Cryoelectron microscope workstation and use method of cryoelectron microscope

Abstract

The invention discloses a cryoelectron microscope work station and a using method of a cryoelectron microscop.The cryoelectron microscope work station comprises a sample preparation module, the sample preparation module is used for loading a carrying net, the carrying net is used for bearing a sample, and the sample preparation module comprises a first heat sink used for sealing at least one end of the carrying net, and a second heat sink used for sealing at least one end of the carrying net; an interaction force is formed between the first heat sink and the grid, and the first heat sink is used for conducting cold to the end part of the grid; the second heating component is used for being connected to the bearing surface of the carrying net, and the second heating component is used for heating the bearing surface of the carrying net; and the cold source is connected to the first heat sink, the cold source is used for conducting cold to the first heat sink, and the cold source is used for freezing the bearing surface of the carrier network when the second heating component stops heating the bearing surface of the carrier network. By the adoption of the sample preparation module, the freezing speed can be increased, and overspeed freezing of the sample is achieved.

Description

Technical Field

[0001] This application relates to the field of electron microscopy, and in particular to a cryo-electron microscopy workstation and a method for using a cryo-electron microscope. Background Technology

[0002] In protein structure research, cryo-electron microscopy is a commonly used method. Before placing the sample in a cryo-electron microscope, it needs to be prepared to be sufficiently thin to ensure clarity of observation; for example, the thickness of a typical protein sample should be controlled to around 10-30 nm. Furthermore, rapid freezing within a sufficiently short time is necessary to prevent structural damage during prolonged cooling and freezing; the freezing rate needs to reach 10E4 kJ / s.

[0003] In related technologies, frozen sample preparation typically employs the filter paper adsorption method. Specifically, the sample is first dropped onto a grid, and then filter paper adsorbs the sample from the grid, thereby thinning the sample. The sample thickness is controlled by adjusting the contact position, contact angle, and contact duration between the filter paper and the sample. However, the filter paper adsorption method relies heavily on the operator's skill level, thus exhibiting significant uncontrollability and making it difficult to meet the requirements of sample quality consistency and sample preparation efficiency.

[0004] In cryo-electron microscopy, the pole piece system is the core component of the objective lens, consisting of two pole pieces forming an electromagnetic lens system. A strong magnetic field can be generated between the pole pieces, and the movement of the electron beam is controlled within this magnetic field using the Lorentz force.

[0005] Traditional cryo-electron microscopy requires a large first heat sink (cold shielding) at the pole pieces. Because this heat sink occupies a significant amount of space, it reduces the magnetic field strength between the pole pieces, resulting in lower imaging resolution and hindering subsequent observation. Furthermore, to observe larger sample areas, traditional cryo-electron microscopy requires a larger electron beam channel, which increases the pole piece spacing, further reducing the magnetic field strength and gradient between the pole pieces, ultimately leading to lower imaging resolution.

[0006] In related technologies, the narrow pole piece space of transmission electron microscopes is adapted to improve the accuracy by improving the sample carrier or chip technology. However, if applied to cryo-electron microscopes, it still cannot solve the problems of low magnetic field strength between pole pieces and low imaging resolution caused by the large volume of the first heat sink.

[0007] In addition, to improve the resolution of electron microscopes, optimization algorithms and multi-lens focusing are usually used, but the implementation of these methods is very complicated. Summary of the Invention

[0008] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a cryo-electron microscopy workstation capable of controllably thinning samples, thereby improving the accuracy of thickness control during sample preparation.

[0009] This application also proposes a method for using cryo-electron microscopy.

[0010] According to a first aspect embodiment of the cryo-electron microscopy workstation, the cryo-electron microscopy workstation includes a sample preparation module, the sample preparation module being used to load a mesh carrier, the mesh carrier being used to hold a sample, the sample preparation module including:

[0011] A first heat sink is used to encapsulate at least one end of a carrier mesh. An interaction force is formed between the first heat sink and the carrier mesh. The first heat sink is used to conduct heat to the end of the carrier mesh.

[0012] The second heating element is used to connect to the bearing surface of the netting and to heat the bearing surface of the netting.

[0013] A cold source is connected to the first heat sink. The cold source is used to conduct cold to the first heat sink and to freeze the bearing surface of the net when the second heating component stops heating the bearing surface of the net.

[0014] According to a second aspect embodiment of the cryo-electron microscopy workstation, the cryo-electron microscopy workstation includes a sample preparation module, the sample preparation module comprising:

[0015] A carrying net having a bearing surface for supporting a sample;

[0016] A first heat sink, at least one end of the carrier mesh is encapsulated in the first heat sink, the bearing surface is exposed from the first heat sink, an interaction force is formed between the first heat sink and the carrier mesh, and the first heat sink is used to conduct heat to the end of the carrier mesh;

[0017] A second heating element is disposed in the carrier net, and the second heating element is used to surround the sample and to heat the carrier surface;

[0018] A cold source is connected to the first heat sink, the cold source is used to conduct cold to the first heat sink, and the cold source is used to freeze the bearing surface when the second heating component stops heating the bearing surface.

[0019] According to some embodiments of this application, the sample preparation module is provided with a first film, the surface of the first film is formed as a bearing surface, and the first film is provided with a through structure that penetrates through both sides of the first film.

[0020] According to some embodiments of this application, the sample preparation module is provided with a light-transmitting second film, the surface of which is formed as a bearing surface, and the second film is continuously disposed.

[0021] According to some embodiments of this application, the first heat sink includes a mounting portion and a side end portion connected in sequence. The mounting portion is used to encapsulate the carrier mesh, and the mounting portion is provided with an observation window. The bearing surface is exposed through the observation window, and the side end portion is used to connect to a cold source.

[0022] According to some embodiments of this application, the thickness of the side end portion is greater than the thickness of the mounting portion.

[0023] According to some embodiments of this application, the thickness of the side end portion and the mounting portion gradually transitions.

[0024] According to some embodiments of this application, the mounting part is provided with a mounting groove for enclosing the carrier net so that an interaction force is formed between the mounting part and the carrier net. The observation window is connected to the mounting groove. The bottom surface of the mounting part is also provided with a clearance window connected to the mounting groove. The clearance window is used for a fixing member with a second heating component to extend into the mounting groove.

[0025] According to some embodiments of this application, the mounting portion includes a support structure connecting each of the side ends, the support structure being used to support the carrier net.

[0026] According to some embodiments of this application, the mounting portion further includes a clamping structure, each of the side ends is connected to the clamping structure, the interval between the clamping structures defines the observation window, and the clamping structure is spaced apart from the support structure to form the mounting groove for inserting the carrier net.

[0027] According to some embodiments of this application, the clamping structure is formed as a clamping plate, which is located on top of the carrier net.

[0028] According to some embodiments of this application, the two side ends are symmetrically arranged with respect to the mounting portion.

[0029] According to some embodiments of this application, the avoidance window is disposed on the support structure, and the projection of the area of ​​the carrier net used to carry the sample on the support structure is within the range of the avoidance window.

[0030] According to some embodiments of this application, the sample preparation module further includes a second heat sink connected to the outer periphery of the first heat sink, a cold source connected to the second heat sink, the cold source being in detachable contact with the second heat sink, and the cold source conducting cold to the first heat sink through the second heat sink.

[0031] According to some embodiments of this application, the sample preparation module further includes a cooling component and a second heat sink, wherein the cold source conducts cooling to the first heat sink through the second heat sink;

[0032] The cooling component includes a support, a movable contact, and a refrigerant cavity. A cooling channel is formed in the support. The second heat sink is installed at one end of the cooling channel, and the other end is connected to the refrigerant cavity. The refrigerant cavity is used to introduce a cold source medium. The movable contact is located between the second heat sink and the refrigerant cavity. The movable contact is movable in the cooling channel to connect or disconnect the second heat sink from the cold source medium.

[0033] According to some embodiments of this application, one end of the movable contact is fixedly connected to the second heat sink, and the other end is radially extendable along the cooling channel. The other end of the movable contact can extend to contact the side wall of the refrigerant cavity, or can retract to separate from the side wall of the refrigerant cavity.

[0034] According to some embodiments of this application, the second heat sink is provided with an assembly slot for installing the first heat sink, the opening edge of the assembly slot is flush with the opening edge of the cooling channel, and the support wraps around the second heat sink.

[0035] According to some embodiments of this application, the second heat sink is provided with an assembly slot for installing the first heat sink, the end of the first heat sink is accommodated in the assembly slot, and the volume of the second heat sink is larger than the volume of the side end of the first heat sink.

[0036] According to some embodiments of this application, when the cryo-electron microscopy workstation is in operation, the temperature of the first heat sink is lower than the temperature of the carrier mesh.

[0037] According to some embodiments of this application, the sample preparation module includes a carrier mesh, the carrier mesh having a slit region for holding the sample, the slit region having a width of 1μm-10μm.

[0038] According to some embodiments of this application, the first heat sink drives the sample in the slit region to the position where the electron beam of the cryo-electron microscopy workstation is focused by unidirectional movement.

[0039] According to some embodiments of this application, the carrier includes a substrate supporting the first thin film, the substrate having an imaging window, and the projection of the slit region onto the substrate being within the range of the imaging window.

[0040] According to some embodiments of this application, the sample preparation module includes a thinning component, which includes a first heating component and / or an air supply component, and the thinning component is used to thin the sample on the bearing surface of the carrier mesh.

[0041] According to some embodiments of this application, the sample preparation module includes a fixing member, the second heating member is disposed on the fixing member, and when the first heat sink is installed on the fixing member, the second heating member is in contact with the bottom surface of the first heat sink.

[0042] According to some embodiments of this application, the air supply component is used to provide a plasma flow.

[0043] According to some embodiments of this application, the polarity of the plasma gas flow provided by the air supply component is the same as the polarity of the sample.

[0044] According to some embodiments of this application, the sample preparation module further includes a flow guiding component, the air supply component is used to supply air to the carrier net in a direction perpendicular to the bearing surface, the flow guiding component is disposed on the outer periphery of the carrier net, the flow guiding component is used to disperse the airflow supplied from the air supply component along the flow guiding surface, and the flow guiding surface is arranged parallel to or coplanar with the bearing surface.

[0045] According to some embodiments of this application, the first heat sink includes a mounting portion and a side end located at the end of the mounting portion;

[0046] Along the thickness direction of the first heat sink, the mounting portion is recessed relative to the side end to form a clearance space. The air supply component is movable to approach or move away from the carrier net. When the air supply component approaches the carrier net, the air supply component extends into the clearance space.

[0047] According to some embodiments of this application, the sample preparation module further includes a thickness detection module and a control device. The thickness detection module and the air supply component are both electrically connected to the control device. The thickness detection module is used to detect the thickness of the sample, and the control device is used to control the air supply component to stop supplying air when the thickness detection module detects that the sample has reached the target thickness.

[0048] According to some embodiments of this application, the thickness detection module includes a light generating structure and a light receiving structure, the light generating structure being disposed toward the bearing surface, and the light receiving structure being disposed in the optical path of the reflected light from the sample.

[0049] According to some embodiments of this application, the thickness detection module includes a detection electrode disposed on the bearing surface and used to contact the sample.

[0050] According to some embodiments of this application, the bearing surface includes a central region for bearing the sample and an edge region on the outer periphery of the central region. The second heating element is disposed corresponding to the central region, and the first heating element is disposed corresponding to the edge region. The heating temperature of the first heating element is greater than the heating temperature of the second heating element, so that the temperature of the edge region is higher than the temperature of the central region. The sample can diffuse and thin from the central region to the edge region under the action of the temperature difference between the central region and the edge region.

[0051] According to some embodiments of this application, the cryo-electron microscopy workstation further includes an observation module, which includes an electron microscope tube with a cylindrical body. The cylindrical body has a pair of objective pole pieces arranged in a cylindrical body, and an observation gap is formed between the objective pole pieces to allow the sample to pass through.

[0052] According to some embodiments of this application, the objective lens pole piece includes a first pole piece and a second pole piece. The first pole piece is used to focus an electron beam and has a first channel. The second pole piece is used to further focus the electron beam and has a second channel. The electron beam can be focused by passing through the first channel and the second channel in sequence. The distance between the first pole piece and the second pole piece is less than 4 mm, and the aperture of the first channel and the second channel is less than 2 mm.

[0053] According to some embodiments of this application, the first heat sink includes a mounting portion and a side end located at the end of the mounting portion, the thickness of the mounting portion being less than the thickness of the side end, and the first pole shoe and the second pole shoe being able to extend into the space between each of the side ends.

[0054] According to some embodiments of this application, the radial dimension of the first pole shoe gradually decreases in the direction approaching the second pole shoe; the radial dimension of the second pole shoe gradually decreases in the direction approaching the first pole shoe.

[0055] According to some embodiments of this application, the observation module further includes a coil that generates a magnetic field between the objective lens pole pieces by being energized.

[0056] According to a third aspect embodiment of this application, the cryo-electron microscopy workstation includes:

[0057] The sample preparation module mentioned above;

[0058] The delivery module includes a delivery tunnel for sample passage, the delivery tunnel being disposed within the observation gap, the space within the delivery tunnel being isolated from the cylindrical body, and a through observation port located on the side wall of the delivery tunnel, the observation port being located on the line connecting the two objective lens pole pieces;

[0059] A first vacuum system and a second vacuum system, wherein the first vacuum system provides a vacuum environment for the cylinder and the second vacuum system provides a vacuum environment for the conveying tunnel.

[0060] According to some embodiments of this application, the conveying module further includes a first buffer chamber, which is connected to the entrance of the conveying tunnel. The first buffer chamber and the conveying tunnel are selectively connected. When the first buffer chamber is evacuated, the first buffer chamber and the conveying tunnel are isolated from each other. When the vacuum degree of the first buffer chamber reaches the vacuum degree of the conveying tunnel, the first buffer chamber and the conveying tunnel are connected, and the sample can enter the conveying tunnel from the first buffer chamber.

[0061] According to some embodiments of this application, the two ends of the conveying tunnel are respectively formed as an inlet and an outlet. The conveying module is provided with at least two conveying mechanisms. One of the conveying mechanisms is movably disposed in the conveying tunnel for moving the sample to the observation port, and the other conveying mechanism is used to send the sample into the inlet of the conveying tunnel or output it from the outlet of the conveying tunnel. The sample can be transferred between the two conveying mechanisms.

[0062] According to some embodiments of this application, the conveying module further includes a first buffer chamber and a second buffer chamber. The first buffer chamber is connected to the entrance of the conveying tunnel, and the second buffer chamber is connected to the exit of the conveying tunnel. The conveying module includes three conveying mechanisms, namely a first conveying mechanism, a second conveying mechanism, and a third conveying mechanism. The first conveying mechanism is movably disposed in the conveying tunnel, the second conveying mechanism is disposed in the first buffer chamber, and the second conveying mechanism is used to transfer samples to the first conveying mechanism. The third conveying mechanism is disposed in the second buffer chamber, and the third conveying mechanism is used to receive samples from the first conveying mechanism.

[0063] According to some embodiments of this application, when at least one of the second conveying mechanism or the first conveying mechanism is working, the third conveying mechanism performs sample feeding.

[0064] When the third conveying mechanism or at least one of the first conveying mechanisms is working, the second conveying mechanism loads the sample.

[0065] According to some embodiments of this application, the first buffer chamber and the conveying tunnel can be selectively connected. When the first buffer chamber is evacuated, the first buffer chamber and the conveying tunnel are isolated from each other. When the vacuum degree of the first buffer chamber reaches the vacuum degree of the conveying tunnel, the first buffer chamber and the conveying tunnel are connected. The second conveying mechanism transfers the sample from the entrance of the conveying tunnel to the first conveying mechanism.

[0066] The second buffer chamber is selectively connected to the conveying tunnel. When the second buffer chamber is evacuated, the second buffer chamber is isolated from the conveying tunnel. When the vacuum level of the second buffer chamber reaches the vacuum level of the conveying tunnel, the second buffer chamber is connected to the conveying tunnel. The third conveying mechanism receives the sample from the first conveying mechanism at the outlet of the conveying tunnel.

[0067] According to some embodiments of this application, a first conveying mechanism disposed in the conveying tunnel includes a track and a first conveying arm. The track is disposed along the extension direction of the conveying tunnel, and the first conveying arm is slidably connected to the track. The first conveying arm is used to connect with at least one side of the sample.

[0068] According to some embodiments of this application, the sample preparation module includes a first heat sink, two tracks are provided, two first conveying arms are provided, the two first conveying arms are respectively arranged in the two tracks, the two first conveying arms are respectively engaged with the first heat sink, and the two first conveying arms synchronously drive the first heat sink to move.

