JP4651473B2 - Method and apparatus for analyzing micro site in sample - Google Patents

Description

  The present invention relates to a fluorescent X-ray analysis method for performing elemental analysis, qualitative analysis, quantitative analysis, chemical bonding state measurement, and the like of a biological sample or a microstructure sample, and an apparatus used therefor.

  As an analysis method for elemental analysis, qualitative analysis, and quantification of a sample surface, FE-AES (electric field) is used to perform elemental analysis in a region several nm deep from the surface by irradiating an electron beam to the surface of a solid. Radiation type Auger electron spectroscopy), TOF-SIMS (time-of-flight secondary ion mass spectrometry) that irradiates a solid sample with a high-speed ion beam and performs elemental analysis of the outermost surface of the sample by sputtering, and irradiates the sample with an electron beam EPMA (X-ray microanalysis) that performs elemental analysis of the sample surface using the generated characteristic X-rays is performed, and X-rays emitted from the object are irradiated by irradiating the measurement object with X-rays. X-ray fluorescence analysis that detects and observes the structure of a sample is also one type of surface analysis technique.

In order to perform elemental analysis inside a sample by fluorescent X-ray analysis, a method is used in which a cross section of the sample is cut out and the surface thereof is measured.
As a method for elemental analysis of the minute space inside the sample in a non-destructive manner by fluorescent X-ray analysis, two X-ray condensing lenses are arranged in a confocal arrangement on the excitation side and the detector side. Has been proposed (see Non-patent Document 1). In that method, X-rays are irradiated from the outside of the sample, and only fluorescent X-rays excited by the X-rays and emitted from the sample to the atmosphere (or vacuum) side are detected. However, due to the significant attenuation of the X-ray intensity by the sample, depending on the X-ray energy and the type of the sample, even in the proposed method, the X-ray penetration depth and the fluorescent X-ray detection depth are about several μm. The shallowness remains as a problem.
Proceeding of SPIE 4144 (2000) 174-182

Even in the X-ray fluorescence analysis method, there is a demand for measuring the inside without destroying the sample, but an appropriate non-destructive measurement method other than the method using the X-ray condenser lens has been proposed so far. Not.
An object of the present invention is to provide a method and an apparatus that enable non-destructive elemental analysis in a minute space inside a sample.

  The method for analyzing a microscopic site in a sample according to the present invention includes an X-ray irradiation guide for irradiating a fine X-ray beam for excitation from the distal end through a fine internal passage with a base end coupled to an X-ray source, The ends of the X-ray detector are coupled to the X-ray detector, and guide the X-rays incident from the tip through the internal passage to the X-ray detector. The axes of the internal passages cross each other on their extension lines. Both guides are inserted into the sample from the respective distal end sides so that the fluorescent X-rays generated from the sample by the X-rays irradiated from the distal end of the X-ray irradiation guide reach the distal end of the X-ray detection guide. The X-ray detector is arranged close to the tip, irradiated with X-rays from the tip of the X-ray irradiation guide, and the X-ray detector generates X-ray fluorescence generated inside the sample by the irradiated X-ray. It is a method of detecting by guiding to.

The sample to be measured of the present invention needs to have a softness that allows the guide to be inserted, but the depth from the sample surface to the measurement target site is determined by the insertion depth of the guide, and therefore, several mm to several cm from the sample surface. It can also be in the position of a degree or deeper.
In addition, since the size of the site to be measured is determined by the distance between the tips of both guides inserted into the sample, it can be a micro site having a diameter of several mm or 1 mm or less.

  Since both guides are inserted into the sample, it is not a non-destructive measurement in the strict sense, but it is closer to non-destructive than the method of cutting the cross section of the sample, and the thinner the guide, the closer to non-destructive state. It becomes. “Non-destructive” in the present invention means that the guide is inserted, but the state is restored to the current state by removing the guide after the measurement. In particular, in the case where the sample is a living biological sample, it can be said that the living body recovers and becomes closer to nondestructive measurement.

