JP3317541B2 - X-ray microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an X-ray microscope for taking a transmission X-ray microscope image of a sample such as a biological sample using X-rays in a soft X-ray region.

[0002]

2. Description of the Related Art In recent years, research and development of X-ray light sources and X-ray optical elements have been progressing, and an X-ray microscope has been proposed as one of systems that apply them. There are many types of such X-ray microscopes, such as a Walta type grazing incidence optical system, a Fresnel zone plate using diffraction, and a Schwarzschild type direct incidence optical system in which two spherical mirrors are coated with a multilayer film. There has been proposed a microscope system using an imaging element. In such an X-ray microscope, especially soft X
In the case of using an electron beam, damage to a biological sample is less than that in the case of using an electron beam, so that it is possible to observe the living body without staining and with high resolution without a living organism. And the needs in the biological field are growing.

In the soft X-ray region, in particular, λ = 43.7 °
The wavelength region of ２３23.6 ° (the wavelength region of the so-called “water window”) is well absorbed by carbon and nitrogen and has a high transmittance to water molecules composed of oxygen and hydrogen. A transmission microscope image of a protein whose main constituent is carbon (such as a living tissue) can be observed with good contrast even in water. At present, research institutes are developing optical elements, light sources, detectors, and the like having high specifications in a wavelength range of λ = 43.7 ° to 23.6 °.

The above wavelength region is suitable for living body observation as described above. However, in order to manufacture an optical element having high specifications in this wavelength region, there are the following problems. The point is that it is difficult to manufacture an X-ray multilayer mirror or filter having excellent characteristics particularly in the above wavelength range, as described later. In other words, when designing a multilayer mirror having a high reflectance, it is necessary to alternately deposit two types of substances having a large difference in refractive index as much as possible. Is close to 1, so that the difference in refractive index becomes large.
It is difficult to select the type of substance. Where Ni
It is also considered to use a material that can be expected to have some reflectance, such as / Sc and Ni / Ti, but in that case, it is difficult to form a uniform film because it is easy to crystallize during vapor deposition. And λ = 43.7 ° to 2 in the current film forming technology.
If it is attempted to manufacture a direct-incidence mirror in the wavelength range of 3.6 °, one cycle of the multilayer film becomes 20 ° or less, and it becomes difficult to manufacture the multilayer film itself. Further, in this wavelength region, an organic material cannot be used as a filter because carbon has a high absorptivity for X-rays, and the selection range of the filter material is narrow.
Therefore, in the X-ray microscope system, the use of the X-ray multilayer mirror or the filter in some form is unavoidable from the viewpoint of the configuration, and the problem of the multilayer mirror or the filter is that the soft X-ray in the above wavelength range is used. An obstacle to doing so.

[0005] Even if the first problem is solved to realize an optical system capable of forming X-rays in the above-mentioned wavelength range which is optimal for capturing a transmission microscope image of a living body, as described below, When observing a biological sample, a second problem of a decrease in contrast due to absorption occurs. That is, the soft X-ray absorption of a biological sample is determined by the thickness of the sample, the density of carbon present in the sample, and the wavelength of the X-ray used. Means that most of the X-rays incident on the biological sample are absorbed, and the obtained biological transmission microscope image
The whole image is dark and has no contrast. Conversely, when observing a biological sample having a low and low carbon density, most of the X-rays incident on the biological sample are transmitted, and the obtained biological transmission microscope image is bright and has no contrast as a whole. It becomes something.

As a measure against the above-mentioned contrast problem,
Since it is impossible to artificially change the carbon density in the biological sample, the thickness of the biological sample is adjusted, or the wavelength of the X-ray used within the wavelength range of λ = 43.7Å to 23.6Å is adjusted. There is a way to do it. Here, the method of adjusting the thickness of the biological sample to be observed requires cutting the sample using a precision processing machine requiring skill such as a microtome, and determining the optimal sample thickness by trial and error. Absent. Also, the method of adjusting the wavelength of the X-ray to be used can avoid a significant design change or layout change of the optical element when using an optical element having wavelength dispersion such as a zone plate or a Schwarzschild type optical system. Not realistic.

