JPH09218300A - Observing method using x-ray microscope and sample holder used in the method - Google Patents

Abstract

PURPOSE: To observe a sample in a vigorous living state. CONSTITUTION: In this method for observing a biological sample floating in a medium with the use of an X-ray microscope, the biological sample in a sample chamber 13 is observed while light is irradiated to the biological sample, in the periphery of the sample or to the medium, or immediately after the irradiation of light. A sample holder is provided with an illumination means 2 which can illuminate a part or the whole of the sample chamber 13 or the periphery of the chamber 13. The illumination means 2 is, for example, constituted of an external light source and an optical fiber.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an observation method using an X-ray microscope and a sample holder used therefor.

[0002]

2. Description of the Related Art 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 a biological sample), for example, Cells, bacteria, sperm, chromosomes,
The demand for high-resolution microscopes that show mitochondria, flagella, etc. is increasing day by day. The reason is,
This is because no biological sample can be seen with a high-resolution electron microscope. Therefore, an X-ray microscope using soft X-rays having a wavelength λ = 2 to 5 nm instead of visible light is being developed. X in Figure 3
The simple structure and optical system of a line microscope are shown. In FIG.
The X-ray emitted from the X-ray generator G is condensed by the condenser optical system C and illuminates the sample capsule B. Then, the X-ray transmitted through the sample capsule B forms an image of the sample on the image pickup device K by the image forming 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 imaging device K is, for example, about 2 m. R
Is a vacuum container for a lens barrel, V is an exhaust system for evacuating the inside of the container R to a vacuum, and H is a sample holder for holding the sample capsule B. However, the X-ray microscope is still in the prototype stage.

Soft X-rays are absorbed by a substance when transmitted through the substance, and the absorptance per unit length of the transmitted optical path in the substance, that is, the linear absorption coefficient, is proportional to the density of the substance,
Generally, the longer the wavelength, the higher the wavelength. FIG. 4 shows the linear absorption coefficient spectra of some substances. The biological sample is observed by utilizing the fact that the linear absorption coefficient varies depending on the substance.

By the way, since soft X-rays are absorbed by nitrogen, which occupies most of the air, from the X-ray generator G to the biological sample and from the biological sample to the image pickup device K in a vacuum space (for example, 4.8 × 10 4). ^-2 Pa). On the other hand, since biological samples die in a vacuum space, it is necessary to suspend them in a suitable liquid medium (for example, water, culture solution, body fluid, physiological saline, etc.) and put them in a suitable container for protection. Such a container is generally called a sample capsule. As shown in FIG. 5, the initial sample capsule has a ring-shaped spacer and a pair of X-ray transmission films (for example,
It is made of a silicon nitride thin film having a thickness of about 0.05 to 0.1 μm). The space surrounded by the spacer and one X-ray permeable film sandwiching the spacer is called a sample chamber, and is filled with the "medium and the biological sample suspended therein". The sample chamber is, of course, a closed space isolated from the vacuum space. However, since the sample chamber is small and the X-ray permeable membrane is thin, the X-ray permeable membrane tends to be destroyed when the medium containing the biological sample is filled in the sample chamber. Therefore, a through hole is provided in a part of the spacer, or the spacer is not divided into rings but is divided into several divided spacers, and a gap is provided between the divided spacers (see FIG. 6).
As a result, a sample capsule having a sample chamber as an open space has been developed (for example, Japanese Patent Application No. 1-3336471). Such a sample capsule is called an open sample capsule.

When the sample chamber of the open-type sample capsule is filled with the "medium containing the biological sample", a part of the medium is exposed to the outside, so that the medium is predicted to flow out of the capsule and be lost. . However, in reality, the distance between the pair of X-ray transparent films is very narrow (for example, about 1 to 10 μm),
The surface tension of the medium prevents the medium from flowing out of the capsule.

Since the sample capsule is small and therefore inconvenient to handle, it is generally held in a sample holder and set in an X-ray microscope. Generally speaking, the sample holder is generally composed of at least two members, a main holder main body (1a) having a bottomed cylindrical shape and a disc-shaped holding holder main body (1b). When joined, a capsule storage chamber (12) for the sample capsule is formed between them. The capsule storage chamber is larger than the sample capsule, so that a space is created in the lateral direction of the sample capsule. Each of the main holder and the pressing holder has an opening for transmitting X-rays at substantially the center thereof. X-rays enter and exit the sample capsule through this opening. An example of the sample holder is shown in FIG. 8 (cross-sectional view). In the open sample capsule, the medium contacts the vacuum space. Therefore, the sample holder has a structure in which the capsule storage chamber (12) for storing the sample capsule is isolated from the surrounding vacuum space. In an actual example, an O-ring (5) is placed between the substrate (8) of the sample capsule and the holder body (1a, 1b) to seal the capsule storage chamber and isolate it from the vacuum space. However, the capsule storage room communicates with the sample room.

