JP6537032B2 - Observation system of scanning electron microscope image - Google Patents

Description

  The present invention relates to an observation technique using a scanning electron microscope. More specifically, the present invention relates to an observation technique using a scanning electron microscope that enables living samples of a biological sample in an aqueous solution to be observed.

  Scanning electron microscopes are widely used as tools for observing biological samples and organic materials with high resolution. Conventionally, when observing a biological sample or an organic material with a scanning electron microscope, the sample is fixed with formaldehyde or the like to reduce the electron beam damage of the sample to be observed and obtain an image with high contrast, Treatments such as coating with gold, platinum, carbon, etc. or dyeing with heavy metals have been essential.

  However, in recent years, methods have been developed in which biological samples can be observed with high contrast without coating or staining (see Patent Document 1 and Non-patent Document 1). In this novel method, a sample is attached to the lower surface of a thin sample support film such as a carbon film, and an electron beam with an acceleration voltage lower than that of the upper part of the sample support film is irradiated. The irradiated electron beam diffuses and diffuses inside the sample support film, reaches near the lower surface of the film, and secondary electrons are emitted from the lower surface of the sample support film. The secondary electrons are absorbed by the sample immediately below the sample support film, whereby an extremely high contrast image can be obtained.

  In this method, conditions are set so that the energy of the above-mentioned secondary electrons is about 10 eV. With such extremely weak secondary electrons, the electron beam damage to the sample to be observed is significantly reduced, so that even the sample that is easily damaged like a biological sample has high contrast with the original shape and structure of the sample. It is possible to observe or analyze with the image of

  Such observation conditions are called "indirect secondary electron contrast conditions". A method has also been developed in which such an observation method is further developed, a conductive film is formed under the insulating thin film layer, and the resolution and contrast are further improved by utilizing the charging effect by electron beam incidence (patent document 2 and Non-Patent Document 2).

  Furthermore, recently, a thin heavy metal thin film is formed on the upper part of the pressure-resistant insulating thin film, and a local electric potential change is generated by irradiating the heavy metal thin film with an electron beam. A transmission observation technique has also been developed (see Patent Document 3 and Non-Patent Document 3).

  In this method, the above-mentioned local potential change (fluctuating potential) images the attenuation state when passing through a biological sample in an aqueous solution, and performs sample observation. Water has a high relative dielectric constant of about 80, and therefore is permeable to changes in potential. On the other hand, a biological sample has a low relative dielectric constant of about 2 to 3 and inhibits the permeation of potential change. Therefore, it is possible to observe a biological sample in an aqueous solution with high contrast without staining.

Unexamined-Japanese-Patent No. 2010-097844 Patent No. 5115997 specification Specification of Japanese Patent Application No. 2013-080490

T. Ogura “A high contrast method of biological biological samples under a thin carbon film by scanning electron microscopy” Biochem. Biophys. Res. Commun. Vol. 377, p 79-84 (2008) T. Ogura "Direct observation of the inner structure of unstained atmospheric cells by low-energy electrons" Meas. Sci. Technol. Vol. 23, 085402 (8 pp) (2012) T. Ogura "Direct observation of unstained biological species in water by the frequency transmission electric-field method using SEM" PLOS ONE Vol. 9, e92780 (6 pp) (2014)

  According to the above-mentioned variable potential transmission observation technique, it is possible to observe a living biological sample, an organic material or the like in an aqueous solution with high contrast and high resolution without electron beam damage. However, it is necessary to install an amplifier for detecting potential fluctuation, a mechanism for holding an aqueous solution observation holder, and the like inside the scanning electron microscope, and it is necessary to greatly remodel the inside of the sample chamber. Such modifications make it difficult to install a general sample holder and make it impossible to use a scanning electron microscope as a general-purpose device.

  Since a scanning electron microscope is a general-purpose device that is often used not only for biological samples but also for observation of semiconductors and metal materials, it is desirable that both a general-purpose holder and an aqueous solution observation holder can be used.

  In addition, in a high resolution field emission scanning electron microscope, it is common to place the sample through a sample exchange chamber in order to introduce the sample into the interior of the mirror. Therefore, it is not possible to directly open the sample chamber and manually couple the aqueous solution observation holder to the amplifier on the stage.

  Furthermore, when it is decided to install an amplifier for detecting potential fluctuations, a mechanism for holding an aqueous solution observation holder, etc. inside the scanning electron microscope, it takes a lot of time for work when it is necessary to replace or repair the amplifier. And cost is also required.

  The present invention has been made in view of such problems, and the object of the present invention is to observe a biological sample in a living state simply with high resolution and high contrast using a scanning electron microscope. It is about providing the technology that makes it possible.

