JPH07253500A - X-ray microscope - Google Patents

Abstract

PURPOSE:To provide an X-ray microscope which can reduce the influence on a biological sample during observation by providing a visible-light observing mechanism which can observe the sample without dyeing. CONSTITUTION:An X-ray microscope is provided with a light source section which generates X rays and visible light, lighting optical system 300 which condenses the X rays and visible light, enlarging optical system which forms the images of the X rays and visible light transmitted through a sample 4, and image pickup section which takes the pictures of the formed images of the X rays and visible light and the lighting optical system 300 is constituted by combining and uniting two kinds of visible light lenses 10 and 11 having different NAs and an X-ray mirror 3. The enlarging optical system is equipped with two objective lenses 8 and 9 which have different magnifications and can be switched to each other and an X-ray mirror 5 corresponding to the system 300 and a switching section which switches the lenses 8 and 9 and mirror 5.

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, and more particularly to an X-ray microscope capable of observation using X-rays and visible light.

[0002]

2. Description of the Related Art In the fields of medicine and bio-optics, which are rapidly advancing in recent years, ordinary visible light (wavelength λ = about 400 nm to 800 nm) is used.
The resolution is higher than that of a microscope using m), and it is also a high resolution that allows live samples (hereinafter referred to as biological samples) such as cells, bacteria, sperms, chromosomes, mitochondria, and flagella to be clearly observed. The demand for a microscope is increasing day by day.

The reason for this is that such a biological sample could not be seen with a conventional high resolution electron microscope. Therefore, in order to enable the observation of such biological samples, 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 developed specifically.

For example, FIG. 4 shows a simple structure and optical system of such an X-ray microscope. In FIG. 4, the X-rays emitted from the X-ray generator 1 are condensed by the X-ray illumination optical system 3 and irradiated on the sample capsule 27 holding the sample 4. Then, the X-ray transmitted through the sample 4 forms an image of the sample 4 on the X-ray imaging device 7 by the X-ray magnifying optical system 5. Here, the optical path length from the X-ray generator 1 to the X-ray imaging device 7 is, for example, about 2 m. Further, 26 is a vacuum container for a lens barrel, 25 is an exhaust device for evacuating the container, and 24 is a display device for displaying an image of the sample 4 detected by the imaging device 7.

However, the X-ray microscope based on such a structure has some problems at present.

In the soft X-ray region, the absorption of X-rays by a substance changes as shown in FIG. 5, depending on the wavelength of the X-rays, the atomic number of the substance, and the like. Generally, the longer the X-ray wavelength, the easier the absorption, and the larger the atomic number of the substance, the easier the absorption. Particularly, in the X-ray wavelength range of 23 to 40 Å, oxygen, that is, water, has a higher transmittance than organic substances containing carbon such as proteins.

In other words, the difference in absorption rate between water and protein gives contrast, and the structure of cells in water can be distinguished without staining. This wavelength region is called a water window and is a very useful region for a biological microscope. Therefore, the X-ray microscope has 2
If X-rays of ~ 5 nm are used, organisms have various characteristics such as high resolution and no staining.

On the other hand, when observing a sample such as a living thing with an X-ray, the photon energy of the X-ray is larger than that of visible light by one digit or more, so that high resolution can be obtained, but one photon has a large energy. Therefore, the sample is easily damaged. Therefore, when actually observing a living thing or the like with X-rays, the X-ray microscope and the visible light optical microscope are arranged at the same time in order to bring the desired portion into the visual field of the X-ray microscope in advance. That is proposed by, for example, Japanese Patent Laid-Open No. 4-346100, and is actually being carried out.

[0009]

However, in the construction of the conventional X-ray microscope which also has an optical observation means for visible light, when observed with visible light, the biological sample looks almost colorless and transparent, and It is very difficult to find the part you want to observe with a microscope. It may be possible to stain a biological sample for clear observation with an optical microscope, but this does not take advantage of the unstained observation of an X-ray microscope, and the staining affects the biological sample. There was a problem.

In view of the above problems, the object of the present invention is to
It is an object of the present invention to provide an X-ray microscope capable of reducing the influence of a sample during observation by providing a visible light observing means capable of observing a biological sample without any treatment.

[0011]

The above-mentioned object is to provide an X-ray source for generating X-rays, an X-ray illumination optical system for condensing the generated X-rays at a predetermined condensing position, and the condensing position. A visible light source for generating visible light in an X-ray microscope including an X-ray magnifying optical system for imaging X-rays transmitted through a sample arranged in , A visible light illumination optical system that focuses the generated visible light at the same position as the focus position of the X-ray illumination optical system and a visible light that forms an image of the visible light that has passed through the sample placed at the focus position. A light-magnifying optical system, a visible-light imaging device that captures the formed visible-light image, and a generated light for observation using the converted light that responds to and is affected by the intrinsic optical properties of the sample. It absorbs visible light to generate converted light and transmits visible light through the sample. It is achieved by X-ray microscope and having a viewing aids mechanism for re-converting the converted light to be imaged to the image device.