[0069] According to some embodiments of this application, the first heat sink includes a mounting portion and a side end portion, the thickness of the side end portion being greater than the thickness of the mounting portion, the mounting portion being recessed relative to the side end portion to form a clearance space, the second conveying mechanism and the third conveying mechanism both including a claw, the claw including at least a pair of spaced-apart teeth, the claw extending into the clearance space, and the mounting portion being located between the two teeth.

[0070] According to some embodiments of this application, the conveying mechanism includes a clamping structure with a groove and a movable part in the groove. The movable part extends to abut against and hold a first heat sink when energized, and retracts and moves away from the first heat sink when de-energized to release the first heat sink.

[0071] According to some embodiments of this application, the conveying module further includes a first buffer chamber, a first end of which is connected to the entrance of the conveying tunnel, and a second end of which is used to feed in a sample;

[0072] The conveying module includes a second conveying arm, which includes a first sub-arm and a second sub-arm. The second sub-arm is sleeved on the outer periphery of the first sub-arm. The first sub-arm is telescopically movable relative to the second sub-arm along its own axial direction. When the second conveying arm delivers the sample into the first buffer chamber, the outer periphery of the second sub-arm abuts against the opening at the second end of the first buffer chamber and is sealed to the opening at the second end. The first sub-arm extends relative to the second sub-arm and can pass through the opening at the first end of the first buffer chamber to enter the conveying tunnel.

[0073] According to some embodiments of this application, the cryo-electron microscopy workstation further includes a storage module for storing samples. The storage module is disposed at the second end of the first buffer chamber and is connected to the second end of the first buffer chamber.

[0074] The method of using a cryo-electron microscope according to the fourth aspect embodiment of this application includes:

[0075] Add the sample to the sample preparation module;

[0076] The sample is fed into the delivery module, the delivery module and the electron microscope tube are evacuated, and the delivery module transports the sample to the observation port.

[0077] Connect the first heat sink to the cold source;

[0078] The second heating element heats the bearing surface to a temperature suitable for the sample, keeping the sample in a liquid state;

[0079] The thinning component reduces the sample on the bearing surface to the target thickness;

[0080] The second heating element stops heating the bearing surface, and the cold source freezes the sample.

[0081] The electron microscope tube is used to observe the sample through the observation port.

[0082] According to some embodiments of this application, the second heating element heats the bearing surface to a temperature suitable for the sample, thereby maintaining the sample in a liquid state, including:

[0083] The second heating element heats the bearing surface to 4°C to 20°C.

[0084] According to some embodiments of this application, the thinning component thins the sample on the bearing surface to a target thickness, including:

[0085] The air supply component delivers air to the bearing surface, causing the sample to thin to the target thickness.

[0086] According to some embodiments of this application, the thinning component delivers air to the bearing surface, blowing the sample to thin it to the target thickness, including:

[0087] The thickness detection module detects the thickness of the sample;

[0088] When the sample is thinned to the target thickness, the control device controls the air supply component to stop supplying air.

[0089] According to some embodiments of this application, the air supply component supplies air to the bearing surface, blowing the sample to thin it to a target thickness, including:

[0090] The air supply component supplies air to the carrier in a direction perpendicular to the bearing surface, and the air guide component disperses the airflow supplied by the air supply component along the air guide surface. The air guide surface is arranged parallel to or coplanar with the bearing surface.

[0091] According to some embodiments of this application, the thinning component thins the sample on the bearing surface to a target thickness, including:

[0092] The first heating element and the second heating element heat the bearing area simultaneously, and the heating temperature of the first heating element is greater than that of the second heating element.

[0093] According to some embodiments of this application, the sample is fed into the transport module, a vacuum is drawn onto the transport module and the electron microscope tube, and the transport module transports the sample to the observation port, including:

[0094] The second transfer mechanism delivers the sample to the first transfer mechanism from the entrance of the transport tunnel;

[0095] The first transmission mechanism moves the sample through the transport tunnel to the observation gap for observation.

[0096] The first transfer mechanism delivers the sample from the exit of the transport tunnel to the third transfer mechanism.

[0097] According to some embodiments of this application, the sample is fed into the transport module, a vacuum is drawn onto the transport module and the electron microscope tube, and the transport module transports the sample to the observation port, including:

[0098] The third transfer mechanism performs the unloading of the previous sample; and / or, the second transfer mechanism performs the loading of the previous sample.

[0099] According to some embodiments of this application, the electron microscope tube observes the sample through the observation port, including:

[0100] A magnetic field is formed between the pole pieces of the objective lens;

[0101] The electron beam is focused by the objective lens pole piece;

[0102] The sample passes through the observation gap between the objective lens pole pieces.

[0103] According to some embodiments of this application, the sample preparation unit includes a carrying mesh, the carrying mesh having a slit region for carrying the sample; the sample passes through the observation gap between the objective lens pole pieces, including:

[0104] The carrier net moves in the direction of extension of the slit area;

[0105] The carrier net is moved to focus the electron beam at different locations within the slit region.

[0106] According to some embodiments of this application, the sample preparation unit includes a carrying net and a first heat sink; a cold source freezes the sample, including:

[0107] The cold source reduces the temperature of the first heat sink through heat transfer, making the temperature of the first heat sink lower than the temperature of the carrier network.

[0108] The first heat sink reduces the temperature of the carrier network through heat transfer.

[0109] The embodiments of this application have at least the following beneficial effects: By encapsulating at least one end of the carrier mesh in a first heat sink, at least three surfaces of the end of the carrier mesh—the side, top, and bottom—are wrapped by the first heat sink, increasing the contact area between the first heat sink and the carrier mesh and improving the cooling effect of the first heat sink on the carrier mesh. The cold source can then conduct heat to the end of the carrier mesh using the first heat sink. Simultaneously, heating the supporting area using a heating element keeps the sample in the supporting area liquid. Therefore, operations such as adding or thinning the sample can be performed during the window period when the sample remains liquid, further improving sample preparation and observation results by controlling the sample thickness. When the heating element stops heating the supporting area, the cold source can quickly freeze the sample in the supporting area.

[0110] Because the portion of the mesh other than the supporting area (e.g., the ends) is connected to the cold source via a first heat sink, the temperature at the ends of the mesh can be maintained at a sufficiently low level. Once the heating element stops heating, the low temperature can be rapidly transferred to the supporting area, achieving ultra-rapid freezing of the sample. Using this configuration, heating is performed on one part of the mesh (the supporting area), while cooling is conducted to another part (the ends). Since the freezing effect of the cold source is already acting on the ends of the mesh, when heating ceases, the freezing effect can be rapidly conducted along the mesh itself to the supporting area. Due to the small size of the mesh, the time required for the freezing effect to transfer from the ends to the supporting area is extremely short, thus shortening the freezing transfer path from the cold source to the supporting area and greatly increasing the freezing rate to 4.7E5 k / s, thereby achieving ultra-rapid freezing of the sample. Under ultra-rapid freezing conditions, it helps to avoid damage to protein sample particles at the gas-liquid interface, ensuring that the sample maintains a good morphology during sample preparation and improving the accuracy of observation.

[0111] By utilizing the encapsulation effect of the first heat sink on the carrier mesh, an interaction force can also be formed between the two. That is, the first heat sink exerts a compressive force on the end of the carrier mesh. This compressive force can improve the heat conduction between the two, thereby quickly transferring the heat of the carrier mesh to the first heat sink, achieving a rapid freezing effect on the carrier mesh and the sample. Attached Figure Description

[0112] The present application will be further illustrated below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments illustrated in the following drawings are exemplary and are only used to explain the present application, and should not be construed as limiting the present application.

[0113] Figure 1 This is a schematic diagram of the sample preparation unit of the cryo-electron microscopy workstation in an embodiment of this application;

[0114] Figure 2 This is a schematic diagram illustrating an example of the sample preparation module of a cryo-electron microscopy workstation according to an embodiment of this application;

[0115] Figure 3 This is a schematic diagram illustrating another example of the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application.

[0116] Figure 4 for Figure 3 A magnified view of part A;

[0117] Figure 5 This is a schematic diagram illustrating another example of the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application.

[0118] Figure 6 This is a schematic diagram illustrating another example of the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application.

[0119] Figure 7 This is a schematic diagram illustrating another example of the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application.

[0120] Figure 8 This is a schematic diagram illustrating another example of the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application.

[0121] Figure 9 This is a schematic diagram of one embodiment of the second heating component in the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application;

[0122] Figure 10 This is a schematic diagram of another embodiment of the second heating component in the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application;

[0123] Figure 11This is a schematic diagram illustrating another example of the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application.

[0124] Figure 12 This is a schematic diagram showing an example layout of the first heating element and the second heating element in the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application;

[0125] Figure 13 This is a schematic diagram showing another example of the layout of the first heating element and the second heating element in the sample preparation module of the cryo-electron microscopy workstation according to an embodiment of this application.

[0126] Figure 14 This is a schematic diagram showing the cross-section of the grid and the first heat sink after assembly in the cryo-electron microscopy workstation of this application embodiment;

[0127] Figure 15 This is a schematic diagram illustrating one embodiment of the heating component of the cryo-electron microscopy workstation according to an embodiment of this application;

[0128] Figure 16 This is a schematic diagram of the cryo-electron microscopy workstation according to an embodiment of this application;

[0129] Figure 17 This is a schematic diagram showing the transfer of samples from the second conveying mechanism to the first conveying mechanism at the entrance of the transport tunnel of the cryo-electron microscopy workstation in an embodiment of this application.

[0130] Figure 18 This is a schematic diagram of the sample passing through the observation gap in the transport tunnel of the cryo-electron microscopy workstation according to an embodiment of this application;

[0131] Figure 19 This is a schematic diagram showing the first conveying mechanism delivering a sample to the third conveying mechanism at the exit of the conveying tunnel of the cryo-electron microscopy workstation according to an embodiment of this application.

[0132] Figure 20 This is a schematic diagram of the structure of the first buffer chamber in the cryo-electron microscopy workstation of this application embodiment;

[0133] Figure 21 This is a schematic diagram of the storage module in the cryo-electron microscopy workstation of this application embodiment;

[0134] Figure 22 This is a cross-sectional view of the objective pole piece in the cryo-electron microscopy workstation of this application embodiment;

[0135] Figure 23 for Figure 22 A magnified view of part B in the middle section.

[0136] Figure label:

[0137] 1000, Sample preparation module; 1001, Body; 1002, Objective lens pole piece; 1003, Observation gap; 1004, First pole piece; 1005, First channel; 1006, Second pole piece; 1007, Second channel;

[0138] 110. Carrier net; 1101. Slit area; 1103. Imaging window; 1104. Inclined surface; 1105. Support structure; 112. Bearing surface; 1121. Central area; 1122. Edge area; 113. First film; 1131. Through structure; 114. Second film; 120. First heat sink; 121. Mounting part; 1211. Observation window; 1212. Mounting groove; 1213. Clearance window; 122. Side end; 130. Clearance space;

[0139] 200. Second heating component; 201. Fixing component;

[0140] 300. Cold source; 301. Storage tank; 302. Cooling component; 3021. Support; 3022. Cooling channel; 3023. Moving contact; 3024. Refrigerant cavity;

[0141] 400. First heating element;

[0142] 600. Air supply component; 610. First air supply section; 620. Second air supply section;

[0143] 700. Airflow guiding component; 701. Second heat sink;

[0144] 2000, Conveying module; 2100, Conveying tunnel; 2110, Observation port; 2111, Entrance; 2112, Exit; 2200, First buffer chamber; 2210, First end; 2220, Second end; 2300, Clamping structure; 2301, Groove; 2302, Moving part; 2310, First conveying mechanism; 2311, First conveying arm; 2312, Track; 2320, Second conveying mechanism; 2321, Claw; 2322, Gear; 2323, First sub-arm; 2324, Second sub-arm; 2330, Third conveying mechanism; 2400, Second buffer chamber; 3000, Storage module. Detailed Implementation

[0145] The embodiments of this application are described in detail below with reference to the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0146] In the description of this application, it should be understood that the terms "center", "middle", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc., indicating the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0147] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0148] In the description of this application, unless otherwise expressly specified and limited, the terms "set up," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0149] In the description of this application, the use of terms such as "as one implementation," "an embodiment," "some examples," "some embodiments," "illustrative embodiment," "example," "specific example," "some examples," etc., indicates that the specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0150] This application provides a cryo-electron microscopy workstation, which includes a sample preparation module 1000. The sample preparation module 1000 can prepare samples that meet observation requirements, and the samples can be stored or immediately transferred to the observation module for observation. The observation module and the sample preparation module 1000 can be set up independently. After the sample is frozen and prepared in the sample preparation module 1000, it can be stored in the sample preparation module 1000 or directly transferred to the observation module for observation. Setting up the sample preparation module 1000 and the observation module independently improves the independence of their operation, reduces mutual interference, and improves the reliability of both the sample preparation module 1000 and the observation module. Optionally, the sample preparation module 1000 and the observation module can be connected via a sample delivery channel or other structure.

[0151] Please see Figures 1 to 7 The sample preparation module 1000 includes a first heat sink 120, a second heating element 200, and a cold source 300. In some embodiments, the sample preparation module 1000 also includes a carrier mesh 110. The carrier mesh 110 is connected to the first heat sink 120, which conducts heat to the carrier mesh 110. The carrier mesh 110 has a bearing surface 112 for bearing the sample. The second heating element 200 is disposed in or connected to the carrier mesh 110 and is used to maintain the sample in a liquid state when the sample is thinned; specifically, the sample can be maintained at 4℃-20℃. The cold source 300 is connected to the first heat sink 120 and conducts heat to the first heat sink 120. The cold source 300 can freeze the bearing surface 112 when the second heating element 200 stops heating.

[0152] The cold source 300, through the first heat sink 120 and the refrigeration carrier 110, can keep the first heat sink 120 at a low temperature. Specifically, the cold source 300 can quickly conduct away the heat from the first heat sink 120 and the sample placed on the bearing surface 112, thereby keeping the sample and the first heat sink 120 at a low temperature.

[0153] Optionally, see Figure 15 The cold source 300 can be a substance such as liquid nitrogen or liquid ethane, which can be stored in storage tank 301. No specific limitation is made here.

[0154] In some embodiments, the sample preparation module 1000 is provided with a first film 113, the surface of which is formed as a bearing surface 112, and a through structure 1131 extending through both sides of the first film 113. By providing the first film 113, it can be used to bear the sample, allowing the sample preparation module 1000 to conduct heat to the sample through the first film 113; on the other hand, the through structure 1131 on the first film 113 allows the spaces on both sides of the first film 113 to be interconnected, thus providing conditions for air supply to both sides of the first film 113. For example, the first film 113 is usually set approximately horizontally, with the upper surface of the first film 113 used to hold the sample. Since the through structure 1131 is provided on the first film 113, if air is supplied from the lower surface of the first film 113, the airflow can also penetrate to the upper surface of the first film 113 through the through structure 1131.

[0155] Optionally, the through structure 1131 can be a through hole, through groove, etc.

[0156] In some embodiments, please refer to Figure 9 The first film 113 is disposed on the carrier 110, and the second heating component 200 is disposed in the carrier 110. The second heating component can directly heat the carrier 110, and the carrier 110 does not need to rely on other external devices for heating, making the structure of the sample preparation module 1000 simpler.

[0157] Optionally, the second heating element 200 can be disposed on the bottom surface of the carrier mesh 110. This avoids occupying or interfering with the space of the bearing area of ​​the carrier mesh 110, making the structure of the carrier mesh 110 itself more compact.

[0158] In some embodiments, please refer to Figure 7 and Figure 10 The sample preparation module 1000 includes a fixing member 201, and a second heating member 200 is disposed on the fixing member 201. When the first heat sink 120 is installed on the fixing member 201, the second heating member 200 contacts the bottom surface of the first heat sink 120, facilitating the heating of the bearing surface 112 of the carrier mesh 110 by the second heating member 200. The second heating member 200 is separately disposed on the fixing member 201 other than the first heat sink 120, which helps to simplify the structure and reduce the cost of the first heat sink 120 itself. On the other hand, the fixing member 201 can also provide support and stability for the first heat sink 120, improving the stability of the first heat sink 120 and the carrier mesh 110 during the sample preparation process.

[0159] In some cases, when a first film 113 with a through structure 1131 is used to carry the sample, and a first heat sink 120 is supported by a fastener 201, a portion of the sample may flow to the lower surface of the first film 113 due to the presence of the through structure. When the sample is frozen, there is a risk of adhesion between the sample and the surface of the fastener 201. In this case, removing the first heat sink 120 or the carrier net 110 from the fastener 201 may result in sample damage.

