JPH083560B2 - Sample container for soft X-ray microscope observation - Google Patents

Description

TECHNICAL FIELD The present invention relates to a sample container for microscopic observation using soft X-rays as illumination light.

[Conventional technology]

For the future development of medicine and biotechnology, it is necessary to observe the dynamics of living microstructures such as subcellular organelles smaller than cells, viruses, proteins, etc. , It is difficult to realize with existing optical microscopes and electron microscopes. However, by using X-rays (wavelength is as short as several hundredths or less of visible light and resolution of several tens of times that of an optical microscope can be achieved), a living body can be observed in a living state.

Approximately 80% of organisms are water, and intracellular organelles made of proteins float in them. Wavelength 23-44 in the X-ray range
It is also called the Å water window, and there are areas where the absorption of X-rays by water is small. Within this range, the protein nucleic acid that constitutes the living body is X
Since it absorbs the lines, it looks like contrast. See Solid State Physics 20 (11) 1985 p.865.

X-rays are electromagnetic waves between ultraviolet light and gamma rays, and the boundaries are not so clear, but the wavelength is about 1 pm to 10 nm.
Soft X-rays having a wavelength of 0.1 nm or more are mainly used for X-ray microscopes.

Since soft X-rays are absorbed by air, it is necessary to create a vacuum inside the X-ray microscope.

When the biological sample is dried, the microstructure is destroyed. Therefore, in order to observe the biological sample as it is, it is necessary to enclose the sample in a closed container having a sample observation window that transmits soft X-rays. The window also needs to be transparent to visible light for easy handling.

[Problems to be solved by the invention]

Until now, polypropylene or Si was used as the window of the sample container.
Membranes of ₃ N ₄ have been used (Solid State Physics 20 (11) p865; 1985
However, these films show X-ray transmittance, especially at wavelengths of 23-
44 Å X-ray transmittance and strength were insufficient. When a window material having a low X-ray transmittance is used, the window thickness must be reduced, and the window size is inevitably prevented because the window is not destroyed by the pressure difference when the container is placed in the vacuum chamber. The size of the sample that can be observed is limited. Alternatively, if the thickness is large enough to create a large window, the intensity of transmitted X-rays will be reduced, and a larger X-ray source will be required. Not only that, but also a large amount of X-ray irradiation may destroy the sample.

 The same can be said when the strength of the window material is insufficient.

An object of the present invention is to improve these drawbacks and to provide a sample container having a high X-ray transmission performance and a larger observable area.

[Means for solving problems]

The present invention provides a sample container for soft X-ray microscope observation, comprising a space for containing a sample, a housing capable of sealing the space, and a sample observation window provided in at least a part of the housing. The atomic ratio of silicon: carbon: oxygen is 1:
A container characterized by being a thin film having a specific gravity in the range of 0.1: 0.1 to 1: 4: 2 and a specific gravity of 1.6 or more.

[Action]

The window material, which is a feature of the present invention, is a thin film (polymerized film) containing carbon, oxygen and silicon as main components, and has a self-supporting property. The thin film preferably has a specific gravity of 1.6 or more. If the specific gravity is less than 1.6, the airtightness and strength will be reduced.

In particular, in order to maintain a high transmittance of soft X-rays in a water window and obtain a thin film having necessary airtightness and strength, the composition of the film is preferably within the following range.

Number of silicon atoms: Number of carbon atoms = 1: 0.1 to 1: 4 Number of silicon atoms: Number of oxygen atoms = 1.0.1 to 1: 2 If there is too much carbon, absorption of X-rays in the water window will occur. If it is too large and too small, the strength will be insufficient. Further, when the oxygen content is small, the X-ray absorption becomes large, and when the oxygen content is large, the airtightness becomes insufficient.

Thin films can be made by plasma polymerization of organosilicon compounds. In this case, the thin film may contain other elements such as H, N, F, etc. to the extent that they do not affect the X-ray transmissivity.

When the thin film is formed by the plasma polymerization method, the thin film may be thin because it is pinhole-free and has high airtightness. The thinner the film, the higher the X-ray transmittance.