[0012]

The reason why the X-ray microscope according to the present invention can overcome the above problems and achieve the above objects will be described below.

When observing a biological sample such as a cell with an X-ray microscope, the biological sample such as the cell is sandwiched by a film, such as SiN, which is transparent to X-rays and visible light. . Since X-rays naturally cause radiation damage and are harmful to the biological sample to be observed,
When observing with an X-ray microscope, it is desirable to minimize the damage by minimizing the X-ray irradiation dose.

Therefore, among the biological samples to be observed,
It is desirable to specify the region to be observed with the X-ray microscope in advance and bring it into the field of view of the X-ray microscope. That is, the sample is aligned by an X-ray microscope and an optical observation means using the visible light coaxially arranged, for example, an optical microscope.

However, biological samples such as untreated (non-stained) cells which are observed by making use of the characteristics of an X-ray microscope are
When observed with an ordinary visible light optical microscope, most of them are transparent and very difficult to observe.

Therefore, by using the visible light optical microscope function provided in the X-ray microscope according to the present invention, the observation region can be aligned with visible light without dyeing the sample. In the present invention, this optical microscope function is realized by a visible light source, a visible light illumination optical system, a visible light magnifying optical system, a visible light imaging device, and an observation assisting mechanism.

The observation assisting mechanism executes observation using visible light using any one of differential interference observation, phase difference observation, modulation contrast observation, or a combination thereof. Therefore, even for biological samples such as transparent cells, it is possible to add contrast by using visible light interference, phase difference, and modulation contrast, which are caused by the structure of the biological sample, without staining treatment. The characteristics of the sample can be observed in detail, and the observation region can be easily specified.

Furthermore, compared with X-rays, visible light causes almost no damage to biological samples, so that it can be observed for a long time.

For example, in the X-ray optical system and the visible light optical system, the same place is observed with respect to the sample,
It can be switched at any time or can be arranged coaxially. According to such a configuration, the sample to be observed is observed using the visible light optical system, the region to be observed with the X-ray optical system is brought to the center of the visual field, and the X-ray optical system is switched to quickly. An X-ray magnified image of the target observation region can be obtained.

Therefore, it is not necessary to observe with an X-ray for a long time in order to find a desired observation region, and damage due to X-ray can be suppressed to a minimum. In addition, by utilizing the difference between the NA of the optical element of the X-ray optical system and the NA of the optical element of the visible light optical system, these optical elements are integrated and used at the same time, so that the same observation area can be obtained. , X-ray image and visible light image can be observed simultaneously.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment of an X-ray microscope to which the present invention is applied will be described with reference to the drawings. FIG. 6 shows a block configuration of the present embodiment, and FIG. 1 is an explanatory diagram showing a specific configuration example of the X-ray and visible light optical system in the present embodiment.

In this embodiment, as shown in FIG. 6, as an optical system, a light source section 100 for generating X-rays and visible light, and an illumination optics for condensing the X-rays and visible light generated by the light source section 100. The system 300, a sample holder 27 that holds the biological sample 4 so that the collected X-rays and visible light are irradiated to the biological sample 4, and a predetermined focus for the X-rays and visible light that have passed through the sample 4. Enlarging optical system 500 for forming an image at a position
And an imaging unit 700 that takes in the formed X-ray image and visible light image as an image.

This embodiment further includes a vacuum container 26 for accommodating at least a portion of the above optical system related to X-rays and holding it in a vacuum, and an exhaust device 25 for exhausting the inside of the vacuum container. The output device 24 displays an image captured by the image capturing unit 700.

The present embodiment further includes switching units 120, 520, and 720 for switching between X-rays and visible light in the light source unit 100, the magnifying optical system 500, and the image pickup unit 700, which will be described in detail below. Switching unit control device 800 for performing operation control, and the switching unit control device 80
0, the light source unit 100, the imaging unit 700, the exhaust device 25, and the microscope control device 90 that controls the operation of the output device 24.
Has 0 and.

The light source unit 100, as shown in FIG.
X-ray generator 1 for generating a line, a visible light source 16 for generating a visible light, a polarizing means 110 for observing the biological sample 4 without staining by polarizing the generated visible light, an X-ray and a visible light. It has an optical path switching unit 120 for switching to and from light.

In this embodiment, the visible light source 16 is mounted on the outside of the vacuum container 26, and the generated visible light is a visible light introducing window 26a attached to the vacuum container 26.
Is projected into the vacuum container 26.

The polarization means 110 has a polarizer 12 that receives the visible light generated by the visible light source 16 and changes its polarization state, and a Wollaston prism 13. Together with the polarization means 710 of the imaging unit 700 described below, these realize observation by the differential interference method for observing the biological sample 4 without staining.

The optical path switching unit 120 comprises the polarization means 110.
It has a visible light mirror 2 for projecting visible light polarized by the illumination optical system 300, and an actuator (not shown) for changing the installation angle of the visible light mirror 2. In this embodiment, the mirror 2 is rotated along the direction of the arrow near the mirror 2 shown in FIG. 1, and X-rays and visible light are switched as the light projected onto the illumination optical system 300. The switching unit 120 only needs to be able to switch the light projected onto the illumination optical system 300, and may have another structure such as a structure for moving the mirror 2 in the direction perpendicular to the optical axis 1000.

