for determining the element to be analyzed, the type of metal element to be analyzed can be easily determined according to the type and concentration of the metal element in the analysis area.

According to the device for analyzing trace elements in biological tissue according to claim 2 of the present invention, the image synthesis means for synthesizing the stained image of the biological tissue and the trace element distribution image is provided. Trace element analysis images can be compared, and an analysis image that is easy to see when used by a doctor for diagnosis can be provided.

According to the trace element analyzer for biological tissue according to claim 3 of the present invention, since the average roughness (Ra) of the surface of the sample stage is 1 to 5 nm, no section of the biological tissue is attached to the sample stage. The heat generated in the carbon even when irradiated with a high-current electron beam due to the fact that it can be fixed tightly and the sample stage is made of ultra-high purity glassy carbon with excellent thermal conductivity. Diffuses quickly, local temperature rise is prevented, and heat generated in the living tissue is quickly absorbed and diffused by the sample stage, preventing destruction of the living tissue due to heat and being included in the living tissue The trace metal elements to be analyzed can be analyzed with high sensitivity using an X-ray microanalyzer (EPMA).

According to the trace element analyzing apparatus in biological tissue according to claim 4 of the present invention, it is provided with stage coordinate determining means for determining the stage coordinate of the biological tissue placed on the sample stage, and based on the analysis area information. The stage coordinates of the living tissue can be determined, and the analysis can be efficiently performed in a short time and focused on the target analysis area.

According to trace element analysis device in a biological tissue according to claim 5 of the present invention, comprising an electron beam intensity determining means for determining the intensity of the electron beam to be morphism irradiation, and an analysis time determining means for determining the analysis time it is thereby possible depending on the type and concentration of the metal element in the analysis area, the intensity of the electron beam, to easily determine the optimum analysis conditions such as the scanning speed.

  Hereinafter, the trace element analyzer in the biological tissue of the present invention, the sample stage, and the analysis method using the same will be described in detail.

First, one embodiment of the trace element analyzer in living tissue of the present invention will be described. The trace element analyzer in living tissue of this embodiment analyzes trace metal elements by irradiating the tissue with an electron beam. An X-ray microanalyzer (EPMA) is provided. A detailed description of the basic configuration of the X-ray microanalyzer (EPMA) is omitted.

  The block diagram shown in FIG. 1 shows the outline of the structure of the control part which controls the X-ray microanalyzer (EPMA) which is not shown in the trace element analyzer in the biological tissue of a present Example. 21 is an analysis area determining means for determining an analysis area of a biological tissue section to be analyzed by an X-ray microanalyzer (EPMA). When the analysis area of the living tissue is determined by the analysis area determination means 21, the analysis conditions of the X-ray microanalyzer in this analysis area are determined by the analysis condition determination means 31, and the analysis by the X-ray microanalyzer is performed under the optimal analysis conditions. Is configured to be performed.

  When the analysis is performed by the X-ray microanalyzer, the analysis result is sent to the image synthesis means 41 as a trace element distribution image. Further, a stained image of a section adjacent to the analyzed biological tissue section is acquired by the stained image acquisition means 51, and this stained image is combined with the trace element distribution image obtained by the analysis by the image combining means 41. It is like that.

  In addition, the trace element analysis device in a biological tissue of the present invention includes a sample stage on which the biological tissue is placed. Since this sample stage is the same as the sample stage of the present invention described later, description thereof is omitted here.

  FIG. 2 shows the configuration of the analysis area determining means 21. In this embodiment, the analysis area determination means 21 includes a sample image acquisition means 22 for acquiring a sample image of a biological tissue placed on a sample stage, and an analysis area information acquisition means 23 for acquiring analysis area information of the biological tissue. The image correspondence means 24 for associating the sample image of the biological tissue obtained by the sample image acquisition means 22 with the analysis area information of the biological tissue obtained by the analysis area information acquisition means 23, and the image correspondence means 24 Stage coordinate determining means 25 for determining the stage coordinates of the biological tissue placed on the sample table based on the sample image and the analysis area information.

  The sample image acquisition means 22 is constituted by a CCD camera, acquires a sample image of a biological tissue section placed on a sample stage installed in the trace element analyzer, and sends the image data to the image correspondence means 24 It is configured.

