JP7407664B2 - Sample preparation method - Google Patents

Description

The present invention relates to a sample preparation method and a support membrane.

Single particle analysis is known as a method for structural analysis of biological samples using an electron microscope. In single particle analysis, the three-dimensional structure of biopolymers such as proteins and nucleic acids can be analyzed by image processing from an electron microscope image (see, for example, Patent Document 1).

In single particle analysis, for example, first, a sample prepared by an ice embedding method or the like is photographed using a cryo-electron microscope, and a particle image is extracted from the obtained electron microscope image. Next, the extracted particle images are classified by particle orientation, and the classified particle images are averaged to obtain an averaged image for each particle orientation. Using this averaged image, the three-dimensional structure of the particle is calculated.

Japanese Patent Application Publication No. 2007-41738

In the ice embedding method, an aqueous solution in which particles to be analyzed are dispersed is dropped onto a transmission electron microscope grid, the excess aqueous solution is absorbed with filter paper, etc., and then immediately immersed in a coolant such as liquid ethane to quickly freeze. let This allows particles to be embedded in an amorphous ice film.

Here, in single particle analysis, as mentioned above, particles photographed as projection images of a transmission electron microscope are classified by orientation, and the classified particles are averaged, so images of particles oriented in various directions are obtained. It becomes necessary.

However, in the ice embedding method, particles move to the air-liquid interface and exhibit orientation. Therefore, even when a sample prepared by ice embedding is observed using an electron microscope, many particles are observed pointing in the same direction. It also tends to deform the particles.

One aspect of the sample preparation method according to the present invention is
A sample preparation method for observing a plurality of particles with an electron microscope, the method comprising:
preparing a carbon film deposited on mica;
The mica on which the carbon film is vapor-deposited is submerged in a liquid so that the first surface of the carbon film that was in contact with the mica is downward and the second surface opposite to the first surface is upward. , a step of floating the carbon film on the liquid;
scooping the carbon film floating in the liquid with a support having a through hole, and supporting the carbon film with the support with the liquid attached to the first surface;
adsorbing the plurality of particles onto the first surface of the carbon film supported by the support;
embedding the plurality of particles in ice;
including.

In such a sample preparation method, particles can be softly adsorbed on the clean first surface of the carbon film, so that the number of particles moving to the air-liquid interface can be reduced. Particles adsorbed on a carbon film are oriented in various directions, and therefore, according to such a sample preparation method, particles oriented in various directions can be observed. Furthermore, since the particles are softly adsorbed and oriented in various directions, the negative effects of deformation in only specific parts can be reduced.

1 is a flowchart illustrating an example of a sample preparation method according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 3 is a diagram schematically showing a process of preparing a sample according to an embodiment. FIG. 1 is a diagram schematically showing a sample produced by a sample production method according to an embodiment. A diagram schematically showing a sample prepared by an ice embedding method. A diagram for explaining the effect of a carbon film. A diagram for explaining the effect of a carbon film. The figure which shows the measurement result of the atomic force microscope of the carbon film obtained with the vacuum evaporation apparatus which is capable of sparkless evaporation. A diagram for comparing single particle analysis using the backup carbon method and single particle analysis using the ice embedding method. A diagram for comparing single particle analysis using the backup carbon method and single particle analysis using the ice embedding method. A diagram for comparing single particle analysis using the backup carbon method and single particle analysis using the ice embedding method.

Hereinafter, preferred embodiments of the present invention will be described in detail using the drawings. Note that the embodiments described below do not unduly limit the content of the present invention described in the claims. Furthermore, not all of the configurations described below are essential components of the present invention.

1. Sample Preparation Method First, a sample preparation method according to an embodiment of the present invention will be described with reference to the drawings.

In the sample preparation method according to the present embodiment, an electron microscope sample for photographing a cryo-electron microscope image (hereinafter also referred to as "CryoEM image") can be prepared. A CryoEM image is an image obtained by cryo-electron microscopy. Cryo-electron microscopy is a method in which a sample is introduced into an electron microscope in a frozen state and observed without staining.

The sample preparation method according to this embodiment is used, for example, in single particle analysis. Specifically, first, a sample containing a large number of particles is prepared using the sample preparation method according to the present embodiment, and a cryoEM image is obtained by photographing the prepared sample with a cryo-electron microscope. Particle images are extracted from the obtained CryoEM image, the extracted particle images are classified by particle orientation, and the classified particle images are averaged to obtain an averaged image for each particle orientation. Using this averaged image, the three-dimensional structure of the particle is calculated.

