JP3537652B2 - 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.

[0002]

2. Description of the Related Art Conventionally, as an optical element used in an X-ray microscope, for example, a grazing incidence optical system and a Schwarzschild optical system are known.
For example, FIG. 1 illustrates a Walter reflection optical system. The Walter-type reflection optical system shown in FIG. 1 is a kind of oblique incidence optical system. By utilizing total reflection on the reflection surface, the light is incident at a slight angle with respect to the reflection surface, that is, at an incident angle of the oblique incidence region. X-rays are reflected.

FIG. 2 exemplifies a Fresnel zone plate utilizing diffraction, and FIG. 3 exemplifies a Schwarzschild optical system. The Schwarzschild optical system illustrated in FIG. 3 is a direct-incidence type in which a multilayer film is coated on two spherical mirrors, and an artificial lattice is formed by the multilayer film.
Reflect lines.

[0004] Recently, soft X-rays have the property of causing less damage to ecological samples, and are expected to be applied to biological microscopes that can observe ecology with high resolution and without staining. I have. In particular, the wavelength band is λ
= 43.7 ° to 23.6 ° is used, the X-ray absorption of carbon is high, and X
Since the transmittance of rays is high, if X-rays in such a wavelength band are applied to a biological microscope, a transmission microscopic image of a protein whose main constituent atoms are carbon can be observed with good contrast even in water. Become like

On the other hand, attempts have been made to obtain microscopic images of proteins using X-rays in a wavelength band other than λ = 43.7 ° to 23.6 °, and various methods have been proposed. Here, one of those methods will be described with reference to FIGS. 4 (a) to 4 (f) and FIG.
(A) to (d) respectively exemplify the state of electrons and the state of transition when a carbon atom absorbs X-rays.

FIG. 4A exemplifies the electron configuration of a carbon atom in the ground state, and two electrons E exist in each of the 1S orbit, the 2S orbit, and the 2P orbit. When X-rays are incident on this ground state, as shown in FIG.
The electrons in the S orbital are excited and ionized, and the carbon atoms leave vacancies in the 1S orbital. This is called a first transition. However, since the first transition state is extremely unstable in energy as illustrated in FIG. 4C, the electrons in the 2P orbit transition to the 1S orbit as illustrated in FIG. 4D. It will be in a stable state.
This is called a second transition. Furthermore, if this carbon is a constituent of a molecule such as a protein, a vacancy is generated in the 2P orbit as illustrated in FIG. 4 (e). Therefore, the carbon atom is illustrated in FIG. 4 (f). As described above, electrons are captured from surrounding constituent elements and the like, and return to the initial ground state. This is called a third transition.

Usually, observation of a transmission microscopic image of a protein using an X-ray microscope is performed by utilizing energy absorption by a first transition when X-rays are irradiated. In this case,
If the wavelength of X-rays is not shorter than the absorption edge of carbon, X
The line is not absorbed by the protein, and the contrast of the transmitted image is deteriorated. However, when the electron transition process described above is considered in reverse, even if the wavelength of the X-ray to be incident is longer than the absorption edge of carbon, a microscopic image with excellent contrast can be observed.

That is, in the electron configuration of the carbon atom in the ground state illustrated in FIG. 5A, first, as illustrated in FIG. Is ionized or excited to form a hole in the 2P orbital. This is the reverse process of the third transition. Further, as illustrated in FIG. 5C, in this state, electrons 2 in 1S orbital are excited into vacancies in 2P orbital by X-rays. This is the reverse process of the second transition. This transition can be realized by an X-ray having a lower photon energy than the absorption edge of carbon, that is, a wavelength longer than the absorption edge of carbon. This state is the same as the state in which electrons in the 1S orbit are directly ionized from the ground state by the above-described first transition, as illustrated in FIG. 5D.

In this method, the energy required to ionize the electrons in the 2P orbit is about 5 eV (200 n
m), and ultraviolet laser light or the like can be used as a means for ionizing. Also, the energy required to excite electrons in the 1S orbital into the 2P orbital is lower by about 10 eV to 20 eV than when excited by the first transition.
FIG. 6 illustrates this state, and it can be seen that the ionization of electrons due to normal absorption, that is, the reverse process of the second transition occurs at a slightly lower energy than the first transition. In the case of a protein carbon atom, the absorption wavelength due to the reverse process of the second transition is approximately in the range of 43 ° to 65 °.

