JPH08327511A - Sample capsule for x-ray microscope - Google Patents

Abstract

PURPOSE: To provide a sample capsule for X-ray microscope, enabling the whole of a sample chamber to be observed under a visible light and a whole sample image to be grasped. CONSTITUTION: This capsule is equipped with chips 11 facing each other as a pair, each having an X-ray transmission window 104 and a support section 101b for the reinforcement thereof, and a sample chamber formed between the chips 11 kept at a preset distance. In addition, the section 101b is made of a substance allowing the transmission of a visible light.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a sample capsule for an X-ray microscope.

[0002]

2. Description of the Related Art In the fields of medicine and biotechnology, which have been rapidly advancing in recent years, ordinary visible light (wavelength λ = about 400-80) is used.
A high-resolution microscope that has a higher resolution than a microscope that uses 0 nm) and that can clearly observe living samples (hereinafter referred to as biological samples) such as cells, bacteria, sperms, chromosomes, mitochondria, and flagella. The demand for voice is increasing day by day. However, in an electron microscope having a high resolution, the biological sample had to be placed directly in a vacuum, and the biological sample could not be observed alive. Therefore, in order to enable observation of such a biological sample, an X-ray microscope using soft X-rays having a wavelength λ = 2 to 5 nm instead of visible light has been studied and is being specifically developed.

In the soft X-ray region, as shown in FIG. 9, the absorption of X-rays by a substance changes depending on the wavelength of the X-ray and the substance. Generally, the longer the wavelength of X-rays, the easier the absorption, and the larger the atomic number of the substance, the easier the absorption. Especially 2.3
In the X-ray wavelength range of up to 4.4 nm, water has a higher transmittance than organic substances containing carbon such as proteins, and contrast occurs due to the difference in the X-ray absorption rate between water and proteins. It is possible to observe the structure of cells without any. This X-ray wavelength range is called the Water Window,
This is a very useful area for biological microscopes. In addition, in FIG. 9, O-edge, N-edge and C-edge are absorption edges of oxygen, nitrogen and carbon, respectively, and have wavelengths at which the X-ray absorption coefficient becomes extremely small.

FIG. 10 shows a schematic structure of such an X-ray microscope. In FIG. 10, the X-ray generator 1
The X-ray illumination optical system 2, the sample container 3 in which the sample capsule 4 is housed, the X-ray magnifying optical system 5 and the X-ray imaging device 6 are disposed inside the vacuum container 8 provided with the exhaust device 9. Are arranged in series. The vacuum container 8 is kept in vacuum by the exhaust device 9 in order to avoid absorption of X-rays by air. The X-rays emitted from the X-ray generator 1 are condensed by the X-ray illumination optical system 2 and applied to the sample capsule 4. The X-rays pass through the sample in the sample capsule 4, the transmitted image is magnified by the X-ray magnifying optical system 5, and the magnified image is formed on the imaging device 6. The optical path length from the X-ray generator 1 to the X-ray imaging device 6 is about 2 m. Reference numeral 7 denotes a photographing monitor, and the magnified image formed on the X-ray imaging device 6 is observed by the photographing monitor 7.

Since soft X-rays are easily absorbed in the atmosphere (having an absorptance of about 2 × 10 ^-3 μm ^-1 at 1 atmosphere), X
It is necessary to keep the entire optical system of the line microscope at a high degree of vacuum according to the optical path length. Further, the sample capsule 4 that seals the observation sample to be inserted into the soft X-ray optical path suppresses the absorption of soft X-rays by reducing the thickness of the sample layer, and also serves as an observation window material that is also airtight. It is necessary to select a material that absorbs soft X-rays less. Generally, a thin film of silicon nitride or the like having a small linear absorption coefficient for soft X-rays and a high film strength is used as the observation window material.

