JP2007279010A - Method for preparing observation sample used for scanning electronic microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new method for preparing an observation sample used for electronic microscopes of scanning, a transmitting, and the like. <P>SOLUTION: Adjustment is conducted by atomizing or applying treated water prepared by solving a metal ion-containing liquid at a predetermined concentration and then by diffusing the surface water of the observation material. According to the preparation method, vacuuming in the pretreatment can be omitted and preparation in a short time can be carried out. Thus, biological samples (raw things) are not broken up and the samples can be observed with time and in real time. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for preparing an observation sample in a microscope, and more particularly to a novel method for preparing an observation sample used in an electron microscope such as a scanning type or a transmission type.

Today, as a means of magnifying and observing a minute object, use of an electron microscope using an electron lens is spreading in addition to an optical microscope and a digital microscope using an optical lens.
An electron microscope is an electro-optic configuration of an imaging system such as an optical microscope by freely refracting the traveling direction of electrons, and forms an image of electrons transmitted through an observed sample using an electron lens. In addition to the transmission electron microscope, a reflection electron microscope that forms an image of electrons reflected from the sample surface, and a scanning electron that scans a focused electron beam on the sample surface and forms an image using secondary electrons from each scanning point. A microscope, a surface emission type field ion microscope that forms an image of electrons emitted from a sample by heating or ion irradiation, and the like are used depending on the intended application.

  Among these types of electron microscopes, a transmission electron microscope (TEM) transmits an electron beam through a thin film sample, and at that time, electrons scattered and diffracted by atoms in the sample are electron diffraction patterns or By obtaining a transmission electron microscope image, the internal structure of the substance can be mainly observed.

  On the other hand, a scanning electron microscope (SEM) scans secondary electrons and reflected electrons generated when a target sample is irradiated with a thin electron beam (electron probe) into secondary electron detectors, reflected electrons. It is an apparatus that is taken out using each detector such as a detector and displayed on a display screen such as a cathode ray tube or an LCD, and mainly observes the surface form of the sample.

  When an electron beam irradiates a solid sample, it is transmitted through the solid by the energy of the electrons. At that time, due to the interaction with the nuclei and electrons that make up the sample, there is non-elastic collision, elastic scattering, and energy loss. Causes elastic scattering. The inelastic shell excites electrons in the shell of the sample element, excites X-rays, etc., and emits secondary electrons, thereby losing the corresponding energy. The amount of secondary electrons emitted varies depending on the angle of collision. On the other hand, the reflected electrons scattered back by elastic scattering and emitted again from the sample are emitted in an amount specific to the atomic number. A scanning electron microscope (SEM) uses these secondary electrons and reflected electrons. The SEM irradiates a sample with electrons and detects emitted secondary electrons and reflected electrons to form an observation image.

  There are various factors that hinder the observation of the sample by the SEM, but the main factor due to the sample is a charging (charge-up) phenomenon that occurs during the observation of the sample. Charge-up is a phenomenon in which the irradiated surface is charged positively or negatively due to the difference in charge between incident charged particles and emitted charged particles. When this charge-up occurs, the emitted secondary electrons are accelerated. Or pulled back, no good imaging properties can be obtained, and in rare cases it does not image at all.

When an electron beam having a negative charge is incident on a bulk sample, if the sample is conductive, the charge is grounded through the sample, but if the sample is non-conductive, the charge of the incident electrons is The sample cannot escape from the surface of the sample, and the sample itself is charged up. As a charge-up phenomenon, abnormal contrast of light and darkness occurs in the observation visual field, or light and dark appear in a band shape, making it difficult to observe the secondary electron image.
When the charge-up is further intense, a so-called observation visual field drift occurs in which the visual field moves slowly or suddenly moves greatly.

Problems to be solved by the invention

  Under such circumstances, as a technique for removing the charge of the charged sample, for example, a method of irradiating an electron beam with a low acceleration voltage such that the generation efficiency of electrons from the sample exceeds 1, or accumulation on the sample surface There has been proposed a method of erasing by irradiating an electron beam having an accelerating voltage that generates a charge having a polarity opposite to the generated charge.

However, in the former method, when the energy charged in the sample exceeds the energy of the electron beam irradiated for static elimination, there is a problem that the irradiated electron beam is rebounded and cannot reach the sample, and cannot be eliminated.
In addition, this method has a problem that it is difficult to set the acceleration voltage. Although the acceleration voltage must be set so that the generation efficiency of secondary electrons is 1, the acceleration voltage at which the generation efficiency is 1 differs depending on the material and shape of the sample. An accelerating voltage such as 1 must be found.
When the sample is charged up, it is difficult to set an optimum acceleration voltage because the initial velocity of primary electrons is different from the landing velocity when entering the sample. Even if an accelerating voltage with a generation efficiency of 1 is found, if the sample is irradiated with electrons with a high accelerating voltage in the observation process so far, the sample is already charged with a negative charge, and therefore, due to charge-up. When an image disorder occurs. Although it may be possible to eliminate the charge by continuing to irradiate with an acceleration voltage of which the generation efficiency is 1 for a long time (for example, about several hours), it is not practical.

  On the other hand, the latter method is a method of observing before charging starts to accumulate, that is, a measure for preventing charging, and is not an active charge eliminating method of removing charged charges.

  As another method, a technique has been developed in which it is detected whether or not a sample is charged up, and when it is determined that the sample is charged up, a countermeasure against charging is taken. However, there are problems with charge-up detection, such as the need for dedicated equipment for detection, and various charge-up phenomena, making it difficult to detect correctly.

  In addition, there is a method of removing static electricity by bringing the sample chamber to atmospheric pressure and letting the sample touch the air, but not only is it not possible to remove electricity sufficiently, but it is necessary to re-evacuate to perform observation again. Another drawback is that it takes time and effort.

  In addition, methods for observation under low vacuum, methods using an electronic shower generator or ion shower generator, and methods for providing a control electrode have also been proposed. However, these methods require separate equipment and are easy to implement. It was difficult to do.

As described above, since any method has a problem, a method for easily removing the charge of the sample has been intensively developed. However, as a practical means for preventing the charge-up itself, A method of depositing (sputtering) a conductive thin film made of gold Au or the like on the surface is employed.
However, this method is not only suitable for the preparation of biological samples (raw foods) such as foods, because the sample needs to be dried and evacuated during vapor deposition. There was a problem that it was not able to return to its original state after observation due to the collapse of the image.

Means for solving the problem

By the way, the present inventor has developed an innovative method for dissociating (ionizing) a metal element contained in plant seeds as an electrolyte by utilizing fermentation technology, and has been registered as Japanese Patent No. 2865412.
Further, the metal element (mineral) -containing material fermented with gonococcus in a container formed with an electrostatic magnetic field and an organic acid solution having an alkyl group are mixed and stirred, and the atmosphere in the container is maintained under a predetermined condition for a predetermined period. We have also developed a method for efficiently dissociating (ionizing) and extracting metal elements from metal element-containing substances by holding them.

  And, by using the above method, a technique that has not been considered in the past, such as gold, silver, copper, magnesium, zinc, manganese, aluminum, stainless steel, platinum, etc. In addition, since each metal element in the extract obtained by this method has an angstrom size (10 to the 10th power to 9th power), the quality or function of the material can be greatly improved. In addition, it has been confirmed that it is also effective as an antistatic processing means for clothes, for example.

  Therefore, in the present invention, the treated water in which a metal ion-containing liquid (extract) containing a metal element as an ion is dissolved or sprayed on the sample to be observed, and then the surface water of the sample to be observed is diffused. By using the metal element contained in the treated water as a substitute for the conductive thin film, the charge-up can be prevented.

  In this case, the concentration of the metal ion-containing liquid with respect to the treated water is preferably set in the range of 0.2% to 100.0%. If the concentration of the metal ion-containing liquid is less than 0.2%, charge-up with a metal element is performed. It becomes difficult to prevent, and if it is set to 100.0%, a problem arises in terms of cost effectiveness.