Generally, a film thickness of 0.1 to 100 μm is sufficient for the thin film.

〔Example〕

FIG. 1 is a schematic vertical cross-sectional view of the container of this embodiment, in which a thin film (polymerized film) 1 attached to a support frame 2 sandwiches a spacer / pressure sealant 3 to form a space 4 for containing a sample. . At the time of observation, the sample is put in the space 4 and sealed.

FIG. 2 shows an example of the plasma polymerization apparatus used for producing the polymerized film of this example. A plasma generation chamber 6 is connected to a vacuum chamber 5 that is evacuated by a pump. Argon gas 11 and oxygen gas 12 are supplied to the plasma generation chamber 6 through the flow controller 10, and plasma is generated by the microwave (2.45 GHz) electric power guided by the microwave waveguide 7. The plasma is sent to the chamber 5, and the monomer (hexamethyldisiloxane) 14 sent by the flow controller 13 is
It is activated and polymerized by plasma to form a polymerized film on the substrate 9 on the substrate holder 8.

FIG. 3 shows an example of the sample container manufacturing process, and each process will be described below.

First step: A thin film 16 having a thickness of 500 nm is formed on a silicon substrate 15 by plasma polymerization using hexamethyldisiloxane (abbreviated as HMDSO) as a raw material.

Step II: A resist 17 is applied to the back surface of the silicon substrate 15, and the pattern of the mask 18 is exposed.

Step III: Develop.

Step IV: The silicon substrate 15 is etched with an etching solution.

Step V: The resist 17 is removed.

Thus, the first member in which the window (thin film) 16 is stretched on the window material 15 is created. A container is completed by preparing two first members and joining them through a spacer. The container is held by another holder. Table 1 shows an example of conditions during plasma polymerization, and Table 2 shows soft X-ray transmittances of the plasma polymerization film and the SiN film.

[Advantages of the Invention] As described above, according to the present invention, a wavelength of 23
It is transparent to soft X-rays up to 44Å and is effective as a sample container for X-ray microscopes for living organisms. The sample container according to the invention is a soft X
The transmittance of rays is high, the X-ray light source can be made small, and the sensitivity of the X-ray detector can be small. Further, since the amount of X-ray irradiation to the sample can be suppressed, the damage is small and the observation of the biological sample can be continued for a long time. Further, the sample container according to the present invention has an extremely high transparency to visible light, and the sample can be observed with visible light. Further, since the transmittance is high, the window material can be made thick, and the strength of the window can be increased accordingly.

The window material (soft X-ray transparent film) used in the present invention is also useful for X-ray optical elements such as a mask substrate for X-ray linography and a substrate for an X-ray Fresnel zone plate.

[Brief description of drawings]

FIG. 1 is a schematic vertical sectional view of a sample container according to an example. FIG. 2 is a conceptual diagram for explaining the overall configuration of the microwave plasma polymerization apparatus for creating the window of the example. FIG. 3 is a process diagram illustrating a process of manufacturing a sample container. [Description of symbols of main parts] 1 ... Window or thin film, 11 ... Argon gas 2 ... Support frame, 12 ... Oxygen gas 3 ... Spacer, 13 ... Gas flow rate controller (flow rate controller) 4 ... Space for sample, 14 …… Hexamethyldisiloxane 5 …… Vacuum chamber, 15 …… Substrate or support frame 6 …… Plasma generation tube (plasma generation chamber) 7 …… Microwave waveguide, 16 …… Window or thin film (Polymerized film) 8 ... Substrate holder, 17 ... Resist 9 ... Substrate, 18 ... Photomask 10 ... Gas flow controller (flow controller)

Claims (2)

[Claims] 1. A sample container for soft X-ray microscope observation, which comprises a space for containing a sample, a housing capable of sealing the space, and a sample observation window provided in at least a part of the housing, Window silicon: carbon: oxygen atomic ratio of 1: 0.1: 0.1 to 1:
A container characterized by being a thin film in the range of 4: 2 and having a specific gravity of 1.6 or more. 2. The container according to claim 1, wherein the thin film is formed by plasma polymerization of an organic silicon compound.