At present, as a method different from the above method, λ
= X-rays in a region other than the wavelength region of 43.7 ° to 23.6 ° (for example, a wavelength region of λ = 65 to 43.7 °) have a very high contrast, and a microscopic image of a specific protein molecule is obtained. A method that can be selectively obtained has been proposed, and its principle will be described with reference to FIGS. 6 and 7. FIG.
(A) to (f) respectively show the transition of the state of the electron when the carbon atom absorbs the X-ray with time, and FIG.
Indicates the electronic arrangement of carbon atoms in the ground state. When 1S electrons are ionized by X-ray irradiation from this state, the carbon atoms become vacant in the 1S orbital as shown in FIG. Since this state is extremely unstable in terms of energy as shown in FIG. 3C, the stable state shown in FIG. 4D is obtained by the second transition in which the 2P electrons transition to the 1S orbit. Further, when the carbon atom is a constituent element of a molecule such as a protein, for example, as shown in FIG. 3E, a vacancy is generated in the 2P orbital, so that the carbon atom is changed by the third transition. Electrons are captured from surrounding elements and the like, and return to the same ground state as the initial state as shown in FIG. In the course of such a transition, a transmission microscope image of a protein is usually imaged and observed using absorption by the first transition. At this time, if the X-ray wavelength is longer than the absorption edge wavelength of carbon, X Since the lines are not absorbed by the protein, the contrast of the obtained transmission microscope image is extremely deteriorated.

Considering the case where the order of the above-mentioned transition is reversed, as described below, even when the X-ray wavelength is longer than the absorption edge wavelength of carbon, transmission with extremely good contrast is achieved. A microscope image can be taken. That is, first, 2P electrons are ionized or excited from the ground state shown in FIG. 7A by the reverse process of the third transition to realize the electronic state shown in FIG. 7B. Next, from this state, 1S electrons are excited by X-rays to the still free 2P orbit by the reverse process of the second transition as shown in FIG. This transition process can be realized by X-rays having a photon energy lower than the absorption edge wavelength of carbon (that is, X-rays having a longer wavelength than the absorption edge wavelength of carbon). ).

This state is exactly the same as that shown in FIG. 6B in which 1S electrons are directly ionized from the ground state by the first transition, but the energy required to ionize or excite electrons in this method is as follows. Since it is about several eV to 20 eV (100 nm to 300 nm in terms of wavelength), an ultraviolet laser or the like can be used. Meanwhile, 1
The energy required to excite the S electrons into the 2P orbital is about several eV to 20 eV lower than in the case of ionization. Therefore, there are two processes of ionizing the 2P electrons of the carbon atom in the ground state and exciting the 1S electrons to the 2P orbit.
Through the process of the step, even when X-rays having a wavelength longer than the absorption edge wavelength of carbon are used, a transmission microscope image of the protein can be obtained.

The method of reversing the order of the above transition is described in JHKl
The following advantages are quantitatively confirmed by ems (see JKKlems Phys. Rew Vol 43 (1991) p2041-2045). First, since X-rays having a wavelength longer than the wavelength of the carbon absorption edge can be used, W / C and other materials having excellent optical constants and easy film formation can be selected when a multilayer film is manufactured.
In addition, research on the properties and the like of these materials has been advanced, and they have been used. Second, depending on the type of protein, 2P
Since the ionization or excitation energies of the electrons are different, only the carbon atoms of a specific protein can be selectively ionized or excited. Furthermore, 1S to 2P following the ionization
Since the energy of the electron transition to is uniquely determined in a quantum way, a transmission microscope image of a specific protein can be observed by using an X-ray having photon energy of this magnitude as a probe. At that time, the excitation cross-section of carbon, which determines the contrast, with respect to X-rays has a characteristic as shown in FIG. 8, and is higher by one digit or more than the conventional method using the wavelength region of λ = 43.7 ° to 23.6 °. .

A method using the above-described principle can be easily realized by making some modifications to the conventional X-ray microscope system proposed so far. That is, the conventional X-ray microscope has a light source 1, a condenser lens 2, a sample 3, an objective lens 4, a filter 5, and a detector 6 arranged on the same optical path as shown in the basic conceptual diagram of FIG. . As the objective lens used in this, generally,
Wavelength dispersion type such as zone plate and Schwarzschild optics, and oblique incidence mirror type that condenses white light such as Walta type are divided into two types.In the case of a system using a white light source and a Walta type objective lens, It is necessary to provide a spectroscope in the optical path leading to the detector. As a detector,
An imaging device such as a micro channel plate (MCP) or a CCD is used. When a white light source is used,
Generally, a thin film filter such as Be is provided to cut stray light having a wavelength longer than the ultraviolet region. The optical system is housed in a vacuum container.

