JP4145690B2 - X-ray spectroscopic microscopic analysis method and photoelectric conversion X-ray microscope apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an X-ray spectroscopic microscopic analysis method for measuring a distribution of a composition by identifying and quantifying constituent elements at various points of a sample using a photoelectric conversion X-ray microscope apparatus. In particular, the present invention relates to measurement of trace constituent elements for biological samples and the like.
[0002]
[Prior art]
Conventionally, as a method for measuring trace elements, for example, there is a method for measuring element content using an X-ray absorption edge as disclosed in Patent Document 1, and measuring sulfur content of 1 to several tens of ppm. is doing. However, in this disclosed method, an X-ray detector is disposed at an X-ray spectroscopic angle position corresponding to the X-ray absorption edge of a specified known element such as sulfur, and the transmittance before and after the X-ray absorption edge is detected. The content ratio is measured and applied to a predetermined element, and the elemental composition distribution on the sample cannot be measured.
[0003]
Further, as a trace element analysis method, there is a fluorescent X-ray analysis method (for example, see Patent Document 2). This method performs composition analysis by irradiating a sample with X-rays and detecting characteristic X-rays generated by the contained elements. This method is mainly suitable for the measurement of heavy metal elements, and since the excitation efficiency of fluorescent X-rays is low for relatively light elements such as C, O, and N existing in the living body, the accuracy required for the measurement can be satisfied. It is not possible in principle.
[0004]
Further, as a method for measuring the element content based on the difference in absorption near the absorption edge of the element, a method using the difference in absorption at wavelengths on both sides of the absorption edge has been proposed (see, for example, Non-Patent Document 1). . In this method, the content of an element is calculated from the difference in transmittance at both ends of the absorption edge of the element. If the ratio of the absorption rate on the short wavelength side (T _S ) and the absorption rate on the long wavelength side (T _L ) observed for the absorption edge of an element is T _S / T _L , Since there is also a contribution, it is calculated as follows.
[0005]
T _S / T _L = Exp [− (μ _{CS -Μ CL) · ρ C · x-} (μ N S _{-Μ N L) · ρ N ·} x- (μ O S −μ _{O L} ) · ρ _O · x −...]
Here, μ _CS , μ _CL , μ _{N S} , μ _{N L} Etc. represent the mass absorption coefficient on the short wavelength side and the long wavelength side of carbon, nitrogen, etc., ρ _C , ρ _N, etc. represent the density of carbon, nitrogen, etc., respectively, and x represents the thickness of the region.
Note that ρ · x represents the surface density, which is the element content per unit area determined by the surface density.
[0006]
However, in this method, as shown in the above equation, the contribution of absorption by other elements contained in the absorption edge portion of a certain element cannot be ignored, and the measurement accuracy is insufficient in measuring the content of the element. there were. In particular, in the measurement of trace elements, there is a problem that even the existence of the absorption edge cannot be observed.
[0007]
Furthermore, conventionally, as an attempt to remove the contribution of absorption by other elements contained in the absorption edge portion of an element, there is a molecular analysis using the wavelength in the absorption fine structure (XANES) near the absorption edge region. This method is a method of solving simultaneous equations of X-ray absorption using the element amounts of contained elements obtained at several wavelengths as unknowns.
For example, if the composition of an element of a biological sample composed of oxygen O, nitrogen N, carbon C, and calcium Ca is obtained, first, X-ray absorption rates at wavelengths on both sides of the absorption edge of each element are obtained. The incident and transmitted X-ray doses at a certain wavelength are respectively I ₀ , I, and transmittance T, transmittance T and absorption A can be expressed as follows.
[0008]
T = I / I ₀ = Exp [− (μ _O ρ _O x + μ _N ρ _N x + μ _C ρ _C x + μ _Ca ρ _Ca x)
A = −lnT = μ _O ρ _O x + μ _N ρ _N x + μ _C ρ _C x + μ _Ca ρ _Ca x
Here, μ represents a mass absorption coefficient, and ρ represents density. Subscripts O, N, C, and Ca indicate oxygen, nitrogen, carbon, and calcium, respectively. x represents the thickness of the region, and ρx represents the surface density. Then, since ρx is the element content per unit area, it is the element content per unit area determined by the surface density.