  In addition, the metal ion-containing liquid is obtained by refining a mineral substance containing a metal element by mechanical means, etc., and then fermenting the refined mineral substance with koji mold, and further adding alkyl such as tartaric acid, citric acid, lactic acid or acetic acid. It is preferable to use an aqueous solution in which a metal element contained in the finely divided mineral substance is dissociated as ions in the organic acid solution by adding an organic acid solution having a group and stirring the mixture.

  Furthermore, examples of the metal element contained in the metal ion-containing liquid include gold, silver, copper, titanium, germanium, silicon, aluminum, iron, calcium, potassium, magnesium, zinc, manganese, vanadium, and the like. It is preferable to use a metal ion-containing liquid containing one or more metal elements.

The invention's effect

According to the method of the present invention, it is possible not only to prevent charge-up sufficiently by simply applying a metal ion-containing aqueous solution to the sample to be observed and then dissipating the surface water of the sample to be observed. Since it can be performed in a short time of about 10 minutes, the sample can be observed quickly and in real time.
In addition, there is no reflection or dispersion due to charge-up, so that clear imaging can be obtained, and biological samples such as food (raw products) do not need to be evacuated in the pretreatment, so that they do not collapse, Observation can also be suitably performed.
Furthermore, angstrom-order metal elements penetrate into the fine structure of the sample, so that the depth of focus becomes deeper. Therefore, it is possible to observe finely and clearly to some extent inside the target sample. The effect of this is achieved.

Next, the best mode of the preparation method of the sample to be observed used in the electron microscope according to the present invention will be exemplified and described in detail below.
That is, in the method for preparing a sample to be observed according to the present invention, as shown in FIG. 1, first, a gold (Au) ion-containing liquid 12 is dropped into purified water stored in a container 10, thereby processing at a predetermined concentration. Prepare water 14.
In this case, for example, purified water from which impurities are removed as much as possible by filtering the clean water through a reverse osmosis membrane (RO) is used.

The gold (Au) ion-containing liquid 12 is preferably an extract obtained by the following method.
That is, a mineral material containing gold foil and / or gold (Au) is finely pulverized in advance, and approximately equal amounts of this refined gold foil and / or refined mineral material and purified water are prepared and mixed. % To 20% koji mold is added and fermented at 35 to 40 degrees Celsius and allowed to stand for a certain period of time.
Next, the fermented raw material of the refined gold foil and / or refined mineral substance obtained by the pretreatment in this way and the organic acid solution having an alkyl group are mixed at a weight ratio of 10: 1. In this case, as the organic acid having an alkyl group, for example, tartaric acid, citric acid, lactic acid, or acetic acid can be appropriately used.

  Subsequently, the mixture of the refined gold foil and / or the fermented raw material of the refined mineral substance and the organic acid solution having an alkyl group is stirred and mixed in a container by an appropriate means. At this time, the mixture of the refined gold foil and / or the fermented raw material of the refined mineral substance and the organic acid having an alkyl group is utilized by utilizing the far-red effect radiated by the heater disposed in the container and the material of the container itself. Gold (as a metal element) is maintained by maintaining the liquid at 45 to 55 degrees Celsius and slowly circulating it for a predetermined time under the control of an electrostatic magnetic field, an ultraviolet sterilizer, or a sunlight irradiation mechanism by a circulation system such as a pump device. An extract obtained by dissociating (ionizing) Au) by about 0.3% to 0.5%, that is, a gold (Au) ion-containing solution 12 is used.

  In addition, when the gold (Au) ion-containing liquid 12 is dropped into the treated water 14, if the concentration of the gold ion-containing liquid 12 is 0.2% or less with respect to the treated water 14, prevention of charge-up by the metal element gold. It becomes difficult to expect sufficient functions. Moreover, although a gold ion containing liquid itself (100%) can also be used, in this case, it is not preferable in terms of cost effectiveness.

Next, the prepared sample 16 to be observed, such as a biological sample (raw material), is washed by a conventional method, and then treated water 14 is applied to the sample 16 to be observed using a brush or the like. In this case, it goes without saying that the treated water 14 may be sprayed by spraying or the like.
The observed sample 16 coated with treated water in this way is dried for about 2 to 5 minutes using a known drying method such as air drying, and the surface water is diffused to maintain the shape. This may be transferred to a SEM sample stage and observed by a conventional method.
When the surface morphology of the obtained sample 16 to be observed was observed with an SEM, it had the same conductivity as a conventional preparation method in which a conductive thin film of gold Au was deposited (sputtered) on the surface of the sample, and charge-up was performed. However, fine and high resolution imaging was confirmed. This is because the angstrom-sized metal element (gold Au in this embodiment) contained in the treated water 14 penetrates between the fine structures of the sample.

Example 1
A sample (prepared sample according to the method of the present invention) obtained by washing the longitudinally cut puffer myofibrils by a conventional method, brushing the treated water 14 and then diffusing the surface water by air drying for 3 minutes, and the same puffer A myofiber was washed by a conventional method, and a sample (comparative sample) by a conventional method prepared by vacuum-depositing and depositing gold Au (sputtering) was prepared, and an SEM electron microscope (manufactured by Keyence Corporation, VE-9800) was prepared. ) Were obtained (FIGS. 2a and 2b).
A comparison of the obtained surface morphology imaging photographs shows that the prepared sample by the method of the present invention has a better contrast than the comparative sample, and the detailed state of the surface is fine and accurate even though the sample contains water. Could be observed.

Example 2
After washing crab myofibrils by a conventional method, the treated water 14 was brushed and the surface water was diffused by air-drying for 5 minutes (prepared sample by the method of the present invention), and the same crab myofibrils were obtained by the standard method And a sample by a conventional method (comparative sample) prepared by vacuum evaporation and deposition of gold Au (sputtering), 300 times by SEM electron microscope (manufactured by Keyence Corporation, VE-9800) and 1000 times surface morphology imaging photographs (FIGS. 3a and 3b) were obtained, and when these surface morphology imaging photographs were compared, not only was the sample prepared according to the method of the present invention observed in the same manner as in the previous examples. Moreover, the fine shape of the outermost surface state could be observed more clearly than the comparative sample.

Example 3
After washing the plastic plate (PTFE) by a regular method, spraying treated water 14 and air-drying the surface water for 5 minutes (prepared sample by the method of the present invention) and the same plastic plate (PTFE) A sample by a conventional method (comparative sample) in which gold Au was vapor-deposited (sputtering) after cleaning by a conventional method was prepared, and 300 times and 1000 times in a SEM electron microscope (manufactured by Keyence Corporation, VE-9800) When comparing the surface morphology imaging photographs (FIGS. 4a and 4b), the sample prepared by the method of the present invention has a better contrast than the comparative sample, and less damage and deformation even at the same acceleration voltage (1.2 kv). The fine shape could be observed clearly.

  The preferred embodiments of the preparation method of the sample to be observed used in the electron microscope according to the present invention have been described above. However, the method of the present invention is not limited to these embodiments. The liquid metal element is not only Au, but also various metal elements such as silver, copper, titanium, germanium, silicon, aluminum, iron, calcium, potassium, magnesium, zinc, manganese, vanadium, and two or more of these metal elements. Needless to say, the contained metal ion-containing liquid can be used, and the target sample is a biological sample derived from a living organism, or a replica image forming material thereof, an organic material derived from a living organism, or an inorganic sample. The form is not limited.

It is a schematic procedure explanatory drawing which shows the best form of the preparation method of the to-be-observed sample used with the electron microscope which concerns on this invention. It is an electron microscope (SEM) imaging photograph of a puffer fibril, a is an imaging photograph of a puffer fibril longitudinal section obtained using the preparation method shown in FIG. 1, and b is prepared by a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of a fugu myofibril longitudinal section. FIG. 2 is an electron microscope (SEM) imaging photograph of a crab myofibril, wherein a is an imaging photograph of a crab myofibril obtained using the preparation method shown in FIG. 1, and b is a crab muscle prepared by a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of a fibril. 1 is an electron microscope (SEM) imaging photograph of a plastic plate (PTFE), wherein a is an imaging photograph of the surface of a plastic plate obtained using the preparation method shown in FIG. 1, and b is a plastic plate prepared by a conventional method. It is an imaging photograph of the surface (magnification rate 300 times, 1000 times).

