Preparation device of sample for ultralow-temperature high-solution transmission electron microscope

Abstract

To provide a preparation device of a sample for an ultralow-temperature high-resolution transmission electron microscope, which easily prepares a sample compared to manual preparation of a sample for a cryo-electron microscope.SOLUTION: A preparation device 10 includes a gas-phase reaction mechanism 12, a liquid-phase reaction mechanism 14, and a freezing mechanism 16. The gas-phase reaction mechanism 12 performs gas phase reaction on a sample grid 28. The liquid-phase reaction mechanism 14 performs liquid phase reaction on the sample grid 28 after the gas phase reaction. The freezing mechanism 16 freezes the sample grid 28 with a refrigerant after the liquid phase reaction. Thus, a sample is prepared by ice embedding.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a sample preparation device for a cryogenic transmission electron microscope. [Background technology]

[0002] A cryogenic transmission electron microscope (i.e., cryo-electron microscope) is known as a device for analyzing the structure of substances such as proteins.

[0003] One method for preparing samples for cryo-electron microscopy is the ice embedding method, a type of rapid freezing fixation method. The ice embedding method involves, for example, dropping a sample solution onto a mesh-like metal grid, removing excess solution from the grid, and then rapidly freezing the resulting thin film of sample using a refrigerant, resulting in the preparation of amorphous ice. The refrigerant used can be, for example, liquid ethane, liquid propane, or liquid nitrogen.

[0004] Patent Document 1 describes a method for producing a graphene grid. In this method, a graphene oxide grid is produced by exposing a grid to which graphene is attached as a support film to the activated gas generated by irradiating chlorous acid radicals with ultraviolet or visible light. Epoxy groups are added to the graphene oxide grid by reacting it with epichlorohydrin. The epoxy groups on the grid react with proteins, allowing samples to be produced with the proteins chemically immobilized on the grid. Chemically immobilizing proteins on the grid can eliminate denatured proteins present at the air-liquid interface. Furthermore, protein crystallization can be prevented, thereby reducing the orientation of the proteins. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2022 / 039251 Summary of the Invention [Problem to be solved by the invention]

[0006] In the fabrication method described in Patent Document 1, each step included in the fabrication method is performed manually by an operator. As a result, sample fabrication often fails, and the result of sample fabrication is influenced by the operator's level of skill. As such, the fabrication method described in Patent Document 1 does not allow for easy fabrication of samples for cryo-electron microscopy.

[0007] An object of the present disclosure is to easily prepare a sample for cryo-electron microscopy compared to manual preparation of the sample. [Means for solving the problem]

[0008] One aspect of the present disclosure is an apparatus for preparing samples for cryogenic transmission electron microscopes, comprising: a gas-phase reaction mechanism for performing a gas-phase reaction on a sample grid; a liquid-phase reaction mechanism for performing a liquid-phase reaction on the sample grid after the gas-phase reaction; and a freezing mechanism for freezing the sample grid with a refrigerant after the liquid-phase reaction.

[0009] In the above configuration, a gas-phase reaction is carried out by the gas-phase reaction mechanism, and a liquid-phase reaction is carried out by the liquid-phase reaction mechanism, without manual operation by an operator. This makes it easier to prepare samples compared to manual sample preparation. The sample is not particularly limited, but an example of a sample is an organic substance such as a protein.

[0010] For example, the liquid phase reaction may be an organic reaction, and the organic reaction may produce a chemical bond. The liquid phase reaction mechanism may produce a first chemical bond and a second chemical bond.

[0011] The liquid-phase reaction mechanism may include a reaction solution container that contains a reaction solution, a sample container that contains a sample solution, and a pipette mechanism. The pipette mechanism may have a function of discharging the reaction solution contained in the reaction solution container onto the sample grid and then aspirating the reaction solution on the sample grid, and a function of discharging the sample solution contained in the sample container onto the sample grid and then aspirating the sample solution on the sample grid. For example, the first chemical bond is formed by discharging the reaction solution onto the sample grid, and after the first chemical bond is formed, the sample solution is discharged onto the sample grid to form the second chemical bond.

[0012] The pipette mechanism may discharge a sample solution onto the surface of the sample grid onto which the reaction solution has been discharged.

[0013] The liquid-phase reaction mechanism may further include a washing liquid container for containing a washing liquid for washing the reaction liquid on the sample grid. After the first chemical bond is formed, the pipette mechanism may wash the reaction liquid by discharging the washing liquid onto the surface of the sample grid onto which the reaction liquid has been discharged.

[0014] The above-mentioned preparation apparatus may further include a control device that controls the time from when the reaction solution is discharged onto the sample grid until when the cleaning solution is discharged onto the sample grid.

[0015] The pipette mechanism may be configured to exchange a pipette tip for discharging and aspirating a reaction liquid with a pipette tip for discharging and aspirating a sample solution, thereby discharging and aspirating the reaction liquid and the sample solution, respectively.

[0016] The pipette mechanism may be configured to eject and aspirate the reaction solution, the washing solution, and the sample solution, respectively, by exchanging a pipette tip for ejecting and aspirating the reaction solution, a pipette tip for ejecting and aspirating the washing solution, and a pipette tip for ejecting and aspirating the sample solution.

[0017] The surface of the sample grid onto which the reaction solution and the sample solution are discharged may be arranged perpendicular to the direction in which gravity acts when facing the pipette mechanism.

[0018] The preparation apparatus may further include a pipette tip holding device and a solution holding device. The pipette tip holding device may hold a plurality of pipette tips arranged in a row. The solution holding device may hold a reaction solution, a sample solution, and a cleaning solution arranged in a row. The pipette mechanism may remove a pipette tip for the reaction solution from the pipette tip holding device, use the removed pipette tip for the reaction solution to aspirate the reaction solution from the solution holding device and dispense it onto the sample grid, discard the pipette tip for the reaction solution after dispensing the reaction solution, remove a pipette tip for the cleaning solution from the pipette tip holding device, use the removed pipette tip for the cleaning solution to aspirate the cleaning solution from the solution holding device and dispense it onto the sample grid, discard the pipette tip for the cleaning solution after dispensing the cleaning solution, remove a pipette tip for the sample solution from the pipette tip holding device, and use the removed pipette tip for the sample solution to aspirate the sample solution from the solution holding device and dispense it onto the sample grid.

[0019] The gas-phase reaction mechanism may include a reagent container containing a reagent for a gas-phase reaction, an irradiation device that irradiates light from a side surface of the reagent container, and a cap that covers the reagent container and has an opening formed on the side surface through which the sample grid can be removed. The gas-phase reaction mechanism may perform a gas-phase reaction on the sample grid by irradiating light into the inside of the reagent container with the irradiation device while the sample grid is inserted into the cap.

