WO2010134282A1

WO2010134282A1 - Sample support member for x-ray microscopes, sample-containing cell, x-ray microscope, and method for observing x-ray microscopic image

Info

Publication number: WO2010134282A1
Application number: PCT/JP2010/003178
Authority: WO
Inventors: 小椋俊彦
Original assignee: 独立行政法人産業技術総合研究所
Priority date: 2009-05-22
Filing date: 2010-05-10
Publication date: 2010-11-25
Also published as: JP2011007766A; JP5317120B2

Abstract

A sample support member (11) is provided with a metal film (11b) which emits characteristic X-rays in a wavelength range of 0.6 to 6.0 nm including the "water window" region when an electron beam strikes the metal film (11b) and which is provided on one main surface of a sample support film (11a) which is a silicon nitride film or a carbon film. Characteristic X-rays in the soft X-ray region are emitted with high efficiency (high intensity) from the metal film (11b) which the electron beam has struck. Since the high-intensity characteristic X-rays strike the sample (10), the SN ratio of the observation image is improved. Since th metal film (11b) has an effect of minimizing the range where electrons are diffused in the sample support film (11a), the damage to the sample is reduced. Further, since the acceleration voltage of the incident electron beam can be increased, the metal film (11b) has also an effect of improving the resolution.

Description

Sample support member for X-ray microscope, sample containing cell, X-ray microscope, and observation method of X-ray microscope image

The present invention relates to an observation technique of an X-ray microscope image. More specifically, the present invention relates to a sample support member and a sample storage cell used when performing morphological observation and the like of a sample using an X-ray microscope, and an X-ray microscopic image observation method using these.

In recent years, development of an X-ray microscope using X-rays as a probe has been advanced as a method of observing the forms of various samples such as biological substances and organic substances. An X-ray microscope has a great feature that it is possible to observe a sample in an aqueous solution with high resolution, as compared with an optical microscope which is a form observation means widely used conventionally.

In particular, soft X-rays in the wavelength range of 2.3 to 4.4 nm (corresponding to the energy range of 284 to 540 eV) are easily transmitted through water and are well absorbed by carbon and nitrogen contained in large amounts in biological samples Therefore, it is extremely effective as a probe for observing a biological sample in an aqueous solution with high contrast.

The wavelength range of the above range is generally referred to as the "water window", but when using the "water window" region X-ray, it is possible to use a water-containing object (a biological sample or a sample in solution) as it is. In addition to being able to observe in the state, since the wavelength is shorter than visible light, high resolution observation beyond the optical microscope is possible, so X-ray microscopes using soft X-rays in the relevant wavelength range Development is underway (see, for example, Hideaki Majima et al. “Seeing cells with an X-ray microscope” Medical Imaging Technology, Vol. 17, No. 3 p. 211-216 (1999) (Non-patent Document 1)) . In addition, soft X-rays with a wavelength range of 5.0 to 4.5 nm (corresponding to an energy range of 240 to 280 eV) called “a window of carbon” which has low absorption by carbon and a shorter wavelength of 0.6 to 2. Soft x-rays in the 3 nm wavelength range are also convenient for observation of biological samples.

The X-ray microscope mainly uses a condensing system such as a zone plate to narrow down the X-ray beam and irradiate the sample (condensing system) and a type to irradiate the X-ray beam from a point light source to the sample ( Point light source system).

Focusing X-ray microscopes are classified into those of radiation transmission type and scanning transmission type (for example, Chris Jacobsen "Soft x-ray microscopy" Trend in Cell Biology, Vol. 9, p. 44- 47 (1999) (Non-Patent Document 2). The resolution of a focusing X-ray microscope depends on the processing accuracy of the zone plate, and the theoretical limit is expected to be about 10 to 15 nm (for example, W. Chao et al., “Soft X-ray microscopy at a spatial resolution better than 15 nm "Nature, Vol. 453, p. 1210-1213 (2005) (see Non-Patent Document 3).

On the other hand, methods using point light sources are classified into methods of generating X-rays by laser and methods of generating X-rays by electron beams. As a method of observing a sample using an X-ray generated by causing an electron beam to be incident on a target, the electron beam is directly incident on a sample support film to generate an X-ray, and this X-ray is incident on the electron beam Techniques have been developed to observe the form of a sample by irradiating the sample attached to the surface opposite to the surface (Japanese Patent Application Laid-Open No. 8-43600 (Patent Document 1) and Japanese Patent Application No. 2009-18290 No. (Patent Document 2: undisclosed).

In such a method, it is possible to keep the diffusion range of the electron beam in the sample in a narrow area by making the very thin and focused low acceleration electron beam incident on the sample support film, and as a result, high resolution It is possible to achieve

Further, according to the method, by arranging a plurality of X-ray detectors at various angles and positions below the sample support film (the side opposite to the electron beam incident surface), each X-ray detector can be As it is possible to obtain “tilted images” depending on the arrangement angle, it is possible to obtain various “tilted images” in one electron beam scan. As a result, it is also possible to observe the three-dimensional structure of the observation sample with only one electron beam scan (see Patent Document 2).