Explanation of symbols

10 ... container,
12 ... Ion-containing liquid (gold Au),
14 ... treated water,
16: Sample to be observed,

The present invention relates to a method for preparing an observation sample in a microscope, and more particularly to a novel method for preparing an observation sample used in a scanning electron microscope .

Today, as a means of magnifying and observing a minute object, use of an electron microscope using an electron lens is spreading in addition to an optical microscope and a digital microscope using an optical lens.
An electron microscope is an electro-optic configuration of an imaging system such as an optical microscope by freely refracting the traveling direction of electrons, and forms an image of electrons transmitted through an observed sample using an electron lens. In addition to the transmission electron microscope, a reflection electron microscope that forms an image of electrons reflected from the sample surface, and a scanning electron that scans a focused electron beam on the sample surface and forms an image using secondary electrons from each scanning point. A microscope, a surface emission type field ion microscope that forms an image of electrons emitted from a sample by heating or ion irradiation, and the like are used depending on the intended application.

  Among these types of electron microscopes, a transmission electron microscope (TEM) transmits an electron beam through a thin film sample, and at that time, electrons scattered and diffracted by atoms in the sample are electron diffraction patterns or By obtaining a transmission electron microscope image, the internal structure of the substance can be mainly observed.

  On the other hand, a scanning electron microscope (SEM) scans secondary electrons and reflected electrons generated when a target sample is irradiated with a thin electron beam (electron probe) into secondary electron detectors, reflected electrons. It is an apparatus that is taken out using each detector such as a detector and displayed on a display screen such as a cathode ray tube or an LCD, and mainly observes the surface form of the sample.

When an electron beam irradiates a solid sample, it is transmitted through the solid by the energy of the electrons. At that time, due to the interaction with the nuclei and electrons that make up the sample, there is non-elastic collision, elastic scattering, and energy loss. Causes elastic scattering. The inelastic shell excites electrons in the shell of the sample element, excites X-rays, etc., and emits secondary electrons, thereby losing the corresponding energy. The amount of secondary electrons emitted varies depending on the angle of collision. On the other hand, the reflected electrons scattered back by elastic scattering and emitted again from the sample are emitted in an amount specific to the atomic number. A scanning electron microscope (SEM) detects the secondary electrons and reflected electrons emitted after irradiating the sample and forms an observation image.

  There are various factors that hinder the observation of the sample by the SEM, but the main factor due to the sample is a charging (charge-up) phenomenon that occurs during the observation of the sample. Charge-up is a phenomenon in which the irradiated surface is charged positively or negatively due to the difference in charge between incident charged particles and emitted charged particles. When this charge-up occurs, the emitted secondary electrons are accelerated. Or pulled back, no good imaging properties can be obtained, and in rare cases it does not image at all.

When an electron beam having a negative charge is incident on a bulk sample, if the sample is conductive, the charge is grounded through the sample, but if the sample is non-conductive, the charge of the incident electrons is The sample cannot escape from the surface of the sample, and the sample itself is charged up. As a charge-up phenomenon, abnormal contrast of light and darkness occurs in the observation visual field, or light and dark appear in a band shape, making it difficult to observe the secondary electron image.
When the charge-up is further intense, a so-called observation visual field drift occurs in which the visual field moves slowly or suddenly moves greatly.

Problems to be solved by the invention

  Under such circumstances, as a technique for removing the charge of the charged sample, for example, a method of irradiating an electron beam with a low acceleration voltage such that the generation efficiency of electrons from the sample exceeds 1, or accumulation on the sample surface There has been proposed a method of erasing by irradiating an electron beam having an accelerating voltage that generates a charge having a polarity opposite to the generated charge.

However, in the former method, when the energy charged in the sample exceeds the energy of the electron beam irradiated for static elimination, there is a problem that the irradiated electron beam is rebounded and cannot reach the sample, and cannot be eliminated.
In addition, this method has a problem that it is difficult to set the acceleration voltage. Although the acceleration voltage must be set so that the generation efficiency of secondary electrons is 1, the acceleration voltage at which the generation efficiency is 1 differs depending on the material and shape of the sample. An accelerating voltage such as 1 must be found.
When the sample is charged up, it is difficult to set an optimum acceleration voltage because the initial velocity of primary electrons is different from the landing velocity when entering the sample. Even if an accelerating voltage with a generation efficiency of 1 is found, if the sample is irradiated with electrons with a high accelerating voltage in the observation process so far, the sample is already charged with a negative charge, and therefore, due to charge-up. When an image disorder occurs. Although it may be possible to eliminate the charge by continuing to irradiate with an acceleration voltage of which the generation efficiency is 1 for a long time (for example, about several hours), it is not practical.

  On the other hand, the latter method is a method of observing before charging starts to accumulate, that is, a measure for preventing charging, and is not an active charge eliminating method of removing charged charges.

  As another method, a technique has been developed in which it is detected whether or not a sample is charged up, and when it is determined that the sample is charged up, a countermeasure against charging is taken. However, there are problems with charge-up detection, such as the need for dedicated equipment for detection, and various charge-up phenomena, making it difficult to detect correctly.

  In addition, there is a method of removing static electricity by bringing the sample chamber to atmospheric pressure and letting the sample touch the air, but not only is it not possible to remove electricity sufficiently, but it is necessary to re-evacuate to perform observation again. Another drawback is that it takes time and effort.

  In addition, methods for observation under low vacuum, methods using an electronic shower generator or ion shower generator, and methods for providing a control electrode have also been proposed. However, these methods require separate equipment and are easy to implement. It was difficult to do.

As described above, since any method has a problem, a method for easily removing the charge of the sample has been intensively developed. However, as a practical means for preventing the charge-up itself, A method of depositing (sputtering) a conductive thin film made of gold Au or the like on the surface is employed.
However, this method is not only suitable for the preparation of biological samples (raw foods) such as foods, because the sample needs to be dried and evacuated during vapor deposition. There was a problem that it was not able to return to its original state after observation due to the collapse of the image.

Means for solving the problem

By the way, the present inventor has developed an innovative method for dissociating (ionizing) a metal element contained in plant seeds as an electrolyte by utilizing fermentation technology, and has been registered as Japanese Patent No. 2865412.
Further, the metal element (mineral) -containing material fermented with gonococcus in a container formed with an electrostatic magnetic field and an organic acid solution having an alkyl group are mixed and stirred, and the atmosphere in the container is maintained under a predetermined condition for a predetermined period. We have also developed a method for efficiently dissociating (ionizing) and extracting metal elements from metal element-containing substances by holding them.

  And, by using the above method, a technique that has not been considered in the past, such as gold, silver, copper, magnesium, zinc, manganese, aluminum, stainless steel, platinum, etc. In addition, since each metal element in the extract obtained by this method has an angstrom size (10 to the 10th power to 9th power), the quality or function of the material can be greatly improved. In addition, it has been confirmed that it is also effective as an antistatic processing means for clothes, for example.

Therefore, in the method for preparing an observation sample used in the scanning electron microscope according to the present invention , treated water in which a metal ion-containing liquid (extract) containing a metal element as an ion is dissolved at a predetermined concentration is used as the observation sample. Spraying or applying, then dissipating the surface water of the sample to be observed, and using the metal element contained in the treated water as a substitute for the conductive thin film, the charge-up can be prevented. .

  In this case, the concentration of the metal ion-containing liquid with respect to the treated water is preferably set in the range of 0.2% to 100.0%. If the concentration of the metal ion-containing liquid is less than 0.2%, charge-up with a metal element is performed. It becomes difficult to prevent, and if it is set to 100.0%, a problem arises in terms of cost effectiveness.

  In addition, the metal ion-containing liquid is obtained by refining a mineral substance containing a metal element by mechanical means, etc., and then fermenting the refined mineral substance with koji mold, and further adding alkyl such as tartaric acid, citric acid, lactic acid or acetic acid. It is preferable to use an aqueous solution in which a metal element contained in the finely divided mineral substance is dissociated as ions in the organic acid solution by adding an organic acid solution having a group and stirring the mixture.

