JP2016072184A - Observation system of scanning electron microscopic image - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique that enables observation of a biological sample with high resolution and high contrast by using a scanning electron microscope while the biological sample is alive.SOLUTION: A sample holder for a scanning electron microscope used in an observation system of a scanning electron microscopic image has an insulation thin film 203 having one principal surface serving as a holding face for an observation sample, and an electrically conductive thin film 201 laminated on the other principal surface of the insulation thin film 203. The one principal surface side of the insulation thin film 203 is provided with a signal detector 210 for detecting a signal based on the potential of the one principal surface of the insulation thin film 201 which occurs due to an electron beam 52 incident from the electrically conductive thin film 201 side. A sample stage 20 for the scanning electron microscope used in the observation system of the scanning electron microscopic image contains a circuit unit (amplifier 21 in this case) for amplifying a detection signal detected by the signal detector 210 provided to the sample holder 10 and outputting the amplified detection signal as a measurement signal.SELECTED DRAWING: Figure 2

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 makes it possible to observe a biological sample in an aqueous solution as it is alive.

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

  However, in recent years, a method has been developed that can observe a biological sample 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 having an acceleration voltage lower than that of the upper part of the sample support film is irradiated. The irradiated electron beam spreads while diffusing 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 directly under the sample support film, and thereby an extremely high contrast image can be obtained.

  In this method, conditions are set so that the energy of the secondary electrons is about 10 eV. With such extremely weak secondary electrons, the electron beam damage to the specimen to be observed is remarkably reduced, so that even if the specimen is susceptible to damage such as a biological specimen, the original shape and structure of the specimen are high in contrast. It is possible to observe or analyze the image.

  Such observation conditions are referred to as “indirect secondary electron contrast conditions”. A method for further improving the resolution and contrast has been developed by further developing such an observation technique, forming a conductive film under the insulating thin film layer, and utilizing the charging effect by electron beam incidence (Patent Literature). 2 and Non-Patent Document 2).

  More recently, a thin heavy metal thin film is formed on a pressure-resistant insulating thin film, and a local potential change is generated by irradiating the heavy metal thin film with an electron beam. Transmission observation technology has also been developed (see Patent Document 3 and Non-Patent Document 3).

  In this method, an attenuation state when the above-described local potential change (fluctuating potential) permeates through a biological sample in an aqueous solution is imaged and the sample is observed. Since water has a high relative dielectric constant of about 80, water can easily pass through potential changes. On the other hand, the biological sample has a low relative dielectric constant of about 2-3, and inhibits the transmission of potential changes. Therefore, it is possible to observe a biological sample in an aqueous solution with high contrast without staining treatment.

JP 2010-097844 A Patent No. 5115997 Description of Japanese Patent Application No. 2013-080490

T. Ogura “A high contrast method of unstained biological samples under a thin carbon film by scanning electron microscopy” Biochem. Biophys. Res. Commun. Vol.377, p79-84 (2008) T. Ogura "Direct observation of the inner structure of unstained atmospheric cells by low-energy electrons" Meas. Sci. Technol. Vol.23, 085402 (8pp) (2012) T. Ogura "Direct observation of unstained biological specimens in water by the frequency transmission electric-field method using SEM" PLOS ONE Vol.9, e92780 (6pp) (2014)

  With the above-described variable potential transmission observation technique, it is possible to observe a living biological sample or an organic material 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 fluctuations, a mechanism for holding an aqueous solution observation holder, and the like inside the scanning electron microscope, and thus the inside of the sample chamber needs to be significantly modified. If such modification is made, it becomes difficult to install a general sample holder, and the scanning electron microscope as a general-purpose apparatus cannot be used.

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

  Further, in a high-resolution field emission scanning electron microscope, it is common to install the sample through a sample exchange chamber in order to introduce the sample into the mirror body. Therefore, it is impossible to directly open the sample chamber and manually connect the aqueous solution observation holder to the amplifier on the stage.

  Furthermore, if an amplifier that detects potential fluctuations or a mechanism that holds the aqueous solution observation holder is installed inside the scanning electron microscope, a great deal of time is required for work when the amplifier needs to be replaced or repaired. However, there is a problem that it requires labor and costs.