Particles targeted by the sample preparation method according to the present embodiment include, for example, biopolymers such as proteins, lipids, and nucleic acids, complexes thereof, and viruses.

FIG. 1 is a flowchart showing an example of the sample preparation method according to this embodiment. FIGS. 2 to 10 are diagrams schematically showing the steps of preparing a sample according to this embodiment.

1.1. Step S10 of preparing a carbon film
As shown in FIG. 2, a carbon film 2 deposited on mica (mica) 4 is prepared. The carbon film 2 is formed on a clean surface (cleaved surface) of the mica 4 immediately after it is cleaved. The carbon film 2 is deposited by vacuum deposition.

The carbon film 2 is deposited under a vacuum condition higher than 2×10 ⁻⁴ Pa, for example. Further, the carbon film 2 is deposited using a vacuum evaporation apparatus that is capable of performing sparkless deposition without sparks from the evaporation source. Thereby, it is possible to prevent the clusters emitted from the evaporation source from being taken into the carbon film 2, and it is possible to obtain a carbon film 2 with a smooth surface.

Carbon film 2 has a first surface 2a and a second surface 2b. The first surface 2a is a surface in contact with the cleavage plane of mica 4, and the second surface 2b is a surface opposite to the first surface 2a.

The thickness of the carbon film 2 is, for example, 10 nm or less. The thickness of the carbon film 2 is, for example, 5 nm.

1.2. Step S12 of floating the carbon film on distilled water
As shown in FIG. 3, the mica 4 with the carbon film 2 deposited thereon is submerged in distilled water 6, and the carbon film 2 is floated on the distilled water 6. At this time, the carbon film 2 is floated so that the first surface 2a in contact with the mica 4 is at the bottom and the second surface 2b is at the top.

By submerging the mica 4 on which the carbon film 2 has been deposited, the carbon film 2 is peeled off from the mica 4 and the carbon film 2 floats on the water surface. As a result, the first surface 2a that was in contact with the mica 4 becomes the bottom, and the second surface 2b becomes the top.

In this manner, by submerging the mica 4 on which the carbon film 2 has been deposited and floating the carbon film 2 in the distilled water 6, the first surface 2a of the carbon film 2 does not come into contact with air.

Although the case where the carbon film 2 is floated on the distilled water 6 has been described here, the liquid that floats the carbon film 2 is not limited to the distilled water 6.

1.3. Step S14 of supporting the carbon film with a support body
As shown in FIGS. 3 and 4, the carbon membrane 2 floating on the distilled water 6 is scooped up with the support 10, and the carbon membrane 2 is placed on the support with the distilled water 6 attached to the first surface 2a of the carbon membrane 2. I support it with 10. Distilled water 6 adheres to the first surface 2a of the carbon film 2 due to surface tension. The first surface 2a of the carbon film 2 is covered with distilled water 6 and does not come into contact with air.

The support body 10 has a through hole 12 . In the illustrated example, the support body 10 has a plurality of through holes 12 . As the support 10, for example, a microgrid, Quantifoil (registered trademark), etc. can be used. Note that the support 10 is not particularly limited as long as it is a grid for a transmission electron microscope that can support the carbon film 2 and can be introduced into a cryo-electron microscope.

1.4. Step S16 of replacing distilled water with buffer solution
As shown in FIGS. 5 and 6, the distilled water 6 adhering to the first surface 2a of the carbon film 2 is replaced with a buffer solution 8. For example, as shown in FIG. 5, the buffer solution 8 is dropped onto the Parafilm 20, and the carbon film 2 supported on the support 10 is brought close to the buffer solution 8 on the Parafilm 20. When the distilled water 6 attached to the carbon membrane 2 comes into contact with the buffer solution 8, the distilled water 6 is replaced by the buffer solution 8. Then, as shown in FIG. 6, the carbon film 2 supported by the support 10 is moved away from the buffer solution 8 on the Parafilm 20. As a result, the carbon film 2 supported by the support body 10 is in a state where the buffer solution 8 is attached. The first surface 2a of the carbon film 2 is covered with a buffer solution 8 and is not exposed to air.

Note that the buffer solution 8 can be selected as appropriate depending on the type of particles 3.