[0020] The above-described preparation apparatus may further include a rotation mechanism that rotates the sample grid around a rotation axis after the liquid phase reaction, thereby causing the sample grid to face the freezing mechanism. [Effects of the Invention]

[0021] According to the present disclosure, samples for cryo-electron microscopy can be prepared more easily than when samples are prepared manually. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of a manufacturing apparatus according to an embodiment. [Figure 2] FIG. 1 is a diagram schematically illustrating an example of a manufacturing apparatus according to an embodiment. [Figure 3] FIG. 1 is a diagram schematically illustrating an example of a manufacturing apparatus according to an embodiment. [Figure 4] FIG. 1 is a diagram schematically illustrating an example of a manufacturing apparatus according to an embodiment. [Figure 5] FIG. 1 is a diagram schematically illustrating an example of a manufacturing apparatus according to an embodiment. [Figure 6] FIG. 1 is a diagram schematically illustrating an example of a manufacturing apparatus according to an embodiment. [Figure 7] FIG. 1 is a diagram schematically illustrating an example of a manufacturing apparatus according to an embodiment. [Figure 8] FIG. 2 is a diagram showing the manufacturing apparatus according to the embodiment as viewed from the side (+Y direction). [Figure 9] FIG. 2 is a diagram showing the manufacturing apparatus according to the embodiment as viewed from the side (−Y direction). [Figure 10] FIG. 2 is a diagram showing a part of the fabrication apparatus according to the embodiment as viewed from the side (+Y direction). [Figure 11] FIG. 2 is a perspective view showing a gas-phase reaction mechanism, a containing mechanism, and a gripping mechanism. [Figure 12] FIG. 2 is a perspective view showing a gas-phase reaction mechanism, a containing mechanism, and a gripping mechanism. [Figure 13] FIG. 1 is a cross-sectional view of a gas phase reaction mechanism. [Figure 14] FIG. 2 is a perspective view showing a gas-phase reaction mechanism, a containing mechanism, and a gripping mechanism. [Figure 15] This is a view of the accommodation mechanism as seen from above (+Z direction). [Figure 16] FIG. 2 is a perspective view showing a gas phase reaction mechanism, a pipette mechanism, and a gripping mechanism. [Figure 17]FIG. 2 is a diagram showing the manufacturing apparatus according to the embodiment as viewed from the side (+Y direction). DETAILED DESCRIPTION OF THE INVENTION

[0023] An example of a manufacturing apparatus according to an embodiment will be described with reference to Fig. 1. Fig. 1 is a diagram schematically showing an example of a manufacturing apparatus according to an embodiment.

[0024] The preparation apparatus 10 is an apparatus for preparing samples for a cryogenic transmission electron microscope (i.e., a cryo-electron microscope). The preparation apparatus 10 prepares samples by ice embedding, which is a type of rapid freezing fixation method. Specifically, the preparation apparatus 10 prepares samples on a sample grid 28 by performing gas-phase and liquid-phase reactions, respectively.

[0025] The sample grid 28 is a so-called TEM (Transmission Electron Microscope) grid, which is a metal flat plate (e.g., a disk) having a mesh-like shape. For example, the sample grid 28 is a graphene grid. For example, a graphene grid is produced by attaching a support film made of graphene or amorphous carbon to the surface of a flat plate having a mesh-like shape. The method for producing a graphene grid described here is merely an example, and a graphene grid may be produced by another method (e.g., a known method).

[0026] The production apparatus 10 includes, for example, a gas-phase reaction mechanism 12, a liquid-phase reaction mechanism 14, a freezing mechanism 16, a gripping mechanism 18, an absorbing mechanism 20, a camera 22, and a control device 50. These mechanisms and devices may be housed in a housing or may be arranged in a space surrounded by multiple frames.

[0027] The gas-phase reaction mechanism 12 is a mechanism that performs a gas-phase reaction on the sample grid 28. The gas-phase reaction may be, for example, hydrophilization by vacuum discharge, ozone oxidation by ultraviolet light, or photo-oxidation of chlorous acid radicals as described in Patent Document 1. For example, the gas-phase reaction mechanism 12 includes a container in which the gas-phase reaction is performed. For example, the gas-phase reaction mechanism 12 produces a graphene oxide grid by photo-oxidizing chlorous acid radicals on a graphene grid, which is the sample grid 28.

[0028] The liquid-phase reaction mechanism 14 is a mechanism that performs a liquid-phase reaction on the sample grid 28. After the gas-phase reaction is performed by the gas-phase reaction mechanism 12, a liquid-phase reaction is performed by the liquid-phase reaction mechanism 14. The liquid-phase reaction is an organic reaction that generates chemical bonds. In this embodiment, the liquid-phase reaction mechanism 14 generates a first chemical bond and a second chemical bond on the sample grid 28 as chemical bonds. The liquid-phase reaction mechanism 14 may also clean the sample grid 28. In this embodiment, the steps of first chemical bond, cleaning, and second chemical bond are performed in this order.

[0029] For example, the liquid phase reaction includes epoxidation of the sample grid 28 and chemical fixation of a substance to be structurally analyzed to the sample grid 28, as described in Patent Document 1. The epoxidation of the sample grid 28 corresponds to an example of a first chemical bond. The chemical fixation of a substance to be structurally analyzed to the sample grid 28 corresponds to an example of a second chemical bond.

[0030] The structural analysis target substance may be, for example, an organic substance such as a protein. Of course, the structural analysis target substance may also be a substance other than a protein. For example, the structural analysis target substance may be at least one substance selected from the group consisting of an antibody, a nucleic acid, a sugar chain, a lipid, a virus, a ribosome, a liposome, a mitochondria, an intracellular organelle, an ion channel, an enzyme, a low molecular weight compound, a protein-antibody complex, a protein-nucleic acid complex, a protein-sugar chain complex, a protein-lipid complex, a protein-low molecular weight compound complex, a virus-antibody complex, a virus-low molecular weight compound complex, a liposome-antibody complex, a liposome-nucleic acid complex, a liposome-sugar chain complex, a liposome-low molecular weight compound complex, a liposome-protein complex, and an enzyme complex. The structural analysis target substance may also be a substance derived from a living organism.