[0007]

Generally, when observing with an X-ray microscope, a sample is put in a sample capsule, held by a sample holder, and the sample holder is set on the X-ray microscope.
Then, the inside of the vacuum container R is decompressed to a vacuum, and observation is performed on it. Therefore, it takes a considerable time from placing the sample in the sample capsule to observing. Therefore, in the case where the sample is an organism or a part thereof that is stimulated by light, there is a problem that the sample is dead or loses its energy, and it is not possible to observe a lively life.

[0008]

Means for Solving the Problems As a result of earnest research, the present inventor has conceived that observation can be performed while stimulating a sample by irradiating with light or by irradiating with light in advance, and the present invention Accordingly, the present invention is firstly, in the method of observing a "biological sample suspended in a medium" using an X-ray microscope, applying light to the biological sample or its peripheral portion or the medium. A method (claim 1) is provided, characterized in that the biological sample is observed during irradiation or immediately after irradiation.

Secondly, according to the present invention, in a sample holder for holding a "sample capsule having a sample chamber" and isolating the sample chamber from the surrounding vacuum space, a part or the whole of the sample chamber or its periphery is secured. A sample holder (claim 2) is provided, which is provided with an illumination means capable of illuminating. Thirdly, the present invention provides a sample holder (claim 3) characterized in that, in the sample holder of the second invention (claim 2), the illuminating means is constituted by an external light source and an optical fiber.

Fourth, the present invention is the third invention (claim 3).
The sample holder (1) is characterized in that a condensing optical element is provided at the tip of the optical fiber. Fifth, according to the present invention, in a sample holder that holds a “sample capsule having a sample chamber” and isolates the sample chamber from the surrounding vacuum space, at least a part of the holder body has an optical waveguide structure. A featured sample holder (claim 5) is provided.

Sixth, the present invention provides a fifth invention (claim 5).
In the sample holder of (1), a light source is provided in a part of the optical waveguide structure.

[0012]

According to the observation method of the present invention, a biological sample can be observed while being stimulated with light or immediately before being stimulated, so that a lively and live appearance can be observed. The sample holder of the present invention is extremely useful for this observation. However, the sample holder of the present invention is not only used in the observation method of the present invention, but is, for example, an X incorporating an ordinary optical microscope.
In the case of a line microscope, it may be used to brightly illuminate the sample when observing with a normal optical microscope. In this case, the sample may be dyed in advance with a fluorescent or light absorbing dye. The illumination light referred to in the present invention may be ultraviolet light. Besides, there is also the following usage. In advance, a substance that reacts or decomposes with light to generate a stimulating substance has been added to the medium,
It is also possible to irradiate the medium with illumination light at the time of observation to generate a stimulating substance and observe the dynamic behavior of the sample with respect to the stimulating substance.

Further, by condensing the light in a part of the sample chamber, the phototactic sample is collected in one place or the condensing point is moved to move the sample into the visual field of the X-ray microscope. Alternatively, conversely, by illuminating the peripheral portion of the sample chamber with light, the anaerobic sample can be collected in the center of the sample chamber. Less than,
The present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0014]

[Embodiment 1] FIG. 1 is a schematic vertical sectional view showing a "sample holder holding a sample capsule in a capsule storage chamber" according to this embodiment. The sample capsule is the same as the conventional capsule described above, and includes a ring-shaped spacer (7) and a pair of X-ray transparent films (8a) sandwiching the spacer. Since the X-ray transparent film (8a) made of a silicon nitride thin film is thin, it is integrally formed on the silicon substrate (8). Since the silicon substrate (8) is relatively thick and impermeable to X-rays, it has a window hole (6) for X-ray transmission in the center. The spacer (7) may be a separate body from the pair of X-ray transmissive films (8a), or may be integrally formed with either one. The space surrounded by the pair of X-ray transparent films (8a) and the spacer is the sample chamber (13),
The medium is filled with "medium (not shown) and biological sample suspended therein" (not shown). Here, the medium is water and the biological sample is a phototactic bacterium. After the sample chamber (13) is filled with the “medium and the biological sample suspended therein”, the contact surface between the spacer (7) and the pair of X-ray permeable membranes (8a) may be bonded with an adhesive. That is easier to handle.
Instead of adhering, it may be crimped by an appropriate crimping means.