1.5. Step S18 of adsorbing particles to the carbon film
As shown in FIG. 7, particles 3 to be observed are adsorbed onto the first surface 2a of the carbon film 2 supported by the support 10. As shown in FIG. When the particles 3 are dropped onto the buffer solution 8 attached to the first surface 2a of the carbon film 2, the particles 3 are dispersed in the buffer solution 8. Since the first surface 2a of the carbon film 2 is maintained in a clean state, the particles 3 are more easily adsorbed on the first surface 2a than on the air-liquid interface. Therefore, the particles 3 dispersed in the buffer solution 8 move to the first surface 2a of the carbon film 2 and are adsorbed to the first surface 2a.

1.6. Step S20 of sucking up the buffer solution
As shown in FIGS. 8 and 9, the buffer solution 8 adhering to the carbon membrane 2 and the support 10 is absorbed by a filter paper 30 to adjust the amount of the buffer solution 8 adhering to the first surface 2a of the carbon membrane 2. By adjusting the amount of buffer solution 8 absorbed, the thickness of the ice film formed in the ice embedding process described later can be adjusted.

Note that the means for absorbing the buffer solution 8 is not limited to the filter paper 30, and other paper, cloth, etc. can be used.

1.7. Step S22 of embedding particles in ice
As shown in FIG. 10, the support 10 supporting the carbon film 2 is immersed in liquid ethane 9 and rapidly frozen. As a result, the particles 3 are embedded in the amorphous ice film formed in the through hole 12 of the support 10. That is, the particles 3 are embedded in ice.

Note that the refrigerant for freezing is not limited to liquid ethane 9, and slush liquid nitrogen, liquid propane, or the like may be used.

Through the above steps, an electron microscope sample for observing the particles 3 with an electron microscope can be produced.

2. Sample for Electron Microscopy Next, a sample for electron microscopy produced by the sample production method according to the present embodiment described above will be described. Below, a sample prepared by the sample preparation method according to the present embodiment will be explained while being compared with a sample prepared by the ice embedding method.

FIG. 11 is a diagram schematically showing a sample 100 manufactured by the sample manufacturing method according to the present embodiment. Note that FIG. 11 shows a perspective view schematically showing the sample 100.

As shown in FIG. 11, in the sample 100, particles 3 are embedded in an amorphous ice film 102. The thickness of the ice film 102 is, for example, approximately several tens of nanometers to several hundred nanometers. Further, in the sample 100, the carbon film 2 is in contact with the lower surface 101b of the ice film 102.

FIG. 12 is a diagram schematically showing a sample 200 produced by the ice embedding method. FIG. 12 shows a perspective view schematically showing the sample 200.

Sample 200 is an electron microscope sample prepared by an ice embedding method. Specifically, a buffer solution 8 in which particles 3 are dispersed is dropped onto a support such as a microgrid or Quantifoil, and after absorbing the excess buffer solution 8 with a filter paper, the buffer solution 8 is immersed in a refrigerant such as liquid ethane, and then rapidly frozen. do. Thereby, a sample 200 in which particles 3 are embedded in an ice film 202 can be produced as shown in FIG. 12.

Here, in the ice embedding method, the particles 3 dispersed in the buffer solution 8 move to the gas-liquid interface and are adsorbed to the gas-liquid interface. Even if the excess liquid is quickly frozen after absorbing the excess liquid with a filter paper, many of the particles 3 tend to move to the air-liquid interface and be adsorbed. Therefore, in the sample 200, as shown in FIG. 12, there are particles 3 arranged along the upper surface 201a of the ice film 202 and particles 3 arranged along the lower surface 201b of the ice film 202.

Since the gas-liquid interface is different from the environment inside the buffer solution 8, the particles 3 adsorbed to the gas-liquid interface undergo deformation and denaturation. For example, the particles 3 adsorbed on the air-liquid interface undergo deformation and denaturation under the influence of surface tension, a pH different from that in the buffer solution 8, and exposure to air.

Further, the particles 3 adsorbed on the gas-liquid interface exhibit orientation. Therefore, when the sample 200 is observed with an electron microscope, many particles 3 are observed in the same direction (see left side of FIG. 18).