[0031] For example, the first chemical bond is a reaction in which a reaction solution converts hydroxyl groups of the graphene oxide grid into epoxy groups, oxirane groups, alkene groups, alkyne groups, carboxylic acid groups, aldehyde groups, carboxylic acid chloride groups, acid anhydride groups, carbamic acid groups, ester groups, sulfonyl chloride groups, glyoxal groups, aryl halide groups, imide ester groups, carbodiimide groups, anhydride groups, fluoroester groups, isocyanate groups, isothiocyanate groups, acyl azide groups, NHS ester groups, amide groups, imide groups, ether groups, thioether groups, urea groups, urethane groups, etc. For example, the second chemical bond is a reaction in which, after the first chemical bond, a sample solution containing a substance to be structurally analyzed (e.g., a protein) is dropped onto the sample grid 28, causing the epoxy groups on the sample grid 28 to react with the substance to be structurally analyzed, thereby chemically immobilizing the substance to be structurally analyzed on the sample grid 28. For example, the reaction liquid may contain an epichlorohydrin solution, a glutaraldehyde solution, a dichloroacid solution, an acid anhydride solution, a diisocyanate solution, etc. For example, the cleaning liquid may contain pure water, distilled water, a buffer solution, an organic solvent, or a fluorine-based solvent.

[0032] The liquid phase reaction mechanism 14 includes a pipette mechanism 24 and a container 26 .

[0033] The pipette mechanism 24 is a mechanism for aspirating and dispensing liquids such as reaction liquids, washing liquids, and sample solutions. For example, the pipette mechanism 24 includes a tip 24a and a moving mechanism 24b. A pipette tip is attached to the tip 24a. The moving mechanism 24b is connected to the tip 24a and moves the tip 24a up and down and left and right. For example, the moving mechanism 24b includes a motor, gears, rails, etc., and moves the tip 24a. The movement of the pipette mechanism 24 by the moving mechanism 24b is controlled by the control device 50.

[0034] The containers 26 contain a reaction solution container 26a, a cleaning solution container 26b, and a sample container 26c. The reaction solution container 26a contains a reaction solution that causes a first chemical bond. The cleaning solution container 26b contains a cleaning solution. The sample container 26c contains a sample solution that causes a second chemical bond.

[0035] The pipette mechanism 24 has a function of aspirating each liquid contained in the container 26 and dispensing it onto the sample grid 28. The pipette mechanism 24 also has a function of aspirating liquid (e.g., excess liquid) on the sample grid 28. The amount of each liquid dispensed onto the sample grid 28 and the amount of each liquid aspirated from the sample grid 28 are controlled by the control device 50.

[0036] For example, the pipette mechanism 24 has a function of aspirating the reaction liquid contained in the reaction liquid container 26a and discharging it onto the sample grid 28, and then aspirating the reaction liquid (e.g., excess reaction liquid) on the sample grid 28. The pipette mechanism 24 also has a function of aspirating the reaction liquid contained in the cleaning liquid container 26b and discharging it onto the sample grid 28, and then aspirating the cleaning liquid (e.g., excess cleaning liquid) on the sample grid 28. The pipette mechanism 24 also has a function of aspirating the sample solution contained in the sample container 26c and discharging it onto the sample grid 28, and then aspirating the sample solution (e.g., excess sample solution) on the sample grid 28.

[0037] For example, a pipette tip for a reaction solution, a pipette tip for a cleaning solution, and a pipette tip for a sample solution are provided in advance in the liquid-phase reaction mechanism 14. The pipette tip for a reaction solution is a pipette tip for discharging and aspirating a reaction solution. The pipette tip for a cleaning solution is a pipette tip for discharging and aspirating a cleaning solution. The pipette tip for a sample solution is a pipette tip for discharging and aspirating a sample solution.

[0038] The pipette mechanism 24 has a pipette tip for the liquid to be dispensed attached to the tip 24a, and dispenses and aspirates the liquid using the attached pipette tip. When dispensing and aspirating a different liquid, the pipette mechanism 24 replaces the pipette tip and dispenses and aspirates the different liquid.

[0039] For example, the pipette mechanism 24 attaches a pipette tip for a reaction liquid to the distal end portion 24a and dispenses and aspirates the reaction liquid. The pipette mechanism 24 replaces the pipette tip attached to the distal end portion 24a with a pipette tip for a sample solution and dispenses and aspirates the sample solution.

[0040] More specifically, the pipette mechanism 24 attaches a pipette tip for the reaction liquid to the tip 24a and dispenses and aspirates the reaction liquid. Then, the pipette mechanism 24 replaces the pipette tip attached to the tip 24a from the pipette tip for the reaction liquid to a pipette tip for the cleaning liquid and dispenses and aspirates the cleaning liquid. Then, the pipette mechanism 24 replaces the pipette tip attached to the tip 24a from the pipette tip for the cleaning liquid to a pipette tip for the sample solution and dispenses and aspirates the sample solution.

[0041] The freezing mechanism 16 freezes the sample grid 28 with a refrigerant. After the liquid-phase reaction mechanism 14 performs a liquid-phase reaction, the freezing mechanism 16 performs freezing. For example, the freezing mechanism 16 includes a refrigerant storage unit 16a and a refrigerant container 16b. The refrigerant storage unit 16a is a container that stores a refrigerant such as liquid ethane, liquid propane, or liquid nitrogen. By storing the refrigerant in the refrigerant container 16b, the temperature of the refrigerant container 16b is maintained at an extremely low temperature. The temperature of the refrigerant container 16b is maintained by the refrigerant stored in the refrigerant storage unit 16a. By inserting the sample grid 28 into the refrigerant container 16b, the sample grid 28 is frozen by the refrigerant.

[0042] The gripping mechanism 18 is a mechanism that grips and moves the sample grid 28. For example, the gripping mechanism 18 includes tweezers 18a and a moving mechanism 18b. The tweezers 18a clamp and hold the sample grid 28. The moving mechanism 18b is connected to the tweezers 18a and moves the tweezers 18a up and down and left and right, and also rotates the tweezers 18a. For example, the moving mechanism 18b includes a motor, gears, rails, etc., and moves and rotates the tweezers 18a. The gripping mechanism 18 grips and moves the sample grid 28, thereby performing various processes such as gas-phase reaction, liquid-phase reaction, and freezing. The movement of the tweezers 18a by the moving mechanism 18b is controlled by a control device 50.

[0043] The absorption mechanism 20 is a mechanism that absorbs excess liquid on the sample grid 28. For example, the absorption mechanism 20 includes a pair of filter papers 20a and a holding mechanism 20b. The filter papers 20a are a member that absorbs excess liquid on the sample grid 28. The holding mechanism 20b has the function of holding the pair of filter papers 20a and moving the pair of filter papers 20a toward or away from each other. The sample grid 28 is placed between the pair of filter papers 20a, and the pair of filter papers 20a are brought close to the liquid on the sample grid 28, so that the liquid on the sample grid 28 is absorbed by the filter papers 20a.