Next, the absorption difference A (dif) between the absorption ends of each element is expressed as follows.
[0009]
O absorption edge:
A _o (dif) = (μ ₁ ^O −μ ₂ ^O ) ρ _O x + (μ ₁ ^N −μ ₂ ^N ) ρ _N x + (μ ₁ ^C −μ ₂ ^C ) ρ _C x + (μ ₁ ^Ca −μ ₂ ^Ca ) ρ _Ca x
N absorption edge:
A _N (dif) = (μ ₃ ^O −μ ₄ ^O ) ρ _O x + (μ ₃ ^N −μ ₄ ^N ) ρ _N x + (μ ₃ ^C −μ ₄ ^C ) ρ _C x + μ ₃ ^Ca −μ ₄ ^Ca ) ρ _Ca x
C absorption edge:
A _o (dif) = (μ ₅ ^O −μ ₆ ^O ) ρ _O x + (μ ₅ ^N −μ ₆ ^N ) ρ _N x + (μ ₅ ^C −μ ₆ ^C ) ρ _C x + (μ ₅ ^Ca −μ ₆ ^Ca ) ρ _Ca x
Ca absorption edge:
A _o (dif) = (μ ₇ ^O −μ ₈ ^O ) ρ _O x + (μ ₇ ^N −μ ₈ ^N ) ρ _N x + (μ ₇ ^C −μ ₈ ^C ) ρ _C x + (μ ₇ ^Ca −μ ₈ ^Ca ) ρ _Ca x
Here, μ _i represents a mass absorption coefficient at a wavelength λ _i before and after the absorption edge of the contained element. For example, the one with the subscript 3 is the short wavelength side wavelength of the nitrogen absorption edge, and the one with the subscript 4 is the absorption coefficient at the long wavelength side wavelength. Superscripts O, N, C, and Ca indicate oxygen, nitrogen, carbon, and calcium, respectively.
[0010]
From the above four simultaneous equations, ρ _O x, ρ _N x, ρ _C x, ρ _Ca x Ask for. In this method, if the target of the analysis is known, that is, if the chemical bond species contained in the absorption microstructure is known in the case of molecular analysis, or if the contained element type is known in the case of elemental analysis, The content of the element in it can be determined with high accuracy. For example, in the case of a biological sample, it is possible to quantify major elements such as carbon, nitrogen and oxygen. However, especially in the case of elemental analysis, the purpose is to obtain unknown contained elements in the first place. However, in order to analyze unknown trace elements as well, it is assumed that analysis is performed for all elements that are expected in advance. For example, it is necessary to always solve extremely multiple simultaneous equations, and the calculation load becomes very large, which is not practical.
[0011]
When a scanning X-ray microscope apparatus using a zone plate is used as a method for measuring constituent elements in a living body, it is necessary to perform a wavelength sweep in a two-dimensional range in order to measure the composition of all the main elements in the living body. is there. However, in the zone plate X-ray microscope apparatus, the focal length is different for each wavelength, so that measurement in a two-dimensional range takes a long time to adjust the position of the zone plate and scan the two-dimensional range. It was difficult.
[0012]
[Patent Document 1]
JP 2002-214162 A [Patent Document 2]
JP-A-8-122281 [Non-Patent Document 1]
Ito, Shinohara et al. “Measurement of soft X-ray absorption spectra and elemental analysis in local regions of mammalian cells using an electronic zooming. tube) ", Journal of Microscopy, Vol..181, Pt 1, January 1966, pp.54-60
[0013]
[Problems to be solved by the invention]
Therefore, the problem to be solved by the present invention is that simple X-ray spectroscopy that efficiently and accurately calculates the composition distribution of a sample, particularly a biological sample composed of low atomic weight elements, even if the contained element contains an unknown element. It is to provide a microscopic analysis method and a photoelectric conversion type X-ray microscope apparatus.