Further, the positions of the particles 3 arranged along the upper surface 201a of the ice film 202 and the particles 3 arranged along the lower surface 201b of the ice film 202 in the thickness direction of the ice film 202 are different. Therefore, the focus conditions are different between the particles 3 arranged along the upper surface 201a of the ice film 202 and the particles 3 arranged along the lower surface 201b of the ice film 202. Therefore, it becomes difficult to accurately correct the contrast transfer function (CTF) of the objective lens of the electron microscope. This results in a decrease in resolution in three-dimensional structure analysis.

In contrast, in the sample preparation method according to the present embodiment, the particles 3 in the buffer solution 8 can be mildly adsorbed onto the carbon film 2, so the number of particles 3 adsorbed on the gas-liquid interface can be reduced. Therefore, deformation and denaturation of the particles 3 due to adsorption of the particles 3 to the gas-liquid interface can be reduced (see the right side of FIG. 17).

Further, the particles 3 adsorbed on the carbon film 2 are oriented in various directions. Therefore, in the sample 100, particles 3 oriented in various directions can be observed (see the right side of FIG. 18).

Furthermore, in the sample preparation method according to the present embodiment, as described above, the particles 3 in the buffer solution 8 are adsorbed to the carbon film 2, so the particles 3 are arranged along the carbon film 2. In the example shown in FIG. 12, since the carbon film 2 is in contact with the lower surface 101b of the ice film 102, the particles 3 are arranged along the lower surface 101b of the ice film 102 and not along the upper surface 101a. Therefore, the focal conditions for each particle 3 are approximately equal, allowing accurate correction of the contrast transfer function.

Furthermore, the density of the particles 3 embedded in the ice film 102 formed in the through hole 12 in the sample 100 is higher than the density of the particles 3 embedded in the ice film 202 formed in the through hole 12 in the sample 200. expensive. In the ice embedding method, the particles 3 in the buffer solution 8 gather in the frame portion of the support 10 rather than in the through holes 12 of the support 10. Therefore, in the sample 200, the density of particles embedded in the ice film 202 formed in the through hole 12 is low. On the other hand, in the sample preparation method according to the present embodiment, since the particles 3 in the buffer solution 8 are adsorbed to the carbon film 2, the density of the particles 3 embedded in the ice film 202 formed in the through hole 12 is can be increased.

13 and 14 are diagrams for explaining the effects of the carbon film 2. FIG. 13 is a CryoEM image including a region A where an ice film and a carbon film exist (see FIG. 11) and a region B where only an ice film exists (see FIG. 12). FIG. 14 is an enlarged image of region P in FIG.

As shown in FIGS. 13 and 14, the density of particles 3 in region A is higher than the density of particles 3 in region B. In this way, the density of the particles 3 embedded in the ice film 102 formed in the through hole 12 can be increased by the carbon film 2.

3. Sample Preparation Kit The sample preparation kit used in the sample preparation method according to the present embodiment includes mica 4 and a carbon film 2 deposited on the mica 4, as shown in FIG. The carbon film 2 has a first surface 2a that is in contact with the cleavage plane of the mica 4, and a second surface 2b that is opposite to the first surface 2a. As described above, the first surface 2a of the carbon film 2 is a surface onto which the particles 3 are adsorbed. The carbon film 2 can be used as a support film that supports the sample (particles 3) to be observed in the sample preparation method according to this embodiment. The sample preparation kit may include a support 10.

4. Effects The sample preparation method according to the present embodiment includes a step of preparing the carbon film 2 deposited on the mica 4, and submerging the mica 4 with the carbon film 2 deposited on it in distilled water 6 to form the carbon film 2. A step of floating the carbon film 2 in distilled water 6 so that the first surface 2a that was in contact with the mica 4 is at the bottom and a second surface 2b opposite to the first surface 2a is at the top; A step of scooping the carbon film 2 with a support 10 having a through hole 12 and supporting the carbon film 2 with the support 10 with distilled water 6 attached to the first surface 2a; The method includes a step of adsorbing a plurality of particles 3 onto the first surface 2a of the carbon film 2, and a step of embedding the plurality of particles 3 in ice.

Therefore, in the sample preparation method according to the present embodiment, as described above, the particles 3 can be adsorbed onto the first surface 2a of the carbon film 2, so that the number of particles 3 moving to the gas-liquid interface can be reduced. Therefore, deformation and modification of the particles 3 can be reduced.