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

  In order to solve the above problems, an observation system for a scanning electron microscope image according to the present invention includes 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. And detecting a signal based on a potential of the one main surface of the insulating thin film generated due to an electron beam incident from the conductive thin film side on the one main surface side of the insulating thin film. A scanning electron microscope sample holder provided with a signal detection unit, and a scanning electron microscope sample stage on which the sample holder is placed, and a detection signal detected by the signal detection unit is amplified and used as a measurement signal A scanning electron microscope image is observed using a sample stage having a built-in circuit unit for output.

  In one aspect, the scanning electron microscope sample holder is provided with a pressure-resistant thin film on the one main surface side of the insulating thin film so as to have a predetermined distance from 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 spacer has a thickness of 40 μm or less.

  For example, the spacer is made of a material mainly composed of an adhesive, an adhesive, resin, rubber, and a silicon member.

  In a certain aspect, the said sample holder for scanning electron microscopes is provided with the flow path for perfusing aqueous solution between the said insulating thin film and the said pressure | voltage resistant thin film.

  In a preferred aspect, there is provided an arithmetic unit that processes an output signal obtained by extracting only the frequency component of the reference signal from the measurement signal and forms an image as a scanning electron microscope image.

  Further, in a preferred aspect, the arithmetic device processes the output signal, extracts a signal component having an intensity change frequency from the output signal, and forms an image based on the extracted signal component having the intensity change frequency. .

  Furthermore, in a preferred aspect, 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.

  The sample holder for a scanning electron microscope used in the scanning electron microscope image observation system according to the present invention has an insulating thin film in which one main surface is a holding surface of the observation sample and a conductive layer laminated on the other main surface of the insulating thin film. A signal based on the potential of one main surface of the insulating thin film caused by the electron beam incident from the side of the conductive thin film is detected on 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. By adopting such a configuration, the signal detection unit provided in the sample holder and the circuit unit that amplifies the detection signal detected by the signal detection unit and outputs it as a measurement signal can be arranged very close to each other. As a result, noise can be greatly reduced.

  According to the present invention, it is possible to easily and directly observe a biological sample in an aqueous solution with high resolution. For example, since it is possible to examine the reaction of chemicals and chemical substances while observing biological samples, it is a powerful weapon for research and development of chemicals.

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 into the lens body of a scanning electron microscope. It is a variable potential transmission observation image of unprocessed bacteria in an aqueous solution observed with a sample holder for variable potential transmission observation according to the present 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 a variable potential transmission observation sample holder and a field emission scanning electron microscope according to the present invention. It is the schematic for demonstrating the structural example of the sample holder made into the structure which makes the space | interval of thin films constant by installing a spacer between pressure-resistant thin films, and reduces the background at the time of observation. It is the schematic which shows the structure of the sample holder provided with the flow path for perfusing aqueous solution between an insulating thin film and a pressure | voltage resistant thin film.

  Hereinafter, with reference to the drawings, an observation system for 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 an outline of a variable potential transmission observation method using this observation system will be described. To 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 a target sample in an aqueous solution is introduced into the body of a scanning electron microscope 100 constituting the system. Is set on the sample stage 20 containing the. A sample exchange chamber 30 is provided on the side of the body of the scanning electron microscope 100, and the sample holder 10 is introduced using the sample introduction rod 31.

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

  The measurement signal sent to the lock-in amplifier 310 is outputted to the data recorder 320 and further sent to the PC 330 as an arithmetic unit for data processing.

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

  The beam blanking device 53 is for obtaining a change in the intensity of the incident electron beam 52 on the observation sample. From a function generator 350 provided outside the mirror body, for example, an ON / OFF signal having a frequency of 1 kHz or more is a rectangular wave. As a control signal. The function generator 350 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 (in this case, 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 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 onto 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 is generally set in the range of 20 to 1000 kHz.

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

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

  That is, the lock-in amplifier 310 receives the reference signal from the function generator 350 and the measurement signal from the amplifier 21. 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.

  Then, the PC 330 as an arithmetic unit processes an output signal obtained by extracting only the frequency component of the reference signal from the measurement signal, and forms an image as a scanning electron microscope image.

