JPH06123799A - Sample holder for X-ray microscope - Google Patents

Abstract

PURPOSE:To prevent failure and swell of the X-ray transmission file of a sample capsule. CONSTITUTION:In a sample holder for X-ray microscope which consists of at least a main holder 1b and a pressing holder 1a having concave in one or both of them for forming a sample capsule-containing chamber 12 in between the two by contacting both holders and has an opening 4 for X-ray transmission, the opening 4 is blocked with an X-ray transmission film 14a. By this, a pressurizing chamber 15 is formed and the X-ray transmission film 8a does not directly touch the vacuum so that failure and swell are prevented.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample holder used for observing an X-ray microscope.

[0002]

2. Description of the Related Art In recent years, in the fields of medicine and biotechnology, the resolution is higher than that of a microscope using ordinary visible light (wavelength λ = about 400 nm to 800 nm), and a living sample (hereinafter referred to as biological sample), For example, there is an increasing demand for a new microscope capable of clearly observing cells, bacteria, sperms, chromosomes, mitochondria, flagella, etc. The reason is that conventional high-resolution electron microscopes cannot observe biological samples. Therefore, instead of the conventional microscope using visible light, the wavelength λ =
An X-ray microscope using soft X-rays of 2 to 5 nm has been studied and is being specifically developed. For example, FIG. 4 shows a simple structure and an optical system of such an X-ray microscope. The X-ray emitted from the X-ray generator G is condensed by the condenser optical system C and the sample capsule B is collected. Irradiate. Then, the X-ray transmitted through the sample capsule B forms an image of the sample on the imaging device K by the imaging optical system I. This image is displayed on the monitor device (television screen) M. The optical path length from the X-ray generator G to the image pickup device K is, for example, about 2 m. Further, R is a vacuum container for the lens barrel, and V is an exhaust system for exhausting the inside of the container R to a vacuum. H is a sample holder that holds the sample capsule B.

As described above, although the concept of the basic structure has already been clarified, in reality,
The realization of an X-ray microscope has some problems to be solved, and it is still in the prototype stage. First of all, it is assumed in principle about an X-ray microscope that soft X-rays are absorbed by a substance when they pass through the substance, and the absorption rate per transmission optical path length (unit length) in the substance, that is, the line The absorption coefficient is
It is proportional to the density of the substance, and generally becomes higher as the wavelength becomes longer. 5 (a) and 5 (b) show the linear absorption coefficient spectra of some substances. As described above, a biological sample can be observed by utilizing the fact that the linear absorption coefficient differs depending on the substance.

However, since soft X-rays are absorbed by nitrogen, which occupies most of the air, the X-ray generator G shown in FIG. 4 to the biological sample and the biological sample to the image pickup device K are vacuumed. Space (for example, 4.8 × 10 ^-2 Pa)
I have to On the other hand, since biological samples die in such a vacuum space, they are suspended in an appropriate liquid medium (eg, water, culture solution, body fluid, physiological saline, etc.) and placed in an appropriate container for protection. There is a need. Such a container is generally called a sample capsule, and in the initial sample capsule, as shown in FIG. 6, an X-ray transparent material arranged on a ring-shaped spacer (7) and a pair of substrates (8) sandwiching the spacer (7). Membrane (8a) (for example, having a thickness of 0.05 to 0.1 μm)
Silicon nitride thin film). The space surrounded by the spacer (7) and the pair of X-ray transmissive films (8a) sandwiching the spacer (7) is called a sample chamber (13), and the space is filled with "medium and biological sample suspended therein". This sample chamber (13) is, of course, a closed space isolated from the vacuum space.

In this capsule structure, the spacer (7) is sandwiched between a pair of X-ray permeable membranes (8a), the sample is placed in the sample chamber (13), and the spacer (7) and the X-ray permeable membrane (8a) are separated. The interfaces are glued or glued together. The formed sample capsule is not decomposed, which is convenient. Alternatively, the sample capsules may be integrated with each other by a mechanical joining means in which the spacer (7) is sandwiched between the pair of X-ray transmissive films (8a) from the substrate (8) direction without being adhered.