[0014]
[Means for Solving the Problems]
In order to solve the above problems, the X-ray spectroscopic microscopic analysis method of the present invention acquires and stores an X-ray absorption image for each wavelength by irradiating a sample with X-ray wavelength sweeping in a photoelectric conversion X-ray microscope apparatus. Then, an absorption spectrum is synthesized at a predetermined position on the sample from the obtained X-ray absorption image, the presence or absence of an absorption edge structure of the element is detected based on this absorption spectrum, and the absorption edge of the detected absorption edge structure of each element Establish a multiple linear simultaneous equation with the difference in absorptivity at both wavelengths as the left side, the surface density of each detected element as the independent variable on the right side, and the difference in mass absorption coefficient at each wavelength as the coefficient of each independent variable. Then, by solving this multi-dimensional linear simultaneous equation, the approximate value of the surface density of each element is obtained, and the contribution of the absorption spectrum of each element is calculated using this approximate surface density value, and the difference from the initially obtained absorption spectrum is obtained. The remaining spectrum is formed to detect the presence or absence of an absorption edge structure of a new element from the remaining spectrum.When a new element is detected, the above-mentioned is applied to all the elements detected so far. A similar multi-dimensional simultaneous equation is set up and solved to calculate the approximate value of the surface density of each element. When a new element cannot be detected, the surface density approximate value obtained previously is It is characterized by the surface density of each element at a predetermined position.
[0015]
In the X-ray spectroscopic microscopic analysis method of the present invention, a photoelectric conversion X-ray microscope is used. The photoelectric conversion type X-ray microscope converts an X-ray absorption image into an electron beam, enlarges it at a large magnification, detects it with a CCD detection element, etc., and forms an image. In the analysis method of the present invention, an X-ray image formed by sweeping a wavelength of X-rays and irradiating a sample is stored, and a specified portion is designated using these to read out absorption data at each wavelength and absorb it. Generate a spectrum. Therefore, according to the analysis method of the present invention, it is possible to accurately designate and analyze a specific portion on the sample based on a greatly enlarged image. Since the designation of the position is simple, it is easy to obtain the composition distribution on the sample.
[0016]
According to the X-ray spectroscopic microscopic analysis method of the present invention, an absorption edge unique to an element is found from the shape of an absorption spectrum at a specified portion in a sample, an element contained in that portion is identified, and each identified element Is defined as an independent variable, and a coefficient of the absorption coefficient at the wavelengths on both sides of the absorption edge of each element is used as a coefficient to establish a multi-dimensional simultaneous equation with independent variables for the number of detected elements, and this is solved and identified. Quantify each element.
Using the quantitative value of each element, the absorption spectrum for each element is calculated, and when the remaining absorption spectrum subtracted from the initial absorption spectrum is calculated, the remaining absorption spectrum is the remaining element that has not yet been identified. If the scale is expanded, the structure of the absorption spectrum of the minute element can be seen.
[0017]
Therefore, the remaining absorption spectrum is observed to check the existence of the absorption edge structure, and if there is an absorption edge, an element corresponding to the absorption edge is newly identified. Each element identified in this way is re-identified by establishing and solving a multi-component simultaneous equation using the data of the first absorption spectrum.
Using this method, for example, starting with a small number of elements, such as carbon, oxygen, nitrogen, etc., and detecting other elements one by one, a simultaneous equation is established and solved. It is not necessary to target multiple simultaneous equations with independent variables as many as the number of elements, and it is sufficient to solve multi-dimensional simultaneous equations with a relatively small number of elements with only the number of detected elements. Therefore, the calculation load is small enough for practical use.
[0018]
Select the elements that may be contained, specify the wavelengths on both sides of the absorption edge of those elements, select and store the X-ray absorption data at those specified wavelengths, and absorb by the intermittent data You may make it perform analysis calculation on behalf of a spectrum.