  Furthermore, examples of the metal element contained in the metal ion-containing liquid include gold, silver, copper, titanium, germanium, silicon, aluminum, iron, calcium, potassium, magnesium, zinc, manganese, vanadium, and the like. It is preferable to use a metal ion-containing liquid containing one or more metal elements.

The invention's effect

According to the method for preparing an observation sample used in the scanning electron microscope according to the present invention , only a simple operation of applying a metal ion-containing aqueous solution to the observation sample and then diffusing the surface water of the observation sample. In addition to being able to sufficiently prevent charge-up, the preparation can be performed in a short time of about 10 minutes, so that the sample can be observed quickly and in real time.
In addition, there is no reflection or dispersion caused by charge-up, so clear images can be obtained, and biological samples such as foods (raw foods) can be damaged because they do not need to be evacuated by pretreatment. In addition, observation over time can be suitably and suitably performed.
Furthermore, angstrom-order metal elements penetrate into the fine structure of the sample, so that the depth of focus becomes deeper. Therefore, it is possible to observe finely and clearly to some extent inside the target sample. The effect of this is achieved.

Next, the best mode of the preparation method of the sample to be observed used in the scanning electron microscope according to the present invention is illustrated and described in detail below.
That is, in the method for preparing a sample to be observed according to the present invention, as shown in FIG. 1, first, a gold (Au) ion-containing liquid 12 is dropped into purified water stored in a container 10, thereby processing at a predetermined concentration. Prepare water 14.
In this case, for example, purified water from which impurities are removed as much as possible by filtering the clean water through a reverse osmosis membrane (RO) is used.

The gold (Au) ion-containing liquid 12 is preferably an extract obtained by the following method.
That is, a mineral material containing gold foil and / or gold (Au) is finely pulverized in advance, and approximately equal amounts of this refined gold foil and / or refined mineral material and purified water are prepared and mixed. % To 20% koji mold is added and fermented at 35 to 40 degrees Celsius and allowed to stand for a certain period of time.
Next, the fermented raw material of the refined gold foil and / or refined mineral substance obtained by the pretreatment in this way and the organic acid solution having an alkyl group are mixed at a weight ratio of 10: 1. In this case, as the organic acid having an alkyl group, for example, tartaric acid, citric acid, lactic acid, or acetic acid can be appropriately used.

  Subsequently, the mixture of the refined gold foil and / or the fermented raw material of the refined mineral substance and the organic acid solution having an alkyl group is stirred and mixed in a container by an appropriate means. At this time, the mixture of the refined gold foil and / or the fermented raw material of the refined mineral substance and the organic acid having an alkyl group is utilized by utilizing the far-red effect radiated by the heater disposed in the container and the material of the container itself. The liquid is maintained at 45 to 55 degrees Celsius and is circulated slowly for a predetermined time under the control of an electrostatic magnetic field, an ultraviolet sterilizer, or a sunlight irradiation mechanism by a circulation system such as a pump device, so that gold as a metal element ( An extract obtained by dissociating (ionizing) Au) by about 0.3% to 0.5%, that is, a gold (Au) ion-containing solution 12 is used.

  In addition, when the gold (Au) ion-containing liquid 12 is dropped into the treated water 14, if the concentration of the gold ion-containing liquid 12 is 0.2% or less with respect to the treated water 14, prevention of charge-up by the metal element gold. It becomes difficult to expect sufficient functions. Moreover, although a gold ion containing liquid itself (100%) can also be used, in this case, it is not preferable in terms of cost effectiveness.

Next, the prepared sample 16 to be observed, such as a biological sample (raw material), is washed by a conventional method, and then treated water 14 is applied to the sample 16 to be observed using a brush or the like. In this case, it goes without saying that the treated water 14 may be sprayed by a spray or the like.
The observed sample 16 coated with treated water in this way is dried for about 2 to 5 minutes using a known drying method such as air drying, and the surface water is diffused to maintain the shape. This may be transferred to a SEM sample stage and observed by a conventional method.
When the surface morphology of the obtained sample 16 to be observed was observed with an SEM, it had the same conductivity as a conventional preparation method in which a conductive thin film of gold Au was deposited (sputtered) on the surface of the sample, and charge-up was performed. However, fine and high resolution imaging was confirmed. This is because the angstrom-sized metal element (gold Au in this embodiment) contained in the treated water 14 penetrates between the fine structures of the sample.

Example 1
A sample (prepared sample according to the method of the present invention) obtained by washing the longitudinally cut puffer myofibrils by a conventional method and then brushing the treated water 14 and releasing the surface water by air drying for 3 minutes. A myofiber was washed by a conventional method, and a sample (comparative sample) by a conventional method prepared by vacuum-depositing and depositing gold Au (sputtering) was prepared, and an SEM electron microscope (manufactured by Keyence Corporation, VE-9800) was prepared. ) Were obtained (FIGS. 2a and 2b).
A comparison of the obtained surface morphology imaging photographs shows that the prepared sample by the method of the present invention has a better contrast than the comparative sample, and the detailed state of the surface is fine and accurate even though the sample contains water. Could be observed.

Example 2
After washing crab myofibrils by a conventional method, the treated water 14 was brushed and the surface water was diffused by air-drying for 5 minutes (prepared sample by the method of the present invention), and the same crab myofibrils were obtained by the standard method And a sample by a conventional method (comparative sample) prepared by vacuum-evaporating and depositing gold Au (sputtering) after cleaning with a SEM electron microscope (manufactured by Keyence Corporation, VE-9800) and 300 times 1000 times surface morphology imaging photographs (FIGS. 3a and 3b) were obtained, and these surface morphology imaging photographs were compared. Moreover, the fine shape of the outermost surface state could be observed more clearly than the comparative sample.

Example 3
After washing the plastic plate (PTFE) by a regular method, spraying treated water 14 and air-drying the surface water for 5 minutes (prepared sample by the method of the present invention) and the same plastic plate (PTFE) A sample by a conventional method (comparative sample) in which gold Au was vapor-deposited (sputtering) after cleaning by a conventional method was prepared, and 300 times and 1000 times in a SEM electron microscope (manufactured by Keyence Corporation, VE-9800) When comparing the surface morphology imaging photographs (FIGS. 4a and 4b), the sample prepared by the method of the present invention has a better contrast than the comparative sample, and less damage and deformation even at the same acceleration voltage (1.2 kv). The fine shape could be observed clearly.

As mentioned above, although it demonstrated per suitable embodiment of the preparation method of the sample to be observed used with a scanning electron microscope (SEM) concerning the present invention, the method of the present invention is not limited to these embodiments, For example, the metal element of the metal ion-containing liquid is not only gold Au, but also various metal elements such as silver, copper, titanium, germanium, silicon, aluminum, iron, calcium, potassium, magnesium, zinc, manganese, vanadium, and these Needless to say, a metal ion-containing liquid containing two or more metal elements can be used, and the target sample is a biological sample derived from a living organism, or an organic substance derived from a living organism that is not derived from a living body, or a replica image forming substance thereof. The kind and form of the inorganic sample are not limited.

It is a schematic procedure explanatory drawing which shows the best form of the preparation method of the to-be-observed sample used with the scanning electron microscope (SEM) which concerns on this invention. FIG. 2 is a scanning electron microscope (SEM) imaging photograph of a blowfish fibril, wherein a is a photograph of a fugu myofibril longitudinal section obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of a puffer myofibril longitudinal section prepared by the above. FIG. 3 is a scanning electron microscope (SEM) imaging photograph of a crab myofibril, wherein a is a crab myofibril imaging photograph obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of the crab myofibril. FIG. 2 is an image of a plastic plate (PTFE) formed by a scanning electron microscope (SEM) , wherein a is an image of a plastic plate surface obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an image photograph (magnification rate 300 times, 1000 times) of the plastic plate surface.

Explanation of symbols

10 ... container,
12 ... Ion-containing liquid (gold Au),
14 ... treated water,
16: Sample to be observed,

  The present invention relates to a method for preparing an observation sample in a microscope, and more particularly to a novel method for preparing an observation sample used in a scanning electron microscope.