By the way, silicon nitride films have been widely used as sample support films for soft X-ray microscope observation. Since the silicon nitride support film has high pressure resistance, two silicon nitride films are fixed in parallel with a gap, and a sample containing an aqueous solution is sealed in the gap and sealed in the observation device ( There is also a report on a “sample capsule for X-ray microscope” for introduction into a vacuum for observation (Japanese Patent Application Laid-Open No. 6-180400 (Patent Document 3)).

However, when an electron beam or an ion beam is directly incident on a conventional sample support film made of silicon nitride to perform X-ray microscopic observation, the following problems occur.

That is, the sample support film made of silicon nitride has a relatively low conversion efficiency from charged particle beams to X-rays, and as a result, the SN ratio of the obtained observation image is low. In addition, when observing a biological sample in an aqueous solution, it is necessary to use a material that generates characteristic X-rays in the “water window” region for the sample support film, but the silicon nitride film sufficiently satisfies the requirement. I can not do it. Furthermore, a sample capsule as disclosed in Japanese Patent Application Laid-Open No. 6-180400 (Patent Document 3) seals a sample in an aqueous solution after fixing and fixing two sample support films in parallel at a distance of 10 μm or less. Because of this structure, considerable skill is required for the sample sealing operation.

JP-A-8-43600 Japanese Patent Application No. 2009-18290 JP-A-6-180400

The present invention has been made in view of such problems, and the object of the present invention is to provide a 0.6 to 6 nm window including a “water window” region with high efficiency for charged particle beams such as electron beams and ion beams. Sample support for X-ray microscopes that enables conversion to soft X-rays in the wavelength range and suppression of the damage to the sample caused by charged particle beam irradiation by narrowing the diffusion range of the charged particle beam in the sample Remodeling a device such as a sample storage cell for an X-ray microscope and a general-purpose SEM capable of easily and simply introducing a member, a target observation sample into an observation device even if the observation sample is an aqueous solution sample It is an object of the present invention to provide a technology that makes it possible to obtain a high-resolution X-ray microscope image of a high SN ratio by a simple and inexpensive method.

In order to solve the above-mentioned problems, the sample support member for X-ray microscope of the present invention is provided with a metal film which emits characteristic X-rays by irradiation of charged particles on one main surface of a support film holding a sample. There is.

Preferably, the metal film emits characteristic X-rays in a wavelength range of 0.6 to 6 nm by irradiation of charged particles.

The metal film is, for example, a film containing any of aluminum, scandium, titanium, vanadium, chromium, cobalt, nickel and copper as a main component.

Preferably, the film thickness of the metal film is 200 nm or less.

The sample support member of the present invention may be configured such that a second metal film having low charged particle transmittance and high X-ray transmittance is provided between the support film and the metal film.

The second metal film is, for example, a film containing any of gold, platinum, palladium, osmium, chromium, nickel, tungsten, and lead as a main component.

The support film is, for example, any of a silicon nitride film, a carbon film, and a polyimide film.

In the sample storage cell of the present invention, the upper part of the cell having the sample support member described above and the lower part of the cell facing the sample support membrane side of the upper part of the cell are arranged with a gap of a predetermined distance via the seal member. In the upper part of the cell, an injection hole, an air hole, and an observation window for injecting a sample into the gap are formed.

The sample storage cell may be configured such that a sample adsorbent is provided on the other main surface side of the surface of the sample support film.

In addition, the sample storage cell may be provided with a sealing means for sealing the injection hole and the air hole.

Furthermore, the sample storage cell may be provided with pressure adjusting means for adjusting the pressure in the cell to a predetermined pressure.

The X-ray microscope of the present invention comprises a holder for holding a sample support member provided with a metal film for emitting characteristic X-rays by irradiation of charged particles on one main surface of a support film for holding a sample; A charged particle beam generation unit for irradiating charged particles to an observation sample supported by the sensor, charged particle scanning means for enabling scanning of the charged particles on the observation sample, and a support film surface side of the sample support member A photoelectric conversion means for converting X-rays generated by the irradiation of the charged particles into electrons; an electron beam detector for detecting the electrons photoelectrically converted by the photoelectric conversion means; and an electron beam detected by the electron beam detector An arithmetic unit that processes the line detection signal to form an X-ray image is provided.

The X-ray microscope of the present invention may be an aspect provided with an X-ray transmission filter for shielding charged particles between the observation sample and the photoelectric conversion means.

Further, the X-ray microscope of the present invention may be provided with an electric field or magnetic field generating means between the observation sample and the photoelectric conversion means for blocking incidence of charged particles to the photoelectric conversion means.

According to the first observation method of the X-ray microscope image of the present invention, the observation sample is supported on the other principal surface side of the sample support member of the present invention described above, and the charged particle beam converged from the metal film surface is scanned. The sample support member generates X-rays, which are detected by an X-ray detector disposed on the sample support film side of the sample support member to form an X-ray image.