The illumination optical system 300, as shown in FIG.
A combination of two kinds of visible light lenses having different NAs (numerical apertures) and an X-ray mirror for collecting incident visible light and X-rays to a light collecting position where the biological sample 4 is arranged. , An integrated illumination optical system.

That is, the optical system 300 has the optical axis 10
00, the high NA illumination system lens unit 10 mounted around the X-ray mirror unit 3 so as to have the same optical axis 1000, and the same optical axis 100.
And a low NA illumination system lens unit 11 mounted in the central space of the X-ray mirror 3 so as to have zero.

The sample holder 27 holds a sample capsule for fixing the biological sample 4 having a light entrance window and an exit window made of a member transparent to visible light and X-rays used. is there.

The magnifying optical system 500 corresponds to the illuminating optical system 300, and three magnifying optical systems for imaging visible light and X-rays transmitted through the biological sample 4 and any one of these optical systems. And an optical system switching unit 520 for selecting one of them and placing it on the optical axis 1000.

As these optical systems, an oblique incidence reflection type X-ray mirror 5 corresponding to the X-ray mirror section 3 of the illumination optical system 300 as the X-ray system 5 and a visible light system 510 are provided. The visible light system 510 includes the illumination optical system 300.
High-magnification objective lens 8 corresponding to the lenses 10 and 11 of
And a low-magnification objective lens 9.

An example of the configuration of the optical system switching section 520 is shown in FIG.
Shown in.

The optical system switching section 520 holds the X-ray mirror 5 and the optical system holding member 5 for holding the lenses 8 and 9.
25, a drive mechanism 523 that drives the optical system holding member 525 in the direction perpendicular to the optical axis 1000, and the amount of movement of the drive unit 523 to control the optical axis 1000, the X-ray mirror 5, and the optical components of the lenses 8 and 9. Drive control device 5 that matches the axis
24 and. The arrow shown in the upper part of FIG. 7 indicates the moving direction of the optical system holding member 525.

The optical system holding member 525 is used for aligning the optical axis of the lens 9 with the lens holding member 9a for holding the objective lens 9, and is located at a predetermined relative position with respect to the optical axis center of the lens 9. Position sensor light emitting part 9 to be arranged
and a hole 9b for passing the imaging light of the lens 9 through the optical axis of the lens 9.

The optical system holding member 525 has the same structure as the above with respect to the X-ray mirror 5 and the objective lens 8. That is, the holding member 525 includes the mirror holding member 5a, the objective lens holding member 8a, and the position sensor light emitting units 5c and 8c, and the holes 5b and 8b for passing the imaging light from the respective optical elements. To form.

The drive unit 523 has a drive mechanism 523a having a drive surface for holding the optical system holding member 525 having the above-described structure so as to be movable in a direction perpendicular to the optical axis 1000, and a drive source 523b of the drive mechanism 523a. .

The drive mechanism 523a is the optical axis 10 of this embodiment.
00 (see FIG. 1) and a position sensor light receiving section 522 having a light receiving element installed at a predetermined relative position, and an optical axis 1000 for passing the image forming light by each optical element. A hole 521 formed as the center is formed. In FIG. 7, corresponding to FIG. 1, the X-ray mirror 5 is selected as the magnifying optical system.

In this embodiment, each optical element (mirror 5
Alternatively, in the alignment of the lenses 8 and 9), the drive control unit 524 is configured so that the light receiving unit 522 detects the light emission beam generated by each light emitting unit of the position sensor light emitting units 5c, 8c, and 9c.
However, the amount of movement of the optical system holding member 525 is controlled by controlling the amount of drive of the drive source 523b. In the present embodiment, when the position sensor light emitting portion and the light receiving portion coincide, the holes 5b and 8 are formed.
b or 9b center line (optical axis of each optical element),
The configuration is such that the center line (optical axis 1000) of the hole 521 coincides. Here, by limiting the light receiving area of the light receiving section 522, it is possible to improve the alignment accuracy.

In the present embodiment, the alignment is performed by using the optical means as described above, but the alignment may be performed by a mechanical means such as a limiter or a magnetic means. Further, in the present embodiment, the switching unit 520 as shown in FIG. 7 is used, but the present invention is not limited to this, and an optical element such as an X-ray mirror or a visible light lens arranged along the optical axis 1000. The configuration and the form of the drive mechanism to be used are not limited as long as they can be switched. For example, a revolver mechanism used to replace the objective lens of the optical microscope may be used.

As shown in FIG. 1, the image pickup section 700 includes an optical path switching section 720 for switching the optical path of the image forming light depending on the type of the image forming light received from the magnifying optical system 500, and a polarization for polarizing the image forming light of visible light. It has a means 710, a visible light imaging device 17 for capturing a visible light image, and an X-ray imaging means for capturing an X-ray image.