  In order to solve the above-mentioned subject, the observation system of the scanning electron microscope picture concerning the present invention has conductivity which was laminated on the insulating thin film whose one main surface is a holding surface of observation sample and the other main surface of the insulating thin film. A thin film is provided, and a signal based on a potential of one main surface of the insulating thin film generated due to an electron beam incident from the conductive thin film is detected on the one main surface side of the insulating thin film. A sample holder for a scanning electron microscope provided with a signal detection unit, and a sample stage for a scanning electron microscope on which the sample holder is mounted, wherein a detection signal detected by the signal detection unit is amplified to be a measurement signal It is characterized in that a scanning electron microscope image is observed using a sample stage having a built-in circuit unit to output.

  In one aspect, the sample holder for a scanning electron microscope is provided with a pressure-resistant thin film on the one principal surface side of the insulating thin film so as to have a predetermined distance from the insulating thin film, and the insulating thin film A spacer having a thickness corresponding to the predetermined interval is provided between the pressure-resistant thin film.

  For example, the thickness of the spacer is 40 μm or less.

  For example, the spacer is made of a material having an adhesive, an adhesive, a resin, a rubber, and a silicon member as a main component.

  In one aspect, the sample holder for a scanning electron microscope is provided with a flow path for perfusion of an aqueous solution between the insulating thin film and the pressure-resistant thin film.

  In a preferred embodiment, an arithmetic unit is provided which processes an output signal obtained by extracting only the frequency component of the reference signal from the measurement signal to form an image as a scanning electron microscope image.

  In a preferred embodiment, the arithmetic unit processes the output signal to extract a signal component of the intensity change frequency from the output signal, and forms an image based on the extracted signal component of the intensity change frequency. .

  Furthermore, in a preferred embodiment, the extraction of the signal component of the intensity change frequency is performed by any of a band pass filter method, a lock-in amplifier method, an autocorrelation analysis method, and a Fourier transform analysis method.

  The sample holder for a scanning electron microscope used in the system for observing a scanning electron microscope image according to the present invention has an insulating thin film whose one main surface is a holding surface of an observation sample and a conductive layer laminated on the other main surface of the insulating thin film. Conductive thin film, and the signal based on the electric potential of the one main surface of the insulating thin film generated due to the electron beam incident from the side of the conductive thin film is detected on the one main surface side of the insulating thin film A signal detection unit is provided. On the other hand, the sample stage incorporates a circuit unit that amplifies the detection signal detected by the signal detection unit provided in the sample holder and outputs it as a measurement signal. When such a configuration is adopted, it is possible to extremely closely arrange a signal detection unit provided in the sample holder and a circuit unit that amplifies a detection signal detected by the signal detection unit and outputs it as a measurement signal. As a result, noise can be significantly reduced.

  According to the present invention, direct observation of a biological sample in an aqueous solution can be performed easily and with high resolution. For example, since it is possible to examine reactions of drugs and chemicals while observing a biological sample, it is a powerful weapon for research and development of drugs and the like.

It is a block diagram for demonstrating the structural example of the observation system of the scanning electron microscope image which concerns on this invention. It is the cross-sectional schematic for demonstrating the structural example of the sample holder for scanning electron microscopes used with the observation system of the scanning electron microscope image which concerns on this invention, and the sample stage for scanning electron microscopes which mounts this sample holder. It is the schematic for demonstrating the process of introduce | transducing a sample stage in the mirror body of a scanning electron microscope. It is a fluctuation | variation electric-potential transmission observation image of the non-processed bacteria in the aqueous solution observed by the sample holder for fluctuation | variation electric potential transmission observation concerning this invention, and a field emission scanning electron microscope. It is an observation image of a non-stained / non-fixed protein complex (IgM antibody) in an aqueous solution observed by the sample holder for variable potential transmission observation according to the present invention and a field emission scanning electron microscope. It is the schematic for demonstrating the structural example of the sample holder set as the structure which makes the thin film space | interval constant and reduces the background at the time of observation by installing a spacer between the pressure resistant thin films. It is the schematic which shows the structure of the sample holder in which the flow path for perfusing aqueous solution is provided between an insulating thin film and a pressure resistant thin film.

  In the following, with reference to the drawings, an overview of a system for observing a scanning electron microscope image according to the present invention, a sample holder and a stage for a scanning electron microscope used in this system, and a variable potential transmission observation method by this observation system Do.

  FIG. 1 is a block diagram for explaining a configuration example of a scanning electron microscope image observation system according to the present invention.