The sample holder is roughly divided into at least two members, that is, a main holder body (1a) having a bottomed cylindrical shape and a holding holder body (1b) having a bottomed cylindrical shape. When both members are joined by a suitable joining means (not shown) for tightening from the vertical direction in FIG. 1, a capsule storage chamber (12) for the sample capsule is formed inside. The capsule storage chamber (12) is slightly larger in diameter than the sample capsule. By sandwiching the O-ring (5) between the sample capsule and the holder body (1a, 1b), the sample capsule is firmly held by the holder. As a result, the sample capsule can be crimped. The sample chamber (13) is thus isolated from the surrounding vacuum space.

In this embodiment, the illumination means (2) is arranged in the vicinity of the window hole (4) for X-ray transmission and at a position where the transmission of X-rays is not blocked. The lighting means (2) is
Here, it is one or a plurality of optical fibers, the source of which is connected to an external light source (not shown). Lighting means (2)
The light emitted from the tip of illuminates part or all of the sample chamber (13). A converging optical element (convex lens) or a diverging optical element (concave lens) may be provided at the tip of the optical fiber in order to adjust the illumination area. The illuminating means (2) may also be composed of a light source itself such as a light emitting diode, a semiconductor laser, or incandescent light.

The illumination light (irradiation light) may be continuous light or pulsed light. Light with different wavelengths may be irradiated simultaneously or with a time difference. For example, by irradiating with one continuous light, the phototactic sample is collected in one place in the sample chamber (13), while the photodegradable substance is added in advance in the medium, The sample produced by photolysis may be stimulated by irradiating a light pulse having a wavelength most efficient for the degradation with another optical fiber, and then the sample may be observed with an X-ray microscope.

Next, the illumination light suitable for the sample to be observed will be described in detail. (1) When the sample is a phototaxis organism: When the sample exhibits phototaxis with respect to light of a specific wavelength or wavelength range, the illumination light is the light of that wavelength or wavelength range, or When light having a spectral distribution including a wavelength or a wavelength range is used, the sample gathers in the illumination area. Therefore, if the illumination area is matched with the X-ray observable area, the sample can be observed at a high density. (2) When the sample is stimulated by a stimulating substance generated by reacting or decomposing to light: a substance that reacts or decomposes with light to generate a stimulating substance is added to the medium in advance, and pulsed light (illumination light ) Is irradiated to part or all of the medium. Then, the sample is stimulated by the generated stimulating substance, and therefore, when X-ray observation is performed after a predetermined time, the dynamic behavior of the sample with respect to the stimulating substance can be observed. At this time, it is preferable that the pulsed light (illumination light) has a wavelength with the highest efficiency for causing photoreaction or photolysis, a wavelength in the vicinity thereof, or a spectral distribution including the wavelength.

Next, a specific example will be described. <Caged A
Example of TP> Caged ATP generally does not react with the ATP binding site in a protein molecule (sample).
However, when Caged ATP is irradiated with ultraviolet light, it decomposes into ATP and 2-nitrosoacetophenone. The generated ATP reacts with the ATP binding site and binds. The wavelength effective for this photolysis is 315 nm. Therefore, as a sample, a medium containing protein molecules is pre-caged.
If ATP is added to the medium and the medium is irradiated with "illumination light containing light of 315 nm" from the illumination means and observed with an X-ray microscope after a predetermined time, the dynamic behavior of the sample (protein) with respect to ATP can be observed. it can. (3) When the sample is a photoreactive organism: In this case, a part or all of the medium or a part or all of the X-ray observation region is irradiated with pulsed light (illumination light). Since the sample is stimulated by this, the X-ray observation after a predetermined time allows observation of the dynamic behavior of the sample in response to light. At this time, it is preferable that the pulse light (illumination light) has a wavelength at which the sample reacts most violently, a wavelength in the vicinity thereof, or a spectral distribution including the wavelength. (4) When a fluorescent reagent or an absorptive reagent is administered to the sample or medium: In this case, the spectrum has a wavelength at which the fluorescent efficiency or absorptive efficiency of the reagent is highest, a wavelength in the vicinity thereof, or a wavelength including the wavelength. Illuminating light having a distribution is applied to a sample or a medium. The sample is observed with an optical microscope as it is or immediately after irradiation. Then, with the sample set in the sample holder, the state of the sample can be observed immediately before the observation with the X-ray microscope. (5) When the illumination light is “infrared light or light having a spectral distribution in the infrared region”: In this case, the temperature of the irradiation region (all or part of the X-ray observable region) can be raised. Thus, if the irradiation area is set to a temperature that the sample most prefers, the sample can be collected there. Alternatively, the active sample can be observed by setting the irradiation area to the temperature at which the sample is most active.