[0044] The camera 22 is a device for photographing the gas phase reaction mechanism 12, the liquid phase reaction mechanism 14, the freezing mechanism 16, the absorption mechanism 20, the sample grid 28, and the like.

[0045] The production apparatus 10 may include a temperature and humidity control device that controls the temperature and humidity of each mechanism. For example, the temperature and humidity of each mechanism are maintained at predetermined values.

[0046] The control device 50 controls the operation of each part of the preparation device 10. For example, the control device 50 controls the gripping and movement of the sample grid 28 by the gripping mechanism 18, the dispensing and suction of liquid by the pipette mechanism 24, the movement of the pipette mechanism 24, and the absorption of liquid by the absorption mechanism 20.

[0047] The control device 50 is, for example, a personal computer or a server that includes a processor such as a CPU (Central Processing Unit) and a storage device such as a memory or a hard disk drive. For example, the control by the control device 50 is realized by the processor executing a program stored in the storage device. Of course, the control by the control device 50 may also be realized by an electronic circuit or a programmable logic circuit.

[0048] Below, the general flow of sample preparation using the preparation device 10 will be explained, with steps (1) to (6). (1) First, the gas phase reaction mechanism 12 causes a gas phase reaction on the sample grid 28 . (2) After the gas phase reaction, the liquid phase reaction mechanism 14 performs a liquid phase reaction on the sample grid 28. Specifically, the liquid phase reaction mechanism 14 ejects a reaction liquid onto the sample grid 28 to generate a first chemical bond. (3) After the first chemical bond, the reaction solution used in the first chemical bond is washed with a washing solution, that is, the reaction solution is removed from the sample grid 28. (4) After cleaning, the liquid-phase reaction mechanism 14 discharges the sample solution onto the sample grid 28 to generate a second chemical bond. (5) After the second chemical bond, the absorbing mechanism 20 absorbs excess liquid on the sample grid 28. If there is no excess liquid on the sample grid 28, the absorbing mechanism 20 does not need to suck the liquid. (6) Next, the sample grid 28 is frozen by the freezing mechanism 16. As a result, a sample is formed on the sample grid 28.

[0049] 2 to 7, a detailed flow of sample preparation using the preparation apparatus 10 will be described.

[0050] (1) Gas-phase reaction As shown in Figure 2, a gas-phase reaction is performed on a sample grid 28 inserted into a container of a gas-phase reaction mechanism 12. For example, the gas-phase reaction mechanism 12 produces a graphene oxide grid by photo-oxidizing chlorous acid radicals on a graphene grid, which is the sample grid 28. Figure 2 shows an example of a gas-phase reaction.

[0051] (2) First chemical bond (1) After the gas-phase reaction is completed, as shown in FIG. 3, the gripping mechanism 18 grips the sample grid 28 with the tweezers 18a and moves the sample grid 28 to the liquid-phase reaction mechanism 14. The pipette mechanism 24 has a reaction liquid pipette tip 24c attached to its distal end 24a, and aspirates and dispenses the reaction liquid contained in the reaction liquid container 26a onto the sample grid 28. For example, the pipette mechanism 24 dispenses a reaction liquid 30 onto one side of the sample grid 28. The reaction liquid 30 forms a first chemical bond on the sample grid 28, converting the hydroxyl groups of the graphene oxide grid into epoxy groups. The pipette mechanism 24 may also aspirate excess reaction liquid 30 on the sample grid 28 with the pipette tip 24c. The dispense and aspirate amounts of the reaction liquid are controlled by the control device 50. FIG. 3 illustrates an example of the first chemical bond.

[0052] (3) Cleaning (2) After the reaction solution 30 is dispensed onto the sample grid 28, the gripping mechanism 18 places the sample grid 28 in the liquid-phase reaction mechanism 14 while gripping the sample grid 28 with the tweezers 18a, as shown in FIG. 4. For example, after the reaction solution 30 is dispensed onto the sample grid 28, the gripping mechanism 18 does not move the sample grid 28. The pipette mechanism 24 removes the pipette tip 24c for the reaction solution attached to the tip 24a and attaches the pipette tip 24d for the cleaning solution to the tip 24a. This replaces the pipette tip attached to the tip 24a from the pipette tip 24c for the reaction solution to the pipette tip 24d for the cleaning solution. The pipette mechanism 24 aspirates the cleaning solution contained in the cleaning solution container 26b using the pipette tip 24d for the cleaning solution and dispenses it onto the sample grid 28. For example, the pipette mechanism 24 dispenses a cleaning liquid 32 onto the surface of the sample grid 28 onto which the reaction liquid 30 has been dispensed. As a result, the reaction liquid 30 on the sample grid 28 is washed with the cleaning liquid 32, and the reaction liquid 30 is removed from the sample grid 28. The pipette mechanism 24 may also aspirate excess cleaning liquid 32 from the sample grid 28 using the pipette tip 24d. The amount of cleaning liquid dispensed and aspirated is controlled by the control device 50. The first chemical bond can be controlled by controlling the time until the cleaning liquid 32 is dispensed onto the sample grid 28. This time is controlled by the control device 50. If washing of the reaction liquid 30 is not necessary due to the amount of reaction liquid 30 on the sample grid 28, for example, washing with a cleaning liquid may not be performed.

[0053] (4)Second chemical bond (3) After cleaning, as shown in FIG. 5, the gripping mechanism 18 places the sample grid 28 in the liquid-phase reaction mechanism 14 while gripping the sample grid 28 with the tweezers 18a. For example, after the cleaning solution 32 is dispensed onto the sample grid 28, the gripping mechanism 18 does not move the sample grid 28. The pipette mechanism 24 removes the pipette tip 24d for cleaning solution attached to the tip 24a and attaches the pipette tip 24e for sample solution to the tip 24a. This replaces the pipette tip attached to the tip 24a from the pipette tip 24d for cleaning solution to the pipette tip 24e for sample solution. The pipette mechanism 24 aspirates the sample solution contained in the sample container 26c using the pipette tip 24e for sample solution and dispenses it onto the sample grid 28. For example, the pipette mechanism 24 dispenses the sample solution 34 onto the surface of the sample grid 28 onto which the reaction solution 30 was dispensed. The sample solution 34 forms a second chemical bond on the sample grid 28. As a result, the epoxy groups on the sample grid 28 react with the substance to be structurally analyzed (e.g., a protein), and the substance to be structurally analyzed (e.g., a protein) is chemically immobilized on the sample grid 28. The pipette mechanism 24 may also aspirate excess sample solution on the sample grid 28 using the pipette tip 24e. The amount of sample solution dispensed and aspirated is controlled by the control device 50. Figure 5 shows an example of the second chemical bond. Here, the substance to be structurally analyzed is a protein, as an example.