  In the example shown in this figure, a sample holder 10 capable of observing the target sample in an aqueous solution is introduced into the mirror body of the scanning electron microscope 100 constituting the system. Set on the sample stage 20 containing the A sample exchange chamber 30 is provided on the side of the microscope body of the scanning electron microscope 100, and a sample introduction rod 31 is used to introduce the sample holder 10.

  A terminal portion 22 is provided at an end of the sample stage 20, and the terminal portion 22 is connected to a connector 23 provided in a mirror of the scanning electron microscope 100. The connector 23 is a power receiving unit that receives power supply from the DC power supply 340 to the first-stage amplifier 21 and outputs the output of the first-stage amplifier 21 and the like to the external connection device as a measurement signal. The signal is transmitted to the lock-in amplifier 310 which is an external connection device through the terminal portion 22 and the connector 23.

  The measurement signal sent to the lock-in amplifier 310 is output to the data recorder 320, and further sent to the PC 330 as an arithmetic device to perform data processing.

  The scanning electron microscope 100 is provided with a beam blanking device (deflection plate) 53 for controlling the irradiation of the observation sample with the electron beam 52 emitted from the field emission type electron gun 51.

  The beam blanking device 53 is for obtaining a change in intensity of the incident electron beam 52 to the observation sample, and for example, an ON / OFF signal of a frequency of 1 kHz or more has a rectangular wave shape from the function generator 350 provided outside the mirror. Input as a control signal of The function generator 350 also outputs a reference signal (reference signal) to the lock-in amplifier 310.

  The voltage applied to the deflection plate 53 is controlled by a control signal (here, a rectangular wave signal) output from the function generator 350. As a result of this voltage control, when the electron beam 52 is deflected by the coulomb force, the electron beam 52 is blocked by the aperture 54 and the electron beam 52 is blocked. On the other hand, when the voltage of the rectangular wave is 0 V, the electron beam 52 goes straight without being deflected, passes through the diaphragm 54 and is irradiated to the sample holder 10.

  By repeating ON / OFF by such a control signal, the intensity of the electron beam irradiated to the observation sample changes. The frequency of the control signal at this time is preferably 1 kHz or more, and in general, the frequency is set in the range of 20 to 1000 kHz.

  That is, by the control signal input from the function generator 350, the intensity of the electron beam irradiated to the observation sample (not shown) accommodated in the sample holder 10 changes at the same frequency as the control signal.

  Although details of the configuration of the sample holder 10 will be described later, when the top surface of the pressure-resistant thin film is irradiated with the electron beam 52, a local potential is generated at the incident site. The potential changes according to the same frequency as the control signal, and a signal accompanying the change in the potential is detected by the signal detection unit 210 provided at the lower part of the sample holder 10. The detection signal is amplified by the amplifier 21 built in the sample stage 20, and is output to the lock-in amplifier 310 as a measurement signal.

  That is, the reference signal from the function generator 350 and the measurement signal from the amplifier 21 are input to the lock-in amplifier 310. The lock-in amplifier 310 extracts only the frequency component of the reference signal of the function generator 350 from the measurement signal using the reference signal, and transmits this to the data recorder 320 as an output signal.

  And PC330 as an arithmetic unit processes the output signal obtained by extracting only the frequency component of a reference signal out of a measurement signal, and forms an image as a scanning electron microscope image.

  For example, the PC 330 processes the above-mentioned output signal, extracts the signal component of the intensity change frequency from this output signal, and forms an image according to the scanning signal of the electron beam based on the extracted signal component of the intensity change frequency. Do. Here, extraction of the signal component of the intensity change frequency can be performed by, for example, a method such as a band pass filter method, a lock-in amplifier method, an autocorrelation analysis method, or a Fourier transform analysis method.

  FIG. 2 is a schematic cross-sectional view for explaining a configuration example of a sample holder for a scanning electron microscope used in a system for observing a scanning electron microscope image according to the present invention and a sample stage for a scanning electron microscope on which the sample holder is mounted. It is.

  The sample holder 10 includes an insulating thin film 203 whose one main surface is a holding surface of the observation sample 40 and a conductive thin film 201 laminated on the other main surface of the insulating thin film 203. This laminate has pressure resistance enough to withstand observation in vacuum. That is, even within the electron microscope, the inside of the holder can maintain the atmospheric pressure state. Then, on the one main surface side of the insulating thin film 203, signal detection is performed to detect a signal based on the potential of the one main surface of the insulating thin film 203 generated due to the electron beam 52 incident from the conductive thin film 201 side. A unit 210 is provided.