In such a conventional X-ray microscope system,
It is extremely easy to apply the method proposed by JHKlems, and it is only necessary to add an ultraviolet light source 8, a condenser lens 7, and an ultraviolet reflecting mirror 9, as shown in the basic conceptual diagram of FIG. This system is basically the same as the above-mentioned conventional system except that an ultraviolet reflecting mirror 9 is inserted between the sample 3 and the objective lens 4. The ultraviolet reflecting mirror 9 is made of a thin film of Be or the like. A filter having a sufficient transmittance in the wavelength range of 43.7 ° and a sufficient reflectance for ultraviolet rays is selected, whereby the ultraviolet reflecting mirror 9 has a function as an X-ray filter. Will be. In this system, when capturing a transmission microscope image using X-rays of photon energy corresponding to the reverse process of the second transition in FIG. 6, ultraviolet rays from an external ultraviolet light source 8 are simultaneously reflected by an ultraviolet reflecting mirror 9. By irradiating the sample with carbon atoms, carbon 2P electrons of a specific protein can be ionized or excited, and the X-ray microscopy proposed by JHKlems can be realized. In this case, the ultraviolet reflecting mirror 9
Functions to cut off stray light having a wavelength longer than the ultraviolet region, which becomes noise for the detector 6.

[0013] Further, the above system can artificially and easily adjust the absorption rate of X-rays to a biological sample.
This has the advantage that the contrast of a transmission X-ray microscope image can be arbitrarily selected without adjusting the thickness of the biological sample or the wavelength of the X-ray used. That is, according to the above method, the X-ray absorptivity of the biological sample is proportional to the number of carbons having vacancies in the 2P orbit after passing through ultraviolet irradiation as shown in FIG. Since the number of carbons having is proportional to the intensity of the irradiated ultraviolet light, the X-ray absorption of the biological sample can be easily changed by adjusting the intensity (light amount) of the ultraviolet light applied to the biological sample, By the adjustment, a transmission X-ray microscope image having an optimum contrast is obtained. In addition, a transmission X-ray microscope image when the sample is irradiated with ultraviolet light by the ultraviolet light source and a transmission X-ray microscope image when the sample is not irradiated with ultraviolet light are respectively imaged, and a difference image is created therefrom. The background (noise) caused by absorption by elements other than carbon can be removed, and a transmission X-ray microscope image of pure carbon alone can be obtained with an excellent contrast ratio.

FIGS. 11 and 12 show a conventional example to which the principle of X-ray microscopy proposed by JHKlems is applied. In the conventional example of FIG. 11, a synchrotron radiation light source (SOR light source) 11 is used as an X-ray light source, and a Fresnel zone plate is used as an X-ray optical system. The condenser lens 14 and the objective lens 15 in the X-ray optical path composed of the sample 14, the sample 13, the objective lens 15 and the detector 16 use a Fresnel zone plate. This conventional example further has a thickness of 3 between the sample 13 and the objective lens 15.
A Be thin film 19 of about 000 ° is arranged at 45 ° to the X-ray optical axis, and an ultraviolet light path comprising an ultraviolet laser light source 18, a condensing lens 17 and a wedge 10 is provided outside the microscope, and the ultraviolet laser light is The sample 13 is reflected from the thin film 19 so as to irradiate the sample 13 from the back. At this time, the Be thin film 19 also has a function of blocking stray light such as ultraviolet rays from entering the detector (MCP) 16.

In the above system, a wedge 10 which can move in a direction orthogonal to the ultraviolet optical axis is provided in the ultraviolet light path, and the wedge 10 is made of a material having a sufficiently high absorption rate for ultraviolet light (for example, BK7; glass). By moving the light, the optical path length of the ultraviolet light passing through the wedge 21 can be changed to adjust the amount of the ultraviolet light applied to the sample. Therefore, by irradiating the sample with ultraviolet light whose light amount has been adjusted simultaneously with X-ray irradiation, a transmission X-ray microscope image of the protein can be obtained with a good contrast ratio based on the above principle.