  For example, the PC 330 processes the above-described output signal, extracts a signal component of the intensity change frequency from the 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. To do. Here, the extraction of the signal component of the intensity change frequency can be performed by a technique such as a band-pass filter method, a lock-in amplifier method, an autocorrelation analysis method, a Fourier transform analysis method, or the like.

  FIG. 2 is a schematic cross-sectional view for explaining a configuration example of a scanning electron microscope sample holder used in the scanning electron microscope image observation system according to the present invention and a scanning electron microscope sample stage on which the sample holder is placed. 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 a pressure resistance enough to withstand observation in a vacuum. That is, the atmospheric pressure state can be maintained in the holder even in the electron microscope. Then, on one main surface side of the insulating thin film 203, signal detection for detecting 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 section 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 one main surface of the insulating thin film 203 and the signal detection unit 210. The one main surface of the thin film 204 and the one main surface of the first insulating thin film 203 are arranged so as to have a predetermined gap. 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 in a state of being electrically insulated from the first insulating thin film 203 and the second insulating thin film 204 as the sample support film and separated from them.

  The second insulating thin film 204 is not essential. For example, if the observation sample 40 can be dissolved in an extremely thin water layer, the observation sample is held by the 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 surrounded by the conductive packing 206 and the O-ring 207. It is housed in a sealed conductive outer frame portion 208. That is, it is possible to observe the biological sample together with the aqueous solution.

  As will be described later, the outer frame portion 208 may be provided with a mechanism for adjusting the internal pressure. 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 strength. 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 set to the ground potential of the scanning electron microscope 100, and observation is performed in this state. Note that the sample can be observed even when 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 does not substantially pass through the first insulating thin film 203. Specifically, it is preferable to set the acceleration voltage such that the incident electron beam is substantially scattered or absorbed inside the conductive thin film 201. With such a voltage setting, primary electrons are hardly transmitted to the first insulating thin film 203 side, and electron beam damage to the observation sample 40 can be completely prevented. Generally, the above condition is realized if the acceleration voltage of the incident electron beam is set to 10 kV or less.

  If the first insulating thin film 203 is too thick, the intensity of the signal detected by the signal detection unit 210 is reduced. For this reason, it is preferable that the thickness of the first insulating thin film 203 be 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 intensity of the signal detected by the signal detection unit 210 decreases. For this reason, it is preferable that the thickness of the conductive thin film 201 be 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 efficiently suppress the shielding property of the electron beam and the internal diffusion of incident electrons. Examples of such a metal thin film include those containing tantalum, tungsten, rhenium, molybdenum, osmium, gold, or platinum as a main component.

  Thus, the scanning electron microscope sample holder used in the scanning electron microscope image observation system 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 one of the insulating thin film 203. A conductive thin film 201 laminated on the surface, and on one main surface side of the insulating thin film 203, the insulating thin film 201 caused by the electron beam 52 incident from the conductive thin film 201 side is provided. There is provided a signal detector 210 for detecting 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 in the sample holder 10 and outputs it as a measurement signal. A circuit unit (in this case, an amplifier 21) is built in. As described above, the terminal portion 22 is provided at the end portion of the sample stage 20, and this terminal portion 22 is connected to the connector 23 provided in the body 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) that amplifies the detection signal detected by the signal detection unit 210 and outputs the detection signal as a measurement signal are provided. They can be placed very close together. As a result, noise can be greatly reduced.

  In addition, since the amplification circuit unit is not provided in the main body of the scanning electron microscope but is built in the sample stage 20, a plurality of types of sample stages containing amplification circuit units (amplifiers 21) having different characteristics are prepared. In this case, it is easy to replace the amplification circuit unit (amplifier 21) according to the characteristics of the observation sample. Therefore, it is also easy to obtain an observation image with high contrast and resolution.

  In addition, when the space | interval between the one main surface of the 1st insulating thin film 203 and the one main surface of the 2nd insulating thin film 204 is too thick, the intensity | strength of the signal detected by the terminal part 210 falls. For this reason, it is preferable to set the said space | interval to 40 micrometers or less.

  The observation sample 40 is accommodated in such a sample holder 10, the electron beam 52 is incident while being turned 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. Observation is performed with the frequency set to 52.