  In the configuration example shown in this figure, a second insulating thin film 204 which is also a pressure-resistant thin film is provided between the main surface of the insulating thin film 203 and the signal detection unit 210, and the second insulating property is provided. One main surface of the thin film 204 and one main surface of the first insulating thin film 203 are arranged to have a gap of a predetermined distance. Therefore, in the case of this configuration, the signal detection unit 210 detects the potential of the other main surface of the second insulating thin film 204 as a signal. The signal detection unit 210 is provided to be separated from the first insulating thin film 203 and the second insulating thin film 204 as a sample support film in a state of being electrically insulated.

  The second insulating thin film 204 is not essential. For example, if the observation sample 40 can be dissolved or the like in a very thin water layer, the observation sample is held by its surface tension. Even if the second insulating thin film 204 is not provided, the signal detection unit 210 can detect a signal based on the potential of one main surface of the first insulating thin film 203.

  The observation sample 40 may be a biological sample in the aqueous solution 60, and is enclosed between the first insulating thin film 203 and the second insulating thin film 204, and the inside is sealed by the conductive packing 206 or the O ring 207. It is housed in a sealed conductive outer frame portion 208. That is, observation in a state where the biological sample is supported together with the aqueous solution is possible.

  As described later, the outer frame portion 208 may be provided with a mechanism for adjusting the internal pressure. In addition, reference numeral 209 denotes an insulating member for insulating the terminal portion 210 from the outer frame portion 208, and reference numerals 202 and 205 denote frame portions provided for the purpose of maintaining the strength and the like. Reference numeral 70 denotes a diffusion region of the incident electron beam 52.

  In the example shown in FIG. 2, the packing 206 is conductive, and the conductive thin film 201 is at the ground potential of the scanning electron microscope 100, and observation is performed in this state. Note that observation of the sample is possible even in a state where a predetermined bias voltage is applied without setting the potential of the conductive thin film 201 to the ground potential.

  The acceleration voltage of the electron beam 52 emitted from the electron gun 51 is preferably set to a voltage at which the incident electron beam hardly passes through the first insulating thin film 203. Specifically, it is preferable to set an acceleration voltage at which the incident electron beam is substantially scattered or absorbed inside the conductive thin film 201. With such a voltage setting, the primary electrons are hardly transmitted to the first insulating thin film 203 side, and the electron beam damage to the observation sample 40 can be completely prevented. In general, the above condition is realized by setting the acceleration voltage of the incident electron beam to 10 kV or less.

  If the first insulating thin film 203 is too thick, the strength of the signal detected by the signal detection unit 210 is reduced. Therefore, the thickness of the first insulating thin film 203 is preferably 200 nm or less.

  Examples of the material of the first insulating thin film 203 include a silicon nitride film, a carbon film, and a polyimide film.

  Even if the conductive thin film 201 is too thick, the strength of the signal detected by the signal detection unit 210 is reduced. Therefore, the thickness of the conductive thin film 201 is preferably 100 nm or less.

The conductive thin film 201 is preferably a metal thin film whose main component is a metal having a specific gravity of 10 g / cm ³ or more. This is to effectively suppress the shielding properties of the electron beam and the internal diffusion of the incident electrons. As such a metal thin film, one having tantalum, tungsten, rhenium, molybdenum, osmium, gold or platinum as a main component can be exemplified.

  As described above, the sample holder for a scanning electron microscope used in the system for observing a scanning electron microscope image according to the present invention has the insulating thin film 203 whose one main surface is the holding surface of the observation sample and the other main surface of the insulating thin film 203. And an insulating thin film 201 formed on the one principal surface side of the insulating thin film 203 due to the electron beam 52 incident from the side of the conductive thin film 201. A signal detection unit 210 is provided which detects a signal based on the potential of one of the main surfaces.

  On the other hand, the scanning electron microscope sample stage 20 used in the scanning electron microscope image observation system according to the present invention amplifies the detection signal detected by the signal detection unit 210 provided on the sample holder 10 and outputs it as a measurement signal Circuit (in this case, the amplifier 21). As described above, the terminal portion 22 is provided at the end of the sample stage 20, and the terminal portion 22 is connected to the connector 23 provided in the mirror of the scanning electron microscope 100.

  When such a configuration is adopted, a signal detection unit 210 provided in the sample holder 10, and a circuit unit (here, an amplifier 21) for amplifying a detection signal detected by the signal detection unit 210 and outputting it as a measurement signal It can be placed in close proximity. As a result, noise can be significantly reduced.

  Also, since the amplification circuit section is not provided in the main body of the scanning electron microscope, but is built in the sample stage 20, multiple types of sample stages incorporating amplification circuit sections (amplifiers 21) of different characteristics are prepared If this is done, it is easy to replace the amplification circuit section (amplifier 21) according to the characteristics of the observation sample. Therefore, obtaining an observation image with high contrast and resolution is also facilitated.