  An injection needle can be used for at least the X-ray detection guide among the guides. In that case, when performing analysis by inserting both guides into the sample, it is preferable to set the direction of the tip inclined surface of the injection needle to a position and direction in which excitation X-rays are not directly irradiated.

  The analysis device for a minute part in a sample according to the present invention has an X-ray source and a base end part coupled to the X-ray source, irradiates a fine X-ray beam from the tip through a fine internal passage, An X-ray irradiation guide to be inserted, an X-ray detector for detecting X-rays, and a base end portion coupled to the X-ray detector to guide X-rays incident from the tip through the internal passage to the X-ray detector. And an X-ray detection guide inserted into the sample from the tip.

The guide is preferably a needle having a pointed tip.
Further, when the guide is tube-shaped, it is preferable that an X-ray transmissive cover for preventing the sample from entering the inside is provided at the tip.

  A preferred example of the guide is an injection needle used for medical solution injection or blood collection in the medical field. A commercially available injection needle is a straight, that is, a parallel tube, but a needle with a taper (inclination) can also be used in order to improve X-ray irradiation efficiency and capture efficiency.

  In the present invention, the tip of the X-ray irradiation guide and the tip of the X-ray detection guide are arranged inside the sample so as to form a three-dimensional intersection so that the axes of the internal passages cross each other on the extension line. As a result, elemental analysis of the measurement site inside the sample can be performed non-destructively, and as long as the guide is long, it can measure several centimeters or deeper, Direction restrictions are relaxed.

The size of the region to be analyzed inside the sample depends on the size of the guide tip, but by using an appropriate needle-like one, a minute space having a size of several hundred μm to several mm, or It is possible to make a measurement space smaller than that.
If a needle having a pointed tip is used as a guide, insertion into the sample is facilitated.

  When an injection needle is used as a guide, commercially available injection needles having an inner diameter of about 0.2 mm to 1.0 mm can be easily obtained. An injection needle having a tip diameter of 0.2 mm or less can be prepared. When such a fine injection needle is used as a guide, a micro space on the order of μm can be measured.

When an injection needle is used for the X-ray detection guide, if the direction of the inclined surface of the tip is set to a position and direction so that the excitation X-ray is not directly irradiated, the excitation X-ray is injected. Since it can be prevented from hitting the needle, generation of fluorescent X-rays from the metal of the injection needle can be suppressed, and the S / N (signal to noise) ratio of fluorescent X-ray detection from the target element can be improved. Can do.
When a tube body such as an injection needle is used as a guide, when an X-ray permeable cover is provided at the tip of the tube to prevent the sample from entering the inside, X-rays from the sample entering the guide Attenuation can be prevented.

In the following, an embodiment of the in-sample microscopic region analyzer of the present invention comprising a fluorescent X-ray analyzer will be described.
FIG. 1 is a schematic configuration diagram of the embodiment. The X-ray irradiation unit irradiates a fine X-ray beam from the tip through an X-ray source 1 that generates X-rays for excitation and a proximal end coupled to the X-ray source 1 through a fine internal passage. And an X-ray irradiation guide 3 inserted into the sample. A commercially available X-ray tube is used as the X-ray source 1, and an X-ray transmitting window of the X-ray source 1 is provided with an X-ray transmitting material such as beryllium, boron nitride, or graphite. Of the X-rays emitted from the X-ray emission window of the X-ray source 1, X-rays incident on the inside from the proximal end portion of the guide 3 are emitted from the tip through the internal passage.

  Between the X-ray exit window of the X-ray source 1 and the base end portion of the guide 3, in order to prevent the X-rays derived from the tube from affecting the fluorescent X-ray measurement, measurement of zirconium, aluminum, brass, etc. An appropriate filter may be provided depending on the target element.