The optical path switching unit 720 has the same structure as the optical path switching unit 120, and has a visible light switching mirror 6 and an actuator for rotating the mirror 6. The polarization means 710 has a configuration corresponding to that of the polarization means 110, and includes a Wollaston prism 14 and an analyzer 15 for performing observation by the differential interference method for observing the biological sample 4 without staining. Have.

The visible light image pickup device 17 has a CCD camera, is arranged outside the vacuum container 26, and captures a visible light image that has passed through a visible light emission window 26b provided in the vacuum container 26. The X-ray imaging device 7 has a CCD camera for X-rays and captures an X-ray image.

The output device 24 has an image processing unit for performing image processing on the images captured by the image pickup devices 7 and 17, and a display unit for displaying the processed image data. Alternatively, the image processing unit and the display unit may not be provided, and the data may be output to an external image display device.

The switching unit control device 800 receives from the outside a selection command as to whether to use X-rays or visible light, and based on this, the arrangement of the mirrors 2 and 6 in the optical path switching units 120 and 720 and the optical system switching unit. The arrangement of the optical element used in 520 is switched.

The microscope control device 900 is composed of an information processing device such as a computer, and has an input unit for accepting selection of an optical system to be used, a magnification to be used, an observation method using visible light, etc. from the outside. And a control unit that controls the operation of each unit based on these commands.

Next, the operation of this embodiment will be described with reference to FIG.

First, an X-ray optical system for obtaining an X-ray microscope image of the sample 4 will be described. When the X-ray generator 1 generates soft X-rays, the X-ray illumination system 3 collects the X-rays and illuminates the sample 4. In this embodiment, as shown in FIG. 1, the oblique incidence reflection type X-ray mirror section 3 having high efficiency is used as an illumination system. Similarly, the X-rays that have passed through the sample 4 are magnified by the X-ray magnifying optical system 5 using a grazing incidence reflection type X-ray mirror, and the X-ray image pickup device 7 picks up an image to obtain a magnified image by X-rays. . In this embodiment, an oblique-incidence reflection type X-ray mirror is used as the X-ray optical element in the X-ray optical system, but of course, a direct-incidence multilayer mirror, zone plate, or the like may be used.

The reflectance of the grazing incidence type X-ray mirror depends on the grazing incidence angle, and in general, the larger the grazing incidence angle, the lower the reflectance. Particularly, for X-rays of 2 to 5 nm, the oblique incidence angle of 40 to 50 mrad provides a relatively high reflectance, which is suitable for obtaining a microscope image. If the magnification of the X-ray optical system is set to several tens of times and a double reflection optical system is adopted, the NA will be 0.
It becomes an annular zone opening of about 15 to 0.2.

Next, a visible light optical system for obtaining a visible light image of the sample 4 by visible light in this embodiment will be described.

First, the light used for observation is switched from X-ray to visible light by changing the arrangement of the switching mirrors 2 and 6. Further, the optical system switching section 520 switches the magnifying optical system to be used from the X-ray system 5 to the optical system 510. At this time, the objective lens 8, depending on the magnification used for observation,
Either one of 9 is selected.

When the visible light source 16 generates visible light, the polarizer 12 and the Wollaston prism 13 convert the visible light generated by the visible light source 16 so that the polarization directions are orthogonal to each other and the light beam itself is slightly displaced. 2 wave fronts are formed. This light is reflected by the mirror 2 and is applied to the biological sample 4 via the visible light units 10 and 11 of the illumination optical system 300.

Next, the light, which has undergone a phase change by passing through the sample 4, enters the Wollaston prism 14 and the analyzer 15 via the magnifying optical systems 8 and 9 for visible light and the mirror 6. . Here, when the visible light having these two wavefronts is returned to the original state and interfered with each other, the visible light image pickup device 17 captures an observation image showing the phase change due to the contrast according to the phase change received by the sample 4. .

In the present embodiment, the visible light optical system and the X-ray optical system are coaxially arranged so as to coincide with the optical axis 1000. Therefore, by providing the switching units 120 and 720, the sample 4 can be obtained. Without dyeing the optical axis 10
Both a visible light image and an X-ray image in the same observation region of the sample 4 placed at 00 are obtained. In this embodiment, the case of first obtaining the X-ray image and then the visible light image has been described, but it goes without saying that the order may be reversed.

Further, since the visible light image and the X-ray image are acquired on the same optical axis 1000, the sample can be identified by comparing them with each other, which is very convenient. Of course, a prism or the like may be removed to carry out normal visible light optical microscope observation, or a ring aperture and a ring phase plate may be inserted to perform observation using the phase contrast microscopic method described below.

In this embodiment, when obtaining an optical microscope image with visible light, it is necessary to take a large NA in order to obtain resolution. Therefore, the NA of the objective lens with high magnification, for example, several tens of times, is as large as 0.7 or more. Further, NA of a low-magnification objective lens of about 10 times is generally 0.25 to 0.3. Even under this condition, the working distance is usually sufficient, but NA may be set to 0.2 or less in order to increase the working distance.