  When the distance between the one main surface of the first insulating thin film 203 and the one main surface of the second insulating thin film 204 is too thick, the strength of the signal detected at the terminal portion 210 is reduced. Therefore, the interval is preferably set to 40 μm or less.

  The observation sample 40 is housed in such a sample holder 10, and the electron beam 52 is incident while turning ON / OFF at a desired frequency (for example, 1 kHz or more), and the intensity change frequency extracted by the lock-in amplifier 310 is the electron beam The observation is performed by setting the frequency to 52.

  When an electron beam 52 whose intensity changes in a rectangular wave shape is incident on the sample holder 10, a signal is detected by the signal detection unit 210. In a state where the electron beam 52 is incident on the sample holder 10 without being blocked by the aperture 54 (ON state), almost all incident electrons are scattered or absorbed by the conductive thin film 201. As a result, electrons are accumulated at the site of incidence of the electron beam, and the site becomes a negative potential.

  On the side of the first insulating thin film 203 provided under the conductive thin film 201, an observation sample 40 dissolved in an aqueous solution 60 is sandwiched. Since the water molecules themselves of the aqueous solution 60 are polarized, when the electron beam incident site is negatively charged, the electric dipoles of the water molecules are arranged along the potential gradient. The electric dipole arrangement also generates a charge on the surface of the second insulating thin film 204 below the aqueous solution 60. This charge is detected by the signal detection unit 210 as a potential signal generated on the main surface of the second insulating thin film 204 and becomes a measurement signal.

  On the other hand, in a state where the electron beam 52 is shielded by the diaphragm 54 (OFF state), the incident electrons in the conductive thin film 201 immediately flow into GND, and the above-mentioned negative potential disappears. As a result, the arrangement of the electric dipoles in the aqueous solution 60 breaks apart, the charge on the surface of the first insulating thin film 203 also disappears, and the measurement signal output from the signal detection unit 210 also becomes zero.

  By repeating such ON / OFF of the electron beam at a desired (for example, 1 kHz or higher) frequency, it is possible to extract a signal of the same frequency component from the signal detection unit 210.

  For example, when the electron beam 52 which is repeatedly turned ON / OFF at a frequency of 1 kHz or more is irradiated, the dielectric constant of the biological sample is extremely low compared to water, so the arrangement of electric dipoles becomes weak and the strength of the measurement signal also decreases. .

  Since the dielectric constant of water molecules is as large as about 80, when a potential change occurs on one main surface of the first insulating thin film 203, this can be used as a signal having a strong propagation force in the aqueous solution 60. On the other hand, since the dielectric constant of the biological sample 40 is generally low, for example, the dielectric constant of a protein is 2 to 3, the power of the potential change signal in the biological sample is weak. Therefore, high contrast can be obtained by such a large difference in dielectric constant (difference in propagation force).

  As a result, in the biological sample 40 and in the aqueous solution 60, a difference occurs in the propagation force of the potential change signal due to the difference in the dielectric constant, and this signal is provided on one main surface side of the first insulating thin film 203 The detection by the detection unit 210 makes it possible to observe with high contrast without staining the biological sample. Moreover, since the biological sample 40 is not directly irradiated with the electron beam, the observation sample 40 is not damaged by the electron beam irradiation.

  The resolution of the image obtained by the observation substantially depends on the irradiation diameter of the electron beam. Therefore, if the irradiation diameter of the electron beam is narrowed to about 1 nm, it is also possible to achieve almost the same resolution (1 nm) as this. As a result, biological samples consisting of bacteria, viruses, proteins or protein complexes can also be observed with high resolution and high contrast in a living state in an aqueous solution.

  As described above, in the present invention, the signal detection unit 210 is provided on the sample holder 10, and the detection signal detected by the signal detection unit 210 is amplified on the sample stage 20 on which the sample holder 10 is mounted, and output as a measurement signal. Circuit portion (amplifier) 21 is incorporated. By adopting such a configuration, the detection signal detected by the signal detection unit 210 can be amplified directly by the circuit unit (amplifier) 21 in the shortest distance, and noise mixing from the outside can be minimized. Become.