The X-ray absorption data used for the analysis is only X-ray absorption on both sides of the absorption edge of the contained element. Therefore, if the stored absorption data includes X-ray absorption at the wavelengths on both sides of the absorption edge of the element contained in the sample, there is no problem in the analysis. Therefore, if you select the elements that may be contained in the sample and record only the data at the wavelengths before and after the absorption edge of those elements, the storage capacity is small and it is necessary for the analysis. The amount of computation can be significantly reduced.
[0019]
In order to solve the above problems, the photoelectric conversion X-ray microscope apparatus of the present invention includes an arithmetic processing unit for spectroscopic analysis, and X-rays are wavelength-swept from behind a sample placed in close contact with the photoelectric conversion surface. An electronic image formed by irradiating the image is magnified by an electronic image enlarging device and formed on an image detection unit, and an arithmetic processing unit stores an X-ray absorption image from the image detection unit in an image memory for each sweep wavelength. When an arbitrary position in the line image is designated, an image signal corresponding to the designated position is extracted for a necessary wavelength, and is synthesized as a measured X-ray absorption spectrum of the designated portion.
[0020]
The arithmetic processing unit further detects the presence / absence of the absorption edge structure of the element based on the measured X-ray absorption spectrum, and detects the difference between the absorption rates at the wavelengths on both sides of the absorption edge structure of the detected element as the left side. The surface density of each element was obtained by solving the multi-dimensional simultaneous equations using the surface density of each element as the variable on the right side and the difference in mass absorption coefficient at both wavelengths as the coefficient of each right side variable. The contribution of the absorption spectrum of each element is calculated using the approximate density value and subtracted from the measured absorption spectrum to form a residual spectrum, and the presence or absence of an absorption edge structure of a new element is further examined from the shape of the residual spectrum.
When new elements can be detected, new approximate values of the surface density of each element can be calculated by solving the same simultaneous multiple equations for all the elements detected so far, and new elements can be detected. If not, the surface density of each element at the specified position is taken as the last surface density approximation.
[0021]
By using the photoelectric conversion X-ray microscope apparatus of the present invention, it is possible to accurately designate and analyze a specific portion on a sample based on a greatly enlarged image. Since the designation of the position is simple, it is easy to obtain the composition distribution on the sample. Also, the calculation load is not large, and the calculation processing device can use a personal computer. Furthermore, the element can be easily identified and quantified with respect to a biological sample composed of low atomic weight elements.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
The X-ray spectroscopic microscopic analysis method of the present invention will be described in detail based on the illustrated embodiment.
The method of this example detects the inclusion of an element based on the absorption edge structure appearing in the absorption spectrum obtained by measuring the sample, and solves the multi-dimensional linear simultaneous equations relating to the detected element based on the measured absorption spectrum. Estimate the content of the element, calculate the absorption spectrum of the substance with the estimated content, subtract it from the measured absorption spectrum to obtain the residual spectrum, and add another element contained by the absorption edge structure that appears in this residual spectrum Find out.
[0023]
Furthermore, based on the measured absorption spectrum, a multi-component simultaneous equation relating to the elements detected so far is established and solved to estimate the content of the contained element, and a residual spectrum is generated by the same method as above. Search for the absorption edge structure of another element. This procedure is repeated so that when no new element can be found, the final estimated value is the content of the element contained in the substance.
Therefore, the number of terms in the multiple simultaneous equations is the same as the number of elements found so far, so the number of terms gradually increases with repeated operations, but is kept to the minimum necessary and the computation load is wasted. It doesn't get bigger.
[0024]
FIG. 1 is a conceptual configuration diagram showing an X-ray microscope apparatus used in this embodiment. The X-ray microscope apparatus of FIG. 1 includes an X-ray generation apparatus 1, a photoelectric conversion surface 2, an electronic image enlargement apparatus 3, an image detection unit 4, and an arithmetic processing apparatus 5.