FIG. 11 is a diagram showing the structure of a conventional sample capsule 4 and a sample container 3 used in an X-ray microscope, and FIG. 11 (a) shows the planar structure of the sample encapsulation portion of the sample capsule 4. 11 (b) shows a cross-sectional structure of the sample container 3 and the sample capsule 4 therein. The sample capsule 4 is
An annular spacer 13 is sandwiched between a pair of chips 11 having an X-ray transmission window 11c formed therein, and a sample chamber 10 which is a closed space inside thereof is filled with a culture solution containing an observation sample. It The chip 11 has a silicon nitride (Si ₃ N ₄ ) thin film 11b formed on a silicon substrate 11a,
The silicon substrate in the portion corresponding to the X-ray transmission window 11c is removed by etching. X-ray transmission window 11c
Is a square of 0.2 to 1 mm square and its film thickness is 0.1.
It is 05 to 0.1 μm. The reason for making the film thickness thin in this way is to suppress absorption of soft X-rays as much as possible.

The spacer 13 keeps the thickness of the sample layer in the sample chamber 10 constant, and an appropriate thickness within the range of 1 to 15 μm is selected according to the application. For example, soft X
If soft X-rays having a wavelength of 2.3 nm are used, and the thickness of the silicon nitride thin film 11b is 0.1 μm and the thickness of the sample layer is 10 μm, the respective soft X-ray transmittances are 39%,
The transmittance is 12.3%, and the total transmittance is about 11%.
A sealing surface is provided on each of the surfaces 13a and 13b of the spacer 13, and also has a function of closely contacting the chip 11 and sealing the sample chamber 10. Chip 11 is spacer 1
In some cases, it may be molded integrally with 3. After the sample capsule 4 is housed in the concave portion 15a of the pressing plate 15 which constitutes the sample container 3, the pressing plate 16 is fixed by the screw 17 to apply a pressing force to the sample capsule 4 via the O-ring 18 and thereby the sample container Hold within 3.

X-ray transmission window 11c used for the sample capsule 4
Must be very thin in order to reduce the X-ray absorption rate, and its strength is very weak. In addition, the sample capsule 4 is sealed in a state where the biological sample is contained in a liquid such as a culture solution so that the biological sample can exist in a normal state. Therefore, stress due to the pressure difference between the pressure inside the sample capsule 4 and the pressure inside the vacuum container 8 is applied to the X-ray transmission window 11c, and the X-ray transmission window 11c swells or is damaged. In order to prevent the swelling and damage of the X-ray transmission window 11c,
For example, when silicon nitride is used for the X-ray transmission window 11c, the size of the X-ray transmission window 11c is 0.5 to 0.1 mm.
It is necessary to reduce it to about the corner.

However, when the X-ray transmission window 11c is made smaller, the observation area becomes narrower. For example, in the case of a large sample, only a part of the sample can be observed from the X-ray transmission window 11c, while the sample is small. In this case, the sample filled in the sample capsule 4 may not always come to the portion of the X-ray transmission window 11c. FIG. 12 is a diagram showing a case where the sample is large. In the figure, 703 is a nerve cell, and 703a and 703b are the tip parts of neurons. Further, a region 701 is a region of the X-ray transmission window portion of the sample chamber 10, and a region 702 is a region of the silicon substrate portion.

[0010]

By the way, in order to confirm which part of the sample is the part observed by the X-ray microscope, the silicon substrate part is observed even when the sample capsule 4 is observed by the optical microscope, for example. Has a very small visible light transmittance and is opaque, so that the sample in the region 702 cannot be observed. For example, in observing a biological sample such as a nerve cell, when the nerve cell 703 is arranged as shown in FIG. 12, the whole image of the sample, that is, how many neurons the nerve cell being observed emits It is necessary to understand whether or not the tip 703b of the neuron is connected to another nerve cell. However, in the above-described conventional sample capsule 4, the silicon substrate, which is the support portion of the X-ray transmission window 11c, is opaque to both X-rays and visible light. However, there is a drawback in that the region 702 cannot be observed even by using.