FIG. 7 illustrates the configuration of an X-ray microscope utilizing such a method. In the conventional X-ray microscope illustrated in FIG. 7, an X-ray filter (34) is provided between the sample (33) and the X-ray objective lens (35). The X-ray filter (34) is made of a thin film of Be or the like, has a high transmittance for incident light in a wavelength band of 43 ° to 65 °, and has a high reflectance for incident light in an ultraviolet region. have. Then, the X-ray of the photon energy corresponding to the reverse process of the second transition and the ultraviolet laser light adjusted to the photon energy of the reverse process of the third transition are respectively supplied to the X-ray light source (31) and the ultraviolet light source (38). ), And the X-ray condenser lens (3
2) and the sample (3) through the condenser lens (37).
By simultaneously irradiating 3), only the carbon atoms of the specific protein can be ionized, and a transmission microscopic image of the sample (33) can be obtained using the detector (36).

However, such a conventional X-ray microscope using ultraviolet light assist has the following problems in a technique for simultaneously introducing ultraviolet light. That is, for example, ultraviolet light is irradiated from the back side of the observation sample by a thin film mirror arranged at an angle of 45 degrees,
When irradiating X-ray light from the front side, it transmits X-rays,
45 capable of reflecting ultraviolet light with sufficient reflectance
Although a thin film mirror having a high degree of transmittance is indispensable, a thin film mirror having a high transmittance in the X-ray region and a high reflectance in the ultraviolet region needs to have a film thickness of about 0.1 μm. However, there is a problem that it is extremely difficult to accurately manufacture an extremely thin thin film.

Although it is difficult, it is possible to manufacture a free-standing thin-film mirror by, for example, coating aluminum on a silicon substrate and performing anisotropic etching from the back surface thereof. Since the thin aluminum film is very thin, it does not have a beautiful mirror surface due to non-uniformity of stress, and it is impossible to effectively irradiate an observation sample with ultraviolet light.

Furthermore, for example, when the observation sample is arranged at an angle of 45 degrees with respect to the optical axis of the microscope and the sample is irradiated with ultraviolet light and X-rays simultaneously from the surface side of the sample, the observation sample is placed with respect to the optical axis of the microscope. Since the camera is tilted by 45 degrees, the captured image is distorted, and there is a problem that the distorted image must be corrected by image processing. Therefore, the present invention has been made in view of the circumstances described above, and solves the problems of the related art, can easily introduce ultraviolet light, and obtains a high-precision microscopic image of an observation sample. It is an object of the present invention to provide a new ultraviolet light assisted X-ray microscope that can be manufactured and is easy to manufacture.

[0014]

SUMMARY OF THE INVENTION The claimed invention is as to solve the above problems, there is provided an X-ray microscope for obtaining a microscopic image of the observation sample is irradiated with X-rays and ultraviolet light to the observation specimen, pinholes X-ray and ultraviolet light by the urging mirror being adapted to be irradiated in the same region of the observation specimen saw is passed through on the same optical axis, a mirror with the pinhole mirror
Surface polished silicon substrate and anisotropic silicon substrate
A pinhole for passing X-rays formed by etching
Formed with aluminum coated on the surface of the silicon substrate
An X-ray microscope (Claim 1), comprising: a formed reflective layer for reflecting ultraviolet light .

[0015] The invention of this application, in the above X-ray microscope, with a mirror pinhole is, between the X-ray source and the microscopic image detector, the X-ray and ultraviolet light through the same optical axis , it is provided in a position to irradiate the same region of the observation sample and (claim 2), with a mirror pinholes, between the observation sample and the microscopic image detector, from the back to the observation sample with ultraviolet light The mirror is provided at a position for irradiation (claim 3 ), and the mirror with a pinhole is provided such that the pinhole is located on the optical axis of the microscope and inclined at an arbitrary angle with respect to the optical axis of the microscope. (Claim 4 )
And, full Les panel zone plate, that is provided between the pin with a mirror hole with the observation sample (claim 5) also Hisage <br/> provided.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, an X-ray microscope according to the present invention uses a mirror with a pinhole to pass ultraviolet light and X-rays on the same optical axis to irradiate the same region of an observation sample. Is obtained. FIG. 8 illustrates the basic concept of the X-ray microscope of the present invention.