The X-ray generator 1 emits X-rays while sweeping the wavelength within an appropriate range by a grating, a total reflection mirror, or the like. Synchrotron radiation may be used. X-rays emitted from the X-ray generator 1 irradiate the photoelectric conversion surface 2.
On the photoelectric conversion surface 2, a photoelectric conversion film having a photoelectric conversion function such as a two-layer structure thin film of a gold thin film and a cesium iodide or antimony cesium film is disposed behind the support film. A sample to be measured is adhered and adhered to the surface of the support film, and an X-ray image is formed on the photoelectric conversion surface 2 in which the portion blocked by the sample is shaded. The photoelectric conversion surface 2 emits an amount of photoelectrons corresponding to the intensity of the incident X-rays to the surface at the position where the X-rays are incident, and forms an electron image corresponding to the X-ray image.
[0025]
The electronic image enlarging device 3 extracts photoelectrons from the electron image generated on the surface of the photoelectric conversion surface 2 by the anode, enlarges the image by the objective lens and the projection lens, and enlarges the image to the planar image detection unit 4 at a predetermined distance. Project as an electronic image.
The image detection unit 4 is a functional element that visualizes an electronic image. For example, the image detection unit 4 is configured with a microchannel plate (MCP) and a fluorescent screen provided behind the microchannel plate to make a visible image that can be observed by a person. It can be converted into an electrical signal by an optical system incorporating a relay lens provided behind the fluorescent screen and a CCD camera.
[0026]
The image signal converted into an electrical signal by the image detection unit 4 is displayed on the monitor as an image suitable for the purpose of measurement by performing appropriate image processing, and is sent to the arithmetic processing unit 5 where analysis processing is performed. Is called.
The arithmetic processing unit 5 includes a large number of image memories corresponding to the pixels of the X-ray absorption image formed by the image detection unit 4, and can store the X-ray absorption image as a grayscale image for each wavelength.
[0027]
The X-ray absorption image stored in the image memory has a larger amount of information and a greater degree of freedom in later calculations as the wavelength step is smaller, but requires a large amount of memory capacity. For the identification and quantification of the contained elements by the analysis method of the present invention, data at wavelengths before and after the absorption edge of the contained elements are used, and the others are not used.
Therefore, if elements that may be contained in the sample are determined, it is sufficient to store absorption images at predetermined wavelengths before and after the absorption edge of those elements. Therefore, in an actual analyzer, simplification of the apparatus and calculation can be achieved by storing only an X-ray absorption image at a wavelength related to an element that can be detected.
[0028]
FIG. 2 is a table listing examples of wavelengths at the absorption edge and wavelengths used for analysis on both sides of the absorption edge for main elements constituting a living body. For example, nitrogen has an absorption edge at a position of 3.099 nm, and the difference in absorption rate between 3.0 nm and 3.15 nm before and after the absorption edge can be used for analysis. Carbon has an absorption edge at 4.368 nm. For example, absorption at 4.25 nm and 4.45 nm may be used for analysis.
[0029]
FIG. 3 is a flowchart showing an analysis procedure by the arithmetic processing unit 5.
When performing X-ray spectroscopic analysis, first, a parameter i representing the number of loop operations is reset (S1). Next, when the operator designates a portion to be analyzed via the monitor, the pixel position data on the image memory corresponding to the designated portion is read, and a zeroth-order absorption spectrum having the wavelength as an independent variable is generated. (S2). The zeroth order spectrum is the original X-ray absorption spectrum observed for the sample.
The absorption spectrum may be generated by taking wavelengths at appropriate intervals. However, as described above, the absorption spectrum may be a spectrum in which only other wavelengths are ignored and other wavelengths are ignored. .