  The analysis area information acquisition means 23 is constituted by a scanner, and acquires a stained image by HE staining of a section adjacent to the biological tissue section placed on the sample stage. The stained image is marked with an analysis area instructed by a doctor, and the analysis area information acquisition means 23 acquires the analysis area information of the living tissue by capturing the analysis area information together with the stained image. It is configured as follows. The analysis area information is sent to the image corresponding means 24 together with the image data of the stained image. The analysis area information acquisition unit 23 may also be used as the stained image acquisition unit 51.

  The image correspondence means 24 is adapted to associate the sample image from the sample image acquisition means 22 with the analysis area information sent together with the stained image from the analysis area information acquisition means 23. Specifically, the data of the sample image and the stained image are binarized, and the magnification and direction of the sample image and the stained image are matched by a known pattern recognition method. Then, by matching the sample image with the stained image, information about which area of the sample image should be analyzed can be obtained by associating the sample image with the analysis area information.

  The stage coordinate determination means 25 is configured to determine the stage coordinates of the biological tissue placed on the sample stage based on the sample image and analysis area information associated with each other by the image correspondence means 24. In other words, the analysis area is converted into stage coordinates expressed by the X-axis and Y-axis coordinate axes, and the coordinate data is sent to the next analysis condition determination means 31.

  FIG. 3 shows the configuration of the analysis condition determining means 31. In the present embodiment, the analysis condition determination means 31 is a preliminary that obtains a preliminary quantitative analysis value by causing the X-ray microanalyzer to perform a preliminary quantitative analysis of trace elements in the analysis area of the biological tissue determined by the analysis area determination means 21. Quantitative analysis means 32, analytical element determination means 33 for determining an element to be analyzed by an X-ray microanalyzer based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means 32, and preliminary quantitative analysis obtained by the preliminary quantitative analysis means 32 An electron beam intensity determining means 34 for determining the intensity of the electron beam irradiated by the X-ray microanalyzer based on the value, and an analysis for determining the analysis time by the X-ray microanalyzer based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means 32 Time determining means 35.

  The preliminary quantitative analysis means 32 is configured to perform any three simple automatic quantitative analyzes as preliminary quantitative analysis within the analysis area. Then, the data of the preliminary quantitative analysis value of the trace element obtained by the preliminary quantitative analysis is sent to the analytical element determination means 33. The automatic simple quantitative analysis is not limited to three points, but may be one point, two points, or four points or more. However, considering the balance between the time efficiency and accuracy of the preliminary analysis, three points are desirable.

  The analytical element determination means 33 is configured to determine the analysis for an element whose concentration of a certain level or more is detected by the preliminary quantitative analysis. Specifically, the element to be analyzed is automatically determined by comparing the information accumulated in a database (not shown) and the information on the minimum concentration for analyzing the element with the preliminary quantitative analysis value. ing. In addition, an analysis person can designate an element to be analyzed.

  When the element to be analyzed is determined based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means 32, the electron beam intensity determination means 34 is configured to determine the electron beam intensity suitable for the element. In elemental analysis with an X-ray microanalyzer, the intensity of an electron beam suitable for the type of element is determined, and irradiation is performed from information about the element stored in a database (not shown) and the intensity of the electron beam suitable for the element. The electron beam intensity is automatically determined.

  The analysis time determining means 35 is configured to determine the analysis time based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means 32. For example, based on information accumulated in a database (not shown), if a certain concentration or more of elements is detected by preliminary quantitative analysis and it is determined that sufficient analysis can be performed in normal analysis time, a short analysis time It is decided to perform high-speed mapping. On the other hand, if the preliminary quantitative analysis does not detect an element concentration above a certain level and it is determined that sufficient analysis cannot be performed in the normal analysis time, highly reliable mapping should be performed in a long analysis time. It comes to decide. In the present embodiment, the high-speed mapping and the high-reliability mapping are used. However, it may be configured such that the optimum analysis time can be selected from three or more analysis times by setting more detailed conditions.

  The series of operations in the analysis area determination means 21 and the analysis condition determination means 31 described above are all performed automatically.

