WO2021256412A1 - 走査型電子顕微鏡を用いた観察方法、及びそのための試料ホルダ - Google Patents

走査型電子顕微鏡を用いた観察方法、及びそのための試料ホルダ Download PDF

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Publication number
WO2021256412A1
WO2021256412A1 PCT/JP2021/022443 JP2021022443W WO2021256412A1 WO 2021256412 A1 WO2021256412 A1 WO 2021256412A1 JP 2021022443 W JP2021022443 W JP 2021022443W WO 2021256412 A1 WO2021256412 A1 WO 2021256412A1
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WIPO (PCT)
Prior art keywords
sample
thin film
target sample
target
radiation source
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Ceased
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PCT/JP2021/022443
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English (en)
French (fr)
Japanese (ja)
Inventor
正嶺 新谷
誠二 山口
博朗 ▲たか▼玉
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Chubu University
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Chubu University
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Priority to EP21825836.6A priority Critical patent/EP4167266A4/en
Priority to US18/010,610 priority patent/US20230274905A1/en
Priority to JP2022531786A priority patent/JP7142404B2/ja
Publication of WO2021256412A1 publication Critical patent/WO2021256412A1/ja
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/02—Details
    • H01J37/20—Means for supporting or positioning the object or the material; Means for adjusting diaphragms or lenses associated with the support
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
    • H01J37/28—Electron or ion microscopes; Electron or ion diffraction tubes with scanning beams
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/20—Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated
    • H01J2237/2002—Controlling environment of sample
    • H01J2237/2003—Environmental cells
    • H01J2237/2004—Biological samples

Definitions

  • the present invention relates to an observation method using a scanning electron microscope and a sample holder for that purpose, and can be suitably used for observing a sample in a water-containing or liquid environment such as a living body.
  • Pretreatment includes chemical fixation that forms crosslinks between molecules constituting the sample, and metal coating that applies metal ions to the surface of the sample in a state where it is rapidly cooled and frozen. In either case, the movement of the sample is stopped regardless of whether the sample is alive or dead, and the movement of the sample cannot be observed.
  • Patent Document 1 a composition for suppressing evaporation is applied to the surface of a sample, and a polymer film is formed on the surface by irradiating the surface with an electron beam or plasma to prevent plastic deformation and damage of the sample.
  • Non-Patent Document 1 a hydrated sample was placed in a container together with a solution, the opening of the container was closed with an electron-permeable thin film, and the thin film was reinforced with a lattice rigider than the thin film in order to maintain the flatness of the thin film. Then, the sample is irradiated with an electron beam through a thin film to detect reflected electrons. The sample is in close contact with the thin film. Further, in order to increase the transparency of backscattered electrons, a thin film having a thickness of, for example, 145 nm, which is as thin as possible, is optimal.
  • Patent Document 2 in a state where the sample is housed between the laminated body composed of the first conductive thin film and the insulating thin film and the second conductive thin film, an electron beam is incident from above and transmitted through the sample. The secondary electrons emitted from the lower surface of the second conductive thin film are detected.
  • Patent Document 3 the strength is pulsed from the conductive thin film side in a state where the sample is housed together with the liquid between the laminate composed of the conductive thin film and the first insulating thin film and the second insulating thin film. A changing electron beam is incident, and the change in the outer surface potential of the second insulating thin film corresponding to the difference in the dielectric constants of the sample and the liquid is measured.
  • Non-Patent Document 1 it is necessary to stain the sample with a compound containing a heavy metal element such as osmium in order to detect backscattered electrons. I will stop it. In addition, it is only possible to observe the portion of the sample that is close to the thin film, and it is not possible to obtain a sense of distance in the direction orthogonal to the thin film.
  • a heavy metal element such as osmium
  • the sample is limited to a thickness that fits within the distance between the laminate and the second conductive thin film or the second insulating thin film facing the laminate.
  • the interval is preferably 200 ⁇ m or less (Patent Document 2 [0043]) or 40 ⁇ m or less (Patent Document 3 [0063]).
  • the image obtained by the method of Patent Document 2 is two-dimensional as in Non-Patent Document 1, and the image obtained by the method of Patent Document 3 is not an actual structure but a two-dimensional distribution of permittivity. It's just that.