Since the sample capsule is small, it is inconvenient to handle. Therefore, the sample capsule is generally held by the sample holder and set in the X-ray microscope. As shown in FIG. 7, for example, this sample holder is generally composed of at least two members, that is, a bottomed cylindrical main holder (1a) and a disc-shaped holding holder (1b). They are arranged facing each other and joined. By this joining, a capsule storage chamber (12) for the sample capsule is formed between the two.
Since the capsule storage chamber (12) is larger than the sample capsule, a space is created in the lateral direction of the sample capsule. Further, the main holder (1a) and the pressing holder (1b) are provided with an opening (4) for transmitting X-rays at substantially the center thereof.
There is. X-rays enter and exit the sample capsule through this opening (4). When the sample capsules are integrated without using an adhesive, as shown in FIG. 7, the sample capsules, that is, the substrate (8), the X-ray transparent film (8a), and the spacers (8a) illustrated also in FIG. The material capsule composed of 7) is pressed by the holders (1a) and (1b). At this time, in order to ensure the pressing, the O between the substrate (8) of the sample capsule and the holders (1a) and (1b). Place the ring (5). This ensures isolation from the vacuum space around the sample chamber (13).

[0007]

Although the device for realizing the X-ray microscope has been devised as described above, the structure of the material capsule and the material holder has a problem to be solved. This is because in the case of the conventional sample holder, as is clear from FIG. 7, the outer side of the X-ray permeable membrane (8a) of the sample capsule faces the vacuum space, and conversely, the X-ray permeable membrane ( The inside of 8a) faces the sample chamber (13) containing the sample. For this reason, a thin X-ray permeable film having a thickness of, for example, about 0.05 to 0.1 μm is subject to a pressure difference between the inside and the outside. As a result, when the sample capsule is set in the X-ray microscope, there is a problem that the X-ray transparent film is damaged.

Even if the damage does not occur, the central portion of the X-ray permeable film swells due to the pressure difference, which lengthens the optical path of the X-rays passing through the central portion, so that the X-rays are absorbed by the medium and are not observed. There was a problem that it became difficult.

[0009]

SUMMARY OF THE INVENTION The present invention has been made to solve the drawbacks of the conventional X-ray microscope as described above, and the main holder and the pressing holder having a depression in either one or both of them are provided. For an X-ray microscope, which consists of at least two members, a storage chamber for the sample capsule is formed between the two holders by joining them, and an opening for X-ray transmission is provided in the approximate center of both holders. In the sample holder, the opening is closed by an X-ray transparent film.

That is, the inventor of the present invention has advanced a detailed study on an X-ray microscope, and found that the X
It was found that a pressure chamber of about 1 atm is provided outside the line permeable membrane (8a) to prevent a large pressure difference from occurring in the X-ray structure membrane (8a), and the present invention was completed based on this finding. Let

[0011]

In the present invention, since the X-ray transmitting openings of the main holder and the pressing holder are closed by the X-ray transmitting film, respectively, the X-ray transmitting film of the holder and the X-ray transmitting film of the sample capsule are provided. A space (pressurizing chamber) can be provided. Therefore, no inconvenience due to the pressure difference occurs. In addition, when the sample capsule is stored in the sample holder, if working in the atmosphere,
The pressure chamber is filled with 1 atm of air, but the pressure chamber is 0.3
Since it can be thinned to about 0.6 mm, absorption of X-rays by air (nitrogen) can be ignored.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

[0013]

Embodiment 1 FIG. 1 of the accompanying drawings is a schematic sectional view illustrating a sample holder (H) according to an embodiment of the present invention. The sample capsule (B) is the same as the conventional capsule (see FIG. 6) described above, and the X-ray transparent film (8a) arranged on the ring-shaped spacer (7) and the pair of substrates (8) sandwiching the spacer (7). It consists of X-ray transparent film (8
Since a) is thin, it is formed on a substrate (8) such as silicon. Since the silicon substrate (8) is relatively thick and impermeable to X-rays, a window (6) for X-ray transmission is provided at the center. A space surrounded by the pair of X-ray permeable membranes (8a) and the spacer (7) is a sample chamber (13), and the "medium and biological sample suspended therein" are put therein.

The sample holder (H) is roughly classified into a bottomed cylindrical main holder (1a) and a disc-shaped holding holder (1).
b) and two members. The main holder (1a) and the pressing holder (1b) are each provided with an opening (4) for transmitting X-rays at substantially the center thereof, and X-rays enter the sample capsule through the opening (4). I am trying to emit it. An X-ray transmission film (14a), which is a feature of the present invention, is disposed in the opening (4) to close the sample holder (H). Therefore, the pressurizing chamber (15) is formed between the X-ray permeable membrane (8a) of the sample capsule and the X-ray permeable membrane (14a) of the holders (1a) (b). X-ray transparent film (8a) (14
The O-ring (5) is sandwiched between a) and the main holder (1a)
When the pressing holder (1b) and the holding holder (1b) are joined by an appropriate joining means for tightening in the directions of the arrows X and Y in FIG. 1, the pressurizing chamber (15) becomes a closed space of approximately 1 atm (atmosphere). At the same time, a capsule storage chamber (12) for the sample capsule (B) is formed inside the holder. In this embodiment, the capsule storage chamber (12) has a larger diameter than the sample capsule (B), so that when the sample capsule (B) is put in, a preliminary chamber is formed outside the capsule. That is, the spare room becomes a part of the capsule storage room (12). The pressure chamber (15) is
It is isolated from the external vacuum space by the X-ray transparent film (14a), and is also isolated from the capsule storage chamber (12) by the O-ring (5). In this case, the sample chamber (13)
Since the pressurizing chamber (15) and the pressurizing chamber (15) are completely isolated from each other, there is no special reason for making the capsule storage chamber (12) a closed space.