In the prior art shown in FIG. 12, the light source is Nd: YA.
A laser plasma light source using a G laser or the like, an X-ray condenser lens 22 as a Walter-type mirror, an X-ray objective lens 24 as a Schwarzschild optical system, and a photodetector 26 as a micro-channel plate (MCP) to form an X-ray optical path. The operation of the other parts is basically the same as that of the conventional example shown in FIG. The difference between this conventional example and the above-mentioned conventional example is that a part of the laser light from the Nd: YAG laser 20 that shoots the target 21 via the condenser lens 30 for X-ray generation is branched by the half mirror 27. The ultraviolet light path is configured to use the half mirror 27.
The amount of the laser light branched by is adjusted by the polarizer 32,
After being converted into the fourth harmonic of the ultraviolet wavelength region by the DP crystal 25, the light is irradiated on the ultraviolet reflecting mirror 29 through the mirror 28 and the condenser lens 31. This conventional example has an advantage that a light source such as an ultraviolet laser is not required as compared with the above-described conventional example. In addition, by using a W / C multilayer film having excellent characteristics for the above-described Schwarzschild optical system, a wavelength of 45 ° can be obtained. When 200 layers are stacked at the incidence, a reflectance of approximately 30% is obtained, and a bright X-ray optical system can be configured.

[0017]

Such soft X-ray microscopy (hereinafter referred to as ultraviolet-excited soft X-ray microscopy) for irradiating ultraviolet rays (excitation radiation) and soft X-rays is as described above. However, in order to exert the superiority, it is premised that the following problems are solved. In other words, when observing various different elements with an X-ray microscope or when observing the same element but containing different substances, the wavelengths of the ultraviolet rays to be irradiated are different from each other. It is necessary to change the wavelength according to the target). However, in the related art, no study has been made on a technique for changing the wavelength of ultraviolet light to be irradiated on a sample according to an observation target. Therefore, the related art cannot deal with the change of the observation target as described above.

In view of the above problems, an object of the present invention is to provide a method for changing the wavelength of excitation radiation to be applied to a sample according to an object to be observed, and an X-ray microscope to which the method is applied.

[0019]

For this purpose, the present invention relates to an X-ray microscope for irradiating a sample with excitation radiation and soft X-rays to capture a transmission X-ray microscope image of the sample. The wavelength of the excitation radiation is changed according to the imaging target. Accordingly, even if the wavelength of the excitation radiation applied to the sample is changed, the wavelength becomes suitable for the imaged object, and a transmission X-ray microscope image with good image quality can be obtained.

[0020]

Embodiments of the present invention will be described below in detail with reference to the drawings. FIG. 1 is a system diagram showing the configuration of the first embodiment of the X-ray microscope of the present invention. In the X-ray microscope of this embodiment, a laser light source for generating X-rays is shared without independently providing a laser light source for generating ultraviolet rays as excitation radiation. That is, the laser beam generated by the Nd: YAG laser 51 is condensed by the condensing lens 52 and collided with the target 53 to generate a laser plasma X which generates X-rays.
A line light source, an X-ray optical path having a condenser lens 54, a pinhole plate 106, an objective lens 55, an ultraviolet cut filter 61 and a detector 57, and irradiating the sample 58 obliquely with X-rays to obtain a transmission X-ray microscope image. And a part of the laser light from the Nd: YAG laser 51 is branched by the half mirror 64 and converted into the third harmonic in the ultraviolet wavelength region by the KDP crystal 66.
The ultraviolet wavelength is adjusted (changed) by an optical parametric oscillator (OPO) 90, and further converted to a higher harmonic by a harmonic generator (SHG) 91, passing through a mirror 67, a condenser lens 60, and a wedge-shaped glass (wedge) 92. An ultraviolet light path for obliquely irradiating the sample 58 with ultraviolet light is provided. In addition, among the above components, the Nd: YAG laser 51 and the condenser lens 52
And each component on the ultraviolet light path is installed outside the vacuum vessel, other components are housed in the vacuum vessel 62,
Transmission portions 62a and 62b are provided in portions of the vacuum vessel 62 through which laser light and ultraviolet light are transmitted, respectively. The optical window 62b is made of a material having a high transmittance of ultraviolet light having a long wavelength of about 200 nm or more ( Diamond, fluorite, etc.).