[0020]

Second Embodiment When the sample holder of the first embodiment is used,
An observation method using an X-ray microscope will be described. First, a sample capsule is prepared, and the sample is put in the medium together with the medium. This sample capsule is held in the sample holder via the O-ring. At this time, the center of the window (6) of the sample capsule and the center of the opening (4) of the sample holder are aligned.
Then, the sample holder and the illumination means (2) are set on the X-ray microscope.

The illumination means (2) is turned on, and the sample or medium is illuminated (irradiated) through the opening (4) and the window (6) of the sample holder. In this case, the illumination area may be local (extremely narrowed down to about 1 μ spot diameter) or the whole, if desired.

[0022]

[Embodiment 3 ... Embodiment of the invention of claim 5] FIG. 2 is a schematic vertical sectional view showing a "sample holder holding a sample capsule in a capsule storage chamber" according to the present embodiment. The sample capsule is the same as the capsule of Example 1 (FIG. 1). The feature of this embodiment is that the holder body has an optical waveguide structure. Therefore, the holder has a ring-shaped spacer (1
c) and a main holder body (1a) sandwiching the c) and a holding holder body (1b). These three members are shown in FIG.
When joined by an appropriate joining means (not shown) for tightening from above and below, a capsule storage chamber for the sample capsule is formed inside. There are openings for X-ray transmission in the centers of the main bodies (1a) and (1b). Body (1a), (1
b) is a parallel plate composed of a transparent material such as glass, and has a reflective layer (for example, aluminum, gold,
Metal plating such as silver or vapor-deposited thin film or dielectric multilayer film) is coated on each. Therefore, these main bodies (1a) and (1b) have an optical waveguide structure. The reflective layer is not shown in FIG.

The illuminating means (2) is also the same as that of the first embodiment, and the optical fibers hitting the tip portions are the main bodies (1a), (1).
There are a total of four locations near the end face of b). But,
The lighting means (2) may be provided at one place. The illuminating light (3) emitted from the optical fiber is supplied to the main body (1a), (1
An oblique end face (1f) that penetrates through b) and faces the opening
Emitted from At this time, since the end face (1f) is inclined toward the sample capsule, the light emitted from the end face (1f) illuminates the sample capsule. The diagonal end surface (1f) is a smooth surface or a ground glass surface.

The optical waveguide structure can be formed only by a transparent material without providing a reflective layer. Body (1a),
Each surface of (1b) faces the vacuum space. The transparent material forming the main body has a high refractive index, and the vacuum space has a low refractive index. Therefore, the light transmitted (propagated) through the body is totally reflected at the interface between the transparent material (body) and the vacuum space. Even if total internal reflection does not occur, a part is reflected. Therefore, the body (1
The illumination light incident from the end faces of a) and (1b) can be emitted from the oblique end face (1f) facing the opening while repeating (total) reflection therein. When light leaks out without being reflected at the interface between the transparent material (main body) and the vacuum space, and when this light leakage is inconvenient, a light shielding coating may be provided on the front surface and the back surface. For the light-shielding coating, for example, black paint or black chrome plating is used.

The illuminating means (2) is shown in FIG.
It is installed at the same position as a) and (1b), and light is incident from each end face of the main body. However, the means (2) may be provided at another position for illumination, for example, above or below, and light may be incident from the front surface or the back surface of the main bodies (1a), (1b). The light emitting surface is not limited to the oblique end surface (1f) facing the opening, and may be provided at any position on the back surface of the main body. The number of optical fibers is not limited to one and may be plural. In this case, the incident positions on the main body may be dispersed at a plurality of positions. Further, when a plurality of optical fibers are provided, the wavelength of the light emitted from each may be changed, and the light may be further divided into continuous light and pulsed light.
In FIG. 2, an illuminating means including a light source and an optical fiber (an optical fiber of 29 is shown, but a light source (for example, a light emitting diode, a semiconductor laser, an incandescent lamp) itself may be installed instead of the optical fiber.