Further, since the particles 3 adsorbed on the carbon film 2 are oriented in various directions, it is possible to observe the particles 3 oriented in various directions. Here, in single particle analysis, particles are classified by orientation, and the classified particles are averaged to obtain an averaged image. In the sample preparation method according to this embodiment, particles 3 oriented in various directions can be observed, so that a good averaged image can be obtained in any direction.

Further, in the sample preparation method according to the present embodiment, the particles 3 in the buffer solution 8 are adsorbed to the carbon film 2, so the particles 3 are arranged along the carbon film 2, and the focal conditions of each particle 3 are almost equal. . Therefore, the contrast transfer function can be accurately corrected. Thereby, resolution can be improved in three-dimensional structure analysis.

In addition, in the sample preparation method according to the present embodiment, by adsorbing the particles 3 to the carbon film 2, the density of the particles 3 embedded in the ice film 102 formed in the through-hole 12 can be adjusted in the through-hole in the sample 200. The density of the particles 3 can be higher than that of the particles 3 embedded in the ice film 102 formed in the ice film 102. Therefore, for example, even if highly concentrated particles 3 cannot be prepared, a large number of particles 3 can be embedded in the ice film 102.

For example, in the case of G protein-coupled receptors, which are important targets for drug discovery, the ice embedding method requires that the sample be at an extremely high concentration of 5 mg/ml to 50 mg/ml to prevent formation in the through-holes of Quantifoil. A sufficient number of particles 3 cannot be embedded in the ice film 202. However, it is generally difficult to obtain samples with such high concentrations. On the other hand, in the sample preparation method according to the present embodiment, even if the sample has a low concentration of 1 mg/ml or less, a large number of particles 3 can be embedded in the ice film 102 formed in the through hole of Quantifoil.

In the sample preparation method according to the present embodiment, the first surface 2a of the carbon film 2 is exposed to air from the step S10 of preparing the carbon film 2 deposited on the mica 4 to the step S18 of adsorbing the particles 3 to the carbon film 2. can not touch. Therefore, it is not necessary to perform a hydrophilic treatment on the carbon film 2 by glow discharge or the like.

For example, when the surface of a carbon film comes into contact with air, oily molecules floating in the air are adsorbed, making the surface of the carbon film hydrophobic. Therefore, target particles cannot be adsorbed unless the surface of the carbon film is treated to make it hydrophilic. In this way, when the surface of the carbon film 2 is subjected to hydrophilic treatment, the particles 3 in the buffer solution 8 are strongly adsorbed to the surface of the carbon film 2. As a result, the particles 3 are denatured and deformed.

In the sample preparation method according to the present embodiment, the first surface 2a of the carbon film 2 does not come into contact with air, so there is no need to perform a hydrophilic treatment. The adsorption force of the particles 3 on the first surface 2a of the carbon film 2 that is not exposed to air is smaller than the adsorption force of the particles 3 on the surface of the carbon film that has been subjected to a hydrophilic treatment. Therefore, in the sample preparation method according to this embodiment, the particles 3 can be mildly adsorbed onto the carbon film 2 without causing any denaturation or deformation of the particles 3.

Note that even when graphene is used as the sample support film, the surface of graphene needs to be hydrophilized. Similar to the hydrophilic carbon film, the hydrophilized graphene strongly adsorbs the particles 3 in the buffer solution 8, causing the particles 3 to be denatured or deformed.

The sample preparation method according to the present embodiment includes the step of sucking up the buffer solution 8 attached to the first surface 2a of the carbon film 2 and adjusting the amount of the buffer solution 8 attached to the first surface 2a of the carbon film 2. . Therefore, in the sample preparation method according to this embodiment, the thickness of the ice film 102 can be adjusted.

The sample preparation method according to this embodiment includes a step of vacuum-depositing a carbon film 2 on mica 4. Thereby, a carbon film 2 with a smooth surface can be obtained. Furthermore, in the sample preparation method according to the present embodiment, the carbon film 2 is deposited using a vacuum evaporation apparatus that is capable of sparkless deposition. Thereby, a carbon film 2 with a smooth surface can be obtained.

Figure 15 shows the atomic force microscope measurement results of carbon film 2 (atomically smooth carbon film) obtained using a vacuum evaporation device capable of sparkless evaporation, and the atomic Shows the results of force microscopy measurements. As shown in FIG. 15, by using a vacuum evaporation apparatus capable of sparkless evaporation, a carbon film that is smooth at the atomic level can be obtained.