Considering the above, as the illumination optical system 300, as shown in FIG. 1, the high NA illumination system lens unit 10 for visible light is arranged around the X-ray mirror unit 3, and the illumination system lens for low NA is provided. It is found that the configuration in which the portion 11 is arranged at the center of the X-ray mirror portion 3 is preferable. On the other hand, in the magnifying optical system 500, the X-ray magnifying optical system 5 is moved to the visible light high-magnification objective lens 8 by the optical system switching unit 520 depending on the observation magnification and the application.
Alternatively, a configuration in which the visible light low-magnification objective lens 9 is replaced and used is suitable as a visible light optical system.

That is, according to this structure, the X-ray optical system and the visible light optical system are arranged on the same optical axis 1000, and at least the low-magnification objective lens 9 and the high-magnification objective lens 9 are arranged. It is possible to observe with three or more optical systems of the lens 8 and the X-ray objective mirror 5.

For example, in the configuration of this embodiment, sample 4
Of visible light rays and X-rays that pass through, the NA of 0.2 to 0.7 is distributed to the high-magnification objective lens 8 which requires high NA, and the NA of 0.15 to 0.2 is distributed to the X-ray objective mirror 5. Further, NA of 0.15 or less is distributed to the low magnification objective lens 9.

In this embodiment, in observing visible light, the Wollaston prisms 13 and 14, the polarizer 12 and the analyzer 15 are put in and the differential interference method is executed. As a result, a visible light image can be acquired even for a sample 4 such as a biological sample that is transparent to visible light. Here, the differential interference method is a method of converting information of the phase difference, which is usually invisible to the eyes, into the contrast by the interference method and observing.

Although an example using a Wollaston prism is shown here, a Normaski prism may be used instead. Further, in the present invention, the mechanism for performing the differential interference observation is not limited to the configuration of the present embodiment, and may be any configuration as long as the differential interference observation can be performed in the X-ray microscope.

According to this example, even in the biological sample 4 which is almost transparent to visible light, it is possible to obtain a visible light optical microscope image on the same optical axis as the X-ray image without staining.
Prior to the acquisition of the line image, the observation region can be easily specified, and the X-ray dose applied to the biological sample 4 can be minimized.

In this embodiment, two visible light optical elements having different magnifications and NAs are used, but the type and number of visible light optical elements used are not limited to this. The present invention is capable of observing a sample using visible light without staining, and further, if a visible light optical microscope image can be acquired on the same optical axis as the X-ray optical system, the number of visible light optical elements to be used and its type can be arbitrarily set. Can be set.

Next, another embodiment of the X-ray microscope to which the present invention is applied will be described with reference to FIG.

The present embodiment is an X-ray microscope in which an optical system including an optical element from a light source to an image pickup device, which is independent of each other, for X-ray and for visible light is provided, and which can be switched to each other. is there.

As in the first embodiment, the constitution of this embodiment is the same as that of the first embodiment. As an optical system, a light source unit for generating X-rays and visible light, and an illumination for collecting X-rays and visible light are provided. An optical system, a sample holding unit for holding the biological sample 4 arranged at a condensing position, a magnifying optical system for forming an image of X-rays and visible light transmitted through the sample 4, and an imaged X-ray image And an image pickup unit that takes in a visible light image as an image.

In addition to the first embodiment, this embodiment further includes a vacuum container 26 for accommodating at least the X-ray-related part of the optical system and holding it in a vacuum.
An exhaust device for exhausting the inside of the vacuum container, an output device for displaying an image captured by the imaging unit, and an X described in detail below.
It has a switching unit control device for switching between a line and visible light, and a switching operation control for a visible light microscopic method to be used, and a microscope control device for controlling the operation of each of the above constituent elements.

In this embodiment, as compared with the first embodiment,
Although the configurations of the light source unit, the illumination optical system, the magnifying optical system, and the image capturing unit are different, the same configuration can be used for the remaining portions. Here, the description of the same constituents as those of the first embodiment will be omitted.

The light source unit is, as shown in FIG.
And a visible light source 16 and a light source switching unit that moves one of these light sources and arranges one of them on the optical axis 1000 that passes through the installation position of the sample 4. The light source unit further includes a polarizer 12 and a Wollaston prism 13 for differential interference observation, and a ring aperture 20 for phase difference observation.
And a slit 30 for oblique illumination for modulation contrast observation, and a polarizer 12, a Wollaston prism 13, a ring aperture 20, and a slit 30 for switching between these observation methods, on the optical axis 1000. And a filter switching unit to be arranged.

Here, the phase contrast microscopic method is a method of observing the phase difference caused by the biological sample 4 by changing it into a change in light intensity. In the present embodiment, the ring opening 20 and the ring phase plate 21 described below allow the light that has undergone the phase change to
A reference wave having a phase difference of ± π / 2 is caused to interfere with the sample. In addition, in modulation contrast microscopy,
The sample 30 is obliquely illuminated by the slit 30, and the contrast of the sample 4 is emphasized by the pattern plate 31 described below.

The illumination optical system is such that the oblique incidence reflection type X-ray mirror 3 as the X-ray illumination optical system, the lens 18 as the visible light illumination optical system, and either one of them coincide with the optical axis 1000. And an illumination optical system switching unit for moving.