[0160] Therefore, to resolve this issue, please refer to [link / reference]. Figure 6 In some embodiments, the sample preparation module 1000 is provided with a light-transmitting second film 114, the surface of which is formed as a bearing surface 112, and the second film 114 is continuously disposed. Because the second film 114 is continuously disposed, the sample will not flow to the lower surface of the second film 114. Thus, after the sample is frozen, the first heat sink 120 and the fixing member 201 can be easily separated, avoiding the problem of sample adhesion.

[0161] It should be noted that even if the sample preparation module 1000 uses the first film 113, the sample is not necessarily bound to the surface of the fixture 201. Whether the sample adheres to the surface of the fixture 201 depends on one or more factors, such as the adhesiveness of the sample itself, the pore size of the through-structure 1131 of the first film 113, and the freezing rate of the sample. This application provides two examples of the sample preparation module 1000 being equipped with either the first film 113 or the second film 114, which do not constitute a limitation on the specific implementation of this application. The implementation method using the second film 114 will be further described below.

[0162] In some embodiments, a second film 114 is disposed on a carrier mesh 110, and a second heating element 200 is disposed in the carrier mesh 110. The second heating element 200 can directly heat the carrier mesh 110, so the carrier mesh 110 does not need to rely on other external devices for heating, making the structure of the sample preparation module 1000 simpler.

[0163] Optionally, the second heating element 200 can be disposed on the bottom surface of the carrier mesh 110. This avoids occupying or interfering with the space of the carrier mesh 110's bearing area, making the structure of the carrier mesh 110 itself more compact. In some embodiments, the sample preparation module 1000 includes a fixing member 201, and the second heating element 200 is disposed on the fixing member 201. When the first heat sink 120 is mounted on the fixing member 201, the second heating element 200 contacts the bottom surface of the first heat sink 120. The second heating element 200 is disposed separately on the fixing member 201 other than the first heat sink 120, which helps to simplify the structure and reduce the cost of the first heat sink 120 itself. On the other hand, the fixing member 201 can also provide support and stability for the first heat sink 120, improving the stability of the first heat sink 120 and the carrier mesh 110 during the sample preparation process.

[0164] In some embodiments, the sample preparation module 1000 includes a first heat sink 120, at least one end of the carrier mesh 110 is encapsulated in the first heat sink 120, and the bearing surface 112 is exposed from the first heat sink 120. The first heat sink 120 is used to conduct heat to the end of the heat sink, and a cold source 300 is connected to the first heat sink 120 and is used to conduct heat to the first heat sink 120. By encapsulating at least one end of the carrier mesh 110 in the first heat sink 120, at least three surfaces of the end of the carrier mesh 110—the side surface, the top surface, and the bottom surface—can be wrapped by the first heat sink 120, increasing the contact area between the first heat sink 120 and the carrier mesh 110, improving the cooling effect of the first heat sink 120 on the carrier mesh 110, and enabling the cold source 300 to conduct heat to the end of the carrier mesh 110 using the first heat sink 120. This arrangement avoids direct contact between the carrier mesh 110 and the cold source 300, thus allowing the carrier mesh 110 to be made smaller and thinner. However, the carrier mesh 110 is somewhat fragile. Therefore, the first heat sink 120 is used to encapsulate the carrier mesh 110. This protects the carrier mesh 110 from bending or damage during sample preparation. Furthermore, the thermal conductivity of the first heat sink 120 is used to conduct heat to the carrier mesh 110, achieving a freezing effect on both the carrier mesh 110 and the sample.

[0165] Furthermore, by utilizing the encapsulation effect of the first heat sink 120 on the carrier mesh 110, an interaction force can be formed between them. That is, the first heat sink 120 exerts a compressive force on the end of the carrier mesh 110. This compressive force can improve the thermal conductivity between them, thereby rapidly transferring the heat of the carrier mesh 110 to the first heat sink 120, achieving a rapid freezing effect on both the carrier mesh 110 and the sample. The table below shows the cooling rate of the carrier mesh 110 from 298.15K to below 110K under different pressure conditions, taking a typical processed surface roughness as an example. The second column in the table represents the temperature of the carrier mesh 110 at the end of the cooling process. As can be seen from the table, when the compressive force of the first heat sink 120 on the carrier mesh 110 is less than 1250Pa, the temperature of the carrier mesh 110 cannot be reduced to below 110K. When the pressure is greater than 1500Pa, the cooling rate of the carrier mesh 110 increases with increasing pressure. It is evident that by adopting the structural form of the sample preparation module 1000 provided in this application, a faster cooling effect can be achieved, thereby realizing the purpose of rapid sample freezing.

[0166] Pressure (Pa) Temperature (K) Freezing speed 1500 110 10500 1750 106.3 51275 2000 103.1 93500 2250 100.4 124000 2500 98.2 147500 2750 96.5 157500 3000 95.1 164000 3250 93.7 169750 3500 92.5 175000

[0167] In some embodiments, see Figure 14 The first heat sink 120 includes a mounting portion 121 and a side end portion 122 connected in sequence. The mounting portion 121 is used to encapsulate the carrier mesh 110 and has an observation window 1211 through which the bearing surface 112 is exposed. The side end portion 122 is used to connect to a cold source. By using the side end portion 122 and the mounting portion 121, it is possible to satisfy the requirement that the end of the carrier mesh 110 be indirectly connected to the cold source through the side end portion 122, and also to expose the bearing area of ​​the carrier mesh 110 through the observation window 1211 of the mounting portion 121, which facilitates operations such as adding samples, freezing samples, and observing samples in the bearing area. The bearing area here includes the bearing surface 112.

[0168] Furthermore, the mounting part 121 is provided with a mounting groove 1212, in which the mesh 110 is encapsulated. An observation window 1211 communicates with the mounting groove 1212. The bottom surface of the mounting part 121 is also provided with a clearance window 1213 communicating with the mounting groove 1212. The clearance window 1213 is used for the fixing member 201, which is provided with the second heating component 200, to extend into the mounting groove 1212. By providing the clearance window 1213, the distance between the fixing member 201 and the mesh 110 can be shortened, thereby improving the heating effect of the second heating component 200 on the mesh 110.

[0169] In some embodiments, the thickness of the side end portion 122 is greater than the thickness of the mounting portion 121. In this way, the side end portion 122 can have a larger volume than the mounting portion 121, thereby having better heat absorption capacity and better heat conduction effect. When the side end portion 122 is connected to a cold source and lowered to a sufficiently low temperature, it helps to quickly conduct away the heat on the carrier 110, realize the rapid freezing of the carrier 110, and improve the freezing efficiency.

[0170] In some embodiments, the thickness of the side end portion 122 and the mounting portion 121 gradually transitions. Since the thicknesses of the side end portion 122 and the mounting portion 121 are different, by providing a gradual thickness transition between the two, the stress between the side end portion 122 and the mounting portion 121 can be reduced, thereby reducing the risk of breakage at the junction of the end portion and the mounting portion 121.

[0171] In some embodiments, the first heat sink 120 has two side ends 122, which are symmetrically arranged about the mounting portion 121. In this way, both ends of the carrier net 110 can be connected to the first heat sink 120.

[0172] As an alternative implementation, only one side end 122 may be provided, and the side end 122 may be provided at one end of the mounting portion 121. The side end 122 is installed in the second heat sink 701, and the mounting portion 121 is suspended relative to the second heat sink 701. The side end 122 is provided on one side, which can avoid the problem of easy breakage caused by the asynchronous installation of the two side ends 122 in the second heat sink 701, and improve the reliability of the first heat sink 120.

[0173] In some embodiments, see Figure 7 The sample preparation module 1000 also includes a second heat sink 701, which is connected to the outer periphery of the first heat sink 120. A cold source 300 is connected to the second heat sink 701, and the cold source 300 and the second heat sink 701 are in detachable contact. The cold source 300 conducts cooling to the first heat sink 120 through the second heat sink 701. By setting the second heat sink 701 and the cold source 300 to be in detachable contact, when the cold source 300 is in contact with the second heat sink 701, the freezing effect of the cold source 300 can be conducted to the first heat sink 120 through the second heat sink 701, and then to the carrier mesh 110. When the cold source 300 is separated from the second heat sink 701, the transmission of the freezing effect can be cut off. At this time, the frozen sample can be unloaded, and the carrier mesh 110 carrying the sample to be frozen can be connected to the second heat sink 701. This configuration allows for controllable cooling timing of the cold source 300, enabling the connection or separation of the cold source 300 from the second heat sink 701 as needed.

[0174] In some embodiments, the sample preparation module 1000 further includes a cooling conductive assembly 302, which includes a support 3021, a movable contact 3023, and a refrigerant cavity 3024. A cooling conductive channel 3022 is formed in the support 3021. A second heat sink 701 is installed at one end of the cooling conductive channel 3022, and the other end is connected to the refrigerant cavity 3024. The refrigerant cavity 3024 is used to introduce the cold source 300 medium. The movable contact 3023 is located between the second heat sink 701 and the refrigerant cavity 3024. The movable contact 3023 is movable in the cooling conductive channel 3022 to connect or disconnect the second heat sink 701 from the cold source 300 medium. By utilizing the movement of the movable contact 3023, contact and separation between the second heat sink 701 and the cold source 300 medium can be achieved, thereby controlling the cooling effect of the cold source 300 medium on the carrier network 110 between connected and disconnected states. During the switching process, the second heat sink 701 can remain stationary, improving the stability of the sample preparation module 1000.

[0175] In some embodiments, one end of the movable contact 3023 is fixedly connected to the second heat sink 701, and the other end is radially retractable along the cooling channel 3022. The other end of the movable contact 3023 can extend to contact the sidewall of the refrigerant cavity 3024, or retract to separate from the sidewall of the refrigerant cavity 3024. Utilizing the retraction and extension of the movable contact 3023, the refrigerant cavity 3024 can be connected to the second heat sink 701 when the movable contact 3023 extends, and the connection can be disconnected when the movable contact 3023 retracts, thereby achieving controllable cooling of the cold source 300. As an alternative implementation, one end of the movable contact 3023 can be fixedly connected to the sidewall of the refrigerant cavity 3024, while the other end can be detachably connected to the second heat sink 701 through the retraction and extension of the movable contact 3023 itself. As an alternative implementation, both ends of the movable contact 3023 can be configured to be movable and retractable, so as to contact and connect or separate from the sidewalls of the second heat sink 701 and the refrigerant cavity 3024, respectively.

[0176] In some embodiments, the support 3021 encloses at least a portion of the second heat sink 701. On one hand, the enclosing effect of the support 3021 can fix the second heat sink 701 in the support 3021, improving the stability of the connection between the second heat sink 701 and the support 3021 and preventing the second heat sink 701 from detaching from the support 3021. On the other hand, the cold source 300 medium can also conduct cold to the second heat sink 701 using the support 3021. Therefore, by housing at least a portion of the second heat sink 701 in the support 3021, the support 3021 can conduct cold to the second heat sink 701, improving the freezing effect of the second heat sink 701.

[0177] Optionally, the second heat sink 701 can be partially enclosed in the support 3021, with the remainder exposed outside the support 3021. Alternatively, the entire second heat sink 701 can be enclosed in the support 3021.

[0178] In some embodiments, the second heat sink 701 is provided with an assembly slot for mounting the first heat sink 120. The opening edge of the assembly slot is flush with the opening edge of the cooling channel 3022, and the support 3021 encloses the second heat sink 701. This arrangement allows the support 3021 to completely enclose the second heat sink 701, increasing the contact area between the second heat sink 701 and the support 3021, thereby improving the cooling effect. It also helps to make the structure of the second heat sink 701 after installation on the support 3021 more compact. Furthermore, the assembly slot allows the second heat sink 701 to include the end of the first heat sink 120, increasing the contact area between the two heat sinks, thereby improving the cooling effect of both. Therefore, the stronger the enclosure effect of the support 3021 on the second heat sink 701, and the stronger the enclosure effect of the second heat sink 701 on the first heat sink 120, the better the cooling effect. By setting the above two aspects, the cooling effect between the cold source 300 and the second heat sink 701, and between the second heat sink 701 and the first heat sink 120, can be improved, thereby improving the freezing efficiency of the cold source 300 on the first heat sink 120.

[0179] In some embodiments, the second heat sink 701 is provided with an assembly slot for mounting the first heat sink 120, with the end of the first heat sink 120 accommodated in the assembly slot. The volume of the second heat sink 701 is larger than the volume of the side end 122 of the first heat sink 120. By setting the second heat sink 701 to have a larger volume than the side end 122 of the first heat sink 120, the second heat sink 701 can store more cooling capacity and has a more stable temperature change. When the first heat sink 120 is installed in the second heat sink 701, the second heat sink 701 can quickly conduct away the heat of the first heat sink 120, achieving rapid cooling of the first heat sink 120.

[0180] In some embodiments, the second heat sink 701 is symmetrically arranged about the mesh 110. In this way, both ends of the mesh 110 can be connected to the first heat sink 120, and both ends of the first heat sink 120 can be connected to the second heat sink 701, thereby improving the cooling effect on the mesh 110 and improving the efficiency of cooling and freezing the mesh 110.

[0181] It is understandable that when the second heat sink 701 is symmetrically arranged with respect to the carrier net 110, the support 3021 is also set to two, and the two second heat sinks 701 are respectively arranged in the two supports 3021, so as to achieve the effect of cooling and freezing at both ends of the carrier net 110 simultaneously.

[0182] Optionally, the first heat sink 120, the second heat sink 701, the movable contact 3023, and the refrigerant cavity 3024 can all be made of metal, which helps to improve thermal conductivity.

[0183] In some embodiments, the thinning module includes a thinning component for thinning the sample on the bearing surface 112. Exemplarily, the thinning component may include an air supply component 600.

[0184] When thinning a sample, it needs to remain in a liquid state to prevent freezing and condensation, which would hinder the thinning process. Therefore, the continuous heating of the sample by the second heating element 200 prevents freezing and condensation. While the sample remains liquid, the air supply element 600 blows across the sample surface, spreading the liquid outwards and achieving liquid thinning in the blowing area. Once the sample reaches the target thickness, the heating of the second heating element 200 is stopped. At this point, the cooling effect of the cold source 300 is rapidly transferred from the first heat sink 120 to the supporting surface 112, quickly freezing and condensing the sample to maintain the target thickness. This prevents the sample from returning to its original thickness due to surface tension after the air supply element 600 stops blowing. The second heating element 200 typically only operates on the localized area of ​​the supporting surface 112 containing the sample; therefore, the heating effect generally does not affect the low-temperature state of other areas. By utilizing the combined action of the second heating element 200, the air supply element 600, and the cold source 300, sample thinning can be achieved, enabling controllable sample thickness preparation. Furthermore, the sample can be frozen promptly after thinning to maintain it in a suitable morphology. In addition, the freezing, heating, and thinning operations are all performed in the same space; therefore, these three operations can be performed simultaneously, or the time difference between them can be controlled within a very short range. Compared to schemes where sample thinning and freezing are performed in two different spaces, the sample preparation module provided in this application can rapidly freeze the sample in situ after it has been thinned to the target thickness without moving it, ensuring a good and precise thinning effect. By controlling the sample thickness within a suitable range, it is ensured that the electron beam of the electron microscope can penetrate the sample, thereby obtaining sufficient resolution.

[0185] Understandably, the sample thickness can be controlled by adjusting parameters such as the blowing power and duration of the air supply component 600. Before the sample reaches the target thickness, the second heating component 200 continuously heats the sample to prevent freezing. Heating stops only when the sample reaches the target thickness. At this point, due to the disappearance of heating, the small amount of heat remaining in the local area where the sample is located can be quickly conducted and dissipated to the cold source 300 through the first heat sink 120. Since the first heat sink 120 itself has a low temperature, the freezing and cooling effect of the cold source 300 can be transferred extremely quickly from the first heat sink 120 to the bearing surface 112. This arrangement shortens the freezing action transmission path and increases the rate. Furthermore, because the carrier net 110 itself has a small size, the time required for the freezing action to be transferred from the end of the carrier net 110 to the bearing surface 112 is extremely short. Therefore, the freezing transmission path from the cold source 300 to the bearing surface 112 is shortened, greatly increasing the freezing speed to 4.7E5 k / s, thereby achieving ultra-fast freezing of the sample. Under ultra-fast freezing conditions, it helps to avoid damage to protein sample particles at the gas-liquid interface, ensuring that the sample can maintain a good morphology during sample preparation and improving the accuracy of observation.

[0186] In some embodiments, please refer to Figure 5 The sample preparation module 1000 includes a thinning component, which comprises a first heating component 400 and / or an air supply component 600. The air supply component 600 includes a first air supply section 610 and a second air supply section 620, which are symmetrically arranged on both sides of the first film 113. The first air supply section 610 and the second air supply section 620 are used to simultaneously supply air to both sides of the first film 113. By simultaneously supplying air to both sides of the first film 113, the airflow effect can be improved, the sample thinning speed can be increased, and the thinning operation time can be shortened, thereby improving the sample preparation efficiency.