[0054] (5) Absorption of liquid by the absorption mechanism 20 After the liquid-phase reaction by the liquid-phase reaction mechanism 14 is completed, as shown in FIG. 6 , the gripping mechanism 18, while holding the sample grid 28 with the tweezers 18a, moves the sample grid 28 to the absorption mechanism 20 and places the sample grid 28 between the pair of filter papers 20a. The absorption mechanism 20 closes the pair of filter papers 20a, bringing the pair of filter papers 20a close to the sample grid 28 and bringing them into contact with the sample solution 34 on the sample grid 28. As a result, excess sample solution 34 on the sample grid 28 is sucked by the filter papers 20a and removed from the sample grid 28. Note that if excess sample solution 34 is not present on the sample grid 28, the absorption mechanism 20 does not have to absorb the sample solution 34.

[0055] (6)Freezing After absorption by the absorption mechanism 20, as shown in FIG. 7, the gripping mechanism 18, while holding the sample grid 28 with the tweezers 18a, moves the sample grid 28 to the refrigerant container 16b and inserts it into the refrigerant container 16b. As a result, the sample grid 28 is frozen by the refrigerant. For example, the gripping mechanism 18 immerses the sample grid 28 in the refrigerant in the refrigerant container 16b, thereby rapidly freezing the sample grid 28 and the sample solution on the sample grid 28. This completes the preparation of the sample by the ice embedding method.

[0056] As described above, with the preparation apparatus 10 according to this embodiment, the dispensing and aspirating of each liquid is performed mechanically by the pipette mechanism 24, rather than manually by an operator, thereby improving the accuracy of the dispensing and aspirating of each liquid. Furthermore, the surface modification of the sample grid 28 is performed mechanically by the gas-phase reaction mechanism 12, thereby achieving stable results from the gas-phase reaction. Similarly, the surface modification of the sample grid 28 is performed mechanically by the liquid-phase reaction mechanism 14, thereby achieving stable results from the liquid-phase reaction. According to this embodiment, samples can be prepared more easily for cryo-electron microscopy than when prepared manually.

[0057] <Specific Example of Manufacturing Apparatus 10> A specific example of the manufacturing apparatus 10 will be described below with reference to Figs. 8 to 17. Figs. 8 to 17 show a manufacturing apparatus 100. The manufacturing apparatus 100 is an example of a specific configuration of the above-mentioned manufacturing apparatus 10. Of course, the manufacturing apparatus according to this embodiment is not limited to the manufacturing apparatus 100. In Figs. 8 to 17, a three-dimensional Cartesian coordinate system consisting of an X-axis, a Y-axis, and a Z-axis is defined.

[0058] FIG. 8 is a view of the fabrication apparatus 100 as viewed from the side (+Y direction). FIG. 9 is a view of the fabrication apparatus 100 as viewed from the side (-Y direction). FIG. 10 is a view of a portion of the fabrication apparatus 100 as viewed from the side (+Y direction). FIGS. 11, 12, and 14 are perspective views showing the gas-phase reaction mechanism, the containing mechanism, and the gripping mechanism. FIG. 13 is a cross-sectional view of the gas-phase reaction mechanism. FIG. 15 is a view of the containing mechanism as viewed from above (+Z direction). FIG. 16 is a perspective view of the gas-phase reaction mechanism, the pipette mechanism, and the gripping mechanism. FIG. 17 is a view of the fabrication apparatus 100 as viewed from the side (+Y direction).

[0059] The production apparatus 100 includes a gas-phase reaction mechanism 110, a pipette mechanism 120, a storage mechanism 130, a freezing mechanism 140, a gripping mechanism 150, a rotation mechanism 160, and a frame 170. The gas-phase reaction mechanism 110, the pipette mechanism 120, the storage mechanism 130, the freezing mechanism 140, the gripping mechanism 150, and the rotation mechanism 160 are supported by the frame 170.

[0060] The gas-phase reaction mechanism 110 is an example of the gas-phase reaction mechanism 12 described above. The pipette mechanism 120 is an example of the pipette mechanism 24 described above. The storage mechanism 130 is an example of the container 26 described above. The pipette mechanism 120 and the storage mechanism 130 constitute an example of the liquid-phase reaction mechanism 14 described above. The freezing mechanism 140 is an example of the freezing mechanism 16 described above. The gripping mechanism 150 is an example of the gripping mechanism 18 described above. Although not shown in FIGS. 8 to 17 , the production apparatus 100 includes the control device 50 described above. The operation of each part of the production apparatus 100 is controlled by the control device 50. The production apparatus 100 may also include the absorbing mechanism 20 and camera 22 described above.

[0061] The rotation mechanism 160 is configured to rotate within the XZ plane around a rotation axis 162 extending in the Y direction. The gripping mechanism 150 is supported by the rotation mechanism 160, and is rotated within the XZ plane around the rotation axis 162 as the rotation mechanism 160 rotates.

[0062] The gas-phase reaction mechanism 110 is disposed in front of the rotation mechanism 160 (+X direction). The freezing mechanism 140 is disposed below the rotation mechanism 160 (-Z direction). The rotation mechanism 160 rotates around a rotation axis 162, so that the gripping mechanism 150 can be positioned facing the gas-phase reaction mechanism 110 or the freezing mechanism 140.

[0063] 8 and 9, the rotation mechanism 160 rotates downward, and the gripping mechanism 150 is disposed facing downward. As a result, the gripping mechanism 150 is disposed opposite the freezing mechanism 140.

[0064] 10, the rotation mechanism 160 rotates forward, and the gripping mechanism 150 is disposed facing forward. As a result, the gripping mechanism 150 is disposed opposite the gas-phase reaction mechanism 110. For example, the gas-phase reaction mechanism 110 is disposed at a position rotated 90 degrees from the freezing mechanism 140.

[0065] 10 to 12, the gripping mechanism 150 includes tweezers 152 and a moving mechanism 154. The tweezers 152 and the moving mechanism 154 are mounted on the rotation mechanism 160 and, as described above, are rotated in accordance with the rotation of the rotation mechanism 160. In the example shown in FIGS. 11 and 12, the rotation mechanism 160 rotates forward, and the gripping mechanism 150 is disposed opposite the gas-phase reaction mechanism 110.

[0066] The tweezers 152 are an example of the tweezers 18a described above, and hold the sample grid 28 by clamping it. The moving mechanism 154 is an example of the moving mechanism 18b described above. The tweezers 152 are connected to the moving mechanism 154. The moving mechanism 154 includes a motor, gears, rails, etc., and is a mechanism for moving the tweezers 152. For example, the moving mechanism 154 slides the tweezers 152. The movement of the tweezers 152 by the moving mechanism 154 is controlled by the control device 50.