Today, as a means of magnifying and observing a minute object, use of an electron microscope using an electron lens is spreading in addition to an optical microscope and a digital microscope using an optical lens.
An electron microscope is an electro-optic configuration of an imaging system such as an optical microscope by freely refracting the traveling direction of electrons, and forms an image of electrons transmitted through an observed sample using an electron lens. In addition to the transmission electron microscope, a reflection electron microscope that forms an image of electrons reflected from the sample surface, and a scanning electron that scans a focused electron beam on the sample surface and forms an image using secondary electrons from each scanning point. A microscope, a surface emission type field ion microscope that forms an image of electrons emitted from a sample by heating or ion irradiation, and the like are used depending on the intended application.

  Among these types of electron microscopes, a transmission electron microscope (TEM) transmits an electron beam through a thin film sample, and at that time, electrons scattered and diffracted by atoms in the sample are electron diffraction patterns or By obtaining a transmission electron microscope image, the internal structure of the substance can be mainly observed.

  On the other hand, a scanning electron microscope (SEM) scans secondary electrons and reflected electrons generated when a target sample is irradiated with a thin electron beam (electron probe) into secondary electron detectors, reflected electrons. It is an apparatus that is taken out using each detector such as a detector and displayed on a display screen such as a cathode ray tube or an LCD, and mainly observes the surface form of the sample.

  When an electron beam irradiates a solid sample, it is transmitted through the solid by the energy of the electrons. At that time, due to the interaction with the nuclei and electrons that make up the sample, there is non-elastic collision, elastic scattering, and energy loss. Causes elastic scattering. The inelastic shell excites electrons in the shell of the sample element, excites X-rays, etc., and emits secondary electrons, thereby losing the corresponding energy. The amount of secondary electrons emitted varies depending on the angle of collision. On the other hand, the reflected electrons scattered back by elastic scattering and emitted again from the sample are emitted in an amount specific to the atomic number. A scanning electron microscope (SEM) detects the secondary electrons and reflected electrons emitted after irradiating the sample and forms an observation image.

  There are various factors that hinder the observation of the sample by the SEM, but the main factor due to the sample is a charging (charge-up) phenomenon that occurs during the observation of the sample. Charge-up is a phenomenon in which the irradiated surface is charged positively or negatively due to the difference in charge between incident charged particles and emitted charged particles. When this charge-up occurs, the emitted secondary electrons are accelerated. Or pulled back, no good imaging properties can be obtained, and in rare cases it does not image at all.

When an electron beam having a negative charge is incident on a bulk sample, if the sample is conductive, the charge is grounded through the sample, but if the sample is non-conductive, the charge of the incident electrons is The sample cannot escape from the surface of the sample, and the sample itself is charged up. As the charge-up phenomenon , an abnormality occurs in the contrast of light and darkness in the observation visual field, or the light and dark appear in a band shape, making it difficult to observe the secondary electron image.
When the charge-up is further intense, a so-called observation visual field drift occurs in which the visual field moves slowly or suddenly moves greatly.

Problems to be solved by the invention

  Under such circumstances, as a technique for removing the charge of the charged sample, for example, a method of irradiating an electron beam with a low acceleration voltage such that the generation efficiency of electrons from the sample exceeds 1, or accumulation on the sample surface There has been proposed a method of erasing by irradiating an electron beam having an accelerating voltage that generates a charge having a polarity opposite to the generated charge.

However, the former method has a problem that, for example, when the energy charged in the sample exceeds the energy of the electron beam irradiated for neutralization, the irradiated electron beam is rebounded and cannot reach the sample, and cannot be neutralized. .
In addition, this method has a problem that it is difficult to set the acceleration voltage. Although the acceleration voltage must be set so that the generation efficiency of secondary electrons is 1, the acceleration voltage at which the generation efficiency is 1 differs depending on the material and shape of the sample. An accelerating voltage such as 1 must be found.
When the sample is charged up, it is difficult to set an optimum acceleration voltage because the initial velocity of primary electrons is different from the landing velocity when entering the sample. Even if an accelerating voltage with a generation efficiency of 1 is found, if the sample is irradiated with electrons with a high accelerating voltage in the observation process so far, the sample is already charged with a negative charge, and therefore, due to charge-up. When an image disorder occurs. Although it may be possible to eliminate the charge by continuing to irradiate with an acceleration voltage of which the generation efficiency is 1 for a long time (for example, about several hours), it is not practical.

  On the other hand, the latter method is a method of observing before charging starts to accumulate, that is, a measure for preventing charging, and is not an active charge eliminating method of removing charged charges.

  As another method, a technique has been developed in which it is detected whether or not a sample is charged up, and when it is determined that the sample is charged up, a countermeasure against charging is taken. However, there are problems with charge-up detection, such as the need for dedicated equipment for detection, and various charge-up phenomena, making it difficult to detect correctly.

  In addition, there is a method of removing static electricity by bringing the sample chamber to atmospheric pressure and letting the sample touch the air, but not only is it not possible to remove electricity sufficiently, but it is necessary to re-evacuate to perform observation again. Another drawback is that it takes time and effort.

  In addition, methods for observation under low vacuum, methods using an electronic shower generator or ion shower generator, and methods for providing a control electrode have also been proposed, but these methods require special equipment and are easily implemented. It was difficult to do.

As described above, since any method has a problem, a method for easily removing the charge of the sample has been intensively developed. As a practical means for preventing the charge-up itself, for example, the sample A method of depositing (sputtering) a conductive thin film made of gold Au or the like on the surface is employed.
However, this method is not only suitable for the preparation of biological samples (raw foods) such as foods, because the sample needs to be dried and evacuated during vapor deposition. There was a problem that it was not able to return to its original state after observation due to the collapse of the image.

Means for solving the problem

By the way, the present inventor has developed an innovative method for dissociating (ionizing) a metal element contained in plant seeds as an electrolyte by utilizing fermentation technology, and has been registered as Japanese Patent No. 2865412.
Further, the metal element (mineral) -containing material fermented with gonococcus in a container formed with an electrostatic magnetic field and an organic acid solution having an alkyl group are mixed and stirred, and the atmosphere in the container is maintained under a predetermined condition for a predetermined period. We have also developed a method for efficiently dissociating (ionizing) and extracting metal elements from metal element-containing substances by holding them.

  Then, by utilizing the above method, various metal elements such as gold, silver, copper, magnesium, zinc, manganese, aluminum, stainless steel, and platinum, which have not been considered in the past, are dissociated (ionized) and extracted as an electrolyte. Technology is also established, and each metal element in the extract obtained by this method is angstrom size (10 to the 10th power to 9th power meter), so the quality or function of the material is drastically improved. In addition, it has been confirmed that it is effective as an antistatic processing means for clothing, for example.

  Therefore, in the method for preparing an observation sample used in the scanning electron microscope according to the present invention, treated water in which a metal ion-containing liquid (extract) containing a metal element as an ion is dissolved at a predetermined concentration is sprayed on the observation sample. Or, the surface water of the sample to be observed is diffused, and the charge up itself can be prevented by using the metal element contained in the treated water as a substitute for the conductive thin film. .

  In this case, the concentration of the metal ion-containing liquid with respect to the treated water is preferably set in the range of 0.2% to 100.0%. If the concentration of the metal ion-containing liquid is less than 0.2%, charge-up with a metal element is performed. It becomes difficult to prevent, and if it is set to 100.0%, a problem arises in terms of cost effectiveness.

  In addition, the metal ion-containing liquid is obtained by refining a mineral substance containing a metal element by mechanical means, etc., and then fermenting the refined mineral substance with koji mold, and further adding alkyl such as tartaric acid, citric acid, lactic acid or acetic acid. It is preferable to use an aqueous solution in which a metal element contained in the finely divided mineral substance is dissociated as ions in the organic acid solution by adding an organic acid solution having a group and stirring the mixture.

Furthermore, it is preferable to use a gold (Au) ion-containing liquid as the metal ion-containing liquid.