An object of the present invention is to provide a sample capsule for an X-ray microscope, which can observe the entire sample chamber with visible light and can grasp the entire image of the sample.

[0012]

An embodiment will be described with reference to FIGS. 1 and 6. To be described in association with FIG. 1, the invention of claim 1 includes a pair of substrates 11 each having an X-ray transmission window 104 and a supporting portion 101b that reinforces the X-ray transmission window 104, and a pair of substrates 11 facing each other. It is applied to a sample capsule for an X-ray microscope provided with a sample chamber formed between substrates 11 held at a distance, and the above-mentioned object is achieved by forming the supporting portion 101b from a substance that transmits visible light. Referring to FIG. 6, in the sample capsule of the invention of claim 2, the X-ray transmission window 401a and the support portion 401b are integrally formed of a substance that transmits visible light.
The thickness t of the X-ray transmission window 401a is 0.01 μm ≦ t ≦ 10.
μm.

[0013]

According to the first aspect of the present invention, visible light is transmitted through the X-ray transmission window 10.
Both 4 and the supporting portion 101b are transparent. Claim 2
In the invention, the thickness t of the X-ray is 0.01 μm ≦ t ≦ 10 μ
m is transmitted through the X-ray transmission window 401a, and visible light is transmitted through both the X-ray transmission window 401a and the support portion 401b.

Incidentally, in the section of means and action for solving the above-mentioned problems for explaining the constitution of the present invention, the drawings of the embodiments are used for the sake of easy understanding of the present invention. It is not limited to.

[0015]

Embodiments of the present invention will be described below with reference to FIGS. -First Example-FIG. 1 is a diagram illustrating a first example of a sample capsule according to the present invention, (a) is a plan view of a chip constituting the sample capsule, and (b) is a sectional view. As shown in FIG. 1A, a circular X-ray transmission window 1 is formed at the center of a rectangular chip 11.
04 are formed. The X-ray transmission window 104 is made of a silicon nitride layer 102a and is supported by a glass support portion 101b that transmits visible light. Although silicon nitride layers 102a and 102b are formed on both surfaces of the support portion 101b, the silicon nitride layers 102a and 102b are formed.
Since the thickness of each b is about 0.1 μm, the influence on the transmittance of visible light is very small. As in the conventional case, a pair of chips 11 are arranged opposite to each other, for example, via a ring-shaped spacer to form a sample capsule.

FIG. 2 is a diagram for explaining a method of manufacturing the chip 11 shown in FIG. 1, and (a) to (e) show respective manufacturing steps. After cleaning the surface of the glass substrate 101 in the step shown in FIG. 2A, as shown in FIG.
Silicon nitride layer 102 by P-CVD (low pressure CVD)
Films a and 102b are formed on both surfaces of the glass substrate 101. In the step shown in FIG. 2C, a mask pattern 103 such as a resist or a metal for forming the X-ray transmission window 104 is formed on the silicon nitride layer 102b by using photolithography or the like. Next, as shown in FIG. 2D, the silicon nitride layer 102b is etched based on the mask pattern 103 by dry etching, and then the mask pattern 103 is peeled off. After that, as shown in FIG. 2E, the glass substrate in the X-ray transmission window portion is removed by etching using hydrofluoric acid or the like, and the X-ray transmission window 104 formed by the silicon nitride layer 102a and the supporting portion 101b for supporting it. To form. In this way, the chip 11 constituting the sample capsule is obtained.