  • the sample holder since the sample holder is arranged between the radiation source of electron beam irradiation and the detector, many existing scanning types in which the detector is located on the same side as the radiation source. Cannot be applied to electron microscopes.
  • one object of the present invention is to provide a method for observing a biological sample in a living state without being restricted by the properties of the sample itself or the structure containing the sample. ..
  • Another challenge is to provide a method that can be applied to many existing scanning electron microscopes to observe the three-dimensional structure of biological samples.
  • the observation method of the present invention In a method of observing a target sample on a sample table with a scanning electron microscope having a radiation source of electron beam irradiation, An encapsulation step in which an insulating and electron-permeable thin film is applied to the target sample so as to imitate the surface of the target sample on the radiation source side, and the target sample is enclosed within the space between the thin film and the sample table. , It is characterized by comprising an irradiation step of irradiating the target sample with an electron beam from the radiation source via the thin film.
  • the target sample may coexist with the liquid material or may be in a water-containing state.
  • the liquid material means an ionic liquid, an aqueous solution, a gel, a sol, or the like.
  • a thin film is applied to the target sample so as to imitate the surface of the target sample on the radiation source side.
  • the thin film comes into close contact with the target sample due to the reaction force received by the target sample from the sample table.
  • the target sample is enclosed within the space between the thin film and the sample table.
  • the thin film follows the surface of the target sample.
  • the outer surface of the thin film F corresponds to the outer shape of the target sample S, and the thin film F follows the movement of the target sample S. Therefore, by irradiating the target sample S with an electron beam (thick arrow in the figure) in this state, the three-dimensional structure and movement of the target sample S can be observed.
  • the thin film F since the thin film F is reinforced by the grid G as shown in FIG. 11, the thin film F does not follow the target sample S, and only the plan view shape of the target sample S is obtained. Is significantly different from being able to observe.
  • the target sample may be charged. Since there is no such thing, a clear image can be obtained.
  • the work of covering the target sample with the thin film may be in the atmosphere. Therefore, the water or liquid in the sample does not evaporate. Since the target sample is covered with the thin film and then irradiated with the electron beam, the target sample does not have to have resistance to vacuum.
  • the observation is usually made by detecting secondary electrons emitted from the target sample or the thin film during the electron beam irradiation. Since it is not necessary to stain the target sample and secondary electrons (thin arrows in FIG. 10) are emitted from the position following the target sample S, the direction and amount of displacement of the target sample or thin film should be measured with high accuracy. Because it can be done. Therefore, the observation method of the present invention can be realized by using an existing scanning electron microscope in which the secondary electron detector is fixed on the radiation source side. If the target sample contains both light and heavy elements, or if the outer surface of the thin film is covered with only one layer of metal particles, it is also possible to detect backscattered electrons, which enables secondary electron detection. Similarly, the direction and amount of displacement of the target sample or thin film can be measured.
  • the thin film is preferably elastically deformable according to the movement of the target sample. This is because the thin film is less likely to break even if the displacement of the target sample is large. Further, it is also possible to measure the stress generated between the corresponding points of the target sample via the relative displacement amount of two or more points of the thin film due to the elastic deformation of the thin film. If the Young's modulus with respect to the tensile tension of the thin film is measured in advance and the relative displacement of the two points is divided by the distance between the two points, the "strain” can be obtained, and the "stress” is the product of the "Young's modulus” and the "strain". The stress can be obtained. And the source of the stress is the force generated by the sample.
  • One preferable configuration of the method of the present invention is, in addition to the encapsulation step and the irradiation step, a labeled seeding step of seeding the label on the surface of the thin film opposite to the target sample side before the irradiation step.
  • a labeled seeding step of seeding the label on the surface of the thin film opposite to the target sample side before the irradiation step.
  • the label has a known shape and, in addition to the encapsulation step, the label seeding step and the irradiation step, the non-point aberration of the label is added after the irradiation step. It is provided with an analysis process for analysis.
  • the movement in the height direction of the label corresponds to that in the thickness direction of the film, and by analyzing astigmatism, the movement in the height direction of the label is toward the front with respect to the radiation source. It is possible to identify whether it is a thing or a thing going to the back.