The invention's effect

According to the method for preparing an observation sample used in the scanning electron microscope according to the present invention, only a simple operation of applying a metal ion-containing aqueous solution to the observation sample and then diffusing the surface water of the observation sample. In addition to being able to sufficiently prevent the charge-up itself, the sample can be prepared in a short time of about 10 minutes, so that observation can be performed quickly and in real time.
In addition, there is no reflection or dispersion caused by charge-up, so clear images can be obtained, and even biological samples (raw food) such as food do not require pretreatment such as evacuation. Therefore, observation over time can be performed quickly and suitably.
Furthermore, since the metal element (gold Au ion) in the angstrom order penetrates into the fine structure of the sample, the depth of focus becomes deeper, so that it is possible to observe finely and clearly to some extent inside the target sample. It has excellent effects such as becoming.

Next, the best mode of the preparation method of the sample to be observed used in the scanning electron microscope according to the present invention is illustrated and described in detail below.
That is, in the method for preparing a sample to be observed according to the present invention, as shown in FIG. 1, first, a gold (Au) ion-containing liquid 12 is dropped into purified water stored in a container 10, thereby processing at a predetermined concentration. Prepare water 14.
In this case, for example, purified water from which impurities are removed as much as possible by filtering the clean water through a reverse osmosis membrane (RO) is used.

The gold (Au) ion-containing liquid 12 is preferably an extract obtained by the following method.
That is, a mineral material containing gold foil and / or gold (Au) is finely pulverized in advance, and approximately equal amounts of this refined gold foil and / or refined mineral material and purified water are prepared and mixed. % To 20% koji mold is added and fermented at 35 to 40 degrees Celsius and allowed to stand for a certain period of time.
Next, the fermented raw material of the refined gold foil and / or refined mineral substance obtained by the pretreatment in this way and the organic acid solution having an alkyl group are mixed at a weight ratio of 10: 1. In this case, as the organic acid having an alkyl group, for example, tartaric acid, citric acid, lactic acid, or acetic acid can be appropriately used.

Subsequently, the mixed solution of the refined gold foil and / or the fermented raw material of the refined mineral substance and the organic acid solution having an alkyl group is stirred and mixed in a container by an appropriate means. At this time, the mixed solution is maintained at 45 to 55 degrees Celsius by utilizing the far-red effect radiated by the heater disposed in the container and the material of the container itself, and the mixed solution is supplied to a pump device or the like. Gold (Au) as a metal element is dissociated (ionized) by about 0.3% to 0.5% by slowly circulating for a predetermined time under the control of an electrostatic magnetic field, an ultraviolet sterilizer, or a sunlight irradiation mechanism. ), Ie, a gold (Au) ion-containing liquid 12.
It should be noted that gold is dissociated from the refined gold foil by such a procedure as follows: “Bacteria, yeasts, filamentous fungi, algal fungi themselves, or the yeasts act on organic or inorganic substances to react with organic compounds and inorganic substances. This is because the fermentation that produces a compound and the phenomenon produces some substance, specifically, acetic acid, etc., generated during the fermentation growth of Aspergillus oryzae dissolves and dissociates the fine gold foil in the mixed solution.

  In addition, when the gold (Au) ion-containing liquid 12 is dropped into the treated water 14, if the concentration of the gold ion-containing liquid 12 is 0.2% or less with respect to the treated water 14, the charge-up prevention by the metal element gold is prevented. It becomes difficult to expect sufficient functions. Moreover, although a gold ion containing liquid itself (100%) can also be used, in this case, it is not preferable in terms of cost effectiveness.

Next, the prepared sample 16 to be observed, such as a biological sample (raw material), is washed by a conventional method, and then treated water 14 is applied to the sample 16 to be observed using a brush or the like. In this case, it goes without saying that the treated water 14 may be sprayed by a spray or the like.
The observed sample 16 coated with treated water in this way is dried for about 2 to 5 minutes using a known drying method such as air drying, and the surface water is diffused to maintain the shape. This may be transferred to a SEM sample stage and observed by a conventional method.
When the surface morphology of the obtained sample 16 to be observed was observed with an SEM, it had the same conductivity as a conventional preparation method in which a conductive thin film of gold Au was deposited (sputtered) on the surface of the sample, and charge-up was performed. However, fine and high resolution imaging was confirmed. This is because the angstrom-sized metal element (gold Au in this embodiment) contained in the treated water 14 penetrates between the fine structures of the sample.

Example 1
A sample (prepared sample according to the method of the present invention) obtained by washing the longitudinally cut puffer myofibrils by a conventional method, brushing the treated water 14 and then diffusing the surface water by air drying for 3 minutes, and the same puffer A myofiber was washed by a conventional method, and a sample (comparative sample) by a conventional method prepared by vacuum-depositing and depositing gold Au (sputtering) was prepared, and an SEM electron microscope (manufactured by Keyence Corporation, VE-9800) was prepared. ) Were obtained (FIGS. 2a and 2b).
A comparison of the obtained surface morphology imaging photographs shows that the prepared sample by the method of the present invention has a better contrast than the comparative sample, and the detailed state of the surface is fine and accurate even though the sample contains water. Could be observed.

Example 2
After washing crab myofibrils by a conventional method, the treated water 14 was brushed and the surface water was diffused by air-drying for 5 minutes (prepared sample by the method of the present invention), and the same crab myofibrils were obtained by the standard method And a sample by a conventional method (comparative sample) prepared by vacuum evaporation and deposition of gold Au (sputtering), 300 times by SEM electron microscope (manufactured by Keyence Corporation, VE-9800) and 1000 times surface morphology imaging photographs (FIGS. 3a and 3b) were obtained, and when these surface morphology imaging photographs were compared, not only was the sample prepared according to the method of the present invention observed in the same manner as in the previous examples. Moreover, the fine shape of the outermost surface state could be observed more clearly than the comparative sample.

Example 3
After washing the plastic plate (PTFE) by a regular method, spraying treated water 14 and air-drying the surface water for 5 minutes (prepared sample by the method of the present invention) and the same plastic plate (PTFE) A sample by a conventional method (comparative sample) in which gold Au was vapor-deposited (sputtering) after cleaning by a conventional method was prepared, and 300 times and 1000 times in a SEM electron microscope (manufactured by Keyence Corporation, VE-9800) When comparing the surface morphology imaging photographs (FIGS. 4a and 4b), the sample prepared by the method of the present invention has a better contrast than the comparative sample, and less damage and deformation even at the same acceleration voltage (1.2 kv). The fine shape could be observed clearly.

While there has been explained a preferred embodiment of the preparation process of the observed sample used with a scanning electron microscope according to the present invention (SEM), the method of the present invention be limited to these embodiments rather than , a biological sample of the sample is also derived from organisms the Target, or its replica image forming substance as well further organic substance derived from a living body non its kind and form, such as inorganic sample is not intended to be limiting.

It is a schematic procedure explanatory drawing which shows the best form of the preparation method of the to-be-observed sample used with the scanning electron microscope (SEM) which concerns on this invention. FIG. 2 is a scanning electron microscope (SEM) imaging photograph of a blowfish fibril, wherein a is a photograph of a fugu myofibril longitudinal section obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of a puffer myofibril longitudinal section prepared by the above. FIG. 3 is a scanning electron microscope (SEM) imaging photograph of a crab myofibril, wherein a is a crab myofibril imaging photograph obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of the crab myofibril. FIG. 2 is an image of a plastic plate (PTFE) formed by a scanning electron microscope (SEM), wherein a is an image of a plastic plate surface obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an image photograph (magnification rate 300 times, 1000 times) of the plastic plate surface.

Explanation of symbols

10 ... container,
12 ... Ion-containing liquid (gold Au),
14 ... treated water,
16: Sample to be observed,

  The present invention relates to a method for preparing an observation sample in a microscope, and more particularly to a novel method for preparing an observation sample used in a scanning electron microscope.

Today, as a means of magnifying and observing a minute object, use of an electron microscope using an electron lens is spreading in addition to an optical microscope and a digital microscope using an optical lens.
An electron microscope is an electro-optic configuration of an imaging system such as an optical microscope by freely refracting the traveling direction of electrons, and forms an image of electrons transmitted through an observed sample using an electron lens. In addition to the transmission electron microscope, a reflection electron microscope that forms an image of electrons reflected from the sample surface, and a scanning electron that scans a focused electron beam on the sample surface and forms an image using secondary electrons from each scanning point. A microscope, a surface emission type field ion microscope that forms an image of electrons emitted from a sample by heating or ion irradiation, and the like are used depending on the intended application.