[0030]
FIG. 4 is a zeroth-order absorption spectrum diagram created for a simplified model substance. The absorption spectrum of FIG. 4 includes only carbon, oxygen, nitrogen, calcium, and iron in the sample. The surface density of carbon, oxygen, and nitrogen is 1 × 10 ⁻⁶ g / cm ² , and calcium is 1 × 10 ^−7. When g / cm < ² > and iron are 1 * 10 < ^-8 > g / cm < ² >, it shows. The absorption spectrum shows only the absorptance at wavelengths on both sides of the absorption edge of each element. The arrow indicates the wavelength position of each absorption edge, and the black dot in the graph indicates the absorption at the wavelength used for analysis calculation.
[0031]
The presence or absence of the absorption edge structure is determined by observing such a zeroth-order absorption spectrum (S3). FIG. 4 shows an absorption edge structure in which only oxygen O, nitrogen N, and carbon C are apparent. Therefore, the loop operation count i is incremented to 1 (S4), the newly identified element type (initially O, N, C) is added, and the number of elements identified so far is set to Ni (here, 3). (S5).
[0032]
Then, using the data of the zeroth-order absorption spectrum, the difference A _a (dif) of X-ray absorption by the entire sample at the wavelength on both sides of the absorption edge of each identified element (a represents the identified element) is calculated. 1 on the left side, and on the right side, each element j is a Ni element 1 with the difference in mass absorption coefficient μ _k ^j shown at the wavelength k on both sides of the absorption edge as a coefficient and the surface density ρ _j x as a variable. Ni are generated as the following equations to obtain Ni original simultaneous equations, and the surface density of each element is obtained by solving these simultaneous equations (S6).
[0033]
In the example of FIG. 4, since three elements of O, N, and C are identified, the following ternary linear simultaneous equation is obtained.
O absorption end both sides:
A _o (dif) = (μ ₁ ^O −μ ₂ ^O ) ρ _O x + (μ ₁ ^N −μ ₂ ^N ) ρ _N x + (μ ₁ ^C −μ ₂ ^C ) ρ _C x
N absorption end both sides:
A _N (dif) = (μ ₃ ^O −μ ₄ ^O ) ρ _O x + (μ ₃ ^N −μ ₄ ^N ) ρ _N x + (μ ₃ ^C −μ ₄ ^C ) ρ _C x
C absorption end both sides:
A _o (dif) = (μ ₅ ^O −μ ₆ ^O ) ρ _O x + (μ ₅ ^N −μ ₆ ^N ) ρ _N x + (μ ₅ ^C −μ ₆ ^C ) ρ _C x
The ternary simultaneous equations can be solved relatively easily, and the surface density of each element can be obtained as the first approximate value.
[0034]
Next, an X-ray absorption spectrum that should be exhibited when the identified elements have the areal density obtained as the i-th approximate value is calculated as the i-th absorption spectrum (S7).
A difference spectrum is generated by subtracting the i-th absorption spectrum from the 0th-order absorption spectrum obtained in the experiment (S8).
FIG. 5 shows a difference spectrum generated by subtracting the first absorption spectrum from the zeroth absorption spectrum in the example of FIG. The scale of the vertical axis is magnified 10 times.
[0035]
The ratio of the absorption edge structure is determined from the pattern of the difference spectrum (S3). Since the shape of the absorption edge is remarkable, it can be determined mechanically by pattern recognition. Note that the difference spectrum may be displayed on a monitor, and the operator may observe and judge this.
In FIG. 5, the absorption edge structure of calcium clearly appears and can be easily identified.
[0036]
When there was a newly identifiable element, the data of each element was acquired for the wavelength at both sides of the absorption edge of the newly identified element, and the process progressed from S4 to S8 as before, and was identified so far. A simultaneous equation consisting of Ni original equations for all the number of elements Ni is established and solved to obtain the i-th approximate value of the surface density of each element, and finally the process returns to S3.
[0037]
In the example of FIG. 4, since the presence of calcium is clarified from the difference spectrum of FIG. 5, the following quaternary linear simultaneous equation is established based on the 0th-order absorption spectrum.