[0067] 11 and 12, when the rotation mechanism 160 rotates forward, the tweezers 152 and the movement mechanism 154 are arranged parallel to the X direction. In this case, the movement mechanism 154 can slide the tweezers 152 in the +X direction (forward) or the −X direction (backward), as indicated by the arrow A1.

[0068] As shown in Fig. 11 , movement mechanism 154 can slide tweezers 152 in the -X direction, thereby disposing tweezers 152 at a position away from gas-phase reaction mechanism 110. As shown in Fig. 12 , movement mechanism 154 can slide tweezers 152 in the +X direction, thereby inserting tweezers 152 into gas-phase reaction mechanism 110. In this way, by sliding tweezers 152 with movement mechanism 154, tweezers 152 can be inserted into or removed from gas-phase reaction mechanism 110.

[0069] The tweezers 152 hold the sample grid 28. Therefore, by inserting the tweezers 152 into the gas-phase reaction mechanism 110, the sample grid 28 can be inserted into the gas-phase reaction mechanism 110. Furthermore, by removing the tweezers 152 from the gas-phase reaction mechanism 110, the sample grid 28 can be removed from the gas-phase reaction mechanism 110.

[0070] When a gas-phase reaction is to be carried out on the sample grid 28, the tweezers 152 are slid in the +X direction by the movement mechanism 154, thereby inserting the sample grid 28 into the gas-phase reaction mechanism 110. With the sample grid 28 inserted in the gas-phase reaction mechanism 110, a gas-phase reaction is carried out.

[0071] As will be described later, when a liquid-phase reaction is to be carried out on the sample grid 28, the tweezers 152 are moved in the −X direction by the movement mechanism 154, so that the sample grid 28 is removed from the gas-phase reaction mechanism 110 and placed outside the gas-phase reaction mechanism 110. With the sample grid 28 placed outside the gas-phase reaction mechanism 110, a liquid-phase reaction is carried out.

[0072] The moving mechanism 154 and the rotating mechanism 160 are driven by, for example, motors. The motors are controlled by the control device 50. The speed of the sliding movement of the moving mechanism 154 and the speed of the rotational movement of the rotating mechanism 160 are controlled by the control device 50.

[0073] The configuration of gas phase reaction mechanism 110 will be described with reference to Fig. 13. As shown in Fig. 13, gas phase reaction mechanism 110 includes a reagent container 112, an irradiation device 114, and a cap .

[0074] The reagent container 112 is a container that contains a reagent for a gas-phase reaction. The reagent for a gas-phase reaction is, for example, a hydrochloric acid-acidic NaClO2 aqueous solution. The top of the reagent container 112 is open. The irradiation device 114 is a light source that irradiates light (e.g., ultraviolet light) from the side of the reagent container 112. One or more irradiation devices 114 are arranged around the reagent container 112. The cap 116 is a member that covers the top of the reagent container 112. The cap 116 is hollow. An opening 116a is formed in the bottom surface of the cap 116. The top of the reagent container 112 is inserted into the opening 116a. An opening 116b is also formed in the side surface of the cap 116. The tweezers 152 are slid in the +X direction by the movement mechanism 154, so that the sample grid 28 held by the tweezers 152 is inserted into the cap 116 through the opening 116b. This places the sample grid 28 in the gas-phase reaction mechanism 110. When the irradiation device 114 irradiates light into the reagent container 112 while the sample grid 28 is inserted in the cap 116, the reagent in the reagent container 112 reacts with the light to generate an active gas (e.g., ClO2 radical gas), which then flows into the cap 116 through the opening 116a. The active gas reacts with the sample grid 28, causing a gas-phase reaction on the sample grid 28.

[0075] The liquid phase reaction mechanism will be described below with reference to FIGS. 11, 12, 14, 15 and 16.

[0076] As shown in FIG. 11, the containing mechanism 130 included in the liquid phase reaction mechanism includes a pipette tip containing device 132 and a solution containing device 134.

[0077] The pipette tip storage device 132 stores a plurality of pipette tips arranged in an upright position. The solution storage device 134 stores a reaction solution, a washing solution, and a sample solution separately. For example, the solution storage device 134 stores the reaction solution, the washing solution, and the sample solution arranged in the order in which they are to be used.

[0078] 11 and 15, for example, the pipette tip holding device 132 and the solution holding device 134 each have an annular shape and are arranged concentrically. For example, the pipette tip holding device 132 is arranged outside the solution holding device 134 so as to cover the solution holding device 134.

[0079] The pipette tip holding device 132 and the solution holding device 134 are devices that can rotate around the Z axis as a rotation axis. The rotation of each of the pipette tip holding device 132 and the solution holding device 134 is controlled by the control device 50. For example, the amount of rotation (i.e., the rotation angle) is controlled by the control device 50.

[0080] 15, the pipette tip storage device 132 includes a plurality of storage sections 136 arranged in a circular shape. Each storage section 136 is a hole with a bottom. A pipette tip is stored in each storage section 136 in an upright position.

[0081] As shown in FIG. 15, the solution containing device 134 includes a plurality of containing sections 138 arranged in a circular ring shape. Each containing section 138 is a hole with a bottom. A liquid is contained in each containing section 138. For example, a reaction liquid is contained in a certain containing section 138 (for convenience, referred to as "containing section 138a"). A cleaning liquid is contained in a containing section 138 (for convenience, referred to as "containing section 138b") adjacent to the containing section 138a containing the reaction liquid. A sample solution is contained in a containing section 138 (hereinafter referred to as "containing section 138c") adjacent to the containing section 138b containing the cleaning liquid. In other words, the containing sections 138 are formed side by side in the order of containing sections 138a, 138b, and 138c.

[0082] Additionally, the area 139 inside the solution containing device 134 may be used as a space for discarding used pipette tips.

[0083] As shown in Figures 11 and 12, the pipette mechanism 120 included in the liquid-phase reaction mechanism includes a tip 122 and a moving mechanism 124. The tip 122 is an example of the tip 24a described above. The moving mechanism 124 is an example of the moving mechanism 24b described above. The moving mechanism 124 includes a motor, gears, rails, etc., and is a mechanism that moves the tip 122 in the up-down direction (i.e., the +Z direction or the -Z direction), the front-back direction (i.e., the +X direction or the -X direction), and the left-right direction (i.e., the +Y direction or the -Y direction). The movement of the tip 122 by the moving mechanism 124 is controlled by the control device 50.