For example, in the X-ray microscope shown in FIG. 8, a mirror (2) with a pinhole having a fine pinhole (21) is provided between the X-ray light source (1) and the observation sample (5). The pinhole (21) is provided at an angle of 45 ° with respect to the optical axis of the microscope so that the pinhole (21) is located on the optical axis near the X-ray light source (1). As for the X-rays from the X-ray light source (1), only those that have passed through the pinhole (21) of the mirror (2) with the pinhole enter the reflection objective condenser lens (3), and the surface of the observation sample (5). The light is focused on an arbitrary area on the upper side. On the other hand, the ultraviolet light is incident on the mirror with pinhole (2) from the direction perpendicular to the optical axis of the microscope via the ultraviolet light condensing lens (4) having a long focal length, and travels on the same optical axis as the X-ray. As a result, the X-rays are focused on the same area as the focused area on the observation sample (5). The reason why X-rays and ultraviolet light can be focused on the same sample area through the same optical axis is as follows.

(A) Since the pinhole (21) can be made extremely small because the divergent solid angle of the X-ray used is small, the ultraviolet light is almost reflected by the mirror with pinhole (2) and is not used. The amount of ultraviolet light is extremely small. (B) Since the thickness of the mirror with pinhole (2) can be made extremely thin, X-rays generated from the X-ray light source (1) will not be kicked by the edge of the mirror with pinhole (2).

(C) The ultraviolet light is focused on the observation sample (5) surface in the far field by the ultraviolet light focusing lens (4) arranged at a short distance from the mirror with pinhole (2). The opening of the pinhole (21) having a beam shape is completely closed by the diffraction effect, and the beam shape becomes almost Gaussian, so that the observation sample (5) can be illuminated with sufficient intensity.

The example shown in FIG. 8 corresponds to the case where the light source is a divergent light source. However, other than the divergent light source, a strong white X such as an SOR (synchrotron radiation) light source is used. When a line light source is provided, it is preferable that the Fresnel zone plate (7) is provided between the mirror with pinhole (2) and the observation sample (5) as illustrated in FIG. .

The X-ray microscope illustrated in FIG. 9 is provided with an SOR light source (6) as an X-ray light source.
Since the X-rays emitted from the OR light source (6) are substantially parallel beams and have a diameter of at most about 1 mm, the mirror with pinhole (2) does not directly hit the Fresnel zone plate (7) with ultraviolet light. It is installed as close as possible to the Fresnel zone plate (7). The diameter of the Fresnel zone plate (7) is about 10
X-rays, which are extremely small, about 0 μm, and are substantially parallel beams from the SOR light source (6) are focused at a focal length corresponding to the X-ray wavelength. The diameter of the pinhole (21) of the mirror with pinhole (2) is as close as possible to the effective diameter of the Fresnel zone plate (7).

Then, since the ultraviolet light is irradiated onto the observation sample (5) with a NA as large as possible from a direction perpendicular to the optical axis of the microscope, the beam is irradiated by the diffraction effect similarly to the X-ray microscope of FIG. The opening of the shaped pinhole (21) is completely closed, so that the observation sample (5) can be irradiated with a sufficient intensity with a beam shape substantially similar to Gaussian.

As described above, the provision of the mirror with the pinhole allows ultraviolet light and X-rays to pass through the same optical axis and irradiate the observation sample with direct incidence, thereby deteriorating the resolution and the photon flux. In addition, it is not necessary to use a free-standing mirror which is difficult to process, and complicated image processing is not required.

If the mirror (2) with pinhole can irradiate the same region of the observation sample (5) with the X-ray and ultraviolet light passing through the same optical axis, for example, The ultraviolet light may be provided between the microscopic image detector and the microscopic image detector at a position where the observation sample (5) is irradiated with ultraviolet light from the back surface. Hereinafter, examples are shown along the attached drawings,
Embodiments of the present invention will be described in more detail.