  The fluorescent X-ray light receiving unit guides the X-ray detector 5 that detects X-rays and the X-ray detector 5 having a base end portion coupled to the X-ray detector 5 and incident from the tip to the X-ray detector 5 through an internal passage. And an X-ray detection guide 7 inserted into the sample from the tip, and an X-ray detector position adjustment stage 9 for adjusting the position of the guide 7. As the X-ray detector 5, an energy dispersive X-ray detector is used. X-rays incident from the tip of the guide 7 are guided to the X-ray detector 5 through the internal passage and detected.

  As the guides 3 and 7, a syringe needle is used here. As the injection needle, a commercially available product made of stainless steel having an inner diameter of 0.94 mm and an outer diameter of 1.2 mm is used. The inside of the injection needle becomes an X-ray passage, and X-ray transmissive covers 13 and 15 are provided on the inclined surface of the tip to prevent the sample from entering the inner passage from the tip opening. As the X-ray transparent covers 13 and 15, a polymer film such as a polyimide film or a Kapton film can be used, and can be attached to the tip inclined surfaces of the guides 3 and 7 with an adhesive or the like.

  The sample 4 into which the tips of the guides 3 and 7 are inserted is placed on the sample position adjusting stage 11. The stage 9 and the stage 11 are respectively moved in the XYZ directions (X and Y directions are in-plane directions, and the Z direction is a height direction perpendicular to them), and the positions of the sample 4 and the guide 7 are adjusted. The measurement target area inside the sample 4 can be adjusted.

  In this embodiment, the guides 3 and 7 are inserted into the sample 4 from the respective distal ends so that the axes of their internal passages cross each other on the extended lines, and X-rays irradiated from the distal end of the guide 3 Are arranged so that the tips of both guides 3 and 7 are brought close to a distance where the fluorescent X-rays generated inside the sample 4 reach the tip of the guide 7, and X-rays are irradiated from the tips of the guides 3 by the irradiated X-rays. The fluorescent X-ray generated inside the sample is guided to the X-ray detector 5 by the guide 7 and detected.

The operation of the in-sample microscopic region analyzer of this embodiment will be described below.
As the X-ray source 1, an X-ray tube having molybdenum as a target and having a beryllium X-ray emission window is used, and a zirconium filter is disposed between the X-ray emission window of the X-ray source 1 and the proximal end portion of the guide 3. Then, X-rays for excitation were irradiated.

A jelly-like sample was prepared as a standard sample for obtaining calibration curve data. The standard sample was prepared by (1) dissolving Zn (NO ₃ ) ₂ 6H ₂ O in distilled water to adjust the temperature, (2) mixing it with gelatin, and heating it once to completely dissolve the gelatin (3 ) Created by cooling and solidifying in the refrigerator. In this way, six types of standard samples having different zinc concentrations including those having a zinc concentration of 0 were prepared.
A canned oyster, which is a biological sample, was used as a measurement sample.

  In the measurement, a standard sample or a measurement sample is placed at the position of the sample 4 on the stage 11. The guide 3 is inserted into the sample so that the X-ray from the X-ray source 1 can be guided to the inside of the sample by the guide 3, and the guide 7 is also inserted into the sample and the fluorescent X-ray generated inside the sample is X-rayed by the guide 7. It can be led to the detector 5. At this time, since the tip openings of the guides 3 and 7 are covered with the polymer, the sample does not enter the internal passages of the guides 3 and 7.

  The tips of the guides 3 and 7 have an acute angle of about 12 degrees, and the direction of the inclined surface of the tip is set so that the surfaces with the openings are diagonally opposed as shown in FIG. The excitation X-rays were arranged so that a part of the excited X-rays approached the distance irradiated to the tip of the guide 7. Such an arrangement of the tip portions of the guides 3 and 7 has an advantage that the sensitivity of receiving fluorescent X-rays is increased, and also has a drawback that fluorescent X-rays from the metal of the guide 7 are generated at the same time. Although the angle between the guide 3 and the guide 7 is arranged to be about 90 degrees, it can be changed according to the sample and the measurement situation.