  When the electron beam 52 whose intensity changes in a rectangular wave shape enters 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 diaphragm 54 (ON state), almost all incident electrons are scattered or absorbed by the conductive thin film 201. Thereby, electrons are accumulated in the incident site of the electron beam, and the site becomes a negative potential.

  An observation sample 40 dissolved in the aqueous solution 60 is sandwiched between the first insulating thin film 203 provided below the conductive thin film 201. 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. Due to this electric dipole arrangement, electric charges are also generated on the surface of the second insulating thin film 204 below the aqueous solution 60. This electric charge is detected by the signal detector 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 aperture 54 (OFF state), incident electrons in the conductive thin film 201 immediately flow into GND, and the above-described negative potential disappears. As a result, the arrangement of the electric dipoles in the aqueous solution 60 collapses, the charge on the surface of the first insulating thin film 203 disappears, and the measurement signal output from the signal detection unit 210 becomes zero.

  By repeating ON / OFF of such an electron beam at a desired frequency (for example, 1 kHz or more), a signal having the same frequency component can be extracted from the signal detection unit 210.

  For example, when the electron beam 52 that repeats ON / OFF at a frequency of 1 kHz or higher is irradiated, the dielectric constant of the biological sample is extremely lower than that of water, so that the electric dipole arrangement becomes weak and the intensity 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, it 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 protein is 2 to 3, the propagation force 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, a difference occurs in the propagation force of the potential change signal due to the difference in dielectric constant between the biological sample 40 and the aqueous solution 60, and this is the signal provided on the one main surface side of the first insulating thin film 203. By detecting with the detection part 210, it becomes possible to observe with high contrast, without dye | staining a 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.

  Note that the resolution of an image obtained by observation substantially depends on the irradiation diameter of the electron beam. For this reason, if the irradiation diameter of the electron beam is narrowed down to about 1 nm, it is possible to achieve substantially the same resolution (1 nm). As a result, a biological sample composed of bacteria, viruses, proteins, or protein complexes can be observed with high resolution and high contrast in a living state in an aqueous solution.

  Thus, in the present invention, the signal detection unit 210 is provided in the sample holder 10, and the detection signal detected by the signal detection unit 210 is amplified and output as a measurement signal on the sample stage 20 on which the sample holder 10 is placed. A circuit unit (amplifier) 21 is built in. By adopting such a configuration, it is possible to amplify the detection signal detected by the signal detection unit 210 directly and at the shortest distance by the circuit unit (amplifier) 21, thereby minimizing external noise contamination. Become.

  A terminal portion 22 is provided at the end of the sample stage 20, and this terminal portion 22 is connected to a connector 23 provided in the body 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, when the sample stage 20 is introduced from the sample exchange chamber 30 into the lens body, it is not necessary to connect a power cable or the like. In addition, since it is not necessary to make significant modifications to the scanning electron microscope 100 and it is not necessary to be dedicated to variable potential transmission observation, a general-purpose sample stage is used when performing normal observation not based on variable potential transmission observation. Can be used.

  The sample holder 10 has a configuration in which a sample of an aqueous solution is sealed in a gap between two upper and lower pressure-resistant thin films. As the thickness of the aqueous solution layer changes, the background contrast changes. In addition, the thinner the aqueous solution layer, the greater the intensity of the transmission signal and the S / N ratio and the resolution. Therefore, it is desirable that the aqueous solution layer be maintained at a constant interval that is slightly wider than the size (thickness) of the observation sample. Therefore, it is preferable to form a spacer between the upper and lower pressure-resistant thin films. By setting it as the thickness of such an aqueous solution layer, the removal of the background resulting from the thickness of the water layer becomes easy.

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

  When such a spacer is provided, even when the upper pressure-resistant thin film swells outward in a vacuum, the lower pressure-resistant thin film follows and swells outward, so that these thin films maintain a constant interval. It will be. The thickness of the spacer may be changed depending on the size of the target observation sample. However, if the thickness is too thick, the interval is too wide compared to the size of the observation sample, and noise is likely to occur. Since inconvenience is a concern, it is usually set to 40 μm or less.

  For example, in the observation of bacteria, since the diameter of a cylindrical cell is about 1 μm, the thickness of the spacer is preferably 1 to 2 μm. On the other hand, in the observation of viruses and proteins, about 100 to 200 nm is preferable. Such a spacer can be made of, for example, a material mainly composed of an adhesive, an adhesive, a resin, rubber, or a silicon member.