According to the method of observing an X-ray microscope image, the X-ray detector may be arranged at each of a plurality of positions desired to observe an observation sample supported by the sample support member from different directions to detect the X-ray. Good.

Furthermore, in the observation method of the X-ray microscope image, a three-dimensional X-ray image may be formed by arithmetic processing of X-ray detection signals detected by the plurality of X-ray detectors.

In the second observation method of the X-ray microscope image of the present invention, the observation sample is supported on the other principal surface side of the above-described sample support member of the present invention, and the charged particle beam converged from the metal film surface is scanned. To generate X-rays from the sample support member, the X-rays are converted into electrons by photoelectric conversion means disposed on the sample support film side of the sample support member, and the electrons are detected by an electron beam detector. Detect and form an X-ray image.

In the method of observing the X-ray microscope image, an X-ray transmission filter for shielding charged particles may be provided between the observation sample and the photoelectric conversion means.

Furthermore, according to the observation method of this X-ray microscope image, means for generating an electric field or a magnetic field is provided between the observation sample and the photoelectric conversion means so as to block the incidence of charged particles to the photoelectric conversion means. Good.

The sample support member for an X-ray microscope of the present invention is provided with a metal film that emits characteristic X-rays by irradiation of charged particle beams. Therefore, charged particles such as electron beams and ion beams can be highly efficiently It is possible to convert to soft X-rays in the wavelength range of 0.6 to 6 nm including the "window" region.

As a result, it becomes possible to suppress the damage to the sample due to the charged particle beam irradiation by suppressing the diffusion range of the charged particle beam in the sample narrowly, and it is also possible to obtain an image with a high SN ratio. .

Further, in the sample storage cell for X-ray microscope of the present invention, the introduction of the liquid sample into the sample storage cell is performed by dropping from the injection hole provided around the observation window, and the air hole is provided. Since the configuration is such that the observation window can be easily introduced, even if the target observation sample is an aqueous solution sample, the introduction processing into the observation apparatus can be performed easily and simply.

It is a block diagram for demonstrating the outline | summary of the structural example of the apparatus type at the time of performing X-ray-microscope observation using the sample support member which concerns on this invention. It is a figure for demonstrating notionally the mode of the detection signal of the X-ray detected by the X-ray photodiode. It is a figure for demonstrating the structural example of the scanning X ray microscope provided with two or more X ray photodiodes which are X ray detectors. Based on the signals detected by the three X-ray photodiodes provided in the scanning X-ray microscope of the configuration example shown in FIG. 3, the image data after each angle correction is determined, and based on these image data It is a figure for demonstrating notionally the process which calculates | requires the three-dimensional structure of an object sample. It is the figure which showed the example of a structure which converts the X-ray which permeate | transmitted the observation object sample into the electron by the photoelectric conversion means, and observes an X-ray image by the secondary electron detector in SEM. It is the figure which showed the structural example provided with the filter which shields a charged particle (electron) to the photoelectric conversion means shown in FIG. 5, and permeate | transmits an X-ray. It is the figure which provided the magnet in the photoelectric conversion means shown in FIG. 5, and shielded the charged particle (electron), and made it transmit the X-ray. It is a figure for demonstrating the result of having confirmed the reduction effect of the electron beam damage to the sample by having provided the metal film (in the case without a titanium film). It is a figure for demonstrating the result of having confirmed the reduction effect of the electron beam damage to the sample by having provided the metal film (in the case with a titanium film). It is the image obtained by making the yeast as an observation object sample adhere to the other main surface of the silicon nitride film which formed the titanium film in one main surface, and observing it with an X-ray microscope. It is a figure for demonstrating the result of having confirmed the further reduction effect of the electron beam damage to a sample by having provided the 2nd metal film (in the case without a platinum film). It is a figure for demonstrating the result of having confirmed the further reduction effect of the electron beam damage to a sample by having provided the 2nd metal film (in the case with a platinum film). It is a figure for demonstrating the result of having confirmed the further reduction effect of the electron beam damage to a sample by having provided the gold thin film in the aluminum thin film lower part (in the case without a gold thin film layer). It is a figure for demonstrating the result of having confirmed the further reduction effect of the electron beam damage to a sample by having provided the gold thin film in the aluminum thin film lower part (case with a gold thin film layer). It is an image obtained by attaching yeast as an observation target sample to the other principal surface of a silicon nitride film having an aluminum thin film and a gold thin film formed on one principal surface and observing it with an X-ray microscope (magnification 2000). The yeast image which observed the arrow part in FIG. 12A by 10000 times. It is a figure which shows the structural example of the sample storage cell for, when using the sample in a solution in X-ray-microscope observation. FIG. 6 is a perspective view showing an example of a sample storage cell of a structure that enables easier sealing of a liquid sample. It is an upper side figure which shows the example of a sample holding cell of the structure which enables more easy enclosure of a liquid sample. It is the cross-sectional schematic which shows the example of a sample holding cell of the structure which enables more easy enclosure of a liquid sample. It is a figure which shows the example of the sample accommodation cell of the structure which apply | coated the sample adsorption material thinly to the sample support film of observation window vicinity. It is a figure which shows the example of the sample accommodation cell of the structure which apply | coated the sample adsorption material thinly to the sample support film of observation window vicinity. It is a figure which shows the structural example provided with the adjustment mechanism of the pressure in a solution in the sample storage cell of the structure which enables easy enclosure of a liquid sample.