  In addition, since the form of biological tissue closely matches the analysis image obtained by elemental analysis for nitrogen, elemental analysis is also performed for nitrogen in order to make it easier for a doctor to make a diagnosis corresponding to the analysis image of a metal element. You may comprise as follows.

  Next, an analysis method using the trace element analyzer in the biological tissue of this embodiment will be described with reference to FIG.

  First, in step S 1, a sample image of the biological tissue section placed on the sample stage is acquired by the sample image acquiring unit 22, and the image data is sent to the image corresponding unit 24.

  In step S2, a stained image by HE staining of a section adjacent to the biological tissue section placed on the sample stage is acquired. The stained image is marked with an analysis area instructed by a doctor, and the analysis area information acquisition means 23 acquires the analysis area information of the living tissue by taking the information of the analysis area together with the stained image. This analysis area information is sent to the image corresponding means 24 together with the image data of the stained image.

  In step S3, the image correspondence unit 24 binarizes the sample image from the sample image acquisition unit 22 and the stained image data from the analysis area information acquisition unit 23, respectively. Match the magnification and orientation of the stained image. By matching the sample image with the stained image, the sample image is associated with the analysis area information marked on the stained image to obtain information on which area of the sample image should be analyzed.

  In step S4, the stage coordinate determining means 25 converts the analysis area into stage coordinates expressed by the X-axis and Y-axis coordinate axes based on the sample image and analysis area information associated with each other by the image correspondence means 24. The coordinate data is sent to the next analysis condition determination means 31.

  In step S5, the preliminary quantitative analysis means 32 performs automatic three-point automatic quantitative analysis as preliminary quantitative analysis within the analysis area. Data of the preliminary quantitative analysis value of the trace element obtained by the preliminary quantitative analysis is sent to the analytical element determination means 33.

  In step S6, the analytical element determination means 33 automatically determines the element to be analyzed by comparing the information accumulated in the database with the information on the minimum concentration for analyzing the element and the preliminary quantitative analysis value. To do. In addition, when an analysis person designates an element to be analyzed, it is decided to analyze the element preferentially.

  In step S7, the intensity of the electron beam to be irradiated is automatically determined from the information about the element stored in the database and the intensity of the electron beam suitable for the element.

  In step S8, the analysis time determination means 35 determines that an element concentration of a certain level or more is detected in the preliminary quantitative analysis based on the information accumulated in the database, and that sufficient analysis can be performed in the normal analysis time. Determines to perform high-speed mapping in a short analysis time. On the other hand, if the preliminary quantitative analysis does not detect an element concentration above a certain level and it is determined that sufficient analysis cannot be performed in the normal analysis time, highly reliable mapping should be performed in a long analysis time. decide.

  In step S9, an analysis by an X-ray microanalyzer (EPMA) is performed.

  In step S10, it is determined whether the information obtained by the analysis is sufficient. If the information is not sufficient, the process returns to step S8, and it is determined in step S8 to perform a longer analysis. If the information is sufficient, the analysis result is sent to the image synthesis means 41 as a trace element distribution image.

  In step S11, a stained image of a section adjacent to the analyzed biological tissue section is acquired by the stained image acquisition means 51, and this stained image is sent to the image composition means 41.

  In step S <b> 12, the image synthesizing unit 41 synthesizes the trace element distribution image obtained by the analysis and the stained image acquired by the stained image acquiring unit 51.

  Next, the sample stage of the present invention and the analysis method using the same will be described.

  The sample stage of the present invention is a sample stage for analyzing trace metal elements in a living tissue using an X-ray microanalyzer (EPMA), and is made of only ultra high purity glassy carbon. This ultra high purity glassy carbon is an amorphous carbon having a dense isotropic structure without pores, has conductivity and thermal conductivity, is excellent in heat resistance, chemical resistance and corrosion resistance, It is a material with low expansion, resistance to thermal shock, and high airtightness.

  In the measurement by EPMA, an electron beam penetrates a part of a living tissue section, and characteristic X-rays and continuous X-rays are generated from the sample stage. However, carbon is a light element with atomic number 6, and therefore continuous X-rays that cause noise. Therefore, the characteristic X-ray of the target element can be detected efficiently.