As described above, in the chip 11 constituting the sample capsule of the present embodiment, the X-ray transmission window 104 of the silicon nitride layer 102a is supported by the glass supporting portion 101b. When glass has a thickness of 0.1 mm or more, it hardly transmits X-rays, but it has a sufficiently large transmittance for visible light even if the thickness is several mm or more. for that reason,
When the thickness of the support portion 101b is set to about 0.5 mm in order to have mechanical strength, the entire view can be observed using an optical microscope even for a sample larger than the X-ray transmission window 104. When observing with a line microscope, it is possible to easily grasp which part of the sample is being observed. Also, when silicon is used for the support as in the conventional case,
The shape of the X-ray transmissive window is generally rectangular because anisotropic etching is used, but when glass is used for the support, there is no direction of etching, so not only the conventional rectangular shape but also the rectangular shape is used. As in the embodiment, the shape may be circular, which is a stronger shape. Further, although the shape of the chip 11 is rectangular in this embodiment, the chip 11 may be circular so that it can rotate about the axis of the X-ray optical system in the sample capsule. Although the mask pattern 103 is peeled off in the above-mentioned example, it may not be peeled off if the resist or metal used as the mask is transparent to visible light.

Although glass is used as the supporting portion 101b in the above-described embodiment, a plastic material such as polycarbonate or a plate made of crystal, or a film made of vinyl chloride or polyethylene may be used. Good.
Although silicon nitride is used as the X-ray transmission window material, silicon oxide, boron nitride, silicon carbide, beryllium, or the like may be used.

-Second Embodiment-FIG. 3 is a view for explaining a second embodiment of the sample capsule according to the present invention, (a) is a plan view of a chip constituting the sample capsule, and (b) is a sectional view. is there. Similar to the first embodiment,
A circular X-ray transmission window 206 is formed in the center of the rectangular chip 11.
Are formed. The X-ray transmission window 206 formed by the silicon nitride layer 203 is supported by the support portion 205 made of diethylene glycol bisallyl carbonate, which is a thermosetting resin that is a substance that transmits visible light.

FIG. 4 is a diagram for explaining a method of manufacturing the chip 11 shown in FIG. 3, and (a) to (f) show respective manufacturing steps. In the process of FIG. 4A, the substrate 20 such as silicon or glass is used.
After cleaning the surface of No. 1, as shown in FIG. 4B, a resist 202 is thinly applied to one surface of the substrate 201. At this time, a groove pattern or the like may be formed on the coating surface of the substrate 201 so that the resist 202 can be easily peeled off in a step described later. Then, as shown in FIG.
Silicon nitride layer 20 is formed on resist 202 by CVD or the like.
3 is deposited. Then, as shown in FIG. 4D, after polysilicon (polycrystalline silicon) 204 is formed on a portion of the silicon nitride layer 203 corresponding to the X-ray transmission window,
As shown in (e), diethylene glycol bisallyl carbonate, which is a thermosetting resin, is poured to a uniform thickness and heated and solidified to form the support portion 205. At this time, the portion of the silicon nitride layer 203 corresponding to the X-ray transmission window
Since it is covered with polysilicon 204, diethylene glycol bisallyl carbonate does not flow into this portion. Finally, as shown in FIG. 4F, after removing the polysilicon 204 covering the portion of the X-ray transmission window 206, the resist 202 is peeled off and the substrate 201 is removed to obtain the chip 11. .

When silicon nitride is used for the X-ray transmission window material as in the second embodiment and the first embodiment shown in FIG. 1, the stress generated in the silicon nitride layer of the X-ray transmission window is Since it becomes a tensile stress, the X-ray transmission window can be easily formed. For example, when silicon oxide is used for the X-ray transmission window material, the silicon oxide has a compressive stress in a normal film formation such as sputtering. Therefore, X of the silicon oxide layer
It is difficult to form a line transparent window. However, in the chip manufacturing method of this embodiment, if a resin that expands during solidification is used as the support 205, a tensile stress can be applied to the silicon oxide layer of the X-ray transparent window material when the support 205 solidifies. Therefore, the X-ray transmission window of the silicon oxide layer can be easily obtained. Even when a resin that shrinks when solidified is used, when the support portion shrinks, for example, by forming the support portion in a ring shape and setting the diameter and thickness of the ring to appropriate conditions. The X-ray transparent window material formed inside the ring can be subjected to tensile stress from the support portion.