[0187] In some embodiments, the air supply pressure of the first air supply section 610 and the second air supply section 620 is the same. By controlling the first air supply section 610 and the second air supply section 620 to use the same air supply pressure, the air pressure difference between the two surfaces of the first film 113 can be reduced or even eliminated, thereby reducing the risk of the first film 113 shaking or breaking due to pressure difference and improving the stability and reliability of the sample during the thinning process.

[0188] In some embodiments, the air supply component 600 is used to provide a plasma gas flow. The plasma gas flow can protect the sample, preventing damage or changes in quality or morphology to the sample during the blowing process by the air supply component 600, thereby improving the quality of sample preparation and the accuracy of sample observation.

[0189] Optionally, the polarity of the plasma gas flow is the same as that of the sample. Since the sample surface carries an electric charge (taking a positive charge as an example), using a plasma gas flow of the same polarity to blow the sample can utilize the principle of like charges repelling to protect the sample from damage and improve the sample preparation effect.

[0190] In some embodiments, please refer to Figure 8 The sample preparation module 1000 also includes a flow guiding component 700. An air supply component 600 supplies air to the carrier mesh 110 in a direction perpendicular to the bearing surface 112. The flow guiding component 700 is disposed on the outer periphery of the carrier mesh 110 and disperses the airflow supplied by the air supply component 600 along the guiding surface, which is parallel or coplanar with the bearing surface 112. Utilizing the dispersion effect of the flow guiding component 700 on the airflow, and with the plasma airflow blowing towards the sample in a direction perpendicular to the bearing surface 112, the plasma airflow can form a vortex state. That is, the plasma airflow can be better blown out of the air supply component 600 and diffuse rapidly. The dynamic action of the airflow helps the sample droplets diffuse and thin from the center outwards, thereby achieving the effect of sample thinning.

[0191] In some embodiments, the airflow guiding component 700 includes a vacuum device for absorbing the airflow delivered by the air supply component 600. The negative pressure of the vacuum device enables rapid absorption of the airflow, thus guiding its direction.

[0192] In some embodiments, see Figure 7 The first heat sink 120 includes a mounting portion 121 and a side end portion 122 located at the end of the mounting portion 121. Along the thickness direction of the first heat sink 120, the mounting portion 121 is recessed relative to the side end portion 122 to form a clearance space 130. The air supply component 600 is movable to approach or move away from the carrier mesh 110. When the air supply component 600 approaches the carrier mesh 110, the air supply component 600 extends into the clearance space 130. By utilizing the clearance space 130 formed by the recess of the mounting portion 121 relative to the side end portion 122, the air supply component 600 can enter the clearance space 130, thereby further shortening the distance between the air supply component 600 and the bearing surface 112, allowing the air supply component 600 to get closer to the sample and improving the effect of air blowing and thinning the sample.

[0193] Optionally, the clearance space 130 is symmetrically arranged about the thickness of the carrier net 110. In this way, when the air supply component 600 is provided with the first air supply part 610 and the second air supply part 620, the two air supply parts can enter the two clearance spaces 130 respectively, so as to achieve the effect of air supply to both sides of the carrier net 110.

[0194] In the process of thinning the sample, to further improve the accuracy of thinning, in some embodiments, the sample preparation module 1000 also includes a thickness detection module (not shown) and a control device. The thickness detection module and the air supply component 600 are both electrically connected to the control device. The thickness detection module is used to detect the thickness of the sample, and the control device is used to control the air supply component 600 to stop supplying air when the thickness detection module detects that the sample has reached the target thickness. By using the thickness detection module to detect the degree of sample thinning, the control device can control the air supply component 600 to continue or stop supplying air, achieving controllable thinning of the sample thickness.

[0195] Alternatively, the thickness of the sample can be measured using optical principles or electrical principles, which will be explained below.

[0196] In some embodiments, the thickness detection module includes a light generating structure and a light receiving structure. The light generating structure is disposed facing the bearing surface 112, and the light receiving structure is disposed in the optical path of the reflected light from the sample. The light generating structure illuminates the sample with detection light, which the sample absorbs, reflects, or diffracts. The light receiving structure receives and analyzes the detection light, thereby calculating the thickness of the sample. The operation of using optical principles to detect sample thickness is already described in related technologies for those skilled in the art and will not be described in detail here.

[0197] In some embodiments, the thickness detection module includes a detection electrode disposed on the bearing surface 112 and used to contact the sample. By contacting the detection electrode with the sample, the thickness of the sample can be further analyzed and calculated using signals such as current, resistance, and capacitance detected by the electrode. The operation of using electrical principles to detect sample thickness is already described in related technologies for those skilled in the art and will not be described in detail here.

[0198] Optionally, the methods for detecting sample thickness using optical and electrical principles can be used separately or in combination; no specific limitations are made here.

[0199] In some embodiments, the sample can also be thinned using a temperature difference. See also Figure 11-12In this embodiment, the thinning component may include a first heating component 400, and the bearing surface 112 includes a central region 1121 for bearing the sample and an edge region 1122 on the outer periphery of the central region 1121. A second heating component 200 is provided corresponding to the central region 1121, and the first heating component 400 is provided corresponding to the edge region 1122. The heating temperature of the first heating component 400 is greater than the heating temperature of the second heating component 200, so that the temperature of the edge region 1122 is higher than the temperature of the central region 1121. The sample can diffuse from the central region 1121 to the edge region 1122 and be thinned under the action of the temperature difference between the central region 1121 and the edge region 1122.

[0200] In this embodiment, by setting a central region 1121 and an edge region 1122 on the bearing surface 112, and creating a temperature difference between the central region 1121 and the edge region 1122, i.e., heating the edge region 1122 using the first heating element 400, the temperature of the edge region 1122 is higher than that of the central region 1121. Due to this temperature gradient, the sample at the outer periphery evaporates faster than the sample at the center, thus creating a diffusion force in the liquid from the central region 1121 to the edge region 1122. Under the action of liquid tension, the sample can flow from the low-temperature region to the high-temperature region, thereby causing the sample droplets to diffuse from the center to the surrounding areas, achieving the effect of sample thinning. Throughout the sample thinning process, the second heating element 200 can also heat and keep the sample warm, thus keeping the sample in a liquid state and ensuring that the sample can be thinned by utilizing the fluidity of the liquid. When the second heating element 200 stops heating, the cold source 300 can rapidly freeze the sample.

[0201] In some embodiments, please refer to Figure 12 The first heating element 400 is arranged around the second heating element 200. In this way, the temperature of the edge region 1122 is higher than that of the center region 1121, ensuring that the sample can spread out in all directions.

[0202] In some embodiments, please refer to Figure 13 The thinning component includes at least two spaced-apart first heating components 400, with a second heating component 200 located between the first heating components 400. In this example, the first heating components 400 and the second heating components 200 are arranged discontinuously. For example, the first heating components 400 and the second heating components 200 can be heating chip patches, and the patch arrangement and distribution can be flexibly set according to actual needs to meet the requirements for sample thinning.

[0203] In some embodiments, the first heating element 400 and the second heating element 200 are integrally formed. Both the first heating element 400 and the second heating element 200 include heating units, with the number of heating units in the first heating element 400 being greater than the number in the second heating element 200. For example, the heating unit may be a heating wire. Since the first heating element 400 requires a higher heating temperature, the density of the heating wire can be set higher in the first heating element 400, while the density of the heating wire is reduced in the second heating element 200, where the temperature is lower, thereby meeting the temperature difference requirements between the central region 1121 and the edge region 1122.

[0204] In some embodiments, the thinning component may be provided with a first heating component 400 and an air supply component 600 at the same time. While the sample liquid is thinned by blowing air, the temperature difference between the first heating component 400 and the second heating component 200 is used to achieve the thinning of the sample liquid, thereby obtaining a better sample thinning effect.

[0205] Reference Figures 16 to 21 The cryo-electron microscopy workstation includes a transport module 2000, a first vacuum system (not shown), and a second vacuum system (not shown). The microscope tube of the cryo-electron microscopy workstation includes a tube body 1001, within which a pair of objective pole pieces 1002 are arranged, forming an observation gap 1003 between the objective pole pieces 1002 for sample passage. The transport module 2000 includes a transport tunnel 2100 for sample passage, which passes through the observation gap 1003. The space within the transport tunnel 2100 is isolated from the tube body 1001. A through observation port 2110 is provided on the side wall of the transport tunnel 2100, located on the line connecting the two objective pole pieces 1002. The first vacuum system provides a vacuum environment for the tube body 1001, and the second vacuum system provides a vacuum environment for the transport tunnel 2100.

[0206] By isolating the space within the transport tunnel 2100 from the cylinder 1001, the two spaces are formed as two independent spaces. Two different vacuum systems provide vacuum environments for these two spaces respectively, thus improving the vacuuming efficiency and ensuring good vacuum conditions in both spaces. For example, during sample loading, unloading, and observation within the transport tunnel 2100, even if a small amount of air enters and disrupts the vacuum, the independent arrangement of the transport tunnel 2100 and cylinder 1001 protects the vacuum condition within the cylinder 1001, preventing the transport tunnel 2100 from affecting its vacuum level. Simultaneously, the space within the transport tunnel 2100 can be made very small, just enough to allow sample passage. This helps improve the vacuuming efficiency of the transport tunnel 2100, reduces the difficulty of maintaining a vacuum, and shortens the time required to reach the target vacuum level. This setup can shorten the waiting time for vacuuming the transport tunnel 2100 or cylinder 1001 during sample loading and unloading, thereby improving the efficiency of the cryo-electron microscopy workstation.

[0207] Understandably, the transport tunnel 2100 is equipped with an observation port 2110, which allows the electron beam of the objective lens pole piece 1002 to pass through, ensuring that the sample can be observed in the observation gap 1003. It is evident that the observation port 2110 is the only connection between the transport tunnel 2100 and the cylinder 1001. Therefore, by reducing the size of the observation port 2110, while ensuring sufficient field of view for the sample, the space within the transport tunnel 2100 can be isolated from the space within the cylinder 1001 to the greatest extent possible, improving the independence of these two spaces and reducing the mutual influence of vacuum levels between them.

[0208] In some embodiments, the transport module 2000 further includes a first buffer chamber 2200, which is connected to the inlet 2111 of the transport tunnel 2100. The first buffer chamber 2200 and the transport tunnel 2100 are selectively connected. When the first buffer chamber 2200 is evacuated, it is isolated from the transport tunnel 2100. When the vacuum level of the first buffer chamber 2200 reaches the vacuum level of the transport tunnel 2100, the first buffer chamber 2200 is connected to the transport tunnel 2100, and the sample can enter the transport tunnel 2100 from the first buffer chamber 2200. By setting the first buffer chamber 2200 and the transport channel to be selectively connected, the first buffer chamber 2200 can be evacuated in advance when loading the sample into the transport tunnel 2100. When the first buffer chamber 2200 reaches a sufficient vacuum level, it is then connected to the transport tunnel 2100 to send the sample into the transport tunnel 2100, thus avoiding damage to the vacuum environment in the transport tunnel 2100. The first buffer chamber 2200 can be set to a smaller space. Compared with evacuating the entire transport tunnel 2100, evacuating only the first buffer chamber 2200 can further shorten the vacuum time, reduce the waiting time during the vacuuming process, and thus improve the efficiency of sample loading.

[0209] In some embodiments, a first valve is provided at the connection between the first buffer chamber 2200 and the conveying tunnel 2100. The first valve can connect or disconnect the first buffer chamber 2200 and the conveying tunnel 2100. By using the opening and closing action of the valve, the connection and isolation states between the first buffer chamber 2200 and the conveying tunnel 2100 can be switched.

[0210] In some embodiments, the two ends of the transport tunnel 2100 are respectively formed as an inlet 2111 and an outlet 2112. The transport module 2000 is provided with at least two conveying mechanisms. One conveying mechanism is movably disposed in the transport tunnel 2100 for moving the sample to the observation port 2110, and the other conveying mechanism is used to feed the sample into the inlet 2111 of the transport tunnel 2100 or output it from the outlet 2112 of the transport tunnel 2100. The sample can be transferred between the two conveying mechanisms. On the one hand, the sample enters from one end inlet 2111 of the transport tunnel 2100 and is sent out from the other end outlet 2112. In this way, the sample can form a unidirectional flow in the transport tunnel 2100, which helps to realize continuous sample loading. When the first sample is sent into the transport tunnel 2100 from the inlet 2111 and moved to the observation port 2110 for observation, or when it is sent out of the transport tunnel 2100 from the outlet 2112 after observation, the second sample can continue to be sent into the transport tunnel 2100 from the inlet 2111. The second sample and the first sample enter the transport tunnel 2100 in sequence and move in the same direction. Therefore, the two samples will not interfere with each other. On the other hand, by using at least two conveying mechanisms to transport the samples, while one conveying mechanism is carrying the sample through the observation port 2110 for observation, at least another conveying mechanism can carry the sample for loading or unloading operations. That is, in the three stages of loading, observation and unloading, at least two stages can be carried out simultaneously, thereby reducing the sample waiting time. Compared to the method of sending multiple samples into the cylinder 1001 at once for observation, the cryo-electron microscopy workstation provided in this application allows multiple samples to enter one by one from the inlet 2111 at one end of the transport tunnel 2100 and exit from the outlet 2112 at the other end. Furthermore, while sending out the previous sample, the next sample can continue to be sent in from the inlet 2111. Therefore, it saves the time waiting for sample loading, reduces the waste of sample loading time, and improves the efficiency of sample loading.

[0211] In some embodiments, the transport module further includes a first buffer chamber 2200 and a second buffer chamber 2400. The first buffer chamber 2200 is connected to the inlet 2111 of the transport tunnel 2100, and the second buffer chamber 2400 is connected to the outlet 2112 of the transport tunnel 2100. The transport module includes three transport mechanisms: a first transport mechanism 2310, a second transport mechanism 2320, and a third transport mechanism 2330. The first transport mechanism 2310 is movably disposed in the transport tunnel 2100. The second transport mechanism 2320 is disposed in the first buffer chamber 2200 and is used to transfer samples to the first transport mechanism 2310. The third transport mechanism 2330 is disposed in the second buffer chamber 2400 and is used to receive samples from the first transport mechanism 2310. Using the three transport mechanisms, the sample loading, observation, and unloading stages can be controlled independently, improving the reliability and efficiency of sample delivery between different stages of the cryo-electron microscopy workstation. For example, the transfer mechanism located at inlet 2111 transfers the sample to the transfer mechanism located in transport tunnel 2100, thus completing the sample loading operation. The transfer mechanism in transport tunnel 2100 carries the sample through observation port 2110, enabling the sample observation operation. The transfer mechanism in transport tunnel 2100 transfers the sample to the transfer mechanism located at outlet 2112, completing the sample loading operation. It can be seen that in the three stages of sample loading, observation, and unloading, one or two transfer mechanisms are involved. Simultaneously, there will always be two or one remaining transfer mechanisms that do not participate in the sample delivery process. These remaining transfer mechanisms can then operate on the previous sample (e.g., sample retrieval) or on the next sample (e.g., retrieving a sample from the sample preparation module or storage module 3000), thereby further shortening the sample waiting time and improving the sample transfer efficiency in the cryo-electron microscopy workstation. By setting buffer chambers at the inlet 2111 and outlet 2112 of transport tunnel 2100, the sample loading and unloading processes can be set independently, avoiding mutual interference between the two stages.

[0212] Optionally, the first buffer chamber 2200 and the second buffer chamber 2400 can both be located outside the cylinder 1001 (e.g., Figure 16 As shown, this helps reduce the space occupied in the cylinder 1001. Of course, in other examples, it can also be set inside the cylinder 1001, or one buffer chamber can be set inside the cylinder 1001 and the other outside the cylinder 1001. No specific limitation is made here.

[0213] In some embodiments, when at least one of the second conveying mechanism 2320 or the first conveying mechanism 2310 is operating, the third conveying mechanism 2330 unloads the sample; when at least one of the third conveying mechanism 2330 or the first conveying mechanism 2310 is operating, the second conveying mechanism 2320 loads the sample. This arrangement increases the overlap time of the three conveying mechanisms operating simultaneously, reduces the waiting time between them, thereby reducing wasted time and improving the efficiency of the cryo-electron microscopy workstation in observing samples.