Since the pressurizing chamber (15) has a pressure of about 1 atm (atmospheric pressure), a large pressure difference with the sample chamber (13) does not occur. Therefore, the X-ray transmission film (8a) of the sample capsule (B)
Is not subject to large stresses, which reduces the risk of breakage. Of course, in this embodiment, the X-ray transparent film (14a) is directly bonded and integrated with the main holder (1a), but it may be a separate body. The following example illustrates this. Embodiment 2 FIG. 2 is a schematic sectional view showing a sample holder (H) according to another embodiment of the present invention.

Here, the X-ray transparent film (14a) for closing the opening (4) of the holder is formed separately from the main holder (1a) and the pressing holder (1b). That is,
The X-ray transparent film (14a) is formed on the silicon substrate (14), and a window hole (6) for passing X-rays is opened in the center of the silicon substrate (14). Main holder (1
After stacking a) and the holder (1b),
7) The screw which cut the outer periphery of the main holder (1a) (not shown)
The holders (1a) and (1b) can be joined by screwing into the holder. Also in this case, the X of the sample capsule
A pressurizing chamber (15) is formed between the ray permeable membrane (8a) and the X-ray permeable membrane (14a).

The capsule storage chamber (12) formed in the sample holder (H) is usually larger than the sample capsule (B), and the X-ray transmission region of the sample capsule (B), that is, the window hole (6). Since the diameter of the sample is as small as about 0.2 mm, “positioning of the sample capsule (B) at a position where X-rays penetrate” is troublesome. Therefore, in this embodiment, the ring-shaped positioning jigs (16) are stacked in two stages and placed in the capsule storage chamber (12).

An example of the order of assembling the sample holder (H) will be described. First, the X-ray transparent film (14a) is placed on the bottom of the main holder (1a) via the O-ring (5). Then
The lower part of the alignment jig (16) is placed in the capsule storage chamber (1
2) Put the X-ray transparent film (8a) in the jig (16).
Put. Then, the upper stage of the positioning jig (16) is overlaid on the lower stage. Then, the spacer (7) is placed on the X-ray transparent film (8a). The spacer (7) may be integrally formed on the underlying X-ray transparent film (8a). Next, the sample chamber (1
3) is filled with the medium (9) containing the sample (10). A second X-ray transparent film (8a) is overlaid thereon, and a sample capsule (B) is assembled. The second X-ray transparent film (14
a) is placed, and the pressing holder (1b) is placed on it by way of the O-ring (5). Finally, the fastener (17) is screwed onto the outer periphery of the main holder (1a) to complete the assembly of the sample holder (H).

The X-ray transparent film (14a) integrally formed on the substrate (14) may be placed upside down from that of FIG. Embodiment 3 FIG. 3 is a schematic sectional view showing a sample holder (H) according to yet another embodiment of the present invention.

Here, the X-ray transparent film (14a) integrally formed on the substrate (14) is placed upside down from that of FIG. The sample holder (H) of this embodiment is almost the same as that of the second embodiment, with the major difference that the main holder (1a) has an inlet (2) and an outlet (2) to the inside of the holder (capsule storage chamber). 3) is provided. After accommodating the sample capsule (B), the inlet (2) and the outlet (3) can be used to supply the stimulating substance of the sample to the capsule accommodating chamber.

That is, the sample chamber (13) can be opened by providing a through hole or a gap in a part of the spacer (7) of the sample capsule (B). Such a sample capsule (B) is called an open sample capsule. In this case, the medium is exposed around the sample capsule (B) (that is, the capsule storage chamber). However, since the gap between the pair of X-ray transmission films (8a) is narrow (for example, 1 to 10 μm), the medium does not flow out from the sample capsule (B) due to the surface tension of the medium. Although the medium is exposed to the capsule storage chamber, there is no inconvenience because the capsule storage chamber is isolated from the external vacuum space. Therefore, when a stimulating substance is supplied to the capsule storage chamber, the stimulating substance can stimulate the sample through the medium.