  • the shape of the sample is measured with high resolution by aligning the sample with a certain marker as the center and integrating multiple images. be able to.
  • the structural change and movement of the sample can be precisely analyzed using the marker as a coordinate point.
  • the shape and material of the sign are not limited, but in the case of a spherical shape, a sign having a diameter of 0.5 nm or more and 1 mm or less is preferable. However, it is desirable that the film stays on the thin film due to frictional force or adhesive force, and it is not preferable that the film rolls on the thin film.
  • the scanning electron microscope sample holder of the present invention suitable for the observation method is A sample table on which the target sample can be placed so as to face the radiation source of the electron beam irradiation of the scanning electron microscope, and Insulating and electron-permeable thin film, It is characterized by comprising means for enclosing the target sample within the distance between the thin film and the sample table so that the thin film is applied to the target sample following the surface of the target sample on the radiation source side. do. It is desirable that there is nothing other than the encapsulation means in the vicinity of the surface of the thin film on the radiation source side so that the displacement of the sample and the thin film is not hindered during sample observation.
  • the sample holder of the present invention preferably further includes adjusting means for adjusting the tension applied to the thin film.
  • adjusting the tension applied to the thin film By adjusting the tension applied to the thin film, various properties of the target sample can be observed.
  • the tension is increased, the accuracy of copying the thin film to the target sample is improved, and a finer structure of the target sample can be observed.
  • the tension is lowered, the binding force on the target sample is weakened, and a large movement of the target sample can be observed.
  • the adjusting means is one or more spacers interposed between the target sample and the sample table. This is because even if the target sample is thin, the thin film can be easily imitated by the target sample. It is advisable to prepare a plurality of spacers having different thicknesses and elastic moduluss so that the spacers according to the properties of the target sample can be applied. For example, in addition to cotton and melamine sponge, biomaterial spacers such as heart, liver and kidney can be mentioned. A plurality of spacers may be used at the same time, in which case at least one may have elasticity. The surface pressure and tension applied to the thin film can be quantitatively adjusted according to the performance and height of the spacer to optimize the observation conditions.
  • the thin film has an atmospheric pressure around the target sample during the observation, whereas the upper space of the thin film is a vacuum, so that it can withstand the pressure difference between the vacuum and the atmospheric pressure (100,000 Pa) from the sample. It is required to have a performance that does not break due to a reaction force or the like, and is preferably made of an organic polymer, particularly preferably a polyimide. In addition to the above-mentioned performance, it has optical transparency in addition to the optimum insulating property and electron permeability as the thin film of the present invention, and a commercially available polyimide precursor of an appropriate size can be easily synthesized. Because.
  • the thin film When the thin film is made of polyimide, it preferably has a thickness of 100 nm or more and 5 ⁇ m or less.
  • the reason for setting it to 100 nm or more is that if it is less than 100 nm, the strength is weak, and even if it is 100 nm or more and secondary electrons emitted from the thin film are detected, the shape and movement of the target sample are sufficiently reflected. Is.
  • the reason for setting it to 5 ⁇ m or less is that if it exceeds 5 ⁇ m, it becomes difficult to follow the displacement of the target sample.
  • the sample table is preferably made of a conductive material. This is because the distortion of the image due to the charging of the sample can be prevented only by making the liquid material conductive or using the material of the thin film or the target sample and the component in contact with the sample table as the conductive material.
  • One preferred configuration for the encapsulation means is A sealing member that is interposed between the thin film and the sample table and can be elastically deformed in a direction orthogonal to the thin film side surface of the sample table.
  • a rigid holding member that sandwiches the thin film between the sealing member and the sealing member, It is provided with a fixing member for fixing the holding member to the sample table.
  • thin films can be imitated on samples of various thicknesses and hardnesses by using sealing members having different heights and elasticity.
  • a bolt is used as a fixing member
  • the tension applied to the thin film can be adjusted by adjusting the thickness and elasticity of the sealing member, the thickness of the pressing member, and the screwing amount of the bolt. Therefore, each of these members functions as an element of the encapsulation means and at the same time as the adjusting means.