In the above configuration, the X-ray optical axis and the UV optical axis are orthogonal at the sample irradiation section, and the sample 58
A predetermined angle (for example, 45 °) with respect to the line optical axis and the ultraviolet optical axis.
°) so that the normal of the sample surface is not parallel to the X-ray optical axis. This configuration has a very small distance between the condenser lens, the sample, and the objective lens on the X-ray optical path. Suitable for narrow spaces. Further, in this configuration, the laser light source for generating X-rays is also used for generating ultraviolet rays, so that cost can be reduced. Further, in an image signal processing system described later, it is easy to synchronize the timing of X-ray irradiation with the timing of ultraviolet irradiation. Note that, for example, 4
The transmission X-ray microscopic image of the sample tilted by 5 ° forms a 1: (1 / √2) oblique image of the sample on the image forming plane of the detector 57. After digitizing the captured image, It is necessary to restore the original image to 1: 1 using coordinate transformation or the like by using a hardware or a separately provided arithmetic circuit, and even if the X-ray optical system and the sample are close to each other by performing the restoration process, it is sufficient. It is possible to irradiate the sample with an ultraviolet ray.

Next, each component will be described. As the X-ray light source, a laser plasma light source of a white light source having a compact configuration is used, and as the condenser lens 54, a Fresnel zone plate is used. In that case,
Since the Fresnel zone plate has wavelength dispersibility and focuses X-rays at different positions for each wavelength, if a pinhole plate is placed near the sample, only the X-rays of the desired wavelength will be focused on the sample. X-rays of wavelengths other than the above are blurred on the sample and can be kicked by a pinhole, and the wavelength of the X-ray can be selected and applied to the sample.

As the objective lens 55, a zone plate is used for the following reason. That is, in this embodiment, the wavelength of the ultraviolet light is changed (variable control) to optimize the excitation. Therefore, when the Schwarzschild optical system is used as the objective lens 55, the Schwarzschild optical system is used for wavelengths other than the design reference wavelength. Since the reflectivity becomes extremely small, the X-ray light amount reaching the detector 57 becomes insufficient when the wavelength fluctuation of the X-rays increases to a certain extent or more. On the other hand, since the zone plate has a property (chromatic aberration) having a different focal length depending on the wavelength, the X-ray absorbed by changing the wavelength of X-rays contributing to image formation according to the substance containing carbon is changed. If the wavelength variation of the line becomes larger than a certain extent when the wavelength variation of the line occurs, the position of the zone plate is finely adjusted in the optical axis direction as shown by an arrow in FIG. The image formed on 57 can be prevented from being out of focus, and the above problem does not occur. The position of the zone plate used as the condenser lens 54 is also finely adjusted as needed.

As shown in FIG. 2, the OPO 90 includes a half-wave plate 93, a polarizer 94, beam expanders 95 and 96, a BBO resonance mirror 97, and a BBO crystal from the incident side.
98, comprising a BBO resonant mirror 99 (for example, an OPO is commercially available from BMI, France). Optical parametric oscillation (OP
O) is a nonlinear optical process that oscillates light that can be tuned over a wide range, and is a nonlinear optical crystal BBO
The wavelength can be continuously tuned by changing the temperature or adjusting the angle of the crystal, and the tuning range on the short wavelength side extends to the ultraviolet region (400 nm). Further, by using SHG91 together as shown in FIG. 1, the tuning range can be further expanded to the shorter wavelength side, and in this embodiment, ultraviolet rays in a wide wavelength range (200 nm or more) can be obtained.

The wedge 92 is a mirror from the SHG 91.
67, an element that adjusts the intensity (ultraviolet light amount) of ultraviolet light when irradiating the sample 58 with ultraviolet light through the condenser lens 60;
By moving in the direction orthogonal to the optical axis of the ultraviolet light (in the direction of the arrow in the drawing), the optical path length (glass thickness) of the ultraviolet light passing through the wedge is changed to adjust the amount of ultraviolet light to be applied to the sample. As the wedge 92, a substance (for example, BK7; glass) having a sufficiently high absorptivity to ultraviolet rays is used. In addition,
If the size of the region irradiated with ultraviolet light does not matter, if the condenser lens 60 can be moved in the ultraviolet optical axis direction, the focus state can be changed to adjust the intensity of ultraviolet light, In that case, the wedge 92 is omitted.
Reference numeral 107 denotes a shutter.