[0214] In some embodiments, the first buffer chamber 2200 and the transport tunnel 2100 may be selectively connected. When the first buffer chamber 2200 is evacuated, the first buffer chamber 2200 and the transport tunnel 2100 are isolated from each other. When the vacuum level of the first buffer chamber 2200 reaches the vacuum level of the transport tunnel 2100, the first buffer chamber 2200 and the transport tunnel 2100 are connected. The second conveying mechanism 2320 transfers the sample from the inlet 2111 of the transport tunnel 2100 to the first conveying mechanism 2310. The second buffer chamber 2400 and the transport tunnel 2100 may be selectively connected. When the second buffer chamber 2400 is evacuated, the second buffer chamber 2400 and the transport tunnel 2100 are isolated from each other. When the vacuum level of the second buffer chamber 2400 reaches the vacuum level of the transport tunnel 2100, the second buffer chamber 2400 and the transport tunnel 2100 are connected. The third conveying mechanism 2330 receives the sample from the first conveying mechanism 2310 at the outlet 2112 of the transport tunnel 2100. By setting buffer chambers at the inlet 2111 and outlet 2112 of the conveying tunnel 2100, and selectively connecting the first buffer chamber 2200 and the conveying tunnel 2100, and selectively connecting the second buffer chamber 2400 and the conveying tunnel 2100, the conveying tunnel 2100 can be connected to the outside world through the first buffer chamber 2200 and the second buffer chamber 2400. The first buffer chamber 2200, the second buffer chamber 2400 and the conveying tunnel 2100 can be vacuumed step by step, avoiding the problem that the vacuum level in the conveying tunnel 2100 will drop too much due to direct connection with the outside world, and solving the problem that it takes a long time to reach a sufficient vacuum level each time the conveying tunnel 2100 is vacuumed.

[0215] In some embodiments, please refer to Figures 17 to 19 , Figures 17 to 19The process of transporting a sample in a transport tunnel 2100 is illustrated. A first transport mechanism 2310 disposed in the transport tunnel 2100 includes a track 2312 and a first transport arm 2311. The track 2312 is arranged along the extending direction of the transport tunnel 2100, and the first transport arm 2311 is slidably connected to the track 2312. The first transport arm 2311 is used to connect with at least one side of the sample. By utilizing the track 2312 to follow the extending direction of the transport tunnel 2100, a guiding function can be achieved for the first transport arm 2311, guiding the first transport arm to move along the extending direction of the transport tunnel 2100.

[0216] Optionally, the first conveying arm 2311 can be a gripper, a holding structure, a magnetic attraction structure, etc. The first conveying arm 2311 is connected to one side of the sample, which can both move the sample in the conveying tunnel 2100 and avoid obstructing the observation port 2110 when the sample moves to it, thus facilitating the observation of the sample. The driving structure and transmission structure of the first conveying arm 2311 can also be set in the conveying tunnel 2100.

[0217] In some embodiments, please continue reading Figures 17 to 19 Two tracks 2312 and two first conveyor arms 2311 are provided, each set in one track 2312. The first heat sink 120 includes two side ends 122, which are symmetrically arranged about the mounting part 121. The two first conveyor arms 2311 are respectively engaged with the side ends 122 on both sides, and the two first conveyor arms 2311 synchronously drive the first heat sink 120 to move. By symmetrically arranging the two side ends 122 on both sides of the mounting part 121, the balance of the first heat sink 120 on both sides can be improved. By using the two first conveyor arms 2311 to simultaneously drive the first heat sink 120 to move on both sides, the stability of the first heat sink 120 during the transfer process can be further improved.

[0218] In some embodiments, the thickness of the side end portion 122 of the first heat sink 120 is greater than the thickness of the mounting portion 121. The outer contour of the transport tunnel 2100 is set according to the shape of the first heat sink 120. Along the thickness direction of the first heat sink 120, the mounting portion 121 is recessed relative to the side end portion 122 to form a clearance space 130. A portion of the objective lens pole piece 1002 extends into the clearance space 130. Taking advantage of the first heat sink 120's thin middle and thick sides, a recessed clearance space 130 can be formed at the mounting part 121. When observing the sample, a portion of the objective lens pole piece 1002 extends into the clearance space 130. That is, as the first heat sink 120 moves along the transport tunnel 2100, the first heat sink 120 and the objective lens pole piece 1002 move relative to each other. The clearance space 130 allows the objective lens pole piece 1002 to pass through, thereby further shortening the distance between the objective lens pole piece 1002 and the carrier net 110. On the one hand, it can shorten the distance between the two objective lens pole pieces 1002, thereby reducing the power of the objective lens pole piece 1002 while ensuring sufficient electric field strength, thus achieving the effect of reducing energy consumption. On the other hand, it also helps to realize the miniaturization design of the electron microscope tube.

[0219] In some embodiments, please combine Figure 17 , Figures 19 to 20 The thickness of the side end portion 122 is greater than the thickness of the mounting portion 121. The mounting portion 121 is recessed relative to the side end portion 122 to form a clearance space 130. Both the second conveying mechanism 2320 and the third conveying mechanism 2330 include a claw 2321. The claw 2321 includes at least one pair of spaced-apart teeth 2322. The claw 2321 extends into the clearance space 130, and the mounting portion 121 is located between the two teeth 2322. Utilizing the characteristic that the first heat sink 120 is thin in the middle and thick on both sides, when the claw 2321 clamps the first heat sink 120, the claw 2321 can extend into the clearance space 130, so that the mounting portion 121 is located between the two teeth 2322, and the side end portion 122 is located outside the teeth 2322. The thickness difference between the mounting portion 121 and the side end portion 122 can limit the claw 2321, thereby improving the stability of the claw 2321 when clamping the first heat sink 120.

[0220] In some embodiments, the claw 2321 is magnetically connected to the first heat sink 120. In addition to the mutual locking function, the claw 2321 and the first heat sink 120 can also be magnetically connected to further improve the stability of their connection.

[0221] In some embodiments, the conveying mechanism includes a clamping structure 2300, which has a groove 2301. A movable member 2302 is provided in the groove 2301. The movable member 2302 extends when energized to abut and hold the first heat sink 120. Using the groove 2301 structure, a portion of the first heat sink 120 can be accommodated in the groove 2301, thereby improving connection strength and clamping stability. The clamping and releasing of the first heat sink 120 can be achieved by the extension and retraction of the movable member 2302.

[0222] In some embodiments, the movable component 2302 includes a piezoelectric stack actuator. Utilizing the principle of piezoelectric stacking, when energized, the piezoelectric stack effectively enhances the piezoelectric effect by stacking and combining multiple piezoelectric crystals. Piezoelectric stacking has advantages such as high responsiveness, precise control, and large output force, which helps to realize the clamping and releasing actions of the first heat sink 120 for transportation purposes.

[0223] In some embodiments, the observation port 2110 is disposed on the first sidewall of the conveying tunnel 2100, and the track 2312 is disposed on the second sidewall of the conveying tunnel 2100, with the second sidewall and the first sidewall being adjacent to each other. This satisfies the requirement of simultaneously providing the observation port 2110 and the track 2312 within the conveying tunnel 2100, while also preventing mutual interference between the track 2312 and the observation port 2110, avoiding the track 2312 from obstructing the observation port 2110, and resolving the problem that the observation port 2110 prevents the track 2312 from being continuously installed.

[0224] For example, the paired objective pole pieces 1002 can be spaced apart vertically. In this case, the top wall of the transport tunnel 2100 can serve as the first sidewall, and the observation port 2110 is located on the top wall of the transport tunnel 2100. The left and right side walls of the transport tunnel 2100 can then serve as the second sidewall. When two tracks 2312 are provided in the transport tunnel 2100, the two tracks 2312 can be spaced apart horizontally, and the line connecting the two tracks 2312 is perpendicular to the line connecting the two objective pole pieces 1002. Of course, in other examples, the two objective pole pieces 1002 can also be spaced apart horizontally. In this case, the left and right side walls of the transport tunnel 2100 can serve as the first sidewall, and the top or bottom wall can serve as the second sidewall.

[0225] Besides taking the form of grippers, the first conveying mechanism 2310 can also take the form of a conveyor belt. For example, in some embodiments, the first conveying mechanism 2310 disposed in the conveying tunnel 2100 includes a conveyor belt, which is disposed along the extension direction of the conveying tunnel 2100. The conveyor belt is used to connect with the side wall of the first heat sink 120 to move the sample along the conveying tunnel 2100. The conveyor belt and the first heat sink 120 can abut against each other, and the first heat sink 120 is moved along the conveying tunnel 2100 by static friction. Two conveyor belts can be provided, which are respectively located on both sides of the first heat sink 120 and clamped at the side end 122 of the first heat sink 120. The clamping action of the two conveyor belts is used to move the first heat sink 120.

[0226] In some embodiments, please refer to Figures 20 to 21 The conveying module 2000 also includes a first buffer chamber 2200. The first end 2210 of the first buffer chamber 2200 is connected to the entrance 2111 of the conveying tunnel 2100, and the second end 2220 of the first buffer chamber 2200 is used to deliver a sample. The conveying module 2000 includes a second conveying arm, which includes a first sub-arm 2323 and a second sub-arm 2324. The second sub-arm 2324 is sleeved on the outer periphery of the first sub-arm 2323. The first sub-arm 2323 is telescopically movable relative to the second sub-arm 2324 along its own axial direction. When the second conveying arm delivers the sample into the first buffer chamber 2200, the outer periphery of the second sub-arm 2324 abuts against the opening of the second end 2220 of the first buffer chamber 2200 and is sealed to the opening of the second end 2220. The first sub-arm 2323 extends relative to the second sub-arm 2324 and can pass through the opening of the first end 2210 of the first buffer chamber 2200 to enter the conveying tunnel 2100. The sample is gradually delivered from outside the first buffer chamber 2200 into the first buffer chamber 2200 using the first sub-arm 2323 and the second sub-arm 2324. After sealing the first buffer chamber 2200, the sample is then delivered into the transport tunnel 2100. On the one hand, the sample is evacuated at least twice during its gradual entry into the transport tunnel 2100, gradually reaching the required vacuum level. On the other hand, the sealing connection between the outer periphery of the second sub-arm 2324 and the second end 2220 of the first buffer chamber 2200 simplifies the sealing structure and steps of the first buffer chamber 2200, achieving sealing of the first buffer chamber 2200 simultaneously with sample delivery, thus improving the sealing efficiency of the first buffer chamber 2200 and consequently increasing the efficiency of sample delivery.

[0227] In some embodiments, the cryo-electron microscopy workstation further includes a storage module 3000 for storing samples. The storage module 3000 is disposed at the second end 2220 of the first buffer chamber 2200 and communicates with the second end 2220 of the first buffer chamber 2200. The storage module 3000 can store the prepared samples, thereby providing suitable preservation conditions for the samples and ensuring that the samples maintain their good morphology under suitable conditions (e.g., low temperature) while waiting to be sent into the transport tunnel 2100. By communicating the first buffer chamber 2200 with the storage module 3000, the path of the sample from storage to observation in the electron microscope tube can be shortened, improving sample transport efficiency. Exemplarily, the storage module 3000 can be a sealed, low-temperature chamber, in which suitable temperature, pressure, and other conditions can be provided to maintain the good performance of the sample.

[0228] In some embodiments, the cryo-electron microscopy workstation further includes an observation module, which comprises an electron microscope tube with a barrel 1001. The barrel 1001 contains pole piece modules, specifically paired objective pole pieces 1002, with an observation gap 1003 between the pole pieces 1002 allowing the sample to pass through. The pole piece system is the core component of the objective lens, consisting of two pole pieces forming an electromagnetic lens system. Its working principle involves generating a strong axisymmetric magnetic field between the pole pieces by energizing a coil, using the Lorentz force (F = e(v × B)) to control the movement of the electron beam. When a high-energy electron beam (typically in the range of 60-300 keV) passes through the magnetic field between the pole pieces, the electrons undergo helical motion under the influence of the magnetic field, achieving a focusing effect similar to that of an optical lens. The magnetic field strength can be controlled by precisely adjusting the coil current, thereby achieving precise focusing of the electron beam. To obtain optimal spatial resolution, the pole piece system needs to operate under strictly controlled environmental conditions, including ultra-high vacuum, low temperature, and low humidity (15% RH or lower). Meanwhile, the design of a multi-pole pole shoe system can correct for aberrations such as spherical aberration and chromatic aberration, ultimately achieving atomic-level resolution. This precise electron beam control mechanism makes cryo-electron microscopy an important tool for studying the structure of biological macromolecules.

[0229] Current cryo-electron microscopy has certain limitations, mainly including the following four points: First, the sample needs to be moved in multiple directions; second, the first heat sink 120 in cryo-electron microscopy occupies a large space between the pole pieces; third, cryo-electron microscopy requires a large observation range (about 3 mm), which requires a large pole piece aperture; fourth, due to the limitation of the first heat sink 120, cryo-electron microscopy requires a large pole piece spacing, which affects the imaging quality.

[0230] In principle, the electron beam moves towards the sample because the pole pieces provide a magnetic field. The stronger the magnetic field, the faster the electron beam travels, the shorter the wavelength, and the higher the achievable imaging resolution. There are two ways to increase the magnetic field strength: first, by improving the aperture and gap of the pole pieces; second, by increasing the current intensity. However, increasing the current generates more heat, which needs to be dissipated using water cooling. Water cooling can cause structural vibrations, affecting subsequent imaging results. Therefore, improving the aperture and gap is the preferred method to increase the magnetic field strength.

[0231] Refer to the following magnetic reluctance formula:

[0232] R m = l / μS;

[0233] Among them, R m Let be the magnetic reluctance, l be the magnetic path length, μ be the permeability, and S be the cross-sectional area of ​​the magnetic path. In the upper and lower pole pieces of a cryo-electron microscope, reducing the aperture and the spacing is equivalent to shortening the equivalent length of the magnetic path. Therefore, reducing the aperture and the spacing reduces the magnetic reluctance, as shown in the following Ohm's law formula for magnetic paths:

[0234] Φ=F m / R m ;

[0235] Where Φ is the magnetic flux, F m For magnetomotive force, when the magnetic reluctance R m When the magnetomotive force F decreases, at the same magnetomotive force F m As the magnetic flux Φ increases, refer to the following formula:

[0236] B = μH;

[0237] Φ = BS;

[0238] Where B is the magnetic flux density, and an increase in magnetic flux Φ means an increase in the magnetic field.

[0239] It is understandable that the larger the aperture and spacing of the pole pieces, the weaker the magnetic field strength between the upper and lower pole pieces of the electron microscope, which is not conducive to improving the resolution of electron microscope imaging; while the smaller the aperture and spacing of the pole pieces, the stronger the magnetic field strength between the upper and lower pole pieces of the electron microscope, which can improve the resolution of electron microscope imaging.

[0240] The existing cryo-electron microscope's first heat sink 120 is a metal box. The first heat sink 120 of the metal box needs to be cooled separately to adsorb water molecules in the electron microscope and prevent water molecules from condensing on the sample. However, the current first heat sink 120 needs to have a certain volume, which will hinder the reduction of the distance between the upper and lower pole shoes.

[0241] In addition, the area of ​​the existing sample-bearing grid 110 is relatively large, requiring a large field of view. The field of view is determined by the size of the spot formed by the electron beam on the sample, which in turn is determined by the aperture of the pole pieces. To ensure comprehensive observation of the sample, current cryo-electron microscopes require large apertures for both the upper and lower pole pieces to guarantee the field of view.

[0242] The objective of this application is to reduce the aperture and spacing of the objective pole pieces 1002 through structural design, thereby increasing the magnetic field strength between the pole pieces and ensuring the imaging quality of cryo-electron microscopy. Simultaneously, the grid 110 used in this application carries the sample in a narrow slit region. The electron beam focused by the pole pieces in this application can form a spot in the slit region, and the width of the slit region corresponds to the size of the spot. Even with a small aperture and spacing of the pole pieces, it is possible to ensure that the spot covers a sufficient amount of sample. It is understood that when the grid 110 moves unidirectionally along the length of the slit region, comprehensive observation of the sample can be achieved.

[0243] To this end, this application improves the magnetic field strength between pole pieces by optimizing sample movement, rearranging the first heat sink 120, and optimizing the observation area, thereby ensuring the imaging quality of cryo-electron microscopy.

[0244] Regarding optimizing sample movement, this application restricts sample movement to a single direction, thereby simplifying the mechanical structure of the cryo-electron microscope. In terms of rearranging the first heat sink 120, the larger volume portion of the first heat sink 120 is repositioned, freeing up space between the pole pieces. This facilitates shortening the pole piece spacing, thereby increasing the magnetic field strength between the pole pieces and ensuring the imaging quality of the system.

[0245] In terms of optimizing the observation area, the observation area is reduced from the traditional approximately 3mm to 1μm-10μm to facilitate the reduction of the pole piece aperture, thereby increasing the magnetic field strength between the pole pieces and ensuring the imaging quality of the system.