Hereinafter, with reference to the drawings, configurations of a sample support member for X-ray microscope, a sample storage cell of the present invention, and an observation method and apparatus of an X-ray microscope image using these will be described.

FIG. 1 is a block diagram for explaining an outline of a configuration example of an apparatus system when performing X-ray microscopic observation using a sample support member according to the present invention, and in this figure, the inside of a scanning electron microscope (SEM) Shows an example in which an X-ray detector is provided. Therefore, this scanning X-ray microscope can also have an SEM function.

In the example shown in FIG. 1, the observation sample (10) is supported by the sample support member (11), and the sample support member (11) is held by the sample holder (12). The X-ray microscope observation system generates a signal (scanning signal) for scanning the electron beam emitted from the electron gun (13) and the electron gun (13) which makes the electron beam converge and enters the sample support film (11) Circuit portion (14), a deflection coil (15) for scanning the electron beam based on a scanning signal from the circuit portion (14), and X generated in the sample support film (11) by the incident electron beam X-ray photodiode (16) for detecting a ray, an amplifier (17) for amplifying the detection signal of the X-ray photodiode (16), and an X-ray image for forming an X-ray image based on the detection signal of the X-ray It is comprised by process PC (18).

The observation sample (10) is supported by the sample support film (11a) constituting the sample support member (11), and a metal film (11b) is provided on the main surface opposite to the sample support surface. The observation sample (10) may be a biological sample.

The metal film (11b) is irradiated with charged particle beams such as an electron beam and an ion beam to emit characteristic X-rays in a wavelength range of, for example, 0.6 to 6.0 nm. Examples of the material of such a metal film (11b) include aluminum, scandium, titanium, vanadium, chromium, cobalt, nickel and copper, and a metal film containing a film containing any of these metals as a main component It can be used as (11b). In order to generate characteristic X-rays with high efficiency, the film thickness of the metal film (11b) is preferably 200 nm or less.

A thin carbon film, a silicon nitride film, or a polyimide film can be used as the sample support film (11a). In addition, the sample holder (12) holding the sample support member (11) can be moved in the XY plane by a scanning mechanism (not shown).

The charged particle beam is irradiated from the upper surface (metal film surface) of the sample support member (11). In the example shown in FIG. 1, the charged particle beam is an electron beam. The incident electrons spread while being diffused inside the sample support member (11) and reach near the lower surface (sample support film surface) of the sample support member (11). The acceleration voltage of the electron beam at this time is low enough that electrons incident on the sample support member (11) hardly penetrate the sample support member (11) and reach the lower surface of the sample support member (11). It is adjusted to the acceleration voltage.

In the case of such an accelerating voltage, only the X-ray and low energy secondary electrons generated in the sample support member (11) are emitted from the lower surface of the sample support member (11), and the incident electron beam ( Almost no emission of the primary electrons) out of the sample support member (11) occurs. Therefore, it is possible to prevent the primary electrons from damaging the sample (10) attached to the lower surface of the sample support member (11).

The X-rays and secondary electrons emitted from the lower surface of the sample support member (11) are at least partially absorbed at the site to which the sample (10) is attached, and are transmitted intact at the other site Become.

FIG. 2 is a view for conceptually explaining the state of the X-ray detection signal detected by the X-ray photodiode (16). The X-ray photodiode (16) disposed below the sample support member (11) detects the transmitted X-ray, and the detection signal of the X-ray amplified by the amplifier (17) is detected by the scanning circuit unit (14). The X-ray image processing PC (18) forms an X-ray image on the basis of the detection signal (and the scanning signal).

As described above, the electron beam emitted from the electron gun (13) can be scanned by the deflection coil (15) in a desired range on the sample support film (11), so it is detected by the X-ray photodiode (16). The intensity of the X-rays is relatively weak in the attached area of the sample (10) and relatively strong in the other areas, and the X-ray image formed by the X-ray image processing PC (18) 10) "contrast" including information such as shape and structure will be generated.

In the configuration example shown in FIG. 1, a metal mesh (20) for collecting secondary electrons is provided in the lower lateral direction of the sample support member (11), and a positive voltage (+ V) is applied to this. There is. This is because the secondary electrons are collected laterally so that secondary electrons emitted from the lower surface of the sample support film (11) are not detected by the X-ray photodiode (16) and become noise, and It is for facilitating current collection to the detector (21).