  Further, the ultra-high purity glassy carbon used for the sample stage of the present invention is required to have very high purity from the analysis of trace metal elements. Regarding the impurity elements, 20 kV, 0.8 μA, one in EPMA It is necessary that the number of counts shown in Table 1 be less than or equal to each other under the two-dimensional analysis condition of 0.35 seconds.

  Although the size of the sample stage of the present invention can be set to an arbitrary size, in this embodiment, the outer dimensions of the surface on which the biological tissue is placed are 25 mm long, 20 mm wide, and 1 mm thick. The thickness of the sample stage is at least a thickness that does not allow the transmission of the EPMA electron beam. By doing so, the background noise can be surely kept low.

  In addition, the average roughness (Ra) of the surface on which the biological tissue of the sample stage is placed is preferably 1 to 5 nm. It is important to set the average roughness within this range, and the biological tissue section having a thickness of 1 to 4 μm is closely fixed to the sample table without an adhesive by the appropriate unevenness. A living tissue section that tends to deform even with a slight temperature rise is closely fixed to the sample table without an adhesive, so that the heat generated in the living tissue by the electron beam is quickly absorbed and diffused by the sample table, to the living tissue section. The effect of heat can be minimized.

  The average roughness of the surface of the sample stage can be achieved by polishing the surface of the ultra high purity glassy carbon and raising the mirror surface. In this polishing, it is necessary to pay sufficient attention so that impurities do not adhere to the surface of the sample stage.

  Next, a method for preparing an analysis sample in the analysis of biological tissue using the sample stage will be described with reference to FIG.

  The procedure from the initial tissue collection, formalin fixation, dehydration, paraffin embedding, and excision is exactly the same as a general method for preparing a biological tissue section. In the cutting, a sample section 2 as a biological tissue section is cut out from the sample block 1 with a knife 3. Then, the sample slice 2 is attached to the sample table 4. In addition, 5 is filter paper, 6 is distilled water, the sample section 2 is placed on the sample stage 4 via the distilled water 6, and the excess distilled water 6 is sucked out by the filter paper 5, so that the sample section 2 is removed from the sample stage. 4 is fixed tightly.

  Regarding the thickness of the sample section 2, since the amount of the element to be analyzed is small, a thicker one is desirable from the viewpoint of detection sensitivity. On the other hand, if it becomes too thick, adhesion to the sample stage 4 becomes insufficient, and the sample section 2 is likely to be deformed or peeled off due to the influence of heat by the electron beam of EPMA. Therefore, it is preferable that the thickness of the sample section 2 is 1 to 4 μm. By using this thickness, heat due to the electron beam irradiation of EPMA is quickly absorbed and diffused by the sample stage 4, and the influence of the heat on the sample section 2 is suppressed.

  Next, the sample section 2 placed on the sample stage 4 is dried, and then deparaffinized by being immersed in xylene 8 placed in a beaker 7. When the average roughness (Ra) of the surface of the sample stage 4 is 1 to 5 nm, the sample section 2 is securely fixed to the sample stage 4 and the sample section 2 is not peeled even during deparaffinization. In order to prevent impurities from being mixed in, xylene 8 is always a new one with high purity.

  Then, the xylene remaining on the surface of the sample stage 4 and the sample section 2 is sucked with the filter paper 9 before being evaporated and stored for about 3 hours.

  Finally, in order to ensure conductivity in preparation for electron beam irradiation, carbon 12 is deposited on the sample piece 2. In general, a sputtered gold film is often used in EPMA samples because a thin and uniform film can be formed. However, in order to avoid a slight increase in background noise, it is desirable to deposit carbon.

  The trace metal element in the analytical sample thus prepared is analyzed using an X-ray microanalyzer.

  FIG. 6 shows the result of actual analysis of the copper accumulation site in the living tissue. The left is the result of the conventional staining method (rhodanine staining), which shows that copper is present in the fine dot area. The right is the result of analysis by EPMA by the analysis method using the sample stage of the present invention, and the copper concentration distribution contained only in a minute amount in the living tissue was color-coded and could be clearly confirmed (the attached drawings). Is monochrome, but is actually color). In the case of the conventional staining method, a certain amount of experience is required to determine the copper accumulation site. However, in the analysis method of the present invention, the concentration distribution of the trace metal element can be clearly identified. From this analysis result, it was diagnosed as primary biliary cirrhosis.