[0025]

FIG. 10 illustrates an essential configuration of an X-ray microscope according to an embodiment of the present invention. The X-ray microscope illustrated in FIG. 10 includes an Nd: YAG laser (8) as an ultraviolet light source and an X-ray source.
d: At the output side of the YAG laser (8), a condenser lens for X-rays (9) and an optical window for X-rays (11), a condenser lens for ultraviolet light (10) and an optical window for ultraviolet light (12) And are arranged. In a vacuum vessel (19) in which an optical system is accommodated, a laser plasma rotating target (13) and a pinhole mirror (2) having a minute pinhole (21) are each provided with an X-ray optical window (11). And an oblique incidence condenser mirror (14) as an X-ray condenser lens, an observation sample (5), an X-ray objective A Fresnel zone plate (7) as a lens and an ultraviolet light removing filter (15);
Microchannel plate (16), fluorescent plate (17), and solid-state imaging device (18) constituting a microscopic image detector system
And are arranged.

In the X-ray microscope of the present invention having such a structure, the Nd: YAG laser (8) uses the second harmonic (= 532 nm) and the fourth harmonic (= 266n).
m). The second harmonic is introduced into the vacuum vessel (19) via the X-ray condenser lens (9) and the X-ray optical window (11), and is focused on the laser plasma rotating target (13). Then, the laser plasma rotating target (13) is excited by the collected second harmonic, and X-rays are generated. The generated X-rays pass through the pinhole (21) of the mirror with pinhole (2), and are condensed on the surface of the observation sample (5) by a grazing incidence type condenser mirror (14), for example, a Walter type optical system. You.

On the other hand, the fourth harmonic is used as ultraviolet light for assisting, and the ultraviolet light is passed through a condenser lens for ultraviolet light (10) and an optical window for ultraviolet light (12) to form a vacuum container (19). ), Enters the mirror with pinhole (2), is reflected by the mirror with pinhole (2), passes through the central opening of the oblique incidence type condenser mirror (14), and X-rays The light is focused on the same region on the surface of the observation sample (5) as the focused region.

At the wavelength of the fourth harmonic, it is possible to effectively excite the π-orbital electron of the benzene group of the nucleic acid base or amino acid which is a constituent element of DNA. Therefore, for example, when an X-ray having a wavelength of 4.4 nm is imaged on the microchannel plate (16) by the Fresnel zone plate (7),
The fluorescent plate (17) is excited by photoelectrons from the microchannel plate (16), and an image of a benzene group of a nucleic acid base or amino acid which is a component of DNA is formed.
Then, this image is photographed in real time by the solid-state imaging device (18). Here, the ultraviolet light removal filter (15) removes stray ultraviolet light so as not to enter the solid-state imaging device (18) in order to prevent the image quality of the obtained image from deteriorating.

FIGS. 11A and 11B are a front view and a side sectional view, respectively, illustrating a mirror with a pinhole in the X-ray microscope of the present invention. As exemplified in FIG. 11, in the mirror (2) with a pinhole provided in the X-ray microscope of the present invention, for example, a mirror-polished silicon substrate (22) is used as the mirror substrate, and the silicon substrate ( 22), a pinhole (21) is formed by anisotropic etching. Silicon substrate (1
The surface of 1) is coated with, for example, aluminum having a high reflectivity over a wide range of visible light and ultraviolet light to form a reflection layer (23). Silicon substrate (1
The thickness of 1) is, for example, about 500 to 600 μm,
The mirror is extremely thin and has high reflectivity. The pinhole (21) has, for example, a length of 1.0 mm,
It has a dimension of width = 1.4 mm. Of course, the material to be coated to form the reflective layer is not limited to aluminum, but may be any material having a high reflectance at the wavelength of the assisting ultraviolet light.

In the X-ray microscope of the present invention, a laser plasma light source or an X-ray tube may be provided as the X-ray light source in addition to the above-mentioned synchrotron radiation light source. As an X-ray objective lens, a zone plate or a Schwarzschild type optical system may be provided.

Of course, the present invention is not limited to the above-described example, and it goes without saying that various embodiments are possible in detail.

[0032]

As described above in detail, according to the present invention, the same region of an observation sample can be illuminated by direct incidence of X-rays and ultraviolet light on the same optical axis, and an excellent high-resolution microscopic image can be obtained. A new X-ray microscope is provided which is easy to obtain and easy to manufacture.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating a basic configuration of a Walter reflection optical system.