  Among these types of electron microscopes, a transmission electron microscope (TEM) transmits an electron beam through a thin film sample, and at that time, electrons scattered and diffracted by atoms in the sample are electron diffraction patterns or By obtaining a transmission electron microscope image, the internal structure of the substance can be mainly observed.

  On the other hand, a scanning electron microscope (SEM) scans secondary electrons and reflected electrons generated when a target sample is irradiated with a thin electron beam (electron probe) into secondary electron detectors, reflected electrons. It is an apparatus that is taken out using each detector such as a detector and displayed on a display screen such as a cathode ray tube or an LCD, and mainly observes the surface form of the sample.

  When an electron beam irradiates a solid sample, it is transmitted through the solid by the energy of the electrons. At that time, due to the interaction with the nuclei and electrons that make up the sample, there is non-elastic collision, elastic scattering, and energy loss. Causes elastic scattering. The inelastic shell excites electrons in the shell of the sample element, excites X-rays, etc., and emits secondary electrons, thereby losing the corresponding energy. The amount of secondary electrons emitted varies depending on the angle of collision. On the other hand, the reflected electrons scattered back by elastic scattering and emitted again from the sample are emitted in an amount specific to the atomic number. A scanning electron microscope (SEM) detects the secondary electrons and reflected electrons emitted after irradiating the sample and forms an observation image.

  There are various factors that hinder the observation of the sample by the SEM, but the main factor due to the sample is a charging (charge-up) phenomenon that occurs during the observation of the sample. Charge-up is a phenomenon in which the irradiated surface is charged positively or negatively due to the difference in charge between incident charged particles and emitted charged particles. When this charge-up occurs, the emitted secondary electrons are accelerated. Or pulled back, no good imaging properties can be obtained, and in rare cases it does not image at all.

When an electron beam having a negative charge is incident on a bulk sample, if the sample is conductive, the charge is grounded through the sample, but if the sample is non-conductive, the charge of the incident electrons is The sample cannot escape from the surface of the sample, and the sample itself is charged up. As the charge-up phenomenon, an abnormality occurs in the contrast of light and darkness in the observation visual field, or the light and dark appear in a band shape, making it difficult to observe the secondary electron image.
When the charge-up is further intense, a so-called observation visual field drift occurs in which the visual field moves slowly or suddenly moves greatly.

Problems to be solved by the invention

  Under such circumstances, as a technique for removing the charge of the charged sample, for example, a method of irradiating an electron beam with a low acceleration voltage such that the generation efficiency of electrons from the sample exceeds 1, or accumulation on the sample surface There has been proposed a method of erasing by irradiating an electron beam having an accelerating voltage that generates a charge having a polarity opposite to the generated charge.

However, the former method has a problem that, for example, when the energy charged in the sample exceeds the energy of the electron beam irradiated for neutralization, the irradiated electron beam is rebounded and cannot reach the sample, and cannot be neutralized. .
In addition, this method has a problem that it is difficult to set the acceleration voltage. Although the acceleration voltage must be set so that the generation efficiency of secondary electrons is 1, the acceleration voltage at which the generation efficiency is 1 differs depending on the material and shape of the sample. An accelerating voltage such as 1 must be found.
When the sample is charged up, it is difficult to set an optimum acceleration voltage because the initial velocity of primary electrons is different from the landing velocity when entering the sample. Even if an accelerating voltage with a generation efficiency of 1 is found, if the sample is irradiated with electrons with a high accelerating voltage in the observation process so far, the sample is already charged with a negative charge, and therefore, due to charge-up. When an image disorder occurs. Although it may be possible to eliminate the charge by continuing to irradiate with an acceleration voltage of which the generation efficiency is 1 for a long time (for example, about several hours), it is not practical.

  On the other hand, the latter method is a method of observing before charging starts to accumulate, that is, a measure for preventing charging, and is not an active charge eliminating method of removing charged charges.

  As another method, a technique has been developed in which it is detected whether or not a sample is charged up, and when it is determined that the sample is charged up, a countermeasure against charging is taken. However, there are problems with charge-up detection, such as the need for dedicated equipment for detection, and various charge-up phenomena, making it difficult to detect correctly.

  In addition, there is a method of removing static electricity by bringing the sample chamber to atmospheric pressure and letting the sample touch the air, but not only is it not possible to remove electricity sufficiently, but it is necessary to re-evacuate to perform observation again. Another drawback is that it takes time and effort.

  In addition, methods for observation under low vacuum, methods using an electronic shower generator or ion shower generator, and methods for providing a control electrode have also been proposed, but these methods require special equipment and are easily implemented. It was difficult to do.

As described above, since any method has a problem, a method for easily removing the charge of the sample has been intensively developed. As a practical means for preventing the charge-up itself, for example, the sample A method of depositing (sputtering) a conductive thin film made of gold Au or the like on the surface is employed.
However, this method is not only suitable for the preparation of biological samples (raw foods) such as foods, because the sample needs to be dried and evacuated during vapor deposition. There was a problem that it was not able to return to its original state after observation due to the collapse of the image.

Means for solving the problem

By the way, the present inventor has developed an innovative method for dissociating (ionizing) a metal element contained in plant seeds as an electrolyte by utilizing fermentation technology, and has been registered as Japanese Patent No. 2865412.
Further, the metal element (mineral) -containing material fermented with gonococcus in a container formed with an electrostatic magnetic field and an organic acid solution having an alkyl group are mixed and stirred, and the atmosphere in the container is maintained under a predetermined condition for a predetermined period. We have also developed a method for efficiently dissociating (ionizing) and extracting metal elements from metal element-containing substances by holding them.

  Then, by utilizing the above method, various metal elements such as gold, silver, copper, magnesium, zinc, manganese, aluminum, stainless steel, and platinum, which have not been considered in the past, are dissociated (ionized) and extracted as an electrolyte. Technology is also established, and each metal element in the extract obtained by this method is angstrom size (10 to the 10th power to 9th power meter), so the quality or function of the material is drastically improved. In addition, it has been confirmed that it is effective as an antistatic processing means for clothing, for example.

Therefore, in the method for preparing an observation sample used in the scanning electron microscope according to the present invention, a metal ion-containing liquid containing gold element as ions, that is, a treatment in which a gold ion-containing liquid (extracted liquid) is dissolved at a predetermined concentration. Spraying or applying water to the sample to be observed, then dissipating the surface water of the sample to be observed, and preventing the charge-up itself by using the metal element contained in the treated water as a substitute for the conductive thin film It is something that can be done.

In this case, the concentration of the gold ion-containing liquid with respect to the treated water is preferably set in the range of 0.2% to 100.0%. If the concentration of the gold ion-containing liquid is less than 0.2%, the charge-up with the gold element is performed. It becomes difficult to prevent, and if it is set to 100.0%, a problem arises in terms of cost effectiveness.

As the gold ion-containing liquid , a mineral substance containing gold element is refined by mechanical means, and then fermented by adding koji molds to the refined mineral substance. Further, an alkyl such as tartaric acid, citric acid, lactic acid or acetic acid is used. By adding an organic acid solution having a group and mixing and stirring, an aqueous solution in which the gold element contained in the finely divided mineral material is dissociated as ions in the organic acid solution is used.

The invention's effect

According to the preparation method of the sample to be observed used in the scanning electron microscope according to the present invention, only a simple operation of applying the gold ion-containing aqueous solution to the sample to be observed and then diffusing the surface water of the sample to be observed. In addition to being able to sufficiently prevent the charge-up itself, the sample can be prepared in a short time of about 10 minutes, so that observation can be performed quickly and in real time.
In addition, there is no reflection or dispersion caused by charge-up, so clear images can be obtained, and even biological samples (raw food) such as food do not require pretreatment such as evacuation. Therefore, observation over time can be performed quickly and suitably.
Furthermore, angstrom-order gold elements (gold Au ions) penetrate into the fine structure of the sample, so that the depth of focus becomes deeper. Therefore, it is possible to observe finely and clearly to some extent inside the target sample. It has excellent effects such as becoming.