Similar to the illumination optical system, the magnifying optical system uses an oblique-incidence reflection type X-ray mirror 5 as an X-ray magnifying optical system, a lens 19 as a visible light magnifying optical system, and either one of them. And a magnifying optical system switching unit that moves the optical axis so as to coincide with the axis 1000.

The imaging section stripes at the position of the pupil of the Wollaston prism 13 and the analyzer 15 for differential interference observation, the ring phase plate 21 for phase difference observation, and the lens 19 for modulation contrast observation. Pattern plate 31 on which a circular pattern is arranged, and an analyzer 15 and a Wollaston prism 1 for switching between them.
4, a ring phase plate 21, and a filter switching unit that switches the pattern plate 31 and arranges it on the optical axis 1000. The imaging unit further includes an X-ray imaging device 7, a visible light imaging device 17, and an imaging device switching unit that moves one of these imaging devices and arranges one of them on the optical axis 1000.

A light source switching unit, an illumination optical system switching unit,
The enlarging optical system switching unit and the imaging device switching unit have the same configuration except the optical element to be held.

For example, as shown in FIG. 8, the magnifying optical system switching unit drives the optical system holding member 525 holding the X-ray mirror 5 and the lens 19 and the optical system holding member 525 in the direction perpendicular to the optical axis 1000. The optical axis 1000 is controlled by controlling the driving mechanism 523 that drives the optical axis 1000 and the movement amount of the driving unit 523.
And a drive control device 524 for matching the optical axes of the X-ray mirror 5 and the lens 19.

The optical system holding member 525 includes the X-ray mirror 5,
Lens holding members 5a and 19a for holding the objective lens 19
And the limit switch 5c, 19c, which is used for aligning the optical axis of the lens 19 and is arranged at a predetermined relative position with respect to the optical axis center of the lens 19, The holes 5b and 19b for passing the image formation light by the lens 19 are formed.

The drive unit 523 has a drive mechanism 523a having a drive surface for holding the optical system holding member 525 having the above-described structure so as to be movable in the direction perpendicular to the optical axis 1000, and a drive source 523b of the drive mechanism 523a. . Drive mechanism 523
A forms a hole 521 formed around the optical axis 1000 for allowing image formation light from each optical element to pass therethrough.

In this embodiment, under the control of the drive controller 524, the operation of the drive mechanism 523 is stopped when the limit switch 5c or 19c comes into contact with the optical system holding member 525. The optical axes of the X-ray mirror 5 and the lens 19 are aligned with the optical axis 1000 at the positions where the limit switches corresponding to them actuate and the optical system holding member stops.

Although FIG. 8 shows an example of only the enlarging optical system switching unit, it includes a light source switching unit, an illumination optical system switching unit,
Also, the same configuration can be used for the imaging device switching unit, but the specific configuration of the switching unit is not limited to this in the present invention. Further, these four switching units may be integrated to switch between the X-ray optical system and the visible light optical system.

Further, included in the light source section and the image pickup section,
The two filter switching units can basically have the same configuration as that shown in FIG. It suffices for these filter switching units to be capable of switching in conjunction with optical elements such as prisms according to the selected visible light observation method. Even if two switching units of the same structure are provided, these two are integrated. The structure may be changed.

Since the respective operations of the X-ray optical system and the visible light optical system of this embodiment are the same as those of the first embodiment, the description thereof will be omitted. However, in the present embodiment, the switching of the optical system is executed by replacing all of the light source, the illumination and magnifying optical system used, and the imaging device.

According to this embodiment, it is not necessary to separately use the NA as in the first embodiment. Therefore, the visible light optical system can have a sufficient image forming performance, and can have a simple structure. Furthermore, since the visible light and the X-ray observation are performed on the same optical axis 1000, the same region of the sample 4 can be observed with the X-ray and the visible light, so that the sample 4 can be easily identified. Further, by appropriately replacing the polarizer 12, the Wollaston prisms 13 and 14, the analyzer 15, the ring aperture 20, the ring phase plate 21, etc., optimal observation for a sample to be observed can be performed without performing a dyeing process. It becomes possible.

In this embodiment, the light source, the optical system and the like are moved with respect to the optical axis 1000 in order to switch between the X-ray and the visible light, but instead of this, the X including the light source and the image pickup device is used.
Sample 4 with line optical system and visible light optical system fixed
You may move the sample holding container holding.

Next, the third X-ray microscope to which the present invention is applied.
The embodiment will be described with reference to FIG. In this embodiment, the X-ray image and the visible light image can be observed at the same time without switching the optical path and the switching of the magnifying optical system in the first embodiment.

In this embodiment, in the magnifying optical system 500 of the first embodiment, as in the illumination optical system 300, the high-magnification visible light lens unit 8 on the high NA side and the X-ray of the annular NA are used. The mirror portion 5 and the low-NA low-magnification visible light lens portion 9 are coaxially integrated and used.