[0246] Therefore, the objective pole piece 1002 of this application achieves structural optimization, with a significantly shortened pole piece spacing, a greatly reduced pole piece aperture, and a more compact system structure. The structural improvements enhance the performance of cryo-electron microscopes using the objective pole piece 1002 of this application, specifically in terms of improved electron optical performance, improved imaging quality, and enhanced system stability. The objective pole piece 1002 of this application also achieves space utilization optimization, resulting in a more rational spatial layout, reduced mechanical interference, and optimized temperature control methods.

[0247] like Figure 22As shown, the objective lens pole piece 1002 includes a first pole piece 1004 and a second pole piece 1006. The first pole piece 1004 is used to focus the electron beam, and the second pole piece 1006 further focuses the electron beam. It can be understood that the sample is located between the first pole piece 1004 and the second pole piece 1006. The electron beam focused by the first pole piece 1004 reaches the sample, thereby acquiring the sample's features. The electron beam carrying the sample's features is further focused by the second pole piece 1006, ultimately forming an image of the sample.

[0248] Furthermore, the first pole piece 1004 is provided with a first channel 1005, and the second pole piece 1006 is provided with a second channel 1007, so that the electron beam formed by the first pole piece 1004 can propagate from the first channel 1005 to the second channel 1007.

[0249] The primary function of cryo-electron microscopy is to image frozen samples using an electron beam. The electron beam originates from the electron gun, travels through the space between the pole pieces to reach the sample, and then passes through a series of electromagnetic lenses for imaging. The presence of the first channel 1005 and the second channel 1007 provides a path for the electron beam, ensuring that it accurately reaches the sample's location, thereby achieving image formation.

[0250] In the electron optics system of cryo-electron microscopy, the pole piece plays a role in generating a magnetic field to focus the electron beam. The arrangement of the first channel 1005 and the second channel 1007 helps to precisely control the distribution and intensity of the magnetic field, allowing the electron beam to achieve better focusing as it passes through the channel.

[0251] Specifically, the distance between the first pole piece 1004 and the second pole piece 1006 is less than 4 mm, which is smaller than the distance between pole pieces in a conventional cryo-electron microscope. Simultaneously, the apertures of the first channel 1005 and the second channel 1007 are less than 2 mm, which is smaller than the apertures of pole pieces in a conventional cryo-electron microscope. This application improves the magnetic field strength between the pole pieces by reducing the distance and aperture of the pole pieces, thereby ensuring the imaging quality of the cryo-electron microscope.

[0252] Furthermore, since the carrier grid 110 needs to be positioned between the first pole piece 1004 and the second pole piece 1006, the mounting portion 121 is also positioned between the first pole piece 1004 and the second pole piece 1006. The side end portion 122 functions by utilizing its own low temperature and does not have a specific positional requirement. Therefore, in this application, the side end portion 122 is connected to the opposite sides of the mounting portion 121. That is, the space in the Y-axis direction is used to house the side end portion 122, preventing the thicker side end portion 122 from occupying the space between the first pole piece 1004 and the second pole piece 1006. This facilitates reducing the distance between the first pole piece 1004 and the second pole piece 1006, thereby increasing the magnetic field strength between the first pole piece 1004 and the second pole piece 1006 and ensuring the imaging quality of the electron microscope.

[0253] In some embodiments, the thickness of the mounting portion 121 is less than the thickness of the side end portion 122. The main function of the mounting portion 121 is to support the carrier net 110, and there are no specific requirements for its volume. Since the mounting portion 121 is located between the first pole piece 1004 and the second pole piece 1006, reducing the thickness of the mounting portion 121 helps to reduce the distance between the first pole piece 1004 and the second pole piece 1006, thereby increasing the magnetic field strength between the first pole piece 1004 and the second pole piece 1006. Therefore, while ensuring the supporting strength of the mounting portion 121 for the carrier net 110, it is necessary to minimize the thickness of the mounting portion 121.

[0254] Meanwhile, since the thickness of the mounting portion 121 is less than the thickness of the side end portion 122, the mounting portion 121 and the side end portion 122 located on opposite sides of the mounting portion 121 will form a groove-shaped recessed structure, so there is a certain space between the two side end portions 122, and the pole shoe can extend to the space between the two side end portions 122, thereby further reducing the distance between the first pole shoe 1004 and the second pole shoe 1006.

[0255] In some embodiments, the carrier mesh 110 is mounted after the first heat sink 120, and the common thickness of the carrier mesh 110 and the mounting portion 121 is 0.85mm-2mm, which is much smaller than the thickness of the first heat sink 120 in conventional cryo-electron microscopy. It is understood that, since the carrier mesh 110 and the mounting portion 121 are located between the first pole piece 1004 and the second pole piece 1006, reducing the common thickness of the carrier mesh 110 and the mounting portion 121 helps to reduce the distance between the first pole piece 1004 and the second pole piece 1006.

[0256] In some embodiments, the carrier mesh 110 has a slit region for carrying the sample. The slit region is narrow, so that the electron microscope does not need a large field of view to cover the width of the slit region.

[0257] Furthermore, the field of view of the electron microscope corresponds to the focusing spot size of the electron beam in the slit region. It can be understood that the spot size is positively correlated with the aperture sizes of the first channel 1005 and the second channel 1007. That is, the larger the aperture of the first channel 1005 and the second channel 1007, the larger the spot size; and the smaller the aperture of the first channel 1005 and the second channel 1007, the smaller the spot size. Therefore, a narrower slit region does not require a larger spot size, making it easier to reduce the aperture of the first channel 1005 and the second channel 1007, thereby increasing the magnetic field strength between the first pole piece 1004 and the second pole piece 1006. This design maximizes the magnetic field strength while ensuring observational effectiveness.

[0258] Meanwhile, since the electron microscope's field of view can cover the width of the slit region, the entire sample within the slit region can be observed simply by moving the grid 110 and the first heat sink 120 in a single direction (X-axis direction), without requiring them to move in multiple directions. Therefore, the grid 110 and the first heat sink 120 do not need to move in the Y-axis direction, and the space in the Y-axis direction can be used to set the side end 122. Even if the distance between the first pole piece 1004 and the second pole piece 1006 is small, it can prevent the side end 122 of the first heat sink 120 from colliding with the pole piece during movement.

[0259] Specifically, the width of the slit region is 1μm-10μm, which is much smaller than the width of the sample region on the traditional cryo-electron microscope grid 110, making it easier for the spot formed by the electron beam focusing to cover the width of the slit region.

[0260] In some embodiments, the first heat sink 120 drives the sample in the slit region to the spot position of the electron beam focusing by unidirectional movement, without the need for the first heat sink 120 to move in multiple directions.

[0261] Specifically, since the slit region is quite long, the spot formed by the electron beam focusing cannot completely cover the length of the slit region. The first heat sink 120 needs to move the carrier grid 110 together in the X-axis direction to ensure that the samples in the slit region arrive at the spot position in the X-axis direction in sequence, thus ensuring the comprehensiveness of the observation.

[0262] As the first heat sink 120 moves, the light spot focuses on a certain position within the slit area, forming a sample image at that position. After the image at the current position is captured, the first heat sink 120 continues to move, and the light spot refocuses on another adjacent position within the slit area, forming another sample image. This process is repeated until all sample images within the slit area are acquired, thus ensuring the comprehensiveness of the observation.

[0263] It is understandable that the extension direction of the slit region is the same as the movement direction of the first heat sink 120, that is, the movement direction of the first heat sink 120 is the length direction of the slit region.

[0264] In some embodiments, the first pole piece 1004 and the second pole piece 1006 are distributed vertically, with the first pole piece 1004 located on top of the second pole piece 1006. Therefore, the first channel 1005 and the second channel 1007 are also vertically corresponding, ensuring that the electron beam derived from the first channel 1005 can propagate into the second channel 1007, facilitating the formation of an image of the sample after focusing by the second pole piece 1006.

[0265] In some embodiments, the first pole piece 1004 has a through hole, which is approximately vertical. Correspondingly, the second pole piece 1006 has a through hole, which is also approximately vertical.

[0266] It is understandable that the internal space of the first hole defines the first channel 1005, and the internal space of the second hole also defines the second channel 1007. The first hole and the second hole correspond to each other vertically to ensure the propagation of the electron beam between the first pole piece 1004 and the second pole piece 1006.

[0267] In some embodiments, the distance between the first pole piece 1004 and the second pole piece 1006 is the distance between the position of the first aperture focusing the electron beam and the position of the second aperture receiving the electron beam. To reduce the distance between the first pole piece 1004 and the second pole piece 1006, it is necessary to reduce the distance from the position of the first aperture focusing the electron beam to the position of the second aperture receiving the electron beam, regardless of other positions of the first pole piece 1004 and the second pole piece 1006. Therefore, the specific shapes of the first pole piece 1004 and the second pole piece 1006 can be set according to actual conditions to ensure a stable safe distance between the pole piece and the side end 122, avoiding collision between the pole piece and the side end 122.

[0268] Specifically, the radial dimension of the first pole piece 1004 gradually decreases in the direction close to the second pole piece 1006, that is, the first pole piece 1004 is formed into a cone shape; at the same time, the radial dimension of the second pole piece 1006 also gradually decreases in the direction close to the first pole piece 1004, that is, the second pole piece 1006 is also formed into a cone shape.

[0269] In some embodiments, a coil is also provided around the objective lens pole piece 1002, and the coil can ensure that there is a magnetic field between the first pole piece 1004 and the second pole piece 1006.

[0270] Specifically, the coil forms a magnetic field between the first pole piece 1004 and the second pole piece 1006 by being energized.

[0271] In some embodiments, the first pole piece 1004 and the second pole piece 1006 adopt an asymmetric design. By optimizing their respective conical structural parameters, the magnetic field strength between the pole pieces is guaranteed while providing more room for the first heat sink 120 to move, thereby achieving a balance between high-resolution imaging and sample cooling.

[0272] Since the first pole shoe 1004 and the second pole shoe 1006 are respectively located on the upper and lower sides of the carrier net 110, the first pole shoe 1004 and the second pole shoe 1006 will be referred to as the upper and lower pole shoes in the following text.

[0273] Understandably, the distance between the upper and lower pole shoes needs to be greater than the thickness of the mounting part 121 to ensure that the first heat sink 120 can move between the upper and lower pole shoes and avoid structural interference between the first heat sink 120 and the pole shoes.

[0274] It is worth noting that the distance between the upper and lower pole shoes affects the magnetic field strength between them. Refer to the following reluctance formula:

[0275] R m = l / μS;

[0276] Among them, R m Let denoted as , l as , μ as , and S as , and , be the magnetic reluctance, l as , and μ as , and S as , and , be the magnetic permeability. In the upper and lower pole pieces of a cryo-electron microscope, reducing the distance between the upper and lower pole pieces is equivalent to shortening the equivalent length of the magnetic circuit. Therefore, reducing the aperture reduces the magnetic reluctance.

[0277] According to Ohm's law for magnetic circuits, when the magnetic reluctance R m When the magnetic flux decreases, the magnetic flux increases under the same magnetomotive force, which means that the magnetic field is strengthened.

[0278] It is understandable that the larger the distance between the upper and lower pole pieces of an electron microscope, the weaker the magnetic field strength between them, which is detrimental to improving the resolution of electron microscope imaging; while the smaller the distance between the upper and lower pole pieces, the stronger the magnetic field strength between them, which is beneficial to improving the resolution of electron microscope imaging.

[0279] This application places the thicker side end portion 122 on both sides of the first heat sink 120, without affecting the distance between the upper and lower pole pieces of the electron microscope. Furthermore, the thinner mounting portion 121 is placed in the middle of the first heat sink 120, which facilitates reducing the distance between the upper and lower pole pieces of the electron microscope, thereby increasing the magnetic field strength between the upper and lower pole pieces and improving the resolution of the electron microscope imaging.

[0280] In addition, since the thickness of the side end 122 is greater than the thickness of the mounting part 121, and the mounting part 121 is located between the two side end 122, a certain space is reserved between the two side end 122. This space can be used to set the upper and lower pole pieces, further reducing the distance between the upper and lower pole pieces of the electron microscope.

[0281] In some embodiments, the thickness of the mounting portion 121 on which the carrier mesh 110 is mounted is 0.85mm-2mm. The smaller thickness of the mounting portion 121 facilitates reducing the distance between the upper and lower pole pieces of the electron microscope. The carrier mesh 110 is detachably connected to the mounting portion 121 to improve the flexibility of the connection between the carrier mesh 110 and the mounting portion 121.

[0282] In some embodiments, refer to Figure 14The mounting section 121 includes a support structure 1105, which is formed as a plate. The plate-shaped support structure 1105 is connected between two side ends 122, and the support structure 1105 has an upward-facing support plane. The carrier net 110 is placed on top of the support plane. Therefore, the support structure 1105 provides support for the carrier net 110 through the support plane, so as to ensure the stability of the relative position between the carrier net 110 and the first heat sink 120 during the observation process.

[0283] Meanwhile, the thickness of the support structure 1105 needs to be as small as possible while ensuring structural strength, so that the support structure 1105 also has a small thickness after the carrier grid 110 is placed, which makes it easier to reduce the distance between the upper and lower pole pieces of the electron microscope.

[0284] In addition, to prevent the netting 110 from wobbling in the Y-axis direction after it is placed on top of the support structure 1105, the dimensions of the netting 110 in the Y-axis direction are approximately the same as those of the support structure 1105 in the Y-axis direction. Since the support structure 1105 also has side ends 122 on opposite sides, when the netting 110 is placed on top of the support structure 1105, the netting 110 will come into contact with the side ends 122 on opposite sides, and at this time, the side ends 122 on both sides also provide a certain positioning effect for the netting 110.

[0285] In some embodiments, the support structure 1105 is provided with a clearance window 1213, which can ensure that the electron beam emitted from the upper pole shoe reaches the lower pole shoe.

[0286] In this process, the upper pole piece forms an electron beam, which is first focused on the area of ​​the carrier grid 110 used to support the sample, thereby obtaining the characteristics of the sample. The electron beam with the sample characteristics then passes through the support structure 1105 along the avoidance window 1213 and finally reaches the lower pole piece, which facilitates the formation of an image of the sample by the lower pole piece.

[0287] Furthermore, the area of ​​the carrier 110 used to support the sample is the slit area 1101. In order to facilitate the electron beam to pass through the slit area 1101 and the avoidance window 1213 in sequence, the projection of the slit area 1101 on the support structure 1105 is located within the range of the avoidance window 1213.

[0288] In some embodiments, the mounting portion 121 is provided with an observation window 1211, through which the area of ​​the mesh 110 used to support the sample is exposed. When the electron microscope is in operation, the electron beam emitted from the upper pole piece first passes through the observation window 1211 and reaches the area of ​​the mesh 110 used to support the sample. After passing through the clearance window 1213, the electron beam finally reaches the lower pole piece, thereby completing the imaging of the sample.

[0289] In some embodiments, the mounting portion 121 further includes a clamping structure, which, together with the support structure 1105, limits the carrier net 110 to prevent the carrier net 110 from detaching from the first heat sink 120.

[0290] Both side ends 122 are provided with clamping structures, that is, the number of clamping structures is set to two. Both clamping structures extend a certain distance towards the middle of the first heat sink 120, and the two clamping structures are symmetrical to each other.

[0291] Meanwhile, the two clamping structures are not connected, so a certain gap is formed between the two clamping structures, and this gap is the observation window 1211.

[0292] Furthermore, the clamping structure and the support structure 1105 are spaced apart; specifically, the clamping structure and the support structure 1105 are distributed vertically. When the support structure 1105 supports the netting 110 at its bottom, the clamping structure is located at the top of the support structure 1105. The gap between the clamping structure and the support structure 1105 defines a slot for inserting the netting 110.

[0293] In some embodiments, the clamping structure is formed as a plate-like structure, i.e., a clamping plate, and the support structure 1105 is also formed as a plate-like structure, i.e., a support plate. The clamping plates and support plates spaced apart from each other form slots for inserting the carrier net 110. Since two clamping plates are provided, two slots are also provided accordingly, and the two sides of the carrier net 110 are respectively inserted into the two slots.

[0294] In some embodiments, the first heat sink 120 is made of metal, which has high heat transfer efficiency, making it easier for the first heat sink 120 to conduct heat to the carrier mesh 110, and also making it easier for the first heat sink 120 at a lower temperature to absorb water molecules in the electron microscope tube.

[0295] In some embodiments, the upper pole shoe has a gradually decreasing radial dimension in the direction near the support structure 1105, thus the upper pole shoe is generally formed into a conical structure. Similarly, the lower pole shoe also has a gradually decreasing radial dimension in the direction near the support structure 1105, that is, the lower pole shoe is also generally formed into a conical structure.