  The guide 7 and the X-ray detector 5 are integrated, and the position of the tip of the guide 7 can be adjusted by the stage 9. The size of the minute space to be measured inside the sample is controlled by adjusting the stage 9.

As a specific procedure, the X-ray detection guide 7 is first moved away from the sample 4, and the sample 4 is moved in the direction of the guide 3 so that the guide 3 is inserted into the sample 4 by the stage 11. Next, the guide 7 is inserted into the sample 4 by the stage 9 and the position of the tip of the guide 3 and the tip of the guide 7 in the sample 4 is adjusted. Thereafter, excitation X-rays are generated from the X-ray source 1 and irradiated into the sample 4 from the tip of the guide 3, and the generated fluorescent X-rays are received by the guide 7 and detected by the detector 5. The X-ray source 1 generates X-rays at a voltage of 40 KV and a current of 30 mA, analyzes the energy of the signal detected by the detector 5 through an amplifier and a multi-wave height analyzer, and integrates it for 300 seconds to obtain a fluorescent X-ray spectrum. .
The stage 9 was adjusted so that the relative positional relationship between the tip of the guide 3 and the tip of the guide 7 in the sample 4 was the same for all the standard samples and the measurement samples.

FIG. 2 is an X-ray spectrum when a standard sample prepared with a Zn concentration of 500 ppm is measured, the horizontal axis is energy, and the vertical axis is intensity. The lower spectrum shows the entire spectrum in the X-ray energy range of 1 to 19 KeV, the large spectrum of 16 to 18 KeV on the right end is the excitation X-ray spectrum, and the spectrum surrounded by the 4 to 10 KeV frame on the left is fluorescence. It is a spectrum. The fluorescence spectrum is enlarged and shown on the upper side. A fluorescent X-ray peak due to Zn in the standard sample is seen around 8.5 to 9 keV. The fluorescent X-ray peak due to Fe in the vicinity of 6.5 keV is the fluorescent X-ray generated from the injection needle of the guide 7.
Fluorescent X-ray spectra were measured under the same conditions for other standard samples with different Zn concentrations.

FIG. 3 shows the relationship between the Zn concentration (ppm) and the intensity of the fluorescent X-ray spectrum peak of Zn for a standard sample prepared by variously changing the zinc concentration in the range of 0 to 1000 ppm. The correlation coefficient R is 0.99684, indicating good linearity, indicating that this data can be a calibration curve for measuring the zinc concentration of an unknown sample.
Note that “NET” in FIG. 3 means “raw strength” and means the net strength obtained by subtracting the back brand strength from the total strength.

  FIG. 4 shows an X-ray spectrum in the same manner as FIG. 2 when an oyster as a measurement sample is measured under the same conditions as the standard sample. A fluorescent X-ray peak due to Zn is observed in the vicinity of 8.5 to 9 keV. When the peak intensity is applied to the calibration curve data of FIG. 3, the zinc concentration in oysters can be quantified.

  FIG. 5 shows the arrangement of the tips of the guides 3 and 7 in the sample in another embodiment. The inclined surfaces having openings at the tip portions of the guides 3 and 7 are arranged so as to face opposite sides. Although the angle between the guide 3 and the guide 7 is arranged to be about 90 degrees, it can be changed according to the sample and the measurement situation.

  This arrangement of the guides 3 and 7 prevents the excitation X-ray radiated from the tip of the guide 3 from being irradiated to the tip of the guide 7 and suppresses the generation of fluorescent X-rays generated from the metal of the guide 7. it can. On the other hand, the distance between the site to be measured and the tip openings of the guides 3 and 7 is increased, and both the excitation X-rays and the fluorescent X-rays are greatly attenuated by the sample and the sensitivity is lowered.