5. EXPERIMENTAL EXAMPLES The present invention will be further explained using experimental examples below, but the present invention is not limited by the following experimental examples.

In this experimental example, single particle analysis of leucine dehydrogenase (LDH) was performed. Specifically, a sample for observing LDH with a cryo-electron microscope was prepared using the sample preparation method according to the present embodiment described above, and single particle analysis was performed. Hereinafter, the sample preparation method according to this embodiment will also be referred to as the backup carbon method.

In addition, as a comparative example, a sample for observing LDH with a cryo-electron microscope was prepared using a normal ice embedding method, and single particle analysis was performed.

16 to 18 compare single particle analysis of LDH using the backup carbon method and single particle analysis of LDH using the ice embedding method. Note that in FIGS. 16 to 18, the results obtained using the ice embedding method are described as "Ice embedding method," and the results obtained using the backup carbon method are described as "Backup carbon method."

In FIG. 16, a CryoEM image of a sample prepared by the ice embedding method and a CryoEM image of a sample prepared by the backup carbon method are compared. As shown in FIG. 16, in the ice embedding method, a state in which many particles are oriented in the same direction is observed. In contrast, with the backup carbon method, particles oriented in various directions have been observed. Note that the ice embedding method provides a better S/N image than the backup carbon method.

FIG. 17 compares the results of three-dimensional structure analysis in single particle analysis of LDH. The upper part of FIG. 17 shows the analyzed three-dimensional structure. Further, in the lower part of FIG. 17, cross-sectional images obtained by slicing the three-dimensional structure of LDH at the center are compared.

As shown on the left side of FIG. 17, in the ice embedding method, the upper right portion is deformed and is no longer visible. On the other hand, in the backup carbon method, no structural deformation is observed, as shown on the right side of FIG.

FIG. 18 shows the distribution of particle orientations when the three-dimensional structure of LDH is analyzed. In the conventional ice embedding method shown on the left side of FIG. 18, there is a large distribution of particles in one direction, and there are very few particles in other directions. On the other hand, in the backup carbon method, a relatively uniform distribution of particles was observed through analysis, although there were some differences.

Furthermore, single particle analysis using the ice embedding method had a resolution of 2.92 Å, whereas single particle analysis using the backup carbon method achieved a high resolution of 2.76 Å.

The present invention is not limited to the embodiments described above, and various modifications are possible. For example, the present invention includes configurations that are substantially the same as those described in the embodiments. Substantially the same configurations are, for example, configurations with the same function, method, and result, or configurations with the same purpose and effect. Further, the present invention includes a configuration in which non-essential parts of the configuration described in the embodiments are replaced. Further, the present invention includes a configuration that has the same effects or a configuration that can achieve the same purpose as the configuration described in the embodiment. Further, the present invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

2... Carbon film, 2a... First surface, 2b... Second surface, 3... Particles, 4... Mica, 6... Distilled water, 8
...Buffer, 9...Liquid ethane, 10...Support, 12...Through hole, 20...Parafilm, 30...Filter paper, 100...Sample, 101a...Top surface, 101b...Bottom surface, 102...Ice film, 200...Sample, 201a ...Top surface, 201b...Bottom surface, 202...Ice film

Claims (4)

A sample preparation method for observing a plurality of particles with an electron microscope, the method comprising:
preparing a carbon film deposited on mica;
The mica on which the carbon film is vapor-deposited is submerged in a liquid so that the first surface of the carbon film that was in contact with the mica is downward and the second surface opposite to the first surface is upward. , a step of floating the carbon film on the liquid;
scooping the carbon film floating in the liquid with a support having a through hole, and supporting the carbon film with the support with the liquid attached to the first surface;
adsorbing the plurality of particles onto the first surface of the carbon film supported by the support;
embedding the plurality of particles in ice;
A sample preparation method, including: In claim 1,
A method for preparing a sample, comprising a step of replacing the liquid adhering to the first surface with a buffer solution after the step of supporting the carbon film with the support. In claim 2,
After the step of replacing the liquid with the buffer solution, the sample preparation method includes the step of sucking up the buffer solution adhering to the first surface to adjust the amount of the buffer solution adhering to the first surface. In any one of claims 1 to 3,
A method for preparing a sample, comprising the step of vacuum depositing the carbon film on the mica.

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