  In addition, FIG. 7 shows the result of elemental analysis of fine particles in the alveoli. Since the fine particles in the alveoli are inorganic compounds such as oxides, the element could not be confirmed by the conventional staining method, but the elemental analysis can be performed according to the analysis method of the present invention. The area surrounded by a square that was estimated to be a lesion by HE staining in the lower left was analyzed. Nitrogen distribution can be used to morphologically correspond to HE stained specimens. The concentration distributions of aluminum and silicon were clearly identified by color coding (the attached drawings are monochrome, but are actually in color).

  As described above, according to the trace element analyzing apparatus in the living tissue of the present invention, the sample table 4 on which the living tissue is placed and the living tissue placed on the sample table 4 are irradiated with an electron beam to obtain a trace amount. An X-ray microanalyzer that analyzes a metal element and an analysis area determining means 21 that determines an analysis area of a living tissue to be analyzed by the X-ray microanalyzer are provided. By providing the analysis area determining means 21 for determining the analysis area of the biological tissue, it is possible to efficiently analyze the target analysis area in a short time and from the biological tissue placed on the sample table 4 Only the analysis area instructed by the doctor can be accurately searched and analyzed by the person in charge of analysis.

  Further, an analysis condition determining means 31 for determining an analysis condition of the X-ray microanalyzer in the analysis area of the living tissue determined by the analysis area determining means 21 is provided. By providing the analysis condition determination means 31 for determining the analysis conditions in the analysis area, the optimum analysis of the type of metal element to be analyzed, the intensity of the electron beam, the scanning speed, etc., depending on the type and concentration of the metal element in the analysis area Conditions can be easily determined.

  In addition, a stained image acquiring unit 51 that acquires a stained image of a biological tissue, and a stained image of the biological tissue obtained by the stained image acquiring unit 51 and a trace element distribution image obtained by the X-ray microanalyzer are synthesized. And image synthesizing means 41. By providing the image synthesis means 41 for synthesizing the stained image of the biological tissue and the trace element distribution image, the trace element analysis image can be compared with the stained image familiar to the doctor, and it is easy to see when the doctor uses it for diagnosis. An analytical image can be provided.

  The sample stage 4 is made of ultra-high purity glassy carbon, and the average roughness (Ra) of the surface on which the biological tissue is placed is 1 to 5 nm. Since the average roughness (Ra) of the surface of the sample stage 4 is 1 to 5 nm, it is possible to closely adhere and fix a section of biological tissue to the sample stage 4 without an adhesive, and the sample stage 4 has excellent thermal conductivity. Because it is made of ultra-high-purity glassy carbon, the heat generated in the carbon diffuses quickly even when irradiated with a high-current electron beam, preventing local temperature rise and Since the heat generated inside is quickly absorbed and diffused by the sample stage, the destruction of the living tissue due to heat is prevented, and trace metal elements contained in the living tissue can be increased using an X-ray microanalyzer (EPMA). It can be analyzed with sensitivity.

  The analysis area determination means 21 includes a sample image acquisition means 22 for acquiring a sample image of a biological tissue placed on the sample table 4, and an analysis area information acquisition means 23 for acquiring analysis area information of the biological tissue. The image correspondence means 24 associates the sample image of the biological tissue obtained by the sample image acquisition means 22 with the analysis area information of the biological tissue obtained by the analysis area information acquisition means. Stage coordinate determining means 25 for determining the stage coordinates of the living tissue placed on the sample table 4 based on the sample image and the analysis area information. By providing the stage coordinate determining means 25 for determining the stage coordinates of the living tissue placed on the sample stage 4, the stage coordinates of the living tissue are determined based on the analysis area information, and the target is efficiently and quickly completed. Analyzes can be made focusing on the analysis area.