A signal detected by the secondary electron detector (21) is sent to the control PC (22) which is also a signal processing unit, and based on this signal, an indirect secondary electron contrast (ISEC) image (secondary electron beam image) Can be formed.

In the above configuration example, an X-ray photodiode is illustrated as an X-ray detector, but other detectors having sensitivity to X-rays can also be used.

As illustrated in FIG. 1 and FIG. 2, the sample support member (11) of the present invention can be obtained by irradiating an electron beam onto one principal surface of a sample support film (11a) of silicon nitride film or carbon film or polyimide film. A metal film (11b) is provided which emits characteristic X-rays in the wavelength range of 6 to 6.0 nm. This is because the characteristic X-rays of the soft X region are emitted with high efficiency (high intensity) from the metal film (11b) irradiated with the electron beam, and such high-intensity characteristic X-rays are Can be used to improve the SN ratio of the observation image.

In addition, since the metal film (11b) has the effect of suppressing the diffusion range of electrons in the sample support film (11a), the acceleration voltage of the incident electron beam can be increased, and only the improvement of the SN ratio It is also effective in improving resolution.

As a preferable material of such a metal film (11b), aluminum, scandium, titanium, vanadium, chromium, nickel, and copper can be illustrated. This is because, as shown in Table 1, the wavelength (energy) of the characteristic X-ray excited when the metal material is irradiated with the electron beam is in the soft X-ray region, so that the characteristic of the soft X-ray region is highly efficient. It is because X-rays can be emitted.

Further, as indicated by reference numeral 11c in FIG. 2, a second metal film having a low charged particle transmittance (here, electron beam transmittance) between the sample support film (11a) and the metal film (11b). May be provided.

By providing the second metal film having such low electron transmittance, it becomes difficult to transmit to the sample support film (11a) side even if it is an incident electron beam of high acceleration voltage. It is possible to effectively suppress the damage. In addition, the possibility of electron beam incidence at high acceleration voltage makes it possible to enhance the radiation efficiency of the characteristic X-rays from the metal film (11b), and further, by reducing the diffusion range of electrons, a clear image can be obtained. Make it possible to get

As such a second metal film, a film containing any one of gold, platinum, palladium, osmium, chromium, nickel, tungsten, and lead, which are metal elements having low electron beam transmittance, as a main component can be exemplified. it can. The thickness is preferably about 5 to 100 nm.

In the example shown in FIGS. 1 and 2, only one X-ray detector is provided, but a plurality of X-ray detectors are provided, and the plurality of X-ray detectors are supported by the sample support film The observation sample may be placed at a desired position from different directions.

FIG. 3 is a diagram for explaining a configuration example of a scanning X-ray microscope provided with a plurality of X-ray photodiodes as X-ray detectors, in which three X-ray photodiodes (XPD) are provided (16a to 16c). Corresponding to the above, three amplifiers (AMP) are also provided (17a to c), and detection signals from these amplifiers (17a to c) are transmitted through an A / D converter (23) and a data recorder (19). It is configured to be sent to an X-ray image processing PC (18: here a PC for three-dimensional image processing). The other basic configuration is the same as that shown in FIG.

The X-rays from within the sample support film (11a) are emitted in various directions, but a plurality of X-ray detectors are provided, and these are observed from different directions from the observation sample (10) supported by the sample support film (11a) If placed at the desired position, it is possible to obtain a plurality of X-ray images (tilt images) depending on the angle at which the sample is desired. That is, the same number of tilt images as the number of detectors can be obtained by one electron beam scan, and it is possible to obtain the three-dimensional structure of the sample to be observed from these tilt images, and damage to the sample Is extremely minor.

Although FIG. 3 shows an example in which the number of X-ray detectors is three, it goes without saying that, for example, as many dozens or more X-ray detectors are disposed to acquire a large number of inclined images It is also possible to obtain a high-precision three-dimensional structure.

FIG. 4 shows image data after angle correction based on signals detected by three X-ray photodiodes (16a to 16c) provided in the scanning X-ray microscope of the configuration example shown in FIG. It is a figure for demonstrating notionally the process which calculates | requires and calculates | requires the three-dimensional structure of a target sample based on these image data.

1 to 4 show an embodiment in which the X-ray transmitted through the observation sample (10) is directly detected by the X-ray photodiode and image data is obtained based on this detection. It is also possible to convert lines into electrons and obtain image data based on the intensity (distribution) of the electrons.

FIG. 5 is a view illustrating an aspect of photoelectric conversion in such a case, in which a stage (26) is provided under the sample support member (11), and the photoelectric conversion is provided on the slope part of the stage (26). The surface (27) is designed to be irradiated with X-rays. Note that primary electrons transmitted through the sample support member (11) are blocked by the aperture (26a) by applying a positive potential to the aperture (26a) provided on the stage (26), It is possible not to be incident on the photoelectric conversion surface (27).