Next, an image signal processing system for processing an image signal from the detector (MCP) 57 will be described with reference to FIG. This image signal processing system includes a host computer 71 that controls the entire X-ray microscope system, and a TV that visualizes incident X-rays with an MCP 57 and a phosphor 72 and captures a transmission X-ray microscope image through a lens 73.
Frames for storing image data obtained by digitizing two types of video signals (one with ultraviolet irradiation and one without ultraviolet irradiation) from the camera system 74 and the TV camera system 74 with A / D converters 75-1 and 75-2, respectively. A memory 76-1 and 76-2, a difference circuit 77 for inputting a digital difference signal obtained by taking a difference between each pixel of the image data of the frame memories 76-1 and 76-2 to the host computer 71, and a host computer Frame memory 76 based on command signal from 71
A data selector circuit 78 for outputting a trigger signal for data output timing to the host computer 71 and the host computer 71.
And a CRT 79 for displaying a transmission X-ray microscope image of proteins and the like based on an input signal from the CPU, and further drives a wedge 92 so that a laser light source for generating X-rays can be shared for generating ultraviolet light. A wedge drive circuit 80 for generating a wedge drive signal.

The wedge drive circuit 80 is controlled by a command signal from the host computer 71, and drives the wedge 92 so that the intensity (light amount) of the ultraviolet light applied to the sample becomes appropriate. The host computer 71
OP to OPO90 to adjust (change) the wavelength of ultraviolet light
In addition to outputting the O control signal to change the temperature or angle of the BBO crystal inside the OPO 90, it also outputs the trigger signal of the TV camera system 74 and the Q switch signal of the Nd: YAG laser 51, and also controls the opening and closing of the shutter 107. Do.

Next, a case in which a transmission X-ray microscope image of, for example, carbon is captured by the X-ray microscope of the first embodiment will be described. First, before starting imaging, a substance name (or code number or the like) of a carbon-containing substance to be imaged (observed) is selected from Table 1 and input.

[Table 1] With this input, the host computer 71 automatically adjusts the ultraviolet wavelength so as to have a suitable ultraviolet wavelength according to the target substance as shown in Table 1. That is, the ultraviolet wavelength is
For example, if you want to observe tryptophan,
0 nm, 250-300 nm if you want to observe tyrosine
The angle or temperature of the BBO crystal is adjusted so that In addition to the above-mentioned automatic adjustment function, it is possible to observe a substance whose suitable ultraviolet wavelength is unknown. Therefore, a manual adjustment function is added.

After completing the above adjustment, the host computer 71
Outputs a Q switch signal to the Nd: YAG laser 51,
The Nd: YAG laser 51 receiving the signal generates a laser beam. This laser light is collected by a condenser lens 52 and a transmission window 62a.
Collides with the target 53 through the above, and generates X-rays. On the other hand, the host computer 71 synchronizes with the generation of the X-rays, and the TV camera system 74 and the frame memory 76-1.
Output a trigger signal. In addition, the shutter 107 is driven to open the ultraviolet light path, and a command signal for driving the wedge 92 is issued to the wedge drive circuit 80 so that the intensity of the ultraviolet light applied to the sample 58 becomes appropriate. As a result, the frame memory 76-1 stores image data a, which is a digitized transmission X-ray microscope image of the carbon-containing substance, which is a video signal obtained when ultraviolet light is irradiated.

Next, the host computer 71
107 is closed so that the sample 58 is not irradiated with ultraviolet rays, and the TV camera system 74 is
And a trigger signal is output to the frame memory 76-2. Thus, the frame memory 76-2 stores image data b which is a digitized transmission X-ray microscope image of an element other than carbon of the carbon-containing substance, which is a video signal obtained when no ultraviolet light is irradiated. .

The image data of the frame memory 76-1 is
This is a signal obtained by adding noise to a transmission X-ray microscope image of carbon to be finally extracted, and the image data b of the frame memory 76-2 is a background (noise) signal of the image data a of the frame memory 76-1. . Therefore, the difference circuit 77
By taking the difference between the two for each pixel, the above noise is removed, and pure carbon transmission X
A difference signal (ab) containing only a line microscope image is obtained.
This difference signal (image data) is transmitted to the host computer 71.
Is output to a CRT 79, and a transmission X-ray microscope image of carbon of the carbon-containing substance is displayed. In the above description, an image is first obtained by irradiating X-rays and ultraviolet rays, and then an image is obtained by irradiating only X-rays.
This order may be reversed if 58 is sensitive to ultraviolet light.