  • Another preferred configuration for the encapsulation means is an adhesive that adheres the periphery of the thin film to the sample table. Compared to the above configuration, the number of parts and man-hours are small.
  • the size of the target sample is not limited, but since it is necessary to observe through a thin film, it is difficult to measure with sufficient resolution for a sample with a size of 1 nm or less.
  • a sample with a size of 50 cm or more cannot be enclosed in a sample holder that can be installed in the sample chamber of the current electron microscope.
  • the size of the sample is preferably 1 nm or more and 50 cm or less.
  • the target sample is enclosed by the thin film and the sample table before irradiating the target sample with an electron beam, so that the target sample is not exposed to vacuum or other chemical substances.
  • the thin film is applied following the target sample, there is no strict limitation on the distance between the thin film and the sample table. Therefore, the biological sample can be observed in a living state without being restricted by the properties of the sample itself or the structure containing the sample.
  • secondary electrons emitted from the target sample or the thin film can be detected, it can be applied to many existing scanning electron microscopes to observe the three-dimensional structure of the biological sample. ..
  • 6 is a scanning electron micrograph of a cross section of a myocardial fiber applied with a thin film according to the first embodiment.
  • the sample holder 1 for the scanning electron microscope is made of a conductive material, specifically, a sample table 2 made of aluminum, and an insulating and electron-permeable organic polymer.
  • a thin film 3 an annular or cylindrical waterproof sealing member 4 made of an elastic material, a disk-shaped spacer 5 made of an elastic material, an annular holding member 6 made of a rigid material, and a plurality of pieces corresponding to fixing members.
  • a bolt 7 is provided. All parts can be combined and separated from each other before and after use.
  • the sealing member 4 has an outer circumference sufficiently located inside the edge of the sample table 2.
  • the thin film 3 has a size that closes one opening of the sealing member 4 and extends further outward from the outer circumference of the sealing member 4.
  • the spacer 5 has an outer diameter sufficient to maintain non-contact with the inner circumference of the sealing member 4, and a height sufficiently smaller than the height of the sealing member 4.
  • the pressing member 6 has an inner circumference concentric with the inner circumference of the sealing member 4, and the outer edge thereof protrudes from the sealing member 4.
  • a screw hole (not shown) in which the bolt 7 is fitted is provided at a position protruding from the sealing member 4, and a through hole is provided at a corresponding position in the holding member 6.
  • the sealing member 4 When using the sample holder 1, the sealing member 4 is placed substantially in the center of the sample table 2, and a spacer 5 having an appropriate thickness and elasticity according to the properties of the sample 8 is placed inside the sealing member 4. A sample 8 is placed on the spacer 5, and a liquid material 9 such as an aqueous solution is filled around the sample 8.
  • the thin film 1 is placed on the sealing member 8 so as to close the opening of the sealing member 8, then the pressing member 6 is placed, and the bolt 7 is passed through the through hole of the pressing member 6 and fitted into the screw hole of the sample table 2. With the screw fitting of the bolt 7, the sealing member 4 and the spacer 5 are compressed, and the thin film 3 is lightly pressed against the sample 8 to follow the shape of the upper surface of the sample 8. The excess liquid 9 oozes out from the gap between the sealing member 4 and the thin film 3 or the sample table 2. Since the sealing member 4 is elastically in contact with the thin film 3 pressed by the pressing member 6, the periphery of the sample 8 is kept liquidtight.
  • the above work may be performed outside the sample chamber of the scanning electron microscope, for example, in the normal temperature atmosphere.
  • the sample holder 1 containing the sample 8 and the liquid material 9 is placed in the sample chamber so that the sample 8 faces the radiation source 10 of the electron beam irradiation through the lens.
  • the sample table 2 is electrically connected to the ground.
  • a secondary electron detector 12 is installed above the sample holder 1 at a position that does not obstruct the path of the electron beam 11 irradiated from the radiation source 10. Further, above the portion of the thin film 3 surrounded by the inner circumference of the sealing member 4, there is nothing other than the detector 12 up to the lens.
  • the incident electrons and the reflected electrons that do not hit the sample 8 flow through the liquid body 9 and the sample table 2 and are released to the ground. Therefore, neither the sample 8 nor the thin film 3 is charged.