[0084] As shown in Figure 11, the pipette mechanism 120 uses the moving mechanism 124 to move the tip 122 to a position above the pipette tip storage device 132 (for convenience, referred to as "position α"), and at that position α, moves the tip 122 downward (in the -Z direction), thereby attaching the pipette tip 126 stored in the pipette tip storage device 132 to the tip 122.

[0085] Next, the pipette mechanism 120 uses the moving mechanism 124 to move the tip 122 to a position above the solution containing device 134 (for convenience, referred to as "position β"), and at that position β, moves the tip 122 downward (in the -Z direction) to aspirate the liquid contained in the solution containing device 134 into the pipette tip 126.

[0086] For example, when the reaction liquid is dropped onto the sample grid 28, the solution containing device 134 rotates about the Z axis so that the containing section 138a containing the reaction liquid is positioned below position β. At position β, the pipette mechanism 120 moves the tip 122 downward, and aspirates the reaction liquid contained in the containing section 138a with the pipette tip 126 attached to the tip 122.

[0087] In this state, as shown in FIGS. 14 and 16 , the moving mechanism 124 moves the tip 122, to which the pipette tip 126 that has aspirated the reaction liquid is attached, to the liquid-phase reaction position 128. The position of the liquid-phase reaction position 128 on the X-axis and Y-axis is the position where the tip of the tweezers 152 removed from the gas-phase reaction mechanism 110 is positioned. As shown in FIG. 16 , the moving mechanism 124 moves the tip 122 downward (in the −Z direction) at the liquid-phase reaction position 128 until the tip 122 is positioned near the tweezers 152. In this state, the pipette mechanism 120 drips the reaction liquid aspirated into the pipette tip 126 onto the sample grid 28 held at the tip of the tweezers 152. This causes the first chemical bond described above to occur.

[0088] The moving mechanism 124 then moves the tip 122 to which the pipette tip 126 is attached to a position above the area 139 inside the solution containing device 134, at which point the pipette tip 126 is removed from the tip 122 and discarded into the area 139.

[0089] When the cleaning solution and the sample solution are dropped onto the sample grid 28, the same operation as that for dropping the reaction solution is carried out.

[0090] When dropping cleaning liquid onto the sample grid 28, the pipette mechanism 120 moves the tip 122 to position α above the pipette tip storage device 132 using the movement mechanism 124. The pipette tip storage device 132 rotates around the Z axis so that the storage section 136 storing the pipette tip 126 is positioned below position α. ​​The pipette mechanism 120 attaches the pipette tip 126 stored in the pipette tip storage device 132 to the tip 122 by moving the tip 122 downward (in the −Z direction) at position α.

[0091] Next, the pipette mechanism 120 uses the movement mechanism 124 to move the tip 122 to position β above the solution containing device 134. The solution containing device 134 rotates about the Z axis as the rotation axis so that the containing section 138b containing the cleaning solution is positioned below position β. The pipette mechanism 120 moves the tip 122 downward at position β, and aspirates the cleaning solution contained in the containing section 138b using the pipette tip 126 attached to the tip 122.

[0092] 14 and 16, the moving mechanism 124 moves the tip 122, to which the pipette tip 126 that has aspirated the cleaning solution is attached, to the liquid-phase reaction position 128. As shown in FIG. 16, the moving mechanism 124 moves the tip 122 downward (in the -Z direction) at the liquid-phase reaction position 128 until the tip 122 is positioned near the tweezers 152. In this state, the pipette mechanism 120 drips the cleaning solution aspirated into the pipette tip 126 onto the sample grid 28 held at the tip of the tweezers 152. This completes the cleaning described above.

[0093] The moving mechanism 124 then moves the tip 122 to which the pipette tip 126 is attached to a position above the area 139 inside the solution containing device 134, at which point the pipette tip 126 is removed from the tip 122 and discarded into the area 139.

[0094] Next, when dropping the sample solution onto the sample grid 28, the pipette mechanism 120 moves the tip 122 to position α above the pipette tip storage device 132 using the movement mechanism 124. The pipette tip storage device 132 rotates around the Z axis so that the storage section 136 storing the pipette tip 126 is positioned below position α. ​​The pipette mechanism 120 attaches the pipette tip 126 stored in the pipette tip storage device 132 to the tip 122 by moving the tip 122 downward (in the −Z direction) at position α.

[0095] Next, the pipette mechanism 120 uses the moving mechanism 124 to move the tip 122 to position β above the solution containing device 134. The solution containing device 134 rotates about the Z axis so that the containing section 138c containing the sample solution is positioned below position β. The pipette mechanism 120 moves the tip 122 downward at position β, and aspirates the sample solution contained in the containing section 138c using the pipette tip 126 attached to the tip 122.

[0096] 14 and 16, the moving mechanism 124 moves the tip 122, to which the pipette tip 126 that has aspirated the sample solution is attached, to the liquid-phase reaction position 128. As shown in FIG. 16, the moving mechanism 124 moves the tip 122 downward (in the −Z direction) at the liquid-phase reaction position 128 until the tip 122 is positioned near the tweezers 152. In this state, the pipette mechanism 120 drips the sample solution aspirated into the pipette tip 126 onto the sample grid 28 held at the tip of the tweezers 152. This causes the second chemical bond described above to occur.

[0097] The moving mechanism 124 then moves the tip 122 to which the pipette tip 126 is attached to a position above the area 139 inside the solution containing device 134, at which point the pipette tip 126 is removed from the tip 122 and discarded into the area 139.

[0098] In this manner, the reaction solution, the washing solution, and the sample solution are dropped in sequence onto the sample grid 28. This causes the first chemical bond, washing, and second chemical bond to occur in this order.

[0099] The reaction solution, washing solution, and sample solution are dropped onto the same surface of the sample grid 28. For example, the disk-shaped sample grid 28 is held with the tweezers 152 so that the disk surface faces upward (+Z direction). In this state, the reaction solution, washing solution, and sample solution are dropped onto the same surface of the sample grid 28, one after the other. That is, the surface of the sample grid 28 onto which the reaction solution, washing solution, and sample solution are dropped is positioned perpendicular to the direction of gravity when facing the pipette mechanism 120, and in this state, the reaction solution, washing solution, and sample solution are dropped onto this surface, one after the other. In other words, the surface of the sample grid 28 onto which each liquid is dropped is positioned parallel to the plane formed by the X-axis and Y-axis, and in this state, each liquid is dropped onto this surface.

[0100] Furthermore, excess liquid on the sample grid 28 may be aspirated by the pipette tip 126. That is, if excess reaction liquid is present on the sample grid 28, the excess reaction liquid may be aspirated by the pipette tip 126 after the reaction liquid is dropped onto the sample grid 28. The same applies to the cleaning liquid and the sample solution.