  Thus, according to the present invention, noise reduction and contrast enhancement (an improvement in the SN ratio of the fluctuation potential signal) and the thickness of the water layer during scanning electron microscope observation by the fluctuation potential transmission observation method are achieved. It is possible to remove the background caused by. In addition, it is not necessary to make a significant modification to the scanning electron microscope 100, and the introduction of the sample stage from the sample exchange chamber into the lens body does not require any special procedure.

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

  First, the sample stage 20 on which the observation holder 10 is placed 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 the upper side of the sample stage 20 is fitted and connected to the connector 23 provided in the observation chamber 100b, so that power supply to the first-stage amplifier 21 and transmission of the detection signal are performed. It becomes possible. After the sample stage 20 is introduced into the observation chamber 100b, the sample exchange rod 31 is pulled out to a predetermined position in the sample chamber 30 and the vacuum shutter 32 is closed (FIG. 3 (c)).

  As already described, since the scanning electron microscope 100 does not have to be dedicated to variable potential transmission observation, a general-purpose sample stage may be used when performing normal observation not based on variable potential transmission observation. . Since the general-purpose sample stage is not provided with the terminal portion 22, it is not connected to the connector 23 and is completely electrically insulated from an external signal processing device and an amplifier power source. As a result, it is possible to prevent external noise from being mixed and unexpected voltage addition.

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

  FIG. 4B is an observation image obtained by inverting the contrast and excluding 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 that is a kind of protein complex, reversing the contrast, and removing the background. When an unstained / non-fixed IgM antibody in an aqueous solution was sealed in a sample holder and observed, many particles of about 40 to 50 nm were observed (arrows in FIG. 5). Since the diameter of the IgM antibody is about 40 to 50 nm, it almost matches the size of the particle being observed. For this reason, it was confirmed by observation with the system which concerns on this invention that the protein particle | grains as it is in an aqueous solution can be observed.

  FIG. 6 illustrates a configuration example of the sample holder 10 in which the spacer 212 is provided between the pressure-resistant thin films (203, 204) so that the distance between the thin films is constant and the background during observation is reduced. FIG. FIG. 6A shows a state under atmospheric pressure, and FIG. 6B shows a state in a vacuum (inside 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 film 211 of the sample holder 20 swells outside in the microscope body, and the pressure in the holder decreases. The conductive thin film 201 side also curves outward, but the degree of curvature is relaxed by the effect of the decompression film 211.

  Note that the region below the second insulating thin film 204 is sealed with an O-ring 207 to maintain atmospheric pressure. Therefore, the second insulating thin film 204 is curved upward rather than downward, and a gap between one main surface of the first insulating thin film 203 and one main surface of the second insulating thin film 204 is widened. Thus, the holding of the observation sample 10 is not hindered.

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

  As described above, such a spacer 212 can be made of, for example, a material mainly composed of an adhesive, an adhesive, a resin, rubber, or a silicon member. The spacer 212 is produced by forming a pattern of an adhesive with a spraying device for fine coating, an industrial inkjet printer, or the like, or pasting a thin film after coating with an ultraviolet curable resin or the like, and irradiating with ultraviolet rays to bond them. It may be produced in this way.

  By changing the interval of the spacers 212 according to the size of the sample to be observed, it is possible to perform image observation with high contrast and good SN ratio. For example, it is about 2 to 10 μm for cell and bacteria observation, about 100 nm to 1 μm for virus observation, and 100 nm or less for protein observation.

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

  FIG. 7 shows a flow path for perfusing an aqueous solution into a gap at a predetermined interval between one main surface of the second insulating thin film 204 that 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 corresponding to the three flow paths 214 are formed, and the solution introduced into these flow paths 214 is subjected to the action of surface tension to cause the second insulating thin film 204 to be formed. The first main surface of the first insulating thin film 203 is introduced into a gap at a predetermined interval.

  Further, a damper 215 as a pressure application unit for extruding the solution from the injection hole 213 is provided on the side of the sample holder 10, and a pressure application valve 216 is provided at the tip of these dampers 215. Yes.