  • the thin film 3 has insulating properties and electron permeability, secondary electrons 13 emitted from the surface of the sample 8 and the thin film 3 are detected by the detector 12. Since the thin film 3 has irregularities that imitate the surface of the sample 8, and the thin film 3 follows the unevenness when the sample 8 moves, the secondary electrons 13 detected according to the angle of the surface of the sample 8 and the thin film 3 with respect to the incident electron beam. The strength changes. As a result, the fine structure of the sample 8 and the direction and amount of displacement can be measured.
  • the sample holder 14 for the scanning electron microscope according to the second embodiment does not include any of the sealing member 4, the pressing member 6, and the bolt 7. Instead, as shown in FIG. 2, the adhesive 15 is provided.
  • the spacer 5 When using the sample holder 14, the spacer 5 is placed substantially in the center of the sample table 2, and the adhesive 15 is applied to the peripheral edge of the thin film 3. Before the adhesive 15 dries, the sample 8 wet with the liquid material 9 is placed on the spacer 5, and the thin film 3 is put on the sample 8 while applying tension to the thin film 3 with the coating surface of the adhesive 15 facing downward. The peripheral edge of the thin film 3 is pressed against the sample table 2. After the adhesion of the thin film 3 to the sample table 2 is completed, the sample holder 14 is placed in the sample chamber. Subsequent procedures may be similar to the first embodiment.
  • Biphenyltetracarboxylic dianhydride (U-varnish-S 1001) manufactured by Ube Industries, Ltd. was prepared as a polyimide precursor.
  • the polyimide precursor is thinly coated on a 0.17 mm thick substrate made of optical glass BK7 by a spin coating method, the substrate is heated in an electric furnace at a temperature of 450 ° C. for 20 minutes, and then peeled off from the substrate in water.
  • a polyimide thin film having a size of 32 ⁇ 24 mm and a thickness of 300 nm was obtained, and the thin film 3 was obtained.
  • liquid substance 9 a 1: 1 mixed medium of Dulbecco's Modified Eagle's Medium and Ham's F12 Nutrient Mixture supplemented with 10% calf serum, 100 units / ml of penicillin, and 100 ⁇ g / ml of streptomycin was used.
  • sample 8 a plurality of fluorescent beads manufactured by Polysciences, which were made of polystyrene and had a density of 1.05 g / cm 3 and an average particle size of 1 ⁇ m, were used.
  • an O-ring made of silicon rubber (JIS material code: type 4 C) having a JIS-P11 standard, an inner diameter of 10.8 mm, and a thickness of 2.4 mm is provided so that its center line coincides with the center of the irradiation field. It was used by arranging it in.
  • the spacer 5 was made of melamine sponge, and the holding member 6 was made of a stainless steel plate. Four bolts 7 were evenly arranged in the circumferential direction.
  • FIG. 3 shows the result of observing the sample 8 by detecting the secondary electrons 13 with the detector 12. You can see a single fluorescent bead in the upper left of the image, four in the upper right, and an aggregate of three fluorescent beads in the lower left.
  • FIG. 4 shows the results of observing the sample 8 in the same manner as in Example 1 except for the above.
  • a circular object occupying about two-thirds of the screen area can be seen in the center of the image, and it is recognized as red blood cells.
  • Vesicles / granules derived from other blood tissues are found on and around erythrocytes.
  • FIG. 5 shows the results of observing the sample 8 in the same manner as in Example 1 except for the above. It can be confirmed that each bundle of myocardial myofibrils having a diameter of about 1 ⁇ m is densely formed in a hexagonal or quadrangular shape.
  • FIG. 6 shows the results of observing the sample 8 in the same manner as in Example 1 except for the above.
  • thermoplastic polyimide varnish (Q-AD-X0516) manufactured by PI Technology Research Institute Co., Ltd. was prepared as a polyimide precursor.
  • the polyimide precursor is thinly coated on a 0.17 mm thick substrate made of optical glass BK7 by a spin coating method at 6000 rpm, the substrate is heated on a hot plate at a temperature of 200 ° C. for 3 minutes, and then from the substrate in water. By peeling, a polyimide thin film having a size of 32 ⁇ 24 mm and a thickness of 4 ⁇ m was obtained.