[0101] After the sample solution is dropped onto the sample grid 28 and the second chemical bond is formed, freezing is performed as described above. As shown in Fig. 17, the rotation mechanism 160 rotates downward (-Z direction) around the rotation axis 162. In this way, the tweezers 152 supported by the rotation mechanism 160 can be positioned facing the freezing mechanism 140.

[0102] In this state, the moving mechanism 154 slides the tweezers 152 downward until the sample grid 28 held at the tip of the tweezers 152 is inserted into the freezing mechanism 140. Once the sample grid 28 is inserted into the freezing mechanism 140, the sample grid 28 is frozen in the same manner as the freezing mechanism 16 described above. That is, a refrigerant is contained within the freezing mechanism 140, and the sample grid 28 and the sample solution on the sample grid 28 are rapidly frozen by immersing the sample grid 28 in the refrigerant. This completes the preparation of the sample by the ice embedding method.

[0103] As described above, according to the preparation apparatus 100, the gas phase reaction, cleaning, and liquid phase reaction are each performed mechanically by the preparation apparatus 100, making it easier to prepare samples than if these were performed manually by an operator. [Explanation of symbols]

[0104] 10,100 Preparation device, 12,110 Gas phase reaction mechanism, 14 Liquid phase reaction mechanism, 16,140 Freezing mechanism, 18,150 Holding mechanism, 18a Tweezers, 20 Absorption mechanism, 24 Pipette mechanism, 50 Control device, 120 Pipette mechanism, 130 Storage mechanism, 160 Rotation mechanism.

Claims

1. In a sample preparation device for a cryogenic transmission electron microscope, a gas phase reaction mechanism for performing a gas phase reaction on the sample grid; a liquid phase reaction mechanism for carrying out a liquid phase reaction on the sample grid after the gas phase reaction; a freezing mechanism for freezing the sample grid with a refrigerant after the liquid phase reaction; 1. A cryogenic transmission electron microscope sample preparation device comprising:

2. 2. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 1, The liquid phase reaction is an organic reaction. A sample preparation device for a cryogenic transmission electron microscope, characterized by:

3. 3. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 2, the organic reaction produces a chemical bond; A sample preparation device for a cryogenic transmission electron microscope, characterized by:

4. 4. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 3, the liquid phase reaction mechanism generates a first chemical bond and a second chemical bond; A sample preparation device for a cryogenic transmission electron microscope, characterized by:

5. 5. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 4, The liquid phase reaction mechanism is a reaction liquid container for containing a reaction liquid; a sample container for containing a sample solution; a pipette mechanism; Including, The pipette mechanism includes: a function of discharging the reaction liquid contained in the reaction liquid container onto the sample grid, and then aspirating the reaction liquid on the sample grid; a function of discharging the sample solution contained in the sample container onto the sample grid, and then aspirating the sample solution on the sample grid; and the first chemical bond is generated by discharging the reaction solution onto the sample grid; After the first chemical bond is formed, a sample solution is ejected onto the sample grid, thereby forming the second chemical bond. A sample preparation device for a cryogenic transmission electron microscope, characterized by:

6. 6. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 5, the pipette mechanism dispenses a sample solution onto the surface of the sample grid onto which the reaction solution has been dispensed; A sample preparation device for a cryogenic transmission electron microscope, characterized by:

7. 6. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 5, The liquid phase reaction mechanism further includes: a washing liquid container for containing a washing liquid for washing the reaction liquid on the sample grid; The pipette mechanism includes: After the first chemical bond is formed, the reaction solution is washed by discharging a cleaning solution onto the surface of the sample grid onto which the reaction solution has been discharged. A sample preparation device for a cryogenic transmission electron microscope, characterized by:

8. 8. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 7, a control device that controls the time from when the reaction solution is discharged onto the sample grid to when the cleaning solution is discharged onto the sample grid; A sample preparation device for a cryogenic transmission electron microscope, characterized by:

9. 6. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 5, The pipette mechanism includes: exchanging the pipette tip for discharging and aspirating the reaction solution with the pipette tip for discharging and aspirating the sample solution, and discharging and aspirating the reaction solution and the sample solution, respectively; A sample preparation device for a cryogenic transmission electron microscope, characterized by:

10. 8. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 7, The pipette mechanism includes: exchanging a pipette tip for discharging and aspirating the reaction solution, a pipette tip for discharging and aspirating the cleaning solution, and a pipette tip for discharging and aspirating the sample solution, and discharging and aspirating the reaction solution, the cleaning solution, and the sample solution, respectively; A sample preparation device for a cryogenic transmission electron microscope, characterized by:

11. 6. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 5, a surface of the sample grid onto which the reaction solution and the sample solution are discharged is disposed perpendicular to the direction of gravity when facing the pipette mechanism; A sample preparation device for a cryogenic transmission electron microscope, characterized by:

12. 8. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 7, further comprising a pipette tip receiving device and a solution receiving device; The pipette tip storage device It accommodates multiple pipette tips in a row, The solution containing device is The reaction solution, the sample solution, and the cleaning solution are stored side by side, The pipette mechanism includes: removing a pipette tip for the reaction liquid from the pipette tip storage device, aspirating the reaction liquid from the solution storage device using the removed pipette tip for the reaction liquid and discharging it onto the sample grid; After discharging the reaction liquid, the pipette tip for the reaction liquid is discarded, the pipette tip for the cleaning liquid is removed from the pipette tip storage device, and the cleaning liquid is aspirated from the solution storage device using the removed pipette tip for the cleaning liquid and discharged onto the sample grid; After discharging the cleaning solution, the pipette tip for the cleaning solution is discarded, the pipette tip for the sample solution is removed from the pipette tip storage device, and the sample solution is aspirated from the solution storage device using the removed pipette tip for the sample solution and discharged onto the sample grid. A sample preparation device for a cryogenic transmission electron microscope, characterized by:

13. 2. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 1, The gas phase reaction mechanism is a reagent container for containing a reagent for a gas phase reaction; an irradiation device that irradiates light from a side surface of the reagent container; a cap for covering the reagent container, the cap having an opening formed therein through which the sample grid is inserted and removed; Including, The gas phase reaction mechanism is and irradiating the inside of the reagent container with light by the irradiation device while the sample grid is inserted into the cap, thereby causing a gas phase reaction on the sample grid. A sample preparation device for a cryogenic transmission electron microscope, characterized by:

14. 2. The apparatus for preparing a cryogenic transmission electron microscope sample according to claim 1, a rotation mechanism that rotates the sample grid around a rotation axis after the liquid phase reaction, thereby causing the sample grid to face the freezing mechanism. A sample preparation device for a cryogenic transmission electron microscope, characterized by:

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