Also in this embodiment, since a material having a sufficiently large visible light transmittance is used for the supporting portion, the same effect as that of the first embodiment can be obtained. Further, in the first embodiment, the glass substrate 101 formed in the plate shape in advance is used as the supporting portion 10.
Although the material of 1b is used, in this embodiment, since the resin is cured on the silicon nitride layer 203 to form the support portion 205, the resin as the support member is previously formed into a plate or a film as in the first embodiment. Since it does not need to be formed in a shape, the manufacturing cost can be reduced. Although diethylene glycol bisallyl carbonate was used as the resin in this embodiment, a resin that transmits visible light such as polycarbonate, ultraviolet curable resin, or epoxy resin may be used.

-Modification of Second Embodiment- FIG. 5 is a modification of the second embodiment, in which (a) to (e) show the respective manufacturing steps of the chip 11. As shown in FIG. 5E, the structure of the chip 11 is such that the X-ray transmission window 306 formed by the silicon nitride layer 302 is supported by the supporting portion 305 of polycarbonate which is a thermosetting resin, and is the same as in the second embodiment. However, the manufacturing method of the chip 11 is different from that of the second embodiment. In the process shown in FIG. 5A, the process shown in FIG.
After the surface of the silicon substrate 301 is washed in the same manner as above, a silicon nitride layer 302 is formed on one surface of the substrate 301, and then resists 303 and 304 are applied respectively. Next, as shown in FIG. 5B, the resist 303 in the portion corresponding to the X-ray transmission window is formed by photolithography.
leave only a. Then, as shown in FIG. 5C, the portion of the substrate 301 corresponding to the X-ray transmission window is left in an island shape by etching. Next, as shown in FIG. 5D, polycarbonate is poured into a uniform thickness and heated and solidified to form a support portion 305. Finally, after removing the resists 303a and 304 as shown in FIG. 5E, the silicon substrate 301 is removed by etching with a potassium hydroxide aqueous solution (KOH) of about 10 to 70 wt%.

-Third Embodiment-FIG. 6 is a view for explaining a third embodiment of the sample capsule according to the present invention, (a) is a plan view of a chip constituting the sample capsule, and (b) is a sectional view. is there. The rectangular chip 11 is made of glass and has a sufficiently high transmittance for visible light. Four
Reference numeral 01a denotes a circular X-ray transmission window formed in the central portion of the chip 11, and as shown in FIG. 6B, the plate-shaped glass substrate has a thickness of about 0.1 μm and has a sufficiently large X-ray transmittance. I am trying to become. 401b is an X-ray transmission window 401a
Is a glass substrate in the peripheral portion and forms a support portion for the X-ray transmission window 401a.

FIG. 7 is a diagram for explaining a method of manufacturing the chip 11 shown in FIG. 6, and (a) to (d) show respective manufacturing steps. After cleaning the surface of the glass substrate 401 in the step shown in FIG. 7A, as shown in FIG.
After coating the resists 402a and 402b on both sides of 01, a mask pattern is formed by photolithography in which the portion corresponding to the X-ray transmission window of the resist 402b is removed. Next, as shown in FIG. 7C, the portion of the glass substrate 401 corresponding to the X-ray transmission window is etched with hydrofluoric acid or the like until the thickness reaches about 0.1 μm, and the X-ray transmission window is opened. 401a and the support part 401b are formed. Finally, as shown in FIG. 7D, the resists 402a and 402b are peeled off to remove the X-ray transmission window 401a and the support portion 40.
A chip 11 integrated with 1b is obtained.