  • a polyimide thin film having a thickness of 1.5 ⁇ m was obtained in the same manner as described above except that the rotation speed of the spin coat was changed to 8000 rpm instead of 6000 rpm.
  • FIG. 7 shows an image observed with a scanning electron microscope under the condition of an acceleration voltage of 15 kV after covering a semiconductor chip with a polyimide thin film having a thickness of 4 ⁇ m while lightly applying tension.
  • FIG. 8 shows an image observed under the same conditions by covering the same semiconductor chip with a polyimide thin film having a thickness of 1.5 ⁇ m in the same manner.
  • Example 4 Similar to Example 4, the heart taken out from the mouse was enclosed in the sample holder 1, and the thin film 3 was in close contact with the heart tissue, and a moving image was taken in a state where the heart and the thin film were expanding and contracting. The still image at that moment is shown on the left side of FIG. Then, in the imaging in the figure, among the relative displacement amounts between arbitrary three points surrounded by circles on the thin film, the relative displacement amount in the horizontal direction (distance change amount between the double arrows shown by the solid lines) is determined. The time change of strain was measured by dividing the relative displacement in the vertical direction (the amount of change in distance between the two arrows shown by the broken line) by the time average distance. The measurement result is shown on the right side of the imaging in FIG.
  • the measurement results indicate that the heart tissue is vibrating in all directions, including horizontal, diagonal, and vertical, because the horizontal and vertical changes are oscillating in a consistent or independent manner. .. In other words, it reflects the physical characteristics (restoring force) and behavior of the heart tissue that the myocardial contractile system is oriented so as to twist and can contract and relax in multiple directions.
  • FIG. 13 shows the result of analyzing the movement of the label having a diameter of 1.4 ⁇ m (slightly upper right from the center of FIG. 12) among the spherical labels derived from the microbubbles. As shown in the figure, the trajectory of the movement of the sign can be measured with high accuracy.
  • fine particles may be mixed with the raw material of the thin film and dispersed at the time of film formation, or the label may be printed on the film after the film formation without mixing the fine particles with the raw material of the thin film. ..
  • Dulbecco's Modified Eagle's Medium and Ham's F12 Nutrient Mixture 1 1 mixed medium containing 10% calf serum, 100 unit / ml of penicillin, and 100 ⁇ g / ml of streptomycin were used, and this was enclosed in the sample holder 1. did. Then, the calcium phosphate crystal precipitated from the liquid body 9 in the vicinity of the thin film 3 is used as a sample 8, and the result of imaging the behavior thereof is shown in FIG. In the figure, the upper right and lower right images are taken at the same points as the upper left and lower left images, respectively, after a lapse of time.
  • crystals floating and moving in the liquid body 9 can be observed directly under the thin film 3. That is, it can be confirmed that the crystal indicated by the white arrow in the figure is moving from the upper left image to the upper right image. Further, as the precipitation of the crystal progresses, the thin film 3 is deformed following the shape of the crystal, and the shape of the crystal can be observed three-dimensionally. The shape of the crystal can be confirmed in the lower left part of the upper left image and the upper right image, and the right part of the lower left image and the lower right image.
  • the crystal in the portion indicated by the white arrow in the lower left image is not visible in the lower right image, the crystal deposited in the vicinity of the thin film 3 and deformed the thin film 3 is separated from the thin film 3 to form the liquid body 9. You can also see how it sinks inside.

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  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Analysing Materials By The Use Of Radiation (AREA)
PCT/JP2021/022443 2020-06-16 2021-06-14 走査型電子顕微鏡を用いた観察方法、及びそのための試料ホルダ Ceased WO2021256412A1 (ja)

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EP21825836.6A EP4167266A4 (en) 2020-06-16 2021-06-14 OBSERVATION METHODS WITH SCANNING ELECTRON MICROSCOPE AND SAMPLE HOLDER THEREFOR
US18/010,610 US20230274905A1 (en) 2020-06-16 2021-06-14 Observation method employing scanning electron microscope, and sample holder for the same
JP2022531786A JP7142404B2 (ja) 2020-06-16 2021-06-14 走査型電子顕微鏡を用いた観察方法、及びそのための試料ホルダ

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