That is, the X-rays transmitted through the sample (10) are converted into electrons by the photoelectric conversion surface (27), and the electrons are detected by the secondary electron detector (21) in the SEM apparatus. Then, the electron beam detection signal detected by the secondary electron detector (21) is processed by the operation unit to form an X-ray image.

In such a configuration, it is not necessary to remodel the apparatus such as providing an X-ray detector and an amplifier in a general-purpose SEM apparatus, so simple and inexpensive X-ray image observation can be performed.

In addition, since the photoelectric conversion surface (27) can be obtained by coating gold thinly, etc., the production of the stage is simple and inexpensive, and downsizing is easy. If it is a small stage, there is no hindrance to placing it on a sample stage of a general-purpose SEM apparatus.

FIG. 6 is a view showing another embodiment in which the photoelectric conversion portion is provided. In this embodiment, charged particles (in the area of the diaphragm (26a) located between the observation sample (10) and the photoelectric conversion surface (27) Here, an X-ray transmission filter (28) is provided which shields electrons) and transmits X-rays. With such an X-ray filter (28), transmitted electrons (primary electrons) can be completely blocked, so that the reduction in contrast due to the transmitted electrons can be prevented.

If a silicon nitride film or the like having high pressure resistance is used as the X-ray filter (28), the inside (29) of the space formed above the stage (26) can be maintained at atmospheric pressure. Thus, observation with an aqueous solution sample or the like set inside can also be performed.

FIG. 7 is a view showing another embodiment provided with a photoelectric conversion unit. In this embodiment, a magnet (30) which is a means for generating an electric field or a magnetic field is installed as a mechanism for shielding electrons transmitted through the sample (10). The transmitted electrons are deflected and shielded by the magnetic field generated by the magnet (30). In this configuration, X-rays can be transmitted 100%, so an X-ray image with high contrast can be obtained. In addition, by forming an electric field, electrons can be deflected and shielded.
Example 1

FIGS. 8A and 8B are diagrams for explaining the result of confirming the reduction effect of the electron beam damage to the sample by providing the above-mentioned metal film (11b), and these are silicon nitride having a film thickness of 50 nm. The electron diffusion range (FIG. 8A) in the silicon nitride film when an electron beam (accelerating voltage: 2.6 kV) is incident only on the film (FIG. 8A) and a 20 nm-thick film on one main surface of the 50 nm-thick silicon nitride film It is the result of finding the diffusion range (FIG. 8B) of electrons in the silicon nitride film when a titanium film is provided and an electron beam (accelerating voltage: 2.6 kV) is incident from the titanium film side by Monte Carlo simulation.

When the titanium film is not provided (FIG. 8A), most of the electrons pass through the silicon nitride film (sample support film) under the above-described acceleration voltage (2.6 kV) conditions, and such transmission The electron beam damages the sample to be observed.

On the other hand, when a titanium film with a film thickness of 20 nm is formed on the upper surface of the silicon nitride film (FIG. 8B), the amount of electron beams reaching the lower surface of the silicon nitride film is significantly reduced, suppressing damage to the sample can do.

Further, as described above, a characteristic X-ray having an energy of 453 eV is emitted with high intensity from the titanium film irradiated with the electron beam, so that the SN ratio and contrast of the obtained observation image are improved.

FIG. 9 is an image obtained by attaching yeast as a sample to be observed to the other principal surface of a silicon nitride film having a titanium film formed on one principal surface as described in FIG. 8B and observing it with an X-ray microscope . From this figure, the shape of the yeast can be observed very clearly under the electron beam irradiation condition of the accelerating voltage of 2.6 kV. In addition, even after repeating observation several times on the said conditions, the damage by an electron beam was not confirmed.
(Example 2)

FIG. 10A and FIG. 10B are diagrams for explaining the result of confirming the further reduction effect of the electron beam damage to the sample by providing the above-mentioned second metal film (11c). A 50 nm thick silicon nitride film provided with a 20 nm thick titanium film on one main surface (FIG. 10A) and a 50 nm thick silicon nitride film with a 6 nm thick platinum film on one main surface The electron diffusion range in the silicon nitride film when an electron beam (accelerating voltage: 3.0 kV) is made incident from the titanium film side to the titanium film (FIG. 10B) provided by the Monte Carlo simulation. is there.

As apparent from the comparison of these results, the transmission electron can be further reduced by providing the second metal film of the heavier metal element at the lower part of the titanium film. That is, most of the incident electrons are absorbed by the platinum film, which is the second metal film, and the electrons that reach the lower surface of the silicon nitride film, which is the sample support film, are significantly reduced.

FIGS. 11A and 11B are diagrams showing the state of a transmission electron beam when aluminum is formed to 100 nm as a first metal film and gold is formed to 100 nm as a second metal film. These are formed on one main surface of a silicon nitride film with a film thickness of 100 nm, and when there is no gold thin film layer, almost all electron beams with an acceleration voltage of 10 kV are transmitted (FIG. 11A). On the other hand, when the gold thin film layer is provided, the electron beam is scattered and absorbed by the gold thin film layer, and the transmission electrons are significantly reduced to several% (11B). That is, the electron beam damage on the lower surface of the silicon nitride film can be significantly reduced by providing the gold thin film layer.