This stimulation allows the biological sample to be manipulated to some extent. For example, if oxygen gas (stimulating substance) is put in the capsule storage chamber, the oxygen concentration distribution in the medium can be made thin in the central part and made high in the peripheral part, so when the sample is anaerobic and can run by itself, The sample can be collected in the central observation area. Also, for example,
If a gas containing no oxygen (for example, nitrogen, argon, helium, etc.) is placed in the capsule storage chamber as a stimulant, the oxygen concentration distribution in the medium can be made higher in the central part and thinner in the peripheral part. If the aerobic is self-propelled, the sample can be collected in the central observation area.

Even when the sample consumes oxygen, if oxygen gas (stimulating substance) is put in the capsule storage chamber,
Oxygen is supplied from here to the sample through the medium, so that the sample can be observed for a long period of time while being kept alive. If the sample is acidophilic (or alkaliphilic) and can run on its own, if an alkaline (or acidic) gas or liquid is placed in the capsule storage chamber, the pH distribution in the medium will be high (or low) in the center. ) The sample can be collected in the central part (= observation region) since it can be made lower (or higher) in the peripheral part.

Further, it is possible to observe what kind of stimulant the sample shows and what kind of reaction the sample shows. As stimulants,
Examples include substances that the sample likes, substances that the sample dislikes, substances that the sample repels, and substances that the sample reacts with. Here, after putting the stimulating substance into the capsule storage chamber,
Each outlet is capped with a cap (18). By doing so, the sample holder (H) can be directly attached to the X-ray microscope.
Can be set. Of course, pipes are connected to the inlet (2) and the outlet (3) without a stopper, and the pipes are drawn out of the vacuum vessel R, and the stimulating substance is continuously supplied or circulated through the pipes. Good.

[0025]

As described in detail above, according to the present invention, the "opening for X-ray transmission" of each of the main holder and the pressing holder is closed by the X-ray transmitting film, so that the X-ray of the sample capsule is closed. A pressurizing chamber can be formed outside the permeable membrane. Therefore, since the pressure difference between the outside and the inside of the X-ray permeable membrane of the sample capsule is eliminated or reduced,
The risk of damaging the X-ray transparent film of the sample capsule is reduced. Further, even if the damage does not occur, the phenomenon that the central portion of the X-ray permeable film swells due to the pressure difference is reduced.
The problem that the central part becomes difficult to observe is also solved.

[Brief description of drawings]

FIG. 1 is a schematic sectional view illustrating a sample holder according to an embodiment of the present invention.

FIG. 2 is a schematic cross-sectional view illustrating a sample holder as another embodiment of the present invention.

FIG. 3 is a schematic sectional view illustrating a sample holder as still another embodiment of the present invention.

FIG. 4 is a schematic sectional view showing a conventional sample holder.

FIG. 5 is a conceptual configuration diagram showing a simple structure of an X-ray microscope.

6A and 6B are spectrum diagrams showing spectra of X-ray absorption coefficients.

FIG. 7 is an exploded perspective view of a sample capsule (non-open type).

[Explanation of symbols]

 1a Main holder 1b Pressing holder 2 Inlet 3 Outlet 4 Opening for X-ray transmission 5 O-ring 6 Window hole for X-ray transmission 7 Spacer 8 Substrate 8a X-ray transmission membrane 9 Medium 10 Biological sample 12 Capsule storage chamber 13 Sample chamber 14a X-ray transparent film 14 Substrate 15 Pressurizing chamber 16 Positioning jig 17 Stopper 18 Cap B Sample capsule (7 + 8 + 8a) H Sample holder (1a + 1b) C Condenser optical system I Imaging optical system K Imaging device R Lens barrel Vacuum container M monitor device V vacuum exhaust system G X-ray generator

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[Procedure amendment]

[Submission date] September 7, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Fig. 4

[Correction method] Change

[Correction content]

FIG. 4 is a conceptual configuration diagram showing a simple structure of an X-ray microscope.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 5

[Correction method] Change

[Correction content]

5A and 5B are spectrum diagrams showing spectra of X-ray absorption coefficients.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 6

[Correction method] Change

[Correction content]

FIG. 6 is an exploded perspective view of a sample capsule (non-open type).

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 7

[Correction method] Change

[Correction content]

FIG. 7 is a schematic sectional view showing a conventional sample holder.

Claims (1)

[Claims] 1. A main holder having a depression in one or both of them, and at least two members of a pressing holder, a joint chamber between the two holders forms a storage chamber for the sample capsule, and both holders have a storage chamber. A sample holder for an X-ray microscope having an opening for X-ray transmission in the center, wherein the opening is closed by an X-ray transmission film.
Sample holder for line microscope.