[0296] To accommodate the shapes of the upper and lower electrode shoes and ensure a safe distance between the side end 122 and the electron microscope electrode shoes, the side end 122 has an inclined surface 1104 on the side connecting to the mounting portion 121. This allows for a gradual transition in thickness between the side end 122 and the mounting portion 121, and the inclined surface 1104 maintains the same inclination angle as the sidewall of the electron microscope electrode shoe. Simultaneously, by providing a gradual transition in thickness between the two, the stress between the side end 122 and the mounting portion 121 can be reduced, thus mitigating the risk of breakage at the junction of the side end 122 and the mounting portion 121.

[0297] Additionally, refer to Figure 22 In this application, the carrier grid 110 and the first heat sink 120 only move in the X-axis direction and do not need to move in the Y-axis direction. The space in the Y-axis direction is used to arrange the side end 122, which plays a role in cold shielding, to avoid structural interference between the side end 122 and the pole shoes in the electron microscope. When designing the distance between the upper and lower pole shoes of the electron microscope, only the thickness of the mounting part 121 needs to be considered, which is beneficial to reduce the distance between the upper and lower pole shoes of the electron microscope. The reduction of the distance between the upper and lower pole shoes of the electron microscope is beneficial to improve the magnetic field strength between the pole shoes, and further ensure the imaging resolution of the cryo-electron microscope.

[0298] In some embodiments, the carrier mesh 110 is used to carry a sample. The carrier mesh 110 includes a first film 113 and a substrate. The first film 113 carries the sample, while the substrate supports the bottom of the first film 113 to enhance the structural strength of the carrier mesh 110.

[0299] Furthermore, the first film 113 is provided with a number of pore structures. The distribution of the pore structures can be set according to actual needs. When the sample liquid reaches the first film 113, the sample liquid will be carried in the pore structures.

[0300] In some embodiments, the first film 113 is provided with a slit region 1101, and a perforated structure is provided in the slit region 1101, so that the slit region 1101 is used to hold the sample liquid.

[0301] Furthermore, the substrate is provided with an imaging window 1103. The substrate has a hollow structure, and the hollow region of the substrate defines the imaging window 1103. The electron beam formed by the upper pole piece can pass through the imaging window 1103 to reach the lower pole piece, facilitating the formation of an image of the sample by the lower pole piece.

[0302] Simultaneously, when the first thin film 113 is attached to the top of the substrate, the edge of the first thin film 113 is attached to the upper surface of the substrate, while the middle part of the first thin film 113 covers the hollow area of ​​the substrate. The electron beam formed by the upper pole piece first reaches the first thin film 113 and acquires the characteristics of the sample on the first thin film 113. The electron beam carrying the sample characteristics then propagates to the lower pole piece, thereby initially completing the sample imaging process.

[0303] It is understandable that when the first thin film 113 is attached to the top of the substrate, the slit region 1101 on the first thin film 113 is within the hollow region of the substrate, that is, the projection of the slit region 1101 onto the substrate is within the imaging window 1103. The electron beam is first focused on the sample within the slit region 1101, and then the sample feature information within the slit region 1101 is transmitted to the lower pole shoe.

[0304] Furthermore, the supporting effect of the substrate cannot extend to the first thin film 113 portion within the hollow area of ​​the substrate. To ensure the structural strength of the first thin film 113 portion within the hollow area of ​​the substrate, corresponding support beams can be provided on the surface of the first thin film 113. Specifically, the structural form of the support beams can be set according to actual needs.

[0305] It is understood that the slit region 1101 of the first membrane 113 can be enclosed by a support beam or by other solid structures used to support the first membrane 113.

[0306] In some embodiments, the aperture of the channel used by the upper pole piece to form the electron beam determines the size of the spot formed by the electron beam on the first thin film 113. It is understood that the larger the aperture size of the channel, the larger the spot size formed, i.e., the larger the field of view of the cryo-electron microscope; the smaller the aperture size of the channel, the smaller the spot size formed, i.e., the smaller the field of view of the cryo-electron microscope.

[0307] Meanwhile, the aperture of the channel is also related to the magnetic field strength between the upper and lower pole shoes of the electron microscope, as shown in the following magnetoresistance formula:

[0308] R m = l / μS;

[0309] Among them, R m Let be the magnetic reluctance, l be the magnetic path length, μ be the permeability, and S be the cross-sectional area of ​​the magnetic path. In the upper and lower pole pieces of a cryo-electron microscope, reducing the aperture is equivalent to shortening the equivalent length of the magnetic path. Therefore, reducing the aperture reduces the magnetic reluctance, as shown in the following Ohm's law formula for magnetic paths:

[0310] Φ=F m / R m ;

[0311] Where Φ is the magnetic flux, F m For magnetomotive force, when the magnetic reluctance R m When the magnetomotive force F decreases, at the same magnetomotive force F m As the magnetic flux Φ increases, refer to the following formula:

[0312] B = μH;

[0313] Φ = BS;

[0314] Where B is the magnetic flux density, and an increase in magnetic flux Φ means an increase in the magnetic field.

[0315] It is understandable that the larger the aperture of the channel, the weaker the magnetic field strength between the upper and lower pole pieces of the electron microscope, which is not conducive to improving the resolution of electron microscope imaging; while the smaller the aperture of the channel, the stronger the magnetic field strength between the upper and lower pole pieces of the electron microscope, which can improve the resolution of electron microscope imaging.

[0316] The narrow slit region 1101 of the first thin film 113 in this application allows for comprehensive observation of the sample at its current X-axis position by forming a small spot of electron beam within the slit region 1101, eliminating the need for a large field of view. Therefore, a small aperture of the electron beam channel in the electron microscope pole pieces is sufficient to meet practical observation requirements. Simultaneously, the small aperture of the electron beam channel in the electron microscope pole pieces also ensures a high magnetic field strength between the upper and lower pole pieces, thereby guaranteeing the resolution of the electron microscope imaging.

[0317] In some embodiments, since the slit region 1101 is narrow and matches the aperture of the electron beam channel of the electron microscope pole piece, the first heat sink 120 can be moved in one direction to ensure that the spot formed by the electron beam completes the sweeping process within the slit region 1101 without affecting the comprehensiveness of the observation.

[0318] It is understandable that the size of the electron beam spot corresponds to the width of the slit region 1101. When the spot is focused at a certain position in the X-axis direction of the slit region, it ensures that the sample at that position can be fully observed. The first heat sink 120 moves along the length direction of the slit region 1101, i.e., the X-axis direction. When the first heat sink 120 moves along the X-axis, the focusing position of the spot in the slit region 1101 changes, thereby ensuring that the sample at the focusing position can be fully observed.

[0319] After the first heat sink 120 moves in one direction to drive the light spot to complete the full sweep of the slit region 1101, all samples within the current slit region 1101 have completed the observation process. When the slit region 1101 needs further observation at a certain position in the X-axis direction, the first heat sink 120 can also move in the opposite direction along the X-axis to the corresponding position.

[0320] Understandably, during the observation process, as the first heat sink 120 moves, samples from different areas on the carrier net 110 arrive at the light spot position in sequence for image capture. Then, several captured images are combined and analyzed to ensure the comprehensiveness of the observation.

[0321] In some embodiments, the length of the slit region 1101 is 0.5mm-3mm, that is, the size of the slit region 1101 in the X-axis direction is 0.5mm-3mm, and the width of the slit region 1101 is 1μm-10μm, that is, the size of the slit region 1101 in the Y-axis direction is 1μm-10μm.

[0322] It is worth noting that the cryo-electron microscopy workstation of this application includes at least one sample preparation module 1000, a thinning module, an electrode shoe module, and a transport module. As an example, the following are listed: the cryo-electron microscopy workstation includes a sample preparation module 1000 or a thinning module or an electrode shoe module or a transport system; the cryo-electron microscopy workstation includes a sample preparation module 1000 and a thinning module, or a sample preparation module 1000 and an electrode shoe module, or a sample preparation module 1000 and a transport system, or a thinning module and an electrode shoe module, or a thinning module and a transport system, or an electrode shoe module and a transport system; the cryo-electron microscopy workstation includes a sample preparation module 1000, a thinning module and an electrode shoe module, or a sample preparation module 1000, a thinning module and a transport module, or a sample preparation module 1000, an electrode shoe module and a transport module, or a thinning module, an electrode shoe module and a transport module.

[0323] This application provides a method for preparing cryo-electron microscopy samples, including the following steps:

[0324] Step S100: Add the sample to the sample preparation module 1000;

[0325] Step S200: The sample is sent into the delivery module 2000, the delivery module 2000 and the electron microscope tube are evacuated, and the delivery module 2000 delivers the sample to the observation port 2110.

[0326] Step S300: Connect the first heat sink 120 to the cold source 300;

[0327] Step S400: The second heating element 200 heats the bearing surface 112 to a temperature suitable for the sample, so that the sample remains in a liquid state;

[0328] Step S500: The thinning component thins the sample on the bearing surface 112 to the target thickness;

[0329] Step S600: The second heating element 200 stops heating the bearing surface 112, and the cold source 300 freezes the sample;

[0330] Step S700: The electron microscope tube is used to observe the sample through observation port 2110.

[0331] By isolating the space within the transport tunnel 2100 from the cylinder 1001, the space within the transport tunnel 2100 and the space within the cylinder 1001 are formed as two independent spaces. Two different vacuum systems are used to provide vacuum environments for these two spaces respectively. This shortens the vacuuming time for both spaces, that is, it shortens the execution time of step S200, thereby improving the vacuuming efficiency of these two spaces. By shortening the vacuuming time, the observation cycle for each sample can be shortened, improving the utilization efficiency of the cryo-electron microscopy workstation.

[0332] By connecting the first heat sink 120 with the cold source 300, the cold source 300 can conduct cold to the first heat sink 120 and the carrier mesh 110, thereby lowering the temperature of the first heat sink 120 and the carrier mesh 110 and facilitating the freezing and solidification of the sample.

[0333] Furthermore, one end of the movable contact 3023 is fixedly connected to the second heat sink 701, and the movable contact 3023 is driven to extend so that the other end of the movable contact 3023 contacts the side wall of the refrigerant cavity 3024.

[0334] Before the sample is frozen solidified, the air supply component 600 spreads the liquid sample, reducing its thickness and controlling it within the desired range, which helps improve the observation effect. Once the sample thickness reaches the required thickness, the second heating component 200 can stop heating the bearing surface 112, allowing the sample to be quickly frozen solidified and maintained at the reduced thickness, achieving a sample preparation effect with controllable thickness.

[0335] In some embodiments, step S500 above: thinning the sample on the bearing surface 112 to the target thickness by the thinning component includes:

[0336] Step S510: The air supply component 600 supplies air to the bearing surface 112, blowing the sample to reduce its thickness to the target thickness.

[0337] In some embodiments, step S500 above: thinning the sample on the bearing surface 112 to the target thickness by the thinning component includes:

[0338] Step S511: The thickness detection module detects the thickness of the sample;

[0339] Step S512: When the sample is thinned to the target thickness, the control device controls the air supply component 600 to stop supplying air.

[0340] The thickness detection module can detect the degree of sample thinning, and the control device can control the air supply component 600 to continue or stop air supply, so as to achieve controllable thinning of sample thickness and further improve the accuracy of thinning.

[0341] In some embodiments, the sample preparation module 1000 further includes a flow guiding component 700 disposed on the outer periphery of the carrier net 110. Step S510, where the air supply component 600 supplies air to the bearing surface 112 to thin the sample to the target thickness, includes:

[0342] Step S513: The air supply component 600 supplies air to the carrier net 110 in a direction perpendicular to the bearing surface 112, and the airflow guiding component 700 disperses the airflow supplied by the air supply component 600 along the airflow guiding surface, which is parallel or coplanar with the bearing surface 112.

[0343] By utilizing the dispersion effect of the airflow guide component 700, and with the plasma airflow blowing toward the sample in a direction perpendicular to the bearing surface 112, the plasma airflow can form a vortex state. That is, the plasma airflow can be better blown out from the air supply component 600 and diffuse rapidly. The dynamic effect of the airflow helps the sample droplets to diffuse and spread outward from the center, thereby achieving the effect of sample thinning.

[0344] In some embodiments, step S500 above: thinning the sample on the bearing surface 112 to the target thickness by the thinning component includes:

[0345] Step S520: The first heating element 400 and the second heating element 200 simultaneously heat the bearing area, and the heating temperature of the first heating element 400 is greater than that of the second heating element 200.

[0346] This creates a temperature difference between the central region 1121 and the edge region 1122, with the edge region 1122 having a higher temperature than the central region 1121. This temperature gradient causes the sample at the periphery to evaporate faster than the sample at the center, creating a diffusion force from the central region 1121 to the edge region 1122 within the liquid. Under the influence of liquid surface tension, the sample flows from the lower temperature region to the higher temperature region, causing the sample droplets to diffuse from the center outwards, achieving the effect of sample thinning. Throughout the sample thinning process, the second heating element 200 also heats and maintains the sample temperature, keeping it in a liquid state and ensuring that the sample can be thinned using the fluidity of the liquid. When the second heating element 200 stops heating, the cold source 300 can rapidly freeze the sample.

[0347] In some embodiments, step S200 above, which involves sending the sample into the transport module 2000, evacuating the transport module 2000 and the electron microscope tube, and having the transport module 2000 transport the sample to the observation port 2110, includes:

[0348] Step S210: The second transfer mechanism delivers the sample from the entrance 2111 of the transport tunnel 2100 to the first transfer mechanism;

[0349] Step S220: The first transmission mechanism moves the sample in the transport tunnel 2100 to the observation gap 1003 for observation;

[0350] Step S230: The first transfer mechanism delivers the sample from the outlet 2112 of the transport tunnel 2100 to the third transfer mechanism.

[0351] Three transport mechanisms are used to deliver samples. Each transport mechanism can transport samples to areas outside the entrance 2111 of the transport tunnel 2100, inside the transport tunnel 2100, and outside the exit 2112 of the transport tunnel 2100. When any two of the three transport mechanisms are delivering samples to each other, the remaining transport mechanism can simultaneously operate on another sample (the previous sample or the next sample). This allows multiple transport mechanisms to work simultaneously, improving the efficiency of sample delivery into and out of the transport tunnel 2100, shortening the time samples are occupied by each transport mechanism, and thus improving the efficiency of sample observation.

[0352] In some embodiments, while performing step S210 or S220, the third transfer mechanism unloads the previous sample. This parallel delivery method saves sample waiting time and improves sample transport efficiency.

[0353] In some embodiments, the third transfer mechanism unloading the previous sample includes transporting the sample out of the second buffer chamber 2400. After leaving the buffer chamber, the sample can be further transported to other modules for processing, such as sample recovery. After releasing the sample, the third transfer mechanism can return to an empty state to receive the next sample.

[0354] Similarly, in some embodiments, while performing step S220 or S230, the second transfer mechanism loads the previous sample. This parallel delivery method during sample loading saves sample waiting time and improves sample transport efficiency.

[0355] In some embodiments, the second transfer mechanism loading the previous sample includes transporting the sample into the first buffer chamber 2200. During step S220 or S230, the second transfer mechanism can simultaneously send the sample into the first buffer chamber 2200, for example, by retrieving a sample from the storage module 3000 or taking a sample from the sample preparation module. This way, once the previous sample has been observed and recovered, the second transfer mechanism can immediately deliver the new sample to the first transfer mechanism, shortening sample waiting time, reducing wasted time, and thereby improving sample transport efficiency.

[0356] In some embodiments, step S700 above, where the electron microscope tube observes the sample through the observation port 2110, includes:

[0357] Step S710: Create a magnetic field between the objective lens pole pieces 1002;

[0358] Step S720: The electron beam is focused through the objective lens pole piece 1002;

[0359] Step S730: The sample passes through the observation gap between the objective lens pole pieces 1002.

[0360] In some embodiments, the objective lens pole piece 1002 includes a first pole piece 1004 and a second pole piece 1006. The first pole piece 1004 and the second pole piece 1006 need to ensure that the first hole and the second hole have small apertures. Since the first hole and the second hole form the first channel 1005 and the second channel 1007 respectively, the smaller aperture means that the spot size of the electron beam focusing is also smaller.

[0361] It is worth noting that the apertures of the first and second holes also need to correspond to the width of the slit area of ​​the carrier mesh 110. Specifically, the apertures of the first and second holes are set to 1mm-2mm.

[0362] Meanwhile, during the installation of the first pole shoe 1004 and the second pole shoe 1006, it is necessary to ensure that the first pole shoe 1004 and the second pole shoe 1006 have a small gap.

[0363] The smaller aperture and spacing of the first pole piece 1004 and the second pole piece 1006 facilitate the formation of a larger magnetic field strength between them, thereby ensuring image quality. Specifically, the spacing between the first pole piece 1004 and the second pole piece 1006 is 2mm-4mm.