O absorption end both sides:
A _o (dif) = (μ ₁ ^O −μ ₂ ^O ) ρ _O x + (μ ₁ ^N −μ ₂ ^N ) ρ _N x + (μ ₁ ^C −μ ₂ ^C ) ρ _C x + (μ ₁ ^Ca −μ ₂ ^Ca ) ρ _Ca x
N absorption end both sides:
A _N (dif) = (μ ₃ ^O −μ ₄ ^O ) ρ _O x + (μ ₃ ^N −μ ₄ ^N ) ρ _N x + (μ ₃ ^C −μ ₄ ^C ) ρ _C x + (μ ₃ ^Ca −μ ₄ ^Ca ) ρ _Ca x
C absorption end both sides:
A _o (dif) = (μ ₅ ^O −μ ₆ ^O ) ρ _O x + (μ ₅ ^N −μ ₆ ^N ) ρ _N x + (μ ₅ ^C −μ ₆ ^C ) ρ _C x + (μ ₅ ^Ca −μ ₆ ^Ca ) ρ _Ca x
Both sides of the Ca absorption end:
A _Ca (dif) = (μ ₇ ^O −μ ₈ ^O ) ρ _O x + (μ ₇ ^N −μ ₈ ^N ) ρ _N x + (μ ₇ ^C −μ ₈ ^C ) ρ _C x + (μ ₇ ^Ca −μ ₈ ^Ca ) ρ _Ca x
By solving this quaternary simultaneous equation, the second approximate value of the surface density can be obtained for the four elements.
[0038]
Further, since the last absorption edge structure of iron is found from the difference spectrum obtained by subtracting the second-order absorption spectrum obtained using the second approximate value from the zeroth-order absorption spectrum, based on the zeroth-order absorption spectrum. A ternary simultaneous equation is generated and solved for five elements, and a third approximate value of the surface density is calculated for the five elements.
This procedure is repeated until no absorption edge is observed in the difference spectrum.
[0039]
When the absorption edge structure cannot be observed in the difference spectrum, it is determined that no significant elements other than those already identified are included, and the i-th approximate value obtained last is used as a measurement value of each surface density ( S9).
Since the areal density obtained by this method corresponds to the amount of elements, the relative weight ratio of each element can be known. Further, if the thickness of the sample can be obtained by another method, the density can be obtained by dividing the surface density by the thickness.
By using the method of this embodiment, it is possible to accurately detect an element contained in an unknown sample by solving a multiple simultaneous equation having a minimum number of terms and obtain the element amount as the surface density. Therefore, the relative weight ratio of each element contained in the sample can be known.
[0040]
Since this method uses a photoelectric conversion type X-ray microscope apparatus that generates an X-ray absorption image with a high magnification, it is possible to accurately specify a sample portion and perform component analysis. Further, by selecting and analyzing an arbitrary position in the X-ray absorption image, the two-dimensional distribution of elements contained in the unknown sample can be clarified.
Since the photoelectric conversion type X-ray microscope can also handle a biological sample, it is possible to quantify a relatively light element by specifying a target portion particularly for the biological sample. The technique of the present invention is particularly useful because there is no other suitable technique for detecting trace amounts of light elements.
[0041]
Furthermore, according to the method of the present embodiment, once the X-ray image is generated, the portion to be analyzed can be arbitrarily designated again and again to obtain the result.
In the above description, in order to explain the technical concept in an easy-to-understand manner, the absorption spectrum is synthesized based on the data read from the image memory. However, in the actual configuration, only the data necessary for each calculation is extracted. Needless to say, it is not necessary to treat it as a continuous spectrum.
[0042]
【The invention's effect】
As described above, the composition distribution of a sample, particularly a biological sample composed of a low atomic weight element, even when the contained element contains an unknown element by the X-ray spectroscopic microscopic analysis method and the photoelectric conversion X-ray microscope apparatus of the present invention. Can be calculated efficiently and accurately.
[Brief description of the drawings]
FIG. 1 is a conceptual configuration diagram showing an X-ray microscope apparatus used in one embodiment of the present invention.
FIG. 2 is a table listing absorption edges and wavelengths used for analysis of main elements constituting a living body.