[0026]

[Embodiment 4 ... Embodiment of the invention according to claim 6] Fig. 7 is a schematic vertical sectional view showing a "sample holder holding a sample capsule in a capsule storage chamber" according to the present embodiment. The sample capsule is the same as the capsule of Example 1 (FIG. 1).
The holder is basically the same as that of the third embodiment, but the feature is that the light source itself as the illumination means (2) is embedded in a part of the main body. That is, the holder is a ring-shaped spacer (1
c) and a main holder body (1a) sandwiching the c) and a holding holder body (1b). The main body has a reflective layer (not shown) and has an optical waveguide structure. Here, lighting means (2)
That is, the light source is a light emitting diode. The light emitting diode is driven by a drive circuit (14), and a wiring (15) connects the two.

Light is emitted from the light emitting diode by the drive circuit (14), and the light is a main body (1) having a waveguide structure.
a), propagates inside the main body (1b), exits from the oblique end face (1f) facing the opening, and illuminates the sample chamber (13). The light emitting diode may be provided in one place instead of four places as shown in FIG. The light emitting diode is
a), it may be provided not on the surface of the main body (1b) but on the back surface or the end surface. The light emitting diodes may be distributed and provided in a plurality of places. When providing a plurality of light emitting diodes,
The wavelength of the light emitted from each may be changed, and may be further divided into continuous light and pulsed light. A semiconductor laser may be installed instead of the light emitting diode.

[0028]

According to the present invention, by irradiating with light or by irradiating with light in advance, it is possible to observe while or immediately after stimulating the sample. Therefore, in the case where the sample is an organism or a part thereof which is stimulated by light, the sample can be observed in a lively and live state even if it takes time to observe.

Further, since the observation is performed while stimulating with light, the biological sample can be manipulated to some extent. For example, the samples can be collected (observed at high density) in an observable region and observed. In addition, the present invention makes it possible to observe, for the first time, what kind of light the sample shows and how it reacts. Other effects will be apparent from Examples 1 to 4.

[Brief description of drawings]

FIG. 1 is a conceptual diagram showing a schematic vertical cross section of a sample holder (a state in which a sample capsule is held) according to a first embodiment of the present invention.

FIG. 2 is a conceptual diagram showing a schematic vertical section of a sample holder (a state in which a sample capsule is held) according to the second embodiment.

FIG. 3 is a conceptual diagram showing a simple structure of an X-ray microscope.

FIG. 4 is a graph showing a spectrum of an X-ray absorption coefficient.

FIG. 5 is an exploded perspective view (outline) of a sample capsule (non-open type).

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

FIG. 7 is a conceptual diagram showing a schematic vertical cross section of a sample holder (a state in which a sample capsule is held) according to a third embodiment.

FIG. 8 is a conceptual diagram showing a schematic vertical cross section of a conventional sample holder (a state in which a sample capsule is held).

[Explanation of symbols]

 1a Main Holder Main Body 1b Holding Holder Main Body 1c Spacer 2 Illuminating Means 3 Illuminating Light 4 Opening Port for X-ray Transmission 5 O-ring 6 Window Hole for X-ray Transmission 7 Spacer 8 Substrate 8a X-ray Transmission Film 12 Capsule Storage chamber 13 Sample chamber 14 Drive circuit 15 Wiring B Sample capsule (7 + 8 + 8a) H Sample holder (1a + 1b or 1a + 1b + 1c) 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

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Hideaki Shimizu, 1-4, Umezono, Tsukuba-shi, Ibaraki Prefectural Institute of Industrial Technology Electronic Technology Research Institute (72) Toshikazu Majima 1-4-1, Umezono, Tsukuba-shi, Ibaraki Prefecture Electronic Technology Research Institute, Industrial Technology Institute (72) Inventor Hiroyuki Kondo 1-6-3 Nishiooi, Shinagawa-ku, Tokyo Nikon Oi Manufacturing Co., Ltd.

Claims (6)

[Claims] 1. A method for observing a "biological sample suspended in a medium" using an X-ray microscope, wherein the biological sample or its peripheral portion or the medium is irradiated with light or immediately after irradiation. A method comprising observing the biological sample. 2. Illumination capable of illuminating part or all of the sample chamber or its periphery in a sample holder that holds a "sample capsule having a sample chamber" and separates the sample chamber from the surrounding vacuum space. A sample holder provided with means. 3. The sample holder according to claim 2, wherein the illumination means comprises an external light source and an optical fiber. 4. The sample holder according to claim 3, wherein the optical fiber has a condensing optical element at a tip thereof. 5. A sample holder for holding a “sample capsule having a sample chamber” and isolating the sample chamber from the vacuum space, wherein at least a part of the holder main body has an optical waveguide structure. Sample holder. 6. The sample holder according to claim 5, wherein a light source is provided in a part of the optical waveguide structure.