  When such a plurality of flow paths 214 are provided, an experiment such as newly feeding a reagent or the like into the sample holder 10 in which the observation sample 40 such as a cell or bacteria is previously stored, and observing the reaction by this in detail, etc. Will also be easier. In addition, what is shown with the code | symbol 217 in the figure is a waste liquid tank. Such a solution feeding may be performed electrophoretically without using the pressure application unit.

  As described above, the sample holder for a scanning electron microscope used in the scanning electron microscope image observation system according to the present invention has an insulating thin film in which one main surface is a holding surface of the observation sample and the other main body of the insulating thin film. A conductive thin film laminated on the surface, and on one main surface side of the insulating thin film, one main surface of the insulating thin film caused by an electron beam incident from the conductive thin film side is provided. A signal detector for detecting 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. By adopting such a configuration, the signal detection unit provided in the sample holder and the circuit unit that amplifies the detection signal detected by the signal detection unit and outputs it as a measurement signal can be arranged very close to each other. As a result, noise can be greatly reduced.

  In addition, since the amplification circuit section is not provided in the main body of the scanning electron microscope, but is built in the sample stage, observation is possible by preparing multiple types of sample stages containing amplification circuit sections with different characteristics. It is also easy to replace the amplification circuit unit according to the characteristics of the sample. Therefore, it is also easy to obtain an observation image with high contrast and resolution.

  As described above, according to the present invention, it is possible to simply and directly observe a biological sample in an aqueous solution with high resolution. For example, since it is possible to examine the reaction of chemicals and chemical substances while observing biological samples, it is a powerful weapon for research and development of chemicals.

  As described above, according to the present invention, it is possible to observe a biological sample in an aqueous solution with an extremely high contrast simply and without performing a staining treatment or an immobilization treatment. In addition, because the sample is not damaged by the electron beam, it is possible to know the original form and structure of organic materials that are susceptible to damage, including 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 device 54 Diaphragm 60 Aqueous solution 70 Diffusion area 100 Scanning electron microscope 201 Conduction Conductive 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 Drain tank 310 Lock-in amplifier 320 Data recorder 330 PC
340 DC power supply 350 Function generator

Claims (8)

A scanning electron microscope image observation system,
An insulating thin film whose main surface is a holding surface of the observation sample, and a conductive thin film laminated on the other main surface of the insulating thin film, the conductive thin film on one main surface side of the insulating thin film A sample holder for a scanning electron microscope provided with a signal detection unit for detecting a signal based on the potential of one main surface of the insulating thin film generated due to an electron beam incident from the side;
A sample stage for a scanning electron microscope on which the sample holder is mounted, and a sample stage having a built-in circuit unit for amplifying a detection signal detected by the signal detection unit and outputting it as a measurement signal;
System for observing a scanning electron microscope image using   In the scanning electron microscope sample holder, a pressure-resistant thin film is provided on one main surface side of the insulating thin film so as to have a predetermined distance from the insulating thin film, and the insulating thin film and the pressure-resistant thin film are provided. The scanning electron microscope image observation system according to claim 1, further comprising a spacer having a thickness corresponding to the predetermined interval.   The scanning electron microscope image observation system according to claim 2, wherein the spacer has a thickness of 40 μm or less.   The scanning electron microscope image observation system according to claim 2, wherein the spacer is made of a material mainly composed of an adhesive, an adhesive, a resin, rubber, and a silicon member.   The said sample holder for scanning electron microscopes is provided with the flow path for perfusing aqueous solution between the said insulating thin film and the said pressure | voltage resistant thin film of any one of Claims 1-4. Scanning electron microscope image observation system.   6. The apparatus according to claim 1, further comprising an arithmetic unit that processes an output signal obtained by extracting only a frequency component of a reference signal from the measurement signal to form an image as a scanning electron microscope image. The observation system of the scanning electron microscope image described in 1.   The said arithmetic unit processes the said output signal, extracts the signal component of an intensity | strength change frequency from this output signal, and forms an image based on the extracted signal component of an intensity change frequency. Scanning electron microscope image observation system.   The signal component of the intensity change frequency is extracted by any one of a bandpass filter method, a lock-in amplifier method, an autocorrelation analysis method, and a Fourier transform analysis method. Observation system.