FIG. 3 is a flowchart showing an analysis calculation process in the embodiment.
FIG. 4 is an absorption spectrum diagram of a simplified model substance.
FIG. 5 is a diagram illustrating an example of a difference spectrum used in the present embodiment.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 X-ray generator 2 Photoelectric conversion surface 3 Electronic image expansion apparatus 4 Image detection part 5 Arithmetic processing apparatus

Claims (5)

In a photoelectric conversion type X-ray microscope apparatus, a sample is scanned and irradiated with X-rays to acquire and store an X-ray absorption image for each wavelength, and the obtained X-ray absorption image is absorbed at a predetermined position on the sample. The spectrum is synthesized, the presence or absence of an absorption edge structure of the element is detected based on the synthesized absorption spectrum, and the difference between the absorption rates at the wavelengths on both sides of the absorption edge structure of each detected element is defined as the left side and the detected each Establishing a multiple simultaneous equation with the surface density of the element as the variable on the right side and the difference in mass absorption coefficient at each wavelength as the coefficient of the right side variable, and by approximating the surface density of each element by solving the multiple simultaneous equation And calculating the contribution of the absorption spectrum of each element using the approximate surface density and subtracting from the synthesized absorption spectrum to form a residual spectrum, and further adding a new spectrum from the residual spectrum. If a new element can be detected by checking the presence or absence of the absorption edge structure of a simple element, the same simultaneous equations as described above are set up for all the elements detected so far, and this is solved to solve the surface of each element. An X-ray spectroscopic analysis method characterized in that an approximate value of density is calculated, and when a new element cannot be detected, the surface density approximate value is used as the surface density of each element at the predetermined position. 2. The X-ray spectroscopic microscopic analysis method according to claim 1, wherein the absorption spectrum comprises data at a wavelength position required for calculation. The X-ray spectroscopic microscopic analysis method according to claim 1 or 2, wherein X-ray absorption image data at wavelengths around the absorption edge of an element that may be contained in the sample is selected and stored. A photoelectric conversion X-ray microscope apparatus comprising an X-ray generator, a photoelectric conversion surface, an electronic image enlargement device, an image detection unit, and an arithmetic processing device, wherein a sample is placed in close contact with the photoelectric conversion surface, and the sample When the X-rays generated by the X-ray generator are swept from the back and irradiated, an electron image is formed on the photoelectric conversion surface by the transmitted X-rays, and the electron image enlarger emits electrons emitted from the electron image. The image processing unit is equipped with an image memory, acquires an image signal of an X-ray absorption image from the image detection unit, and stores it in the image memory for each sweep wavelength. When a predetermined position in the X-ray image is designated, an image signal corresponding to the position in the image is extracted from the image memory for a necessary wavelength in the sweep wavelength, and the measurement X of the designated portion is measured. Line absorption spectrum And detecting the presence or absence of the absorption edge structure of the element based on the measured X-ray absorption spectrum, and detecting the difference between the absorption coefficients at the wavelengths on both sides of the absorption edge structure of the detected element as the left side. Establish a multiple simultaneous equation with the surface density of each element as a variable on the right side and the difference in mass absorption coefficient at each wavelength as the coefficient of each right side variable, and approximate the surface density of each element by solving the multiple simultaneous equation A value is obtained, the contribution of the absorption spectrum of each element is calculated using the approximate surface density, and a residual spectrum is formed by subtracting from the measured absorption spectrum, and an absorption edge structure of a new element is further obtained from the residual spectrum. When new elements are detected, the same simultaneous equations as described above are established for all the elements detected so far, and this is solved to approximate the surface density of each element. Calculating a value, when a new element is not detected, the photoelectric conversion type X-ray microscope apparatus characterized by having the surface density approximation and surface density of each element in the predetermined position. 5. The photoelectric conversion X-ray microscope apparatus according to claim 4, wherein the image memory selects and stores X-ray absorption image data at wavelengths around the absorption edge of an element that may be contained in the sample.

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