In this embodiment, visible light is further introduced by a visible light mirror 22 having a ring-shaped hole 22a for passing X-rays in a portion corresponding to the annular zone NA used in the X-ray optical system. An optical system for observing a visible light image through the visible light mirror 23 is adopted.

In this embodiment, the visible light mirror 22 is
The X-ray generated by the X-ray source is transmitted through the ring-shaped hole 22a, and the visible light generated by the visible light source is deflected (9
(0 degree reflection), and the two lights are combined and made incident on the illumination optical system that is coaxially and integrally arranged. In addition, the visible light mirror 23
Of the image-forming light transmitted through the sample 4 and imaged by the magnifying optical system, only the image-forming light by the X-ray mirror unit 5 is transmitted by the ring-shaped hole 23a and the image-forming light by the lens units 8 and 9 is transmitted. The image forming light is separated by deflecting. Each imaging device takes in these and acquires both an X-ray image and a visible light image.

Here, in the observation of visible light, the polarizer 12 and the Wollaston prism 1 are used as in the first embodiment.
Since the differential interference microscopy is used with the analyzers 3 and 14 and the analyzer 15, it is possible to observe even a non-stained sample with visible light.

In the present embodiment, since the visible light optical system and the X-ray optical system are optically separated, the X-ray and the visible light are generated at the same time, so that the position of the sample 4 is completely moved. It is possible to observe the optical image and the X-ray image at the same time without the need.

Even if these lights are not generated in the same manner,
Even if the X-ray and the visible light pulse are sequentially generated at a sufficiently fast time interval with respect to the movement speed of the biological sample itself to acquire the X-ray image and the visible light image, the same observation region of the sample 4 is obtained. On the other hand, it is possible to obtain observation images of X-rays and visible light almost at the same time. Furthermore, by switching the generated light, it is possible to switch between the X-ray image and the visible light image as the observation image to be acquired.

According to the present embodiment, it is possible to simultaneously acquire the observation images by the X-ray and the visible light, which facilitates the identification of the sample 4. Further, it is possible to switch between X-ray and visible light without using a movable part, and as a result, it is not necessary to align the optical axis when switching between X-ray and visible light.

In this embodiment, an optical element in which the NA region is divided into three is used for each optical system, but an optical system in which the low NA part is omitted or the high NA part is omitted is used. Good.

[0094]

According to the present invention, since a biological sample can be observed by visible light without staining, it is possible to provide an X-ray microscope capable of reducing the influence on the sample during X-ray observation. You can Further, the visible light optical system and the X-ray optical system, which are arranged on the same optical axis and have sufficient imaging performance, can easily specify the region to be viewed, and the X-ray irradiation dose can be minimized. it can.

[0095]

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a configuration concept of a first embodiment of an X-ray microscope according to the present invention.

FIG. 2 is an explanatory diagram showing a configuration concept of an X-ray microscope according to a second embodiment of the present invention.

FIG. 3 is an explanatory diagram showing a configuration concept of an X-ray microscope according to a third embodiment of the present invention.

FIG. 4 is an explanatory diagram showing a configuration of a conventional X-ray microscope.

FIG. 5 is a graph showing absorption of X-rays by a substance.

FIG. 6 is a block diagram showing constituent blocks in the first embodiment.

FIG. 7 is an explanatory diagram showing a configuration of an optical system switching unit in the first embodiment.

FIG. 8 is an explanatory diagram showing a configuration of a magnifying optical system switching unit according to a second embodiment.

[Explanation of symbols]

1 ... X-ray generator, 2 ... switching mirror, 3 ...
X-ray illumination optical system, 4 ... sample, 5 ... X-ray magnifying optical system, 6 ... switching mirror, 7 ... X-ray imaging device, 8 ... visible light high-magnification objective lens, 9 ... Visible light low magnification objective lens, 10 ... Visible light high NA illumination system lens, 11 ... Visible light low NA illumination system lens, 12 ... Polarizer, 13, 14 ... Wollast Prism, 15.
..Analyzer, 16 ... Visible light source, 17 ... Visible light imaging device, 18 ... Visible light illumination optical system, 19 ... Visible light magnifying optical system, 20 ... Ring aperture, 21 ... ..Ring phase plates, 22, 23 ... Visible light mirrors, 24 ...
Monitor, 25 ... Exhaust device, 26 ... Vacuum container,
27 ... Sample capsule.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Motohide Kageyama 3 2-3 Marunouchi, Chiyoda-ku, Tokyo Inside Nikon Corporation

Claims (13)