  A terminal portion 22 is provided at an end of the sample stage 20, and the terminal portion 22 is connected to a connector 23 provided in a mirror of the scanning electron microscope 100. When the sample stage 20 is installed inside the scanning electron microscope 100, the terminal portion 22 is directly connected in such a manner that the connector 23 is inserted. Therefore, even when the sample stage 20 is introduced from the sample exchange chamber 30 into the mirror body, it is not necessary to connect a power cable or the like. In addition, since it is not necessary to significantly modify the scanning electron microscope 100 and it is not necessary to make the scanning electron microscope 100 dedicated to fluctuating potential transmission observation, a general-purpose sample stage when performing ordinary observation not based on fluctuating potential transmission observation You can use

  The sample holder 10 is configured to enclose the sample of the aqueous solution in the gap between the upper and lower two pressure-resistant thin films. As the thickness of the aqueous solution layer changes, the background contrast changes. Also, as the aqueous solution layer becomes thinner, the intensity of the transmission signal increases and the SN ratio improves, and the resolution also improves. Therefore, it is desirable that the aqueous solution layer be maintained at a constant interval slightly larger than the size (thickness) of the observation sample. Therefore, it is preferable to form a spacer between the upper and lower two pressure resistant thin films. By setting the thickness of such an aqueous solution layer, it is easy to remove the background due to the thickness of the water layer.

  That is, in the sample holder for a scanning electron microscope used in the present invention, a pressure resistant thin film is provided on the one main surface side of the insulating thin film so as to have a predetermined distance from the insulating thin film. Preferably, a spacer having a thickness corresponding to a predetermined distance is provided between the pressure-resistant thin film and the pressure-resistant thin film.

  When such a spacer is provided, the lower pressure-resistant thin film follows it and expands outward even when the upper pressure-resistant thin film expands outward in a vacuum, so these thin films maintain a constant distance. It will be. The thickness of the spacer may be changed according to the size of the target observation sample, but if it is too thick, the gap is too wide compared to the size of the observation sample, and noise is likely to occur, etc. Usually, the thickness is 40 μm or less because there is a fear of inconvenience.

  For example, in observation of bacteria, since the diameter of cylindrical cells is about 1 μm, it is desirable to set the thickness of the spacer to 1 to 2 μm. On the other hand, in the observation of viruses and proteins, about 100 to 200 nm is suitable. Such a spacer can be made of, for example, an adhesive, an adhesive, a resin, a rubber, and a material having a silicon member as a main component.

  As described above, according to the present invention, noise is reduced and contrast is enhanced at the time of observation with a scanning electron microscope by the fluctuating potential transmission observation method (improvement of the SN ratio of the fluctuating potential signal) and the thickness of the water layer. Background can be removed. Moreover, there is no need to make major modifications to the scanning electron microscope 100, and the introduction of the sample stage from the sample exchange chamber into the mirror does not require any special procedure.

  FIGS. 3A to 3C are schematic diagrams for explaining the process of introducing a sample stage into the observation chamber of the mirror of the scanning electron microscope. The scanning electron microscope is, for example, of the field emission type. The sample stage 20 is introduced into the observation room 100 b through the sample exchange room 30 using the sample introduction rod 31. Therefore, the sample stage 20 can be introduced while maintaining a high vacuum without opening the observation chamber 100b to the atmospheric pressure.

  First, the sample stage 20 with the observation holder 10 mounted thereon is placed in the sample exchange chamber 30, and the sample exchange chamber 30 is evacuated (FIG. 3A). With the sample exchange chamber 30 reaching a predetermined degree of vacuum, the vacuum shutter 32 is opened, and the sample stage 20 is introduced into the observation chamber by the sample introduction rod 31 and fixed on the pedestal 24 (FIG. 3B). At this time, the terminal portion 22 protruding from above the side portion of the sample stage 20 is inserted into and coupled to the connector 23 provided in the observation chamber 100b, and power supply to the first-stage amplifier 21 and transmission of a detection signal are performed. It becomes possible. After introducing the sample stage 20 into the observation chamber 100b, the sample exchange rod 31 is pulled out to a predetermined position of the sample chamber 30, and the vacuum shutter 32 is closed (FIG. 3 (c)).

  As described above, since the scanning electron microscope 100 does not need to be dedicated to variable potential transmission observation, a general-purpose sample stage may be used when performing ordinary observation not based on the variable potential transmission observation. . Since the terminal portion 22 is not provided in the general-purpose sample stage, it is not connected to the connector 23 and is completely electrically insulated from external signal processing equipment and amplifier power supply. Thereby, it is possible to prevent the mixing of external noise and the addition of an unexpected voltage.

  FIG. 4 is a variable potential transmission observation image of untreated bacteria in an aqueous solution observed by the sample holder for variable potential transmission observation according to the present invention and a field emission scanning electron microscope. Bacteria have a black contrast because they inhibit the propagation of the fluctuating potential signal (Fig. 4 (a)). In this observation image, a plurality of elongated bacteria were observed. In the background of the observation image, the upper left is bright and the lower right is dark. This is due to changes in water thickness, and in order to make the image clearer, it is effective to remove this background by image processing.