Next, the best mode of the preparation method of the sample to be observed used in the scanning electron microscope according to the present invention is illustrated and described in detail below.
That is, in the method for preparing a sample to be observed according to the present invention, as shown in FIG. 1, first, a gold (Au) ion-containing liquid 12 is dropped into purified water stored in a container 10, thereby processing at a predetermined concentration. Prepare water 14.
In this case, for example, purified water from which impurities are removed as much as possible by filtering the clean water through a reverse osmosis membrane (RO) is used.

The gold (Au) ion-containing liquid 12 is preferably an extract obtained by the following method.
That is, a mineral material containing gold foil and / or gold (Au) is finely pulverized in advance, and approximately equal amounts of this refined gold foil and / or refined mineral material and purified water are prepared and mixed. % To 20% koji mold is added and fermented at 35 to 40 degrees Celsius and allowed to stand for a certain period of time.
Next, the fermented raw material of the refined gold foil and / or refined mineral substance obtained by the pretreatment in this way and the organic acid solution having an alkyl group are mixed at a weight ratio of 10: 1. In this case, as the organic acid having an alkyl group, for example, tartaric acid, citric acid, lactic acid, or acetic acid can be appropriately used.

Subsequently, the mixed solution of the refined gold foil and / or the fermented raw material of the refined mineral substance and the organic acid solution having an alkyl group is stirred and mixed in a container by an appropriate means. At this time, the mixed solution is maintained at 45 to 55 degrees Celsius by utilizing the far-red effect radiated by the heater disposed in the container and the material of the container itself, and the mixed solution is supplied to a pump device or the like. Gold (Au) as a metal element is dissociated (ionized) by about 0.3% to 0.5% by slowly circulating for a predetermined time under the control of an electrostatic magnetic field, an ultraviolet sterilizer, or a sunlight irradiation mechanism. ), Ie, a gold (Au) ion-containing liquid 12.
It should be noted that gold is dissociated from the refined gold foil by such a procedure as follows: “Bacteria, yeasts, filamentous fungi, algal fungi themselves, or the yeasts act on organic or inorganic substances to react with organic compounds and inorganic substances. This is because the fermentation that produces a compound and the phenomenon produces some substance, specifically, acetic acid, etc., generated during the fermentation growth of Aspergillus oryzae dissolves and dissociates the fine gold foil in the mixed solution.

  In addition, when the gold (Au) ion-containing liquid 12 is dropped into the treated water 14, if the concentration of the gold ion-containing liquid 12 is 0.2% or less with respect to the treated water 14, the function of preventing charge-up by gold is sufficiently expected. It becomes difficult to do. Moreover, although a gold ion containing liquid itself (100%) can also be used, in this case, it is not preferable in terms of cost effectiveness.

Next, the prepared sample 16 to be observed, such as a biological sample (raw material), is washed by a conventional method, and then treated water 14 is applied to the sample 16 to be observed using a brush or the like. In this case, it goes without saying that the treated water 14 may be sprayed by a spray or the like.
The observed sample 16 coated with treated water in this way is dried for about 2 to 5 minutes using a known drying method such as air drying, and the surface water is diffused to maintain the shape. This may be transferred to a SEM sample stage and observed by a conventional method.
When the surface morphology of the obtained sample 16 to be observed was observed with an SEM, it had the same conductivity as a conventional preparation method in which a conductive thin film of gold Au was deposited (sputtered) on the surface of the sample, and charge-up was performed. However, fine and high resolution imaging was confirmed. This is because the angstrom size gold element contained in the treated water 14 penetrates between the fine tissues of the sample.

Example 1
A sample (prepared sample according to the method of the present invention) obtained by washing the longitudinally cut puffer myofibrils by a conventional method, brushing the treated water 14 and then diffusing the surface water by air drying for 3 minutes, and the same puffer A myofiber was washed by a conventional method, and a sample (comparative sample) by a conventional method prepared by vacuum-depositing and depositing gold Au (sputtering) was prepared, and an SEM electron microscope (manufactured by Keyence Corporation, VE-9800) was prepared. ) Were obtained (FIGS. 2a and 2b).
A comparison of the obtained surface morphology imaging photographs shows that the prepared sample by the method of the present invention has a better contrast than the comparative sample, and the detailed state of the surface is fine and accurate even though the sample contains water. Could be observed.

Example 2
After washing crab myofibrils by a conventional method, the treated water 14 was brushed and the surface water was diffused by air-drying for 5 minutes (prepared sample by the method of the present invention), and the same crab myofibrils were obtained by the standard method And a sample by a conventional method (comparative sample) prepared by vacuum evaporation and deposition of gold Au (sputtering), 300 times by SEM electron microscope (manufactured by Keyence Corporation, VE-9800) and 1000 times surface morphology imaging photographs (FIGS. 3a and 3b) were obtained, and these surface morphology imaging photographs were compared. Moreover, the fine shape of the outermost surface state could be observed more clearly than the comparative sample.

Example 3
After washing the plastic plate (PTFE) by a regular method, spraying treated water 14 and air-drying the surface water for 5 minutes (prepared sample by the method of the present invention) and the same plastic plate (PTFE) A sample by a conventional method (comparative sample) in which gold Au was vapor-deposited (sputtering) after cleaning by a conventional method was prepared, and 300 times and 1000 times in a SEM electron microscope (manufactured by Keyence Corporation, VE-9800) When comparing the surface morphology imaging photographs (FIGS. 4a and 4b), the sample prepared by the method of the present invention has a better contrast than the comparative sample, and less damage and deformation even at the same acceleration voltage (1.2 kv). The fine shape could be observed clearly.

  As mentioned above, although it demonstrated per suitable embodiment of the preparation method of the sample to be observed used with a scanning electron microscope (SEM) concerning the present invention, the method of the present invention is not limited to these embodiments, The target sample is not limited to a biological sample derived from a living organism, or a replica image forming material thereof, an organic material derived from a living organism, an inorganic sample, or the like.

It is a schematic procedure explanatory drawing which shows the best form of the preparation method of the to-be-observed sample used with the scanning electron microscope (SEM) which concerns on this invention. FIG. 2 is a scanning electron microscope (SEM) imaging photograph of a blowfish fibril, wherein a is a photograph of a fugu myofibril longitudinal section obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of a puffer myofibril longitudinal section prepared by the above. FIG. 3 is a scanning electron microscope (SEM) imaging photograph of a crab myofibril, wherein a is a crab myofibril imaging photograph obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an imaging photograph (magnification rate 300 times, 1000 times) of the crab myofibril. FIG. 2 is an image of a plastic plate (PTFE) formed by a scanning electron microscope (SEM), wherein a is an image of a plastic plate surface obtained using the preparation method shown in FIG. 1, and b is a conventional method. It is an image photograph (magnification rate 300 times, 1000 times) of the plastic plate surface.

Explanation of symbols

10 ... container,
12 ... Gold (Au) ion-containing liquid ,
14 ... treated water,
16: Sample to be observed,

Claims (4)

  Used in an electron microscope characterized by spraying or applying treated water prepared by dissolving a metal ion-containing solution containing metal elements as ions to a sample to be observed, and then diffusing the surface water of the sample to be observed A method for preparing a sample to be observed.   The method for preparing an observation sample for use in an electron microscope according to claim 1, wherein the concentration of the metal ion-containing liquid with respect to the treated water is set in a range of 0.2% to 100.0%.   The metal ion-containing liquid is obtained by refining a mineral substance containing a metal element by mechanical means, then fermenting the refined mineral substance with koji mold and further having an alkyl group such as tartaric acid, citric acid, lactic acid, and acetic acid. The method for preparing a sample to be observed for use in an electron microscope according to claim 1 or 2, wherein the metal element is dissociated as ions in the organic acid solution by adding an acid and stirring.   As the metal ion-containing liquid, a metal ion-containing liquid containing one or more of gold, silver, copper, titanium, germanium, silicon, aluminum, iron, calcium, potassium, magnesium, zinc, manganese, and vanadium as metal elements is used. The preparation method of the to-be-observed sample used with the electron microscope in any one of Claims 1-3 which consists of this.