As shown in FIG. 11B, yeast as a sample to be observed is attached to the other principal surface of a silicon nitride film having an aluminum thin film and a gold thin film formed on one principal surface as described in FIG. It is an image obtained by sandwiching the yeast with a membrane and sealing the inside at atmospheric pressure and then observing it with an X-ray microscope. The X-rays were converted into electrons by the photoelectric conversion mechanism at the lower part of the sample as shown in FIG. 5 and observed by a secondary electron detector in the SEM apparatus.

When the acceleration voltage of the electron beam is 10 kV and the magnification is 2000 times (FIG. 12A), a large number of yeasts can be observed, and a particulate structure can be confirmed inside the yeasts. FIG. 12B is an X-ray image obtained by observing the yeast indicated by the arrow in FIG. 12A at a magnification of 10,000. From this figure, intracellular organs such as nuclei and vesicles inside the yeast can be observed with high resolution, and resolution beyond the light microscope is clearly achieved.
(Example 3)

FIG. 13 is a view showing a configuration example of a sample storage cell (24) for use in observing a sample in a solution according to this embodiment, which is opposed to the sample support member (11) described above It is a figure which shows a mode that another sample support member (11 ') was provided and the sample (10) was pinched | interposed and supported with a solution between these two sample support members.

The sample (10) is in an aqueous solution and is supported by being sandwiched between two members (11, 11 ') arranged in parallel at an interval of, for example, 10 μm or less. The two members in this state are housed under atmospheric pressure in the sample holder (24) and separated from the vacuum in the SEM apparatus by the O-ring (25). Therefore, even if the sample storage cell (24) is placed in vacuum, evaporation of the aqueous solution can be prevented.

As a member (11a, 11 ') used when performing such a support of a sample, a silicon nitride and a polyimide film | membrane are preferable. This is because the silicon nitride and polyimide films have relatively high mechanical strength (pressure resistance), so it is easy to take an X-ray image in a state separated from the vacuum in the apparatus as illustrated in FIG. It is.

FIGS. 14A to 14C illustrate examples of a sample storage cell having a structure that enables easier sealing of a liquid sample, FIG. 14A is a perspective view, FIG. 14B is a top view, and FIG. 14C is the sample storage cell. FIG.

In this sample storage cell (24), the upper part (24a) and the lower part (24b) are integrated through the sealing material (24c), and a predetermined space formed between the upper part (24a) and the lower part (24b) Inside, a liquid sample is to be injected.

In the upper part (24a) of the sample storage cell (24), a silicon nitride film (11a) as a sample support film is formed on the lower surface of the upper sample holder (12), and the injection hole (24s) and the air hole (24h) And the observation window (24 w) is formed. Then, in the region where the observation window (24w) is formed, titanium is provided on one main surface of the silicon nitride film (11a) as the sample support film via the platinum film (11c) which is the second metal film. A membrane (11b) is formed.

The liquid sample is introduced into the sample storage cell (24) by dropping from the injection hole (24s) provided around the observation window (24w). The liquid sample dropped from the injection hole (24s) can be easily introduced to the observation window (24w) by surface tension because the air hole (24h) is provided on the opposite side. After the sample is introduced, the injection hole (24s) and the air hole (24h) are closed with a sealing member, an airtight tape or the like, and set in the observation device.

In addition, as illustrated in FIG. 15A and FIG. 15B, the sample support material (11d) is thinly applied to the sample support film (11a) in the vicinity of the observation window, and the sample (10) is assured in the observation window (24w). It may be adhered and fixed to prevent sample movement during observation.

For example, when cells or bacteria in a solution are introduced into the sample storage cell (24), the cells are firmly attached and fixed by using fibronectin, polyethylene glycol, concanavalin A or the like as the sample adsorbent (11d). be able to. Of course, other adsorbing substances can be used as well.

When such a sample storage cell is used, as shown in FIG. 15B, the observation sample (10) just adheres to the observation window (24w), which facilitates X-ray microscope observation. After the sample (10) is introduced, the injection hole (24s) and the air hole (24h) are closed with a sealing member (24d), an airtight tape or the like, and set in the observation device.

Furthermore, as shown in FIG. 16, the pressure control valve (31), the adjustment damper (32), etc. make it possible to adjust the pressure inside the sample storage cell, so that samples (10 ) Can be observed. For example, by making the internal pressure lower than the atmospheric pressure, it is possible to easily observe the evaporation process of the water from the sample (10). In addition, the pressure applied to the sample support film can be reduced by reducing the internal pressure, and a thinner support film having a wider observation surface can be used.