In this embodiment, the X-ray transmission window 4 of the chip 11 is used.
01a and the support portion 401b are integrally formed of glass that transmits visible light, and the thickness of the portion of the X-ray transmission window 401a is set so that the X-ray transmittance is sufficiently large (0.1 μm in this embodiment).
Since it is thinned to m), the whole image of the sample can be observed with an optical microscope or the like. Although glass is used as the material of the chip 11 in the present embodiment, it is not limited to glass, and any material that transmits visible light and can sufficiently transmit X-rays when the thickness is set to about 0.1 μm is sufficient. Some examples of such substances are shown in FIG. FIG. 8 shows the X-ray transmittance of a substance transparent to visible light in comparison with that of silicon nitride (Si ₃ N ₄ ) used as an X-ray window member, and the horizontal axis represents the thickness of the substance. . As can be seen from this figure, since the X-ray transmittance becomes sufficiently large when the thickness of each substance is about 0.1 μm, the sample can be observed by the X-ray microscope through the X-ray transmission window 401a. In addition, according to the data in FIG. 8, the X-ray transmittance is considerably small when the thickness of each substance is 1 μm or more, but the low density of each substance makes it possible to obtain the X-ray transmittance even when the thickness is 10 μm. Can be suppressed.

In this embodiment, the resists 402a, 402
A mask pattern using metal or the like may be formed instead of 02b. Note that in the case where the resists 402a and 402b used as masks have sufficient transmittance so as not to affect X-rays and visible light, FIG.
It may be used as a chip in the state shown in (c).

In the correspondence between the embodiment described above and the claims, the chip 11 constitutes a substrate.

[0029]

As described above, according to the present invention,
Since the supporting portion of the sample capsule transmits visible light, the entire image of the sample can be observed using visible light, and which part of the entire sample is being observed with the X-ray microscope can be grasped. .

[Brief description of drawings]

1A and 1B are diagrams illustrating a first embodiment of a sample capsule according to the present invention, FIG. 1A is a plan view of a chip 11 that constitutes the sample capsule, and FIG.

FIG. 2 is a diagram illustrating a method of manufacturing the chip 11 of FIG. 1, in which (a) to (e) show respective manufacturing steps.

3A and 3B are views for explaining a second embodiment of the sample capsule according to the present invention, FIG. 3A is a plan view of a chip 11 constituting the sample capsule, and FIG. 3B is a sectional view.

4A to 4F are diagrams illustrating a method of manufacturing the chip 11 of FIG. 3, in which (a) to (f) show respective manufacturing steps.

FIG. 5 is a diagram illustrating a modified example of the second embodiment, in which the chip 11 is used.
And (a) to (e) show respective manufacturing steps.

6A and 6B are views for explaining a third embodiment of the sample capsule according to the present invention, FIG. 6A is a plan view of a chip 11 constituting the sample capsule, and FIG. 6B is a sectional view.

7A to 7D are diagrams illustrating a method of manufacturing the chip 11 of FIG. 6, and FIGS. 7A to 7D show each manufacturing process.

FIG. 8 is a diagram showing a relationship between a material thickness and X-ray transmittance.

FIG. 9 is a diagram illustrating a linear absorption coefficient of X-rays by a substance.

FIG. 10 is a schematic configuration diagram of an X-ray microscope.

11A and 11B are diagrams showing the structures of a conventional sample capsule and sample container, in which FIG. 11A is a plan view and FIG. 11B is a sectional view.

FIG. 12 is a diagram illustrating an observation area when a conventional sample capsule is used.

[Explanation of reference symbols] 4 sample capsule 10 sample chamber 11 chip 101b, 205, 305, 401b support portion 104, 206, 306, 401a X-ray transmission window

Claims (2)

[Claims] 1. A pair of substrates, each of which has an X-ray transmission window and a support portion that reinforces the X-ray transmission window and faces each other, and a sample chamber formed between the substrates held at a predetermined distance. The sample capsule for an X-ray microscope, comprising the support part formed of a substance that transmits visible light. 2. The sample capsule for an X-ray microscope according to claim 1, wherein the X-ray transmission window and the support portion are integrally formed of a substance that transmits visible light, and the thickness t of the X-ray transmission window is t. To 0.0
A sample capsule for an X-ray microscope, wherein 1 μm ≦ t ≦ 10 μm.