FIG. 2 is a diagram illustrating a basic configuration of a Fresnel zone plate.

FIG. 3 is a diagram illustrating a basic configuration of a direct incidence type Schwarzschild optical system.

FIGS. 4A to 4F are diagrams respectively exemplifying transition states of electrons when a carbon atom absorbs X-rays.

FIGS. 5A to 5D are diagrams illustrating other transition states of electrons when a carbon atom absorbs X-rays, respectively.

FIG. 6 is a diagram comparing and exemplifying energy required for transition of electrons in a 2P orbit of a carbon atom.

FIG. 7 is a main part configuration diagram showing an example of a conventional X-ray microscope.

FIG. 8 is a diagram illustrating the basic concept of the X-ray microscope of the present invention.

FIG. 9 is a diagram showing another example of the basic concept of the X-ray microscope of the present invention.

FIG. 10 is a main part configuration diagram illustrating an X-ray microscope according to an embodiment of the present invention;

FIGS. 11A and 11B are a front view and a side sectional view, respectively, illustrating a mirror with a pinhole in the X-ray microscope of the present invention.

[Explanation of symbols]

1 X-ray light source 2 Mirror with pinhole 21 Pinhole 22 Silicon substrate 23 Aluminum 3 Reflective objective condenser lens 4 Ultraviolet light focusing lens 5 Observation sample 6 SOR light source 7 Fresnel zone plate 8 Nd: YAG laser 9 X-ray focusing lens 10 Condensing lens for ultraviolet light 11 Optical window for X-ray 12 Optical window for ultraviolet light 13 Laser Plasma Rotary Target 14. Grazing incidence condenser mirror 15 UV light removal filter 16 micro channel plate 17 fluorescent screen 18 Solid-state image sensor 19 Vacuum container 31 X-ray light source 32 X-ray condenser lens 33 samples 34 X-ray filter 35 X-ray objective lens 36 detector 37 Condensing lens 38 Ultraviolet light source

────────────────────────────────────────────────── ─── Continuation of front page (56) References JP-A-6-194500 (JP, A) JP-A-5-224000 (JP, A) JP-A-6-230200 (JP, A) JP-A-3-230 207000 (JP, A) JP-A-9-297198 (JP, A) JP-A-8-62398 (JP, A) JP-A-8-248198 (JP, A) JP-A-6-74879 (JP, A) JP-A-5-72400 (JP, A) Jiko 41-14257 (JP, Y1) (58) Fields investigated (Int. Cl. ⁷ , DB name) G21K 7/00 G01N 23/04

Claims (5)

(57) [Claims] 1. A X-ray and ultraviolet light is irradiated to the observation sample to obtain a microscopic image of the observation specimen X-ray microscope, X-rays and ultraviolet light by the mirror with pinholes is passed on the same optical axis watch adapted to be irradiated in the same region of the observation sample
This mirror with a pinhole is a mirror-polished
Anisotropic etching on the silicon substrate and the silicon substrate
Of the silicon substrate and the pinhole through which the X-ray passes
Ultraviolet light formed by coating aluminum on the surface
An X-ray microscope comprising: a reflection layer that reflects light. 2. A mirror with a pinhole, comprising: an X-ray light source;
X-ray and ultraviolet light are the same optical between the image detector
Through the axis to illuminate the same area of the observation sample
Gill is not that X-ray microscope of claim 1. 3. A mirror with a pinhole is provided between an observation sample and a microscopic image detector, for applying ultraviolet light to the observation sample on the back side thereof.
The X-ray microscope according to claim 1, wherein the X-ray microscope is provided at a position where the light is irradiated from above . 4. The mirror with a pinhole is provided with a pinhole.
Is located on the microscope optical axis and is arbitrary with respect to the microscope optical axis.
The X-ray microscope according to any one of claims 1 to 3, wherein the X-ray microscope is provided to be inclined at an angle . 5. The method according to claim 1, wherein the Fresnel zone plate has a pinhole.
The X-ray microscope according to any one of claims 1 to 4, wherein the X-ray microscope is provided between the attached mirror and the observation sample .