  FIG. 4 (b) is an observation image obtained by reversing the contrast and further removing the background. The shape and internal structure of bacteria can be observed more clearly.

  FIG. 5 is an electron microscopic image of the result of observing an IgM antibody, which is a type of protein complex, inverting the contrast and removing the background. When unstained / unfixed IgM antibody in an aqueous solution was enclosed in a sample holder and observed, a large number of particles of about 40 to 50 nm were observed (arrow in FIG. 5). Since the IgM antibody has a diameter of about 40 to 50 nm, it substantially matches the size of the particle under observation. For this reason, it was confirmed by the observation according to the system of the present invention that the intact protein particles in the aqueous solution can be observed.

  FIG. 6 illustrates a configuration example of the sample holder 10 having a configuration in which the thin film interval is kept constant and the background during observation is reduced by installing the spacer 212 between the pressure resistant thin films (203, 204). FIG. FIG. 6 (a) shows the state at atmospheric pressure, and FIG. 6 (b) shows the state in a vacuum (within the microscope). In the example shown in this figure, the pressure reducing film 211 is used as a pressure adjusting mechanism, which is provided on the lower surface side of the outer frame portion 208.

  The decompression membrane 211 of the sample holder 20 bulges outward inside the microscope, and the pressure in the holder decreases. Although the conductive thin film 201 side is also curved outward, the degree of curvature is relaxed by the effect of the pressure reducing film 211.

  The region under the second insulating thin film 204 is sealed by an O-ring 207, and maintains the atmospheric pressure. Therefore, the second insulating thin film 204 is curved upward, not downward, and the gap between the one main surface of the first insulating thin film 203 and the one main surface of the second insulating thin film 204 widens. As a result, there is no problem in holding the observation sample 10.

  In addition to this, a spacer 212 is provided at the center of the thin film, and by adhering to the upper and lower thin films, the distance between the thin films is made constant. That is, since the thin film of the lower part is drawn by the spacer 212 even when the upper thin film is curved, the spacer 212 can maintain a constant distance even when the sample holder 10 is placed in vacuum.

  As described above, such a spacer 212 can be made of, for example, an adhesive, an adhesive, a resin, a rubber, and a material having a silicon member as a main component. The spacer 212 is prepared by forming a pattern of an adhesive by a spray apparatus for fine coating, an industrial inkjet printer, etc., or bonding a thin film after coating with an ultraviolet curing resin or the like, and bonding by irradiation with ultraviolet light. It may be produced in the same manner.

  By changing the distance between the spacers 212 according to the size of the sample to be observed, it is possible to observe an image with high contrast and a good SN ratio. For example, it is about 2 to 10 μm in observation of cells and bacteria, about 100 nm to 1 μm in observation of virus, and 100 nm or less in observation of protein.

  In such a sample holder 10, a flow path for perfusion of an aqueous solution is formed in a gap of a predetermined interval between the one main surface of the first insulating thin film 203 and the one main surface of the second insulating thin film 204. Alternatively, a plurality of solutions may be enclosed in the sample holder 10 and observed while switching the solution.

  FIG. 7 shows a flow path for perfusion of an aqueous solution in a gap of a predetermined distance between one main surface of the second insulating thin film 204 which is a pressure-resistant thin film and one main surface of the first insulating thin film 203. 7A to 7C are a perspective view, a top view, and a cross-sectional view, respectively.

  In the example shown in this figure, three flow paths for perfusing the aqueous solution are provided. The outer frame portion 208 of the sample holder 10 has an upper portion 208a and a lower portion 208b. In the upper portion 208a, injection holes 213 associated with the three flow paths 214 are formed, and the solution introduced into these flow paths 214 has a second insulating thin film 204 by the action of surface tension. It is introduced into a gap of a predetermined distance between the one main surface of the first insulating film 203 and the first main surface of the first insulating thin film 203.

  In addition, dampers 215 as pressure application units for pushing out the solution from the injection holes 213 are provided on the side portions of the sample holder 10, and pressure application valves 216 are provided at the tips of the dampers 215. There is.

  If such a plurality of flow channels 214 are provided, a reagent etc. is newly sent into the sample holder 10 containing the observation sample 40 such as cells and bacteria in advance, and the reaction by this is observed in detail, etc. Is also easy. In addition, what is shown with the code | symbol 217 in the figure is a discharge liquid tank. Such feeding of the solution may be performed electrophoretically regardless of the pressure application unit.