[Claims] 1. An X-ray source for generating X-rays, an X-ray illumination optical system for focusing the generated X-rays at a predetermined focusing position, and a sample placed at the focusing position. In an X-ray microscope including an X-ray magnifying optical system for forming X-rays and an X-ray imaging device for capturing the formed X-ray image, a visible light source for generating visible light, and a visible light source for generating X-rays. Visible light illumination optical system that focuses the light at the same position as the focus position of the line illumination optical system, visible light magnifying optical system that focuses the visible light transmitted through the sample placed at the focus position, and image formation A visible light imager that captures the generated visible light image and accepts the generated visible light for observation with the converted light that responds to and is affected by the intrinsic optical properties of the sample. Transforms the image to be transmitted through the sample and imaged on the visible light imager. X-ray microscope and having a viewing aids mechanism reconverts. 2. The observation assisting mechanism according to claim 1, wherein the observation assisting mechanism is used for any one of differential interference observation, phase difference observation, and modulation contrast observation, or a combination of a plurality of these observations. An X-ray microscope having a configuration for generating the converted light. 3. The observation assist mechanism for performing differential interference observation according to claim 2, further comprising a polarizer and a Wollaston prism arranged in the visible light illumination optical system, and the visible light. An X-ray microscope having a Wollaston prism and an analyzer arranged in a light magnifying optical system. 4. The observation assisting mechanism according to claim 2, which is for performing phase difference observation,
A ring aperture disposed in the visible light illumination optics,
An X-ray microscope having a ring phase plate arranged in the visible light magnifying optical system. 5. The observation assist mechanism for performing modulation contrast observation according to claim 2, wherein a slit for oblique illumination arranged in the visible light illumination optical system and the visible light magnifying device are provided. An X-ray microscope, comprising: a pattern plate having a stripe pattern arranged in an optical system. 6. The switching mechanism according to claim 2, wherein one of the X-ray magnifying optical system and the visible light magnifying optical system is moved so that its optical axis coincides with a predetermined optical axis of the microscope. Further, the X-ray illumination optical system and the visible light illumination optical system have a configuration in which they are coaxially arranged so that their optical axes coincide with the microscope optical axis. An X-ray microscope, characterized in that the optical system and the visible light magnifying optical system are configured to be movably held by a switching mechanism. 7. The X-ray magnifying optical system and the visible light magnifying optical system according to claim 2, wherein one of the X-ray magnifying optical system and the visible light magnifying optical system is moved so that an optical axis thereof coincides with a predetermined microscope optical axis, and A switching mechanism for moving one of the X-ray illumination optical system and the visible light illumination optical system so that the optical axis thereof coincides with the microscope optical axis, corresponding to the optical system coincident with the microscope optical axis. The X-ray magnifying optical system, the X-ray illuminating optical system, the visible light magnifying optical system, and the visible light illuminating optical system are configured to be movably held by a switching mechanism. An X-ray microscope characterized by the above. 8. The optical element used in the visible light magnifying optical system and the visible light illuminating optical system according to claim 6 or 7, wherein the NA of the optical element used in the X-ray magnifying optical system and the X-ray illuminating optical system. An X-ray microscope having an NA that does not match 9. The NA of the optical element for visible light is larger than the NA of the optical element for X-ray according to claim 8, wherein the observation assist mechanism is NA of the optical element for visible light. An X-ray microscope having a configuration in which any one of differential interference observation, phase difference observation, and modulation contrast observation is performed in a region. 10. The NA of the optical element for the visible light is smaller than the NA of the optical element for the X-ray, and the observation auxiliary mechanism has a NA of the optical element for the visible light. An X-ray microscope having a configuration in which any one of differential interference observation, phase difference observation, and modulation contrast observation is performed in a region. 11. The optical element according to claim 8, wherein the NA of the visible light optical element has both a larger area and a smaller area than the NA of the X-ray optical element. Two N of the visible light optical element
An X-ray microscope having a configuration in which any one of differential interference observation, phase difference observation, and modulation contrast observation is performed in the A region. 12. The sample according to claim 2, wherein either the X-ray generated by the X-ray source or the visible light generated by the visible light source is deflected to combine the X-ray and the visible light. Either the X-ray or the visible light that has passed through the sample on the microscope optical axis and the light integrating section that is incident along the predetermined microscope optical axis that passes through are separated to separate the X-rays. X
The optical imaging device further includes a light separation unit that allows visible light to enter the visible light imaging device, and the optical axes of the visible light illumination optical system and the X-ray illumination optical system coincide with the microscope optical axis. X-ray microscope, wherein the visible light magnifying optical system and the X-ray magnifying optical system are coaxially arranged so that their optical axes coincide with the microscope optical axis. . 13. The X-ray illumination optical system and the X-ray magnifying optical system according to claim 12, wherein an optical element having a ring-shaped annular aperture is used, and the visible light illumination optical system and the visible light are used. The light magnifying optics
An optical element having an NA different from the NA of the X-ray optical element is used, and the light integrating unit is arranged coaxially with the X-ray source.
A ring-shaped hole, which is arranged between the line illumination optical system and the visible light illumination optical system and which transmits X-rays, is provided at a portion corresponding to the annular opening of the optical element of the X-ray illumination optical system. A first visible light mirror formed is provided, and the light separation section is arranged between the X-ray magnifying optical system and the visible light magnifying optical system that are coaxially arranged, and the X-ray imaging device. And a second visible light mirror in which a ring-shaped hole for transmitting X-rays is formed in a portion corresponding to the annular zone opening of the optical element of the X-ray magnifying optical system. An X-ray microscope, wherein the X-ray image pickup device and the visible light image pickup device acquire an X-ray image and a visible light image simultaneously or substantially at the same time.