FIG. 6 shows the measurement result of the standard sample when the guides 3 and 7 are arranged as shown in FIG. Except for the difference in the arrangement of the guides 3 and 7, the Zn concentration of the standard sample and the X-ray generation conditions of the X-ray source 1 were the same as the conditions when the X-ray spectrum of FIG. 2 was obtained.
Compared with the result of FIG. 2, although the overall detection sensitivity is lowered, the peak of fluorescent X-rays due to Fe generated from the injection needle itself is not seen.

The present invention is not limited to the above embodiments such as the shape of the guide tip and the direction of insertion into the sample, and other shapes and arrangements are possible. For example, an injection needle is used as the guide, but other shapes can also be used. The material is not particularly limited as long as it can conduct X-rays, and may be a quartz glass capillary.
Also, the guide does not have to be hollow, and may be solid as long as it is a material that transmits X-rays. If it is a solid guide, the sample does not enter the inside. Therefore, it is not necessary to provide a cover for preventing the sample from entering the guide tip.

If the measurement sample is a living organism, a method of releasing after measurement and measuring again after a predetermined time to measure a specific element with time is possible.
Since it is possible to measure any number of deep portions by simply increasing the length of the guide, it is possible to perform measurement at a plurality of locations for one solid biological sample (excluding the human body) .

  INDUSTRIAL APPLICABILITY The present invention can be used for measuring a minute part in a sample for measuring an internal state of a biological sample or a fine structure sample or performing elemental analysis.

It is a schematic block diagram which shows one Example. It is a wave form diagram which shows an X-ray spectrum when a standard sample is measured in the Example. It is a graph which shows the calibration curve of Zn obtained by measuring the standard sample which varied the density | concentration of zinc in the Example. It is a wave form diagram which shows an X-ray spectrum when a measurement sample is measured in the Example. It is a schematic block diagram which shows the guide front-end | tip part in an Example. It is a wave form diagram which shows an X-ray spectrum when a standard sample is measured in the Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 X-ray source 3 X-ray irradiation guide 5 X-ray detector 7 X-ray detection guide 9 X-ray detector position adjustment stage 11 Sample position adjustment stage 13, 15 X-ray transparent cover

Claims (5)

An X-ray irradiation guide for irradiating a fine X-ray beam for excitation from the tip through a fine internal passage with a base end coupled to an X-ray source, and a base end from the tip coupled to an X-ray detector An X-ray detection guide for guiding incident X-rays through the internal passage to the X-ray detector is inserted into the sample from the tip side so that the axes of the internal passages cross each other on the extension lines. ,
Place the tips of both guides close to the distance that the fluorescent X-rays generated from the sample by the X-rays emitted from the tips of the guides for X-ray irradiation reach the tips of the guides for X-ray detection,
X-ray is irradiated from the tip of the X-ray irradiation guide, and fluorescent X-rays generated inside the sample by the irradiated X-ray are guided to the X-ray detector by the X-ray detection guide and detected. Analyzing method of micro site in sample.   Of the guides, at least the X-ray detection guide is an injection needle, and when the two guides are inserted into a sample for analysis, the direction of the tip inclined surface of the injection needle is not directly irradiated with excitation X-rays. The analysis method according to claim 1, wherein the analysis position is set in such a position and direction. An X-ray source for generating excitation X-rays;
A base end portion is coupled to the X-ray source, irradiates a fine X-ray beam from the tip through a fine internal passage, and is inserted into the sample from the tip portion , and comprises a tube, An X-ray irradiation guide provided at the tip with an X-ray transparent cover for preventing the sample from entering the inside ;
An X-ray detector for detecting X-rays;
An X-ray detection guide having a proximal end portion coupled to the X-ray detector and introducing X-rays incident from the distal end through the internal passage to the X-ray detector and inserted into the sample from the distal end portion , An X-ray detection guide provided with an X-ray transmissive cover for preventing the sample from entering the inside at the tip ;
An apparatus for analyzing a minute part in a sample by fluorescent X-rays.   The analyzer according to claim 3, wherein the guide is a needle having a pointed tip. The analyzer according to claim 3 , wherein the guide is an injection needle.

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