  As described above, the sample holder for a scanning electron microscope used in the system for observing a scanning electron microscope image according to the present invention has an insulating thin film whose one main surface is a holding surface of the observation sample and the other main surface of the insulating thin film. A conductive thin film laminated on the surface, and on the one main surface side of the insulating thin film, on the one main surface of the insulating thin film produced due to the electron beam incident from the side of the conductive thin film A signal detection unit that detects a signal based on the potential is provided. On the other hand, the sample stage incorporates a circuit unit that amplifies the detection signal detected by the signal detection unit provided in the sample holder and outputs it as a measurement signal. When such a configuration is adopted, it is possible to extremely closely arrange a signal detection unit provided in the sample holder and a circuit unit that amplifies a detection signal detected by the signal detection unit and outputs it as a measurement signal. As a result, noise can be significantly reduced.

  In addition, since the amplification circuit section is not provided in the main body of the scanning electron microscope but built in the sample stage, observation can be performed by preparing plural types of sample stages incorporating amplification circuit sections having different characteristics. It is also easy to replace the amplification circuit section according to the characteristics of the sample. Therefore, obtaining an observation image with high contrast and resolution is also facilitated.

  As described above, according to the present invention, direct observation of a biological sample in an aqueous solution can be performed easily and with high resolution. For example, since it is possible to examine reactions of drugs and chemicals while observing a biological sample, it is a powerful weapon for research and development of drugs and the like.

  As described above, according to the present invention, it is possible to simply and easily observe a biological sample in an aqueous solution with extremely high contrast without performing a staining process or an immobilization process. In addition, since damage to the sample by the electron beam does not occur, it is possible to know the original form and structure of organic materials that are easily damaged, such as biological samples such as cells, bacteria, viruses and protein complexes. It becomes.

DESCRIPTION OF SYMBOLS 10 sample holder 20 sample stage 21 amplifier 22 terminal part 23 connector 24 base 30 sample exchange chamber 31 sample introduction rod 40 observation sample 51 electron gun 52 electron beam 53 beam blanking apparatus 54 aperture 60 aqueous solution 70 diffusion area 100 scanning electron microscope 201 conduction Thin film 202, 205 frame portion 203 first insulating thin film 204 second insulating thin film 206 conductive packing 207 O ring 208 outer frame portion 209 insulating member 210 signal detecting portion 211 pressure reducing film 212 spacer 213 injection hole 214 flow path 215 Damper 216 Pressure application valve 217 Effluent liquid tank 310 Lock-in amplifier 320 Data recorder 330 PC
340 DC power supply 350 function generator

Claims (4)

An observation system of a scanning electron microscope image,
An insulating thin film whose one main surface is a holding surface of the observation sample, a conductive thin film laminated on the other main surface of the insulating thin film, and a predetermined distance from the insulating thin film on the one main surface of the insulating thin film And a spacer provided at a central portion of the insulating thin film, the spacer maintaining the predetermined distance between the insulating thin film and the pressure resistant thin film. A signal detection unit for detecting a signal based on the potential of the one main surface of the insulating thin film generated on the one main surface side of the insulating thin film due to the electron beam incident from the conductive thin film side A sample holder for a scanning electron microscope provided immediately below the one main surface;
A sample stage for a scanning electron microscope on which the sample holder is mounted, wherein a circuit unit that amplifies a detection signal detected by the signal detection unit and outputs the amplified signal as a measurement signal is incorporated in the vicinity of the signal detection unit. Sample stage, and
A lock-in amplifier that receives the measurement signal;
A function generator that outputs a reference signal to the lock-in amplifier;
An arithmetic device for processing an output signal obtained by extracting only frequency components of the reference signal from the measurement signal to form an image as a scanning electron microscope image;
The lock-in amplifier receives the reference signal from the function generator and the measurement signal from the circuit unit.
The lock-in amplifier extracts only the frequency component of the reference signal of the function generator from the measurement signal using the reference signal, and outputs this as the output signal.
The arithmetic unit processes the output signal to extract a signal component of an intensity change frequency from the output signal, and forms an image based on the extracted signal component of the intensity change frequency.
system. Ri thickness der less 40μm of the spacer, the spacer adhesive, pressure sensitive adhesive, resin, rubber, consists either of material of the silicon member, the observation system of the scanning electron microscope image of claim 1. The scanning electron microscope image according to claim 1 or 2 , wherein the sample holder for the scanning electron microscope is provided with a flow path for perfusion of an aqueous solution between the insulating thin film and the pressure-resistant thin film. Observation system.   The scanning electron microscope image according to claim 1, wherein the signal component of the intensity change frequency is extracted by any one of a band pass filter method, a lock-in amplifier method, an autocorrelation analysis method and a Fourier transform analysis method. Observation system.