As described above, according to the present invention, it is possible to convert charged particle beams such as electron beams and ion beams with high efficiency into soft X-rays in the wavelength range of 0.6 to 6 nm including the "water window" region, and A sample support member for X-ray microscope, which makes it possible to suppress damage to the sample caused by charged particle beam irradiation by narrowing the diffusion range of particle beam in the sample, even if the target observation sample is an aqueous solution sample High S / N ratio by a simple and inexpensive method without modifying a sample storage cell for X-ray microscope having a configuration that can be easily and easily introduced into the observation device and a general purpose SEM or the like The present invention provides a technology that makes it possible to obtain a resolution X-ray microscope image.

The present invention is particularly effective in observing samples of biological samples such as cells, bacteria, viruses, protein complexes and samples of organic materials, as well as samples in aqueous solution.

Claims

A sample support member for an X-ray microscope, in which a metal film that emits characteristic X-rays by irradiation of charged particles is provided on one main surface of a support film that holds a sample.
The sample support member for an X-ray microscope according to claim 1, wherein the metal film emits characteristic X-rays in a wavelength range of 0.6 to 6 nm by irradiation of charged particles.
The sample support member for an X-ray microscope according to claim 1 or 2, wherein the metal film is a film containing any of aluminum, scandium, titanium, vanadium, chromium, cobalt, nickel and copper as a main component.
The sample support member for an X-ray microscope according to claim 1 or 2, wherein the film thickness of the metal film is 200 nm or less.
The sample support for an X-ray microscope according to claim 1 or 2, wherein a second metal film having low charged particle transmittance and high X-ray transmittance is provided between the support film and the metal film. Element.
The sample support member for X-ray microscope according to claim 1 or 2, wherein the second metal film is a film containing any of gold, platinum, palladium, osmium, chromium, nickel, tungsten and lead as a main component. .
The support member for an X-ray microscope according to claim 1 or 2, wherein the support film is any of a silicon nitride film, a carbon film, or a polyimide film.
A cell upper portion having the sample support member according to claim 1 or 2 and a cell lower portion opposite to the surface of the cell upper portion on the sample support membrane side are arranged to have a predetermined gap via a seal member. A sample storage cell for an X-ray microscope, wherein an injection hole for injecting a sample into the gap, an air hole and an observation window are formed in the upper part of the cell.
The sample storage cell for X-ray microscope according to claim 8, wherein a sample adsorbent is provided on the other main surface side of the surface of the sample support film.
9. The sample storage cell for X-ray microscope according to claim 8, further comprising sealing means for sealing the injection hole and the air hole.
9. The sample storage cell for X-ray microscope according to claim 8, further comprising pressure adjusting means for adjusting the inside of the cell to a predetermined pressure.
An observation sample is supported on the other principal surface side of the sample support member according to claim 1 or 2, and the charged particle beam converged from the metal film surface side is scanned and incident from the sample support member X A method of observing an X-ray microscope image, which generates a line and detects the X-ray by an X-ray detector disposed on the sample support film side of the sample support member to form an X-ray image.
The method of observing an X-ray microscopic image according to claim 12, wherein the X-ray detector detects the X-ray by arranging the observation sample supported by the sample support member at each of a plurality of positions desired from different directions.
The method of observing an X-ray microscopic image according to claim 13, wherein a three-dimensional X-ray image is formed by arithmetic processing of X-ray detection signals detected by the plurality of X-ray detectors.
A holder for holding a sample support member provided with a metal film for emitting characteristic X-rays by irradiation of charged particles on one main surface of a support film for holding a sample;
A charged particle beam generation unit configured to irradiate charged particles to the observation sample supported by the sample support member;
Charged particle scanning means capable of scanning the charged particles on the observation sample;
Photoelectric conversion means disposed on the support film side of the sample support member and converting X-rays generated by the irradiation of the charged particles into electrons;
An electron beam detector which detects electrons photoelectrically converted by the photoelectric conversion means;
An X-ray microscope comprising an operation unit that processes an electron beam detection signal detected by the electron beam detector to form an X-ray image.
The X-ray microscope according to claim 15, further comprising an X-ray transmission filter for shielding charged particles between the observation sample and the photoelectric conversion means.
The X-ray microscope according to claim 15, further comprising: an electric field or magnetic field generation means for blocking incidence of charged particles to the photoelectric conversion means between the observation sample and the photoelectric conversion means.
An observation sample is supported on the other principal surface side of the sample support member according to claim 1 or 2, and the charged particle beam converged from the metal film surface side is scanned and incident from the sample support member X X-rays are generated into electrons by photoelectric conversion means disposed on the sample support film surface side of the sample support member, and the electrons are detected by an electron beam detector to form an X-ray image. How to observe a line microscope image.
The method of observing an X-ray microscopic image according to claim 18, wherein an X-ray transmission filter for shielding charged particles is provided between the observation sample and the photoelectric conversion means.
The method of observing an X-ray microscopic image according to claim 18, wherein means for generating an electric field or a magnetic field is provided between the observation sample and the photoelectric conversion means to block incident of charged particles to the photoelectric conversion means.