In the above embodiment, the YAG laser light source for generating X-rays is commonly used for generating ultraviolet light without independently providing an ultraviolet light source. When the output is controlled by using a Ti-sapphire laser or a dye laser as the ultraviolet light source, the scale of the SOR is very large.
The light emitted from the light source may be input to the spectroscope to extract only the necessary ultraviolet component.

In the above embodiment, the image processing is directly performed.
Although the hardware difference circuit 77 is used, the difference circuit is omitted, and the host computer 71 reads out image data from the frame memories 76-1 and 76-2 and performs image processing on software. You may. Further, in the above embodiment, the oblique incidence optical system and the zone plate are used in the X-ray optical path, but the present invention is not limited to this.
The X-ray microscope system may be configured by combining other imaging optical systems such as the Schwarzschild optical system.

FIG. 4 is a system diagram showing the configuration of the second embodiment of the X-ray microscope according to the present invention. The same parts as those in FIG. In this embodiment, the present invention is applied to a scanning X-ray microscope. This scanning microscope is different from the imaging microscope as in the first embodiment in that X
A line is condensed into a minute spot and irradiated onto the sample, and the sample and the beam spot are relatively moved to obtain a signal from a predetermined area on the sample to reproduce an object image.

In this embodiment, X generated at the target 53
The line is condensed as a minute beam spot on the sample 58 via a pinhole plate 106 via a zone plate 101 as an objective lens via an ultraviolet cut filter 100, and the zone plate 101 is irradiated with an X-ray beam by a zone plate driving mechanism 102. It can be slightly moved in the axial direction. The ultraviolet light cut filter 100 prevents unnecessary ultraviolet light or the like radiated from the target 53 from irradiating the sample 58.

The sample 58 is fixed to a stage 103, and the stage 103 is two-dimensionally driven in an XY direction perpendicular to the optical axis by a driving device 104 provided in a vacuum vessel. The X-ray transmitted through the sample 58 enters the detector 57. In the scanning X-ray microscope, only the signal from a minute point on the sample determined by the size of the X-ray beam spot is detected by the detector 57, and the sample is moved to obtain information from the adjacent minute point in time series. To obtain an object image. Therefore, an image sensor type having a large number of pixels is not required as the detector 57, and any detector that can detect X-rays can be used.

The ultraviolet light path of this embodiment is a half mirror.
The portion from 64 to the mirror 67 is the same as that of the first embodiment, but the portions beyond it are different. That is, in this embodiment, since the sample 58 arranged perpendicular to the X-ray optical axis is irradiated with ultraviolet light obliquely through the transmission window 62b, the movable mirror 67, the wedge 92L and the condenser lens 60 used as mirrors are used.
When observing the sample surface, the movable mirror 67 is placed in a solid line as shown in the figure, and the sample surface is irradiated with ultraviolet light from the oblique ultraviolet light path via the pinhole plate 106. In this embodiment, the mirror 105 and the wedge are symmetrical with respect to the sample 58 so that both sides can be observed without turning the sample upside down.
A 92R and a condenser lens 60R are arranged diagonally, and this ultraviolet light path is used to observe the back of the sample.
Is horizontal as shown by the dotted line in the drawing, ultraviolet rays are radiated on the back surface of the sample obliquely through the transmission window 62c.
Note that a wedge driving device 106 is provided to drive the wedges 92L and 92R.

With the above change, the image signal processing system is changed as shown in FIG. In this embodiment, since the image signal is not taken at one time, the phosphor 72, the lens 73,
The TV camera 74 is unnecessary. Output signals from the X-ray detector 57 are passed through gate circuits 74 to A / D converters 75-1 and 75-2.
Supplied to The gate circuit 74 is provided for timing the movement of the sample and the capture of the output signal.
The A / D converted signals are sequentially stored in frame memories 76-1 and 76-2.
When one screen is completed, the process proceeds to the next process.