  Further, the analysis condition determining means 31 is a preliminary that obtains a preliminary quantitative analysis value by causing the X-ray microanalyzer to perform a preliminary quantitative analysis of a trace element in the biological tissue analysis area determined by the analysis area determining means 21. Quantitative analysis means 32, analytical element determination means 33 for determining an element to be analyzed by the X-ray microanalyzer based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means 32, and preliminary data obtained by the preliminary quantitative analysis means 32 An electron beam intensity determination means 34 for determining the intensity of the electron beam irradiated by the X-ray microanalyzer based on the quantitative analysis value, and an analysis by the X-ray microanalyzer based on the preliminary quantitative analysis value obtained by the preliminary quantitative analysis means 32 And an analysis time determining means 35 for determining the time. The analysis element determining means 33 for determining the element to be analyzed, the electron beam intensity determining means 34 for determining the intensity of the electron beam to be irradiated, and the analysis time determining means 35 for determining the analysis time are provided. Depending on the type and concentration of the metal element, it is possible to easily determine the optimal analysis conditions such as the type of metal element to be analyzed, the intensity of the electron beam, and the scanning speed.

  Moreover, the sample stage of the present embodiment is a sample stage 4 for analyzing trace metal elements in a living tissue using an X-ray microanalyzer, and is formed from ultra-high purity glassy carbon, and the living tissue The average roughness (Ra) of the surface on which is placed is 1 to 5 nm. Since the average roughness (Ra) of the surface of the sample stage 4 is 1 to 5 nm, it is possible to directly adhere and fix a section of a biological tissue to the sample stage, and the sample stage 4 has an ultrahigh purity with excellent thermal conductivity. By being formed of glassy carbon, heat is rapidly absorbed and diffused from the living tissue to the sample stage 4 even when irradiated with a large current electron beam, so that destruction of the living tissue due to heat is prevented, Trace metal elements contained in living tissue can be analyzed with high sensitivity using an X-ray microanalyzer (EPMA).

  In the analysis method of this embodiment, the biological tissue section 2 having a thickness of 1 to 4 μm is closely fixed to the sample stage 4 and the trace metal element in the biological tissue is analyzed using an X-ray microanalyzer. By closely fixing the biological tissue section 2 having a thickness of 1 to 4 μm, the destruction of the biological tissue due to heat is prevented, and trace metal elements contained in the biological tissue can be detected with high sensitivity using an X-ray microanalyzer (EPMA). Can be analyzed.

  The trace element analyzer in the biological tissue of this embodiment, the sample stage, and the analysis method using the same are used for diagnosis of diseases in which copper accumulates in the liver and kidneys due to Wilson's disease or the like, or taken into the alveoli in respiratory diseases It is possible to analyze with high sensitivity the trace metal elements present at abnormal concentrations in living tissues due to various causes such as pneumoconiosis and cemented carbide lung diagnosis by elemental analysis of fine particles.

  Moreover, if the distribution of nitrogen is also measured at the same time when measuring trace metal elements in biological tissue, the trace metal element accumulation site can be easily associated with the lesion site of a stained specimen obtained by a conventional method.

  In addition, this invention is not limited to the said Example, It can apply to the metallic element detection in all the biological tissue slices within the range of the thought of this invention.

  Hereinafter, examples in which the pathological diagnosis is confirmed from the results of analysis by the sample stage of the present invention and the analysis method using the same will be listed.

Sample: Lung tissue chief complaint: Dry cough since 10 years Employment history: Enrolled in processing factory until 8-4 years ago. Engaged in solidification by spraying liquid indium.
Results: As a result of mapping analysis of the lung tissue of the lesion, indium deposition was confirmed in a wide range, and it was confirmed that the disease was caused by occupation.

Sample: Thoracoscopic lung biopsy tissue chief: Suspected cemented carbide lung Work history: 12 years of grinding with cemented carbide.
Results: As a result of mapping analysis of the lung tissue of the lesion, tungsten deposition was confirmed, and a cemented carbide lung was diagnosed.

Sample: Lung lavage fluid (macrophages)
Chief complaint: Chest screening abnormal Work history: Welder result: As a result of mapping analysis of macrophages in the lung lavage fluid, macrophages phagocytosing Fe, Si, Al, Mn were confirmed and diagnosed as a welder lung.