A stage drive circuit 82 controlled by the host computer 71 is provided, and the stage 103 is driven via a stage drive device 104 by a stage drive signal from the stage drive circuit 82. In addition, the host computer
A zone plate drive circuit 83 controlled by the control unit 71 is provided, and a zone plate drive signal from the zone plate drive circuit 83 is input to a zone plate drive device 102 in the vacuum chamber 62 so that the zone plate 101 as an objective lens is irradiated with X-ray light. Drive in the axial direction. This allows zone plate 101 to be targeted
When condensing a desired wavelength of the X-rays incident from 53, the focal point is on the sample 58 (when observing the front side, the ultraviolet absorbing portion on the left surface of the sample 58 in FIG. When observing, the sample 58 is located at the ultraviolet absorbing portion on the right surface of the sample 58 in FIG. The wedge drive circuit 80
Is controlled by a command signal from the host computer 71 and operates in the same manner as in the first embodiment in which the intensity of the ultraviolet light applied to the sample is adjusted appropriately, but the wedge to be controlled is selected from the wedges 92L and 92R. This is different from the first embodiment.

The second embodiment has the advantage that the operation and effect of the first embodiment can be obtained and that both sides can be observed without performing the operation of turning the sample over. In this case, when observing the back side,
6 may be removed and another pinhole plate may be placed on the back side of the sample, or the effective luminous flux (X-ray) and ultraviolet light are not kicked even when observing the back surface of the hole of the pinhole plate. With a small size, a separate pinhole plate is not required.

The mechanism for observing both sides of the sample of the second embodiment can be applied to the first embodiment. Also, if the ultraviolet wavelength is changed by controlling the OPO 90 when the observation target is changed, the X-ray wavelength absorbed by the sample also changes. Therefore, the above-described fine adjustment of the axial position of the zone plate 101 is synchronized with the control of the OPO. Automatically (they may be individually controlled).

The above description has been made on the case where ultraviolet light is used as the excitation light. However, depending on the observation object, light in a wavelength region other than the ultraviolet region, such as X-rays or visible light, may be required. The present invention can naturally be applied to such a case, and the necessary excitation light may be selected on a case-by-case basis.

[0043]

As described above, according to the present invention, the wavelength of the excitation radiation irradiating the sample is changed according to the object to be imaged. When an image is also taken, an ultraviolet wavelength suitable for the object to be imaged is irradiated, and a transmission X-ray microscope image with good image quality can be obtained.

[Brief description of the drawings]

FIG. 1 is a system diagram showing a configuration of a first embodiment of an X-ray microscope according to the present invention.

FIG. 2 is a diagram illustrating a configuration of an optical parametric oscillator according to a first embodiment.

FIG. 3 is a diagram illustrating a configuration of an image processing system used in a first embodiment.

FIG. 4 is a system diagram showing a configuration of a second embodiment of the X-ray microscope of the present invention.

FIG. 5 is a diagram illustrating a configuration of an image processing system used in a second embodiment.

FIGS. 6A to 6F are views for explaining the principle of an X-ray microscope.

FIGS. 7A to 7D are diagrams for explaining the principle of an X-ray microscope.

FIG. 8 is a characteristic diagram showing effectiveness of the principle of FIG. 7;

9 is a basic conceptual diagram of the X-ray microscope system corresponding to FIG.

FIG. 10 is a basic conceptual diagram of the X-ray microscope system corresponding to FIG.

FIG. 11 is a diagram showing the configuration of a conventional example that embodies the X-ray microscope system of FIG. 9;

FIG. 12 is a diagram showing a configuration of another conventional example that embodies the X-ray microscope system of FIG. 9;

[Explanation of symbols]

 51 Laser (Nd: YAG laser) 53 Target 54 Condenser lens (Walta condenser lens 55 Objective lens (zone plate) 57 Detector 58 Sample 62 Vacuum container 62a, 62b Transmission window (optical window) 64 Half mirror 66 Nonlinear optical crystal ( KDP crystal) 90 Optical parametric oscillator (OPO) 91 Harmonic generator (SHG) 92 Wedge-shaped glass (wedge)

Claims (1)

(57) [Claims] 1. An X-ray microscope for irradiating a sample with excitation radiation and soft X-rays to capture a transmission X-ray microscope image of the sample, wherein a wavelength of the excitation radiation is changed according to an imaging target. An X-ray microscope, characterized in that it is configured to perform