Specimen: Thoracoscopic lung biopsy tissue chief: Abnormal chest examination Job history: Metal processing industry Result: Mapping of lung tissue in the affected area confirmed tungsten deposition and was diagnosed as cemented carbide lung.

Sample: Liver tissue chief complaint: Suspected Wilson disease Job history: Unknown Result: Mapping analysis of the liver tissue in the lesion revealed that copper was accumulating at a high concentration in the tissue, and confirmed Wilson disease .

It is a block diagram of the control part which shows one Example of the trace element analyzer in the biological tissue of this invention. It is a block diagram which shows the structure of an analysis area determination means same as the above. It is a block diagram which shows the structure of an analysis condition determination means same as the above. It is a flowchart which shows one Example of the analysis method using the trace element analyzer in the biological tissue of this invention. It is explanatory drawing which shows one Example of the analysis method of this invention. The result of having analyzed the accumulation site of copper in living tissue by the analysis method of the present invention is shown. The result of elemental analysis of fine particles in the alveoli by the analysis method of the present invention is shown.

Explanation of symbols

2 Sample section (living tissue section)
4 Sample stage
21 Analysis area determination method
22 Sample image acquisition means
23 Information acquisition means
24 Image handling means
25 Stage coordinate determination means
31 Analytical condition determination means
32 Preliminary quantitative analysis means
33 Analytical element determination means
34 Electron beam intensity determination means
35 Analysis time determination means
41 Image composition means
51 Stained image acquisition means

Claims (5)

A sample stage on which a biological tissue is placed, an X-ray microanalyzer that irradiates the biological tissue placed on this specimen stage with an electron beam and analyzes trace metal elements, and a biological tissue that is analyzed by this X-ray microanalyzer Analysis area determining means for determining the analysis area of the X-ray microanalyzer in the analysis area of the biological tissue determined by the analysis area determining means, and this analysis condition determining means A preliminary quantitative analysis means for obtaining a preliminary quantitative analysis value by causing the X-ray microanalyzer to perform a preliminary quantitative analysis of the trace element in the analysis area of the biological tissue determined by the analysis area determination means, and the preliminary quantitative analysis Element determination means for determining an element to be analyzed by the X-ray microanalyzer based on the preliminary quantitative analysis value obtained by the means This analytical element determination means determines the element to be analyzed by comparing the element accumulated in the database with the information on the minimum concentration for analyzing the element and the preliminary quantitative analysis value, and the distribution of nitrogen. An apparatus for analyzing a trace element in a living tissue, characterized in that it is configured to determine to measure the above . A stained image acquiring means for acquiring a stained image of a biological tissue; an image synthesizing means for combining the stained image of the biological tissue obtained by the stained image acquiring means and the trace element distribution image obtained by the X-ray microanalyzer; trace element analyzer in a biological tissue according to claim 1 Symbol mounting characterized by comprising a. The sample stage, while being formed from ultra-high-purity glassy carbon, the average roughness of the surface of the biological tissue is placed (Ra) of claim 1, wherein it is 1~5nm Trace element analyzer in living tissue. The analysis area determination means includes a sample image acquisition means for acquiring a sample image of a biological tissue placed on the sample stage, an analysis area information acquisition means for acquiring analysis area information of the biological tissue, and the sample image acquisition means The image correspondence means for associating the sample image of the biological tissue obtained in step 1 with the analysis area information of the biological tissue obtained by the analysis area information acquisition means, and the sample image and the analysis area information associated with the image correspondence means. The trace element analysis device in biological tissue according to any one of claims 1 to 3 , further comprising stage coordinate determining means for determining stage coordinates of the biological tissue placed on the sample base based on . The analysis condition determining means, and the electron beam intensity determining means for determining the intensity of the electron beam irradiated by the X-ray microanalyzer on the basis of the preliminary quantitative analysis values obtained in the previous SL preliminary quantitative analysis unit, in the preliminary quantitative analysis means The trace element in the living tissue according to any one of claims 1 to 4 , further comprising an analysis time determining means for determining an analysis time by the X-ray microanalyzer based on the obtained preliminary quantitative analysis value. Analysis equipment.