[0364] As the coil is energized, a magnetic field is formed between the first pole piece 1004 and the second pole piece 1006. Under the influence of the magnetic field, the electron beam is focused by passing through the first pole piece 1004 and the second pole piece 1006 in sequence. The electron beam focused by the second pole piece 1006 finally forms an image of the sample.

[0365] Furthermore, a carrier grid 110 is installed on the first heat sink 120, and the carrier grid 110 carries the sample. The electron beam can form a light spot on the carrier grid 110, thereby capturing the sample image at the position of the light spot.

[0366] As the first heat sink 120 moves, the electron beam will scan the sample on the grid 110, ensuring comprehensive observation.

[0367] In some embodiments, step S730 above, where the sample passes through the observation gap between the objective lens pole pieces 1002, includes:

[0368] Step S731: The carrier net 110 moves in the direction of extension of the slit area;

[0369] Step S732: The carrier net 110 is moved to focus the electron beam at different positions within the slit region.

[0370] The slit region is relatively narrow, which can cover the width of the slit region even when the field of view of the electron microscope is small. In addition, with the unidirectional movement of the first heat sink 120 and the carrier 110, it can ensure that the sample in the slit region can be observed in a comprehensive manner without the need for the first heat sink 120 and the carrier 110 to move in multiple directions, thus simplifying the movement process.

[0371] Meanwhile, the smaller field of view of the electron microscope means that the apertures of the first and second apertures are smaller, which helps to increase the magnetic field strength between the first pole piece 1004 and the second pole piece 1006, thus ensuring the imaging quality of the electron microscope.

[0372] As the first heat sink 120 moves, the electron beam will continuously focus on different positions in the slit region, thus enabling the electron microscope to continuously form sample images at different positions within the slit region. After the electron beam has completed its scanning process of the slit region, the staff can analyze and compare the several images formed by the electron microscope to ensure the comprehensiveness of the observation.

[0373] In some embodiments, step S600 above, in which the second heating element 200 stops heating the bearing surface 112 and the cold source 300 freezes the sample, includes:

[0374] Step S610: The cold source 300 reduces the temperature of the first heat sink 120 through heat transfer, so that the temperature of the first heat sink 120 is lower than the temperature of the carrier mesh 110;

[0375] Step S620: The first heat sink 120 reduces the temperature of the carrier mesh 110 through heat transfer.

[0376] A second heat sink 701 is connected to the cold source 300, and the second heat sink 701 is used to connect to the first heat sink 120, so that the cold source 300 can conduct cold to the first heat sink 120 through the second heat sink 701. Therefore, the first heat sink 120 has a lower temperature. Specifically, the temperature of the first heat sink 120 is lower than the temperature of the carrier 110, which facilitates the reduction of the temperature of the carrier 110 through heat transfer, thereby achieving the freezing of the sample on the carrier 110.

[0377] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

1. A cryo-electron microscopy workstation, characterized in that, The cryo-electron microscopy workstation includes a sample preparation module (1000), which is used to load a carrier grid (110) for carrying samples. The sample preparation module (1000) includes: A first heat sink (120) is used to encapsulate at least one end of a carrier mesh (110). An interaction force is formed between the first heat sink (120) and the carrier mesh (110). The first heat sink (120) is used to conduct heat to the end of the carrier mesh (110). The second heating component (200) is used to connect to the bearing surface (112) of the carrier net (110), and the second heating component (200) is used to heat the bearing surface (112) of the carrier net (110); A cold source (300) is connected to the first heat sink (120). The cold source (300) is used to conduct cold to the first heat sink (120). The cold source (300) is used to freeze the bearing surface (112) of the carrier net (110) when the second heating component (200) stops heating the bearing surface (112) of the carrier net (110).

2. A cryo-electron microscopy workstation, characterized in that, The cryo-electron microscopy workstation includes a sample preparation module (1000), which comprises: The carrier net (110) has a bearing surface (112) for bearing the sample; A first heat sink (120) is provided, at least one end of the carrier mesh (110) is encapsulated in the first heat sink (120), the bearing surface (112) is exposed from the first heat sink (120), an interaction force is formed between the first heat sink (120) and the carrier mesh (110), and the first heat sink (120) is used to conduct heat to the end of the carrier mesh (110); A second heating element (200) is disposed in the carrier net (110). The second heating element (200) is arranged around the sample and is used to heat the carrier surface (112). A cold source (300) is connected to the first heat sink (120). The cold source (300) is used to conduct cold to the first heat sink (120). The cold source (300) is used to freeze the bearing surface (112) when the second heating component (200) stops heating the bearing surface (112).

3. The cryo-electron microscopy workstation according to claim 1 or 2, characterized in that, The sample preparation module (1000) is provided with a first film (113), the surface of the first film (113) is formed as a bearing surface (112), and the first film (113) is provided with a through structure (1131) that penetrates both sides of the first film (113).

4. The cryo-electron microscopy workstation according to claim 1 or 2, characterized in that, The sample preparation module (1000) is provided with a light-transmitting second film (114), the surface of which is formed as a bearing surface (112), and the second film (114) is continuously arranged.

5. The cryo-electron microscopy workstation according to claim 1 or 2, characterized in that, The first heat sink (120) includes a mounting part (121) and a side end (122) connected in sequence. The mounting part (121) is used to enclose the carrier mesh (110). The mounting part (121) is provided with an observation window (1211). The bearing surface (112) is exposed from the observation window (1211). The side end (122) is used to connect to a cold source (300).

6. The cryo-electron microscopy workstation according to claim 5, characterized in that, The thickness of the side end portion (122) is greater than the thickness of the mounting portion (121).

7. The cryo-electron microscopy workstation according to claim 6, characterized in that, The thickness of the side end (122) and the mounting part (121) gradually transitions.

8. The cryo-electron microscopy workstation according to claim 5, characterized in that, The mounting part (121) is provided with a mounting groove (1212), which is used to enclose the carrier net (110) so that there is an interaction force between the mounting part (121) and the carrier net (110). The observation window (1211) is connected to the mounting groove (1212). The bottom surface of the mounting part (121) is also provided with a clearance window (1213) connected to the mounting groove (1212). The clearance window (1213) is used to allow the fixing member (201) with the second heating component (200) to extend into the mounting groove (1212).

9. The cryo-electron microscopy workstation according to claim 8, characterized in that, The mounting part (121) includes a support structure (1105) connecting each of the side ends (122), the support structure (1105) being used to support the carrier net (110).

10. The cryo-electron microscopy workstation according to claim 9, characterized in that, The mounting part (121) further includes a clamping structure, and each of the side ends (122) is connected to the clamping structure. The interval between the clamping structures defines the observation window (1211). The clamping structure is spaced apart from the support structure (1105) to form the mounting groove (1212) for inserting the carrier net (110).

11. The cryo-electron microscopy workstation according to claim 10, characterized in that, The clamping structure is formed as a clamping plate, which is located on top of the carrier net (110).

12. The cryo-electron microscopy workstation according to claim 5, characterized in that, The two side ends (122) are symmetrically arranged with respect to the mounting part (121).

13. The cryo-electron microscopy workstation according to claim 9, characterized in that, The avoidance window (1213) is disposed on the support structure (1105), and the area of ​​the carrier net (110) used to carry the sample is located within the range of the avoidance window (1213) of the projection of the support structure (1105).

14. The cryo-electron microscopy workstation according to claim 1 or 2, characterized in that, The sample preparation module (1000) further includes a second heat sink (701), which is connected to the outer periphery of the first heat sink (120). The cold source (300) is connected to the second heat sink (701), and the cold source (300) is in detachable contact with the second heat sink (701). The cold source (300) conducts cooling to the first heat sink (120) through the second heat sink (701).

15. The cryo-electron microscopy workstation according to claim 1 or 2, characterized in that, The sample preparation module (1000) further includes a cooling component (302) and a second heat sink (701), wherein the cold source (300) conducts cooling to the first heat sink (120) through the second heat sink (701); The cooling component (302) includes a support (3021), a movable contact (3023), and a refrigerant cavity (3024). A cooling channel (3022) is formed in the support (3021). The second heat sink (701) is installed at one end of the cooling channel (3022), and the other end is connected to the refrigerant cavity (3024). The refrigerant cavity (3024) is used to introduce a cold source (300) medium. The movable contact (3023) is located between the second heat sink (701) and the refrigerant cavity (3024). The movable contact (3023) is movable in the cooling channel (3022) to connect or disconnect the second heat sink (701) from the cold source (300) medium.

16. The cryo-electron microscopy workstation according to claim 15, characterized in that, One end of the movable contact (3023) is fixedly connected to the second heat sink (701), and the other end is retractable along the radial direction of the cooling channel (3022). The other end of the movable contact (3023) can extend to contact the side wall of the refrigerant cavity (3024), or can retract to separate from the side wall of the refrigerant cavity (3024).

17. The cryo-electron microscopy workstation according to claim 15, characterized in that, The second heat sink (701) is provided with an assembly slot for installing the first heat sink (120), the opening edge of the assembly slot is flush with the opening edge of the cooling channel (3022), and the support (3021) wraps around the second heat sink (701).

18. The cryo-electron microscopy workstation according to claim 15, characterized in that, The second heat sink (701) is provided with an assembly slot for mounting the first heat sink (120), the end of the first heat sink (120) is accommodated in the assembly slot, and the volume of the second heat sink (701) is larger than the volume of the side end (122) of the first heat sink (120).

19. The cryo-electron microscopy workstation according to claim 2, characterized in that, When the cryo-electron microscope workstation is in operation, the temperature of the first heat sink (120) is lower than the temperature of the carrier mesh (110).

20. The cryo-electron microscopy workstation according to claim 3, characterized in that, The sample preparation module (1000) includes a carrier mesh (110), which has a slit region (1101) for carrying the sample. The width of the slit region (1101) is 1μm-10μm.

21. The cryo-electron microscopy workstation according to claim 20, characterized in that, The first heat sink (120) drives the sample in the slit region (1101) to the electron beam focusing position of the cryo-electron microscope workstation by unidirectional movement.

22. The cryo-electron microscopy workstation according to claim 20, characterized in that, The carrier mesh (110) includes a substrate supporting the first thin film (113), the substrate having an imaging window (1103), and the projection of the slit region (1101) onto the substrate being within the range of the imaging window (1103).

23. The cryo-electron microscopy workstation according to claim 1 or 2, characterized in that, The sample preparation module (1000) includes a thinning component, which includes a first heating component (400) and / or an air supply component (600), and is used to thin the sample on the bearing surface (112) of the carrier net (110).

24. The cryo-electron microscopy workstation according to claim 23, characterized in that, The sample preparation module (1000) includes a fixing member (201), and the second heating component (200) is disposed on the fixing member (201). When the first heat sink (120) is installed on the fixing member (201), the second heating component (200) contacts the bottom surface of the first heat sink (120).

25. The cryo-electron microscopy workstation according to claim 23, characterized in that, The air supply component (600) is used to provide plasma airflow.

26. The cryo-electron microscopy workstation according to claim 25, characterized in that, The polarity of the plasma gas flow provided by the air supply component (600) is the same as that of the sample.

27. The cryo-electron microscopy workstation according to claim 23, characterized in that, The sample preparation module (1000) further includes a flow guiding component (700). The air supply component (600) is used to supply air to the carrier net (110) in a direction perpendicular to the bearing surface (112). The flow guiding component (700) is disposed on the outer periphery of the carrier net (110). The flow guiding component (700) is used to disperse the airflow supplied from the air supply component (600) along the flow guiding surface. The flow guiding surface is parallel to or coplanar with the bearing surface (112).

28. The cryo-electron microscopy workstation according to claim 23, characterized in that, The first heat sink (120) includes a mounting portion (121) and a side end portion (122) located at the end of the mounting portion (121); Along the thickness direction of the first heat sink (120), the mounting portion (121) is recessed relative to the side end (122) to form a clearance space (130). The air supply component (600) is movable to approach or move away from the carrier net (110). When the air supply component (600) approaches the carrier net (110), the air supply component (600) extends into the clearance space (130).

29. The cryo-electron microscopy workstation according to any one of claims 23 to 28, characterized in that, The sample preparation module (1000) also includes a thickness detection module and a control device. The thickness detection module and the air supply component (600) are both electrically connected to the control device. The thickness detection module is used to detect the thickness of the sample. The control device is used to control the air supply component (600) to stop supplying air when the thickness detection module detects that the sample has reached the target thickness.

30. The cryo-electron microscopy workstation according to claim 29, characterized in that, The thickness detection module includes a light generating structure and a light receiving structure. The light generating structure is disposed toward the bearing surface (112), and the light receiving structure is disposed in the optical path of the reflected light from the sample.

31. The cryo-electron microscopy workstation according to claim 29, characterized in that, The thickness detection module includes a detection electrode, which is disposed on the bearing surface (112) and is used to contact the sample.

32. The cryo-electron microscopy workstation according to claim 23, characterized in that, The bearing surface (112) includes a central region (1121) for bearing the sample and an edge region (1122) on the outer periphery of the central region (1121). The second heating element (200) is disposed corresponding to the central region (1121), and the first heating element (400) is disposed corresponding to the edge region (1122). The heating temperature of the first heating element (400) is greater than the heating temperature of the second heating element (200) so that the temperature of the edge region (1122) is higher than the temperature of the central region (1121). The sample can diffuse and thin from the central region (1121) to the edge region (1122) under the action of the temperature difference between the central region (1121) and the edge region (1122).

33. The cryo-electron microscopy workstation according to claim 1 or 2, characterized in that, The cryo-electron microscopy workstation also includes an observation module, which includes an electron microscope tube with a barrel (1001). The barrel (1001) has a pair of objective pole pieces (1002) arranged in it, and an observation gap (1003) is formed between the objective pole pieces (1002) to allow the sample to pass through.

34. The cryo-electron microscopy workstation according to claim 33, characterized in that, The objective lens pole piece (1002) includes a first pole piece (1004) and a second pole piece (1006). The first pole piece (1004) is used to focus the electron beam and has a first channel (1005). The second pole piece (1006) is used to further focus the electron beam and has a second channel (1007). The electron beam can be focused by passing through the first channel (1005) and the second channel (1007) in sequence. The distance between the first pole piece (1004) and the second pole piece (1006) is less than 4 mm, and the aperture of the first channel (1005) and the second channel (1007) is less than 2 mm.

35. The cryo-electron microscopy workstation according to claim 34, characterized in that, The first heat sink (120) includes a mounting portion (121) and a side end portion (122) located at the end of the mounting portion (121). The thickness of the mounting portion (121) is less than the thickness of the side end portion (122). The first pole shoe (1004) and the second pole shoe (1006) can extend into the space between the side ends (122).

36. The cryo-electron microscopy workstation according to claim 34, characterized in that, The first pole piece (1004) has a gradually decreasing radial dimension in the direction of approaching the second pole piece (1006); the second pole piece (1006) has a gradually decreasing radial dimension in the direction of approaching the first pole piece (1004).

37. The cryo-electron microscopy workstation according to claim 33, characterized in that, The observation module also includes a coil that generates a magnetic field between the objective lens pole pieces (1002) by being energized.

38. A cryo-electron microscopy workstation, characterized in that, include: The sample preparation module (1000) as described in any one of claims 1 to 37; The transport module (2000) includes a transport tunnel (2100) for sample passage. The transport tunnel is disposed in the observation gap (1003). The space inside the transport tunnel (2100) is isolated from the cylinder (1001). The side wall of the transport tunnel (2100) is provided with a through observation port (2110). The observation port (2110) is located on the line connecting the two objective lens pole pieces (1002). A first vacuum system and a second vacuum system, wherein the first vacuum system is used to provide a vacuum environment for the cylinder (1001) and the second vacuum system is used to provide a vacuum environment for the transport tunnel (2100).

39. The cryo-electron microscopy workstation according to claim 38, characterized in that, The conveying module (2000) further includes a first buffer chamber (2200), which is connected to the entrance (2111) of the conveying tunnel (2100). The first buffer chamber (2200) and the conveying tunnel (2100) are selectively connected. When the first buffer chamber (2200) is evacuated, the first buffer chamber (2200) and the conveying tunnel (2100) are isolated from each other. When the vacuum degree of the first buffer chamber (2200) reaches the vacuum degree of the conveying tunnel (2100), the first buffer chamber (2200) and the conveying tunnel (2100) are connected, and the sample can enter the conveying tunnel (2100) from the first buffer chamber (2200).

40. The cryo-electron microscopy workstation according to claim 38, characterized in that, The two ends of the transport tunnel (2100) are respectively formed as an inlet (2111) and an outlet (2112). The transport module (2000) is provided with at least two transport mechanisms. One of the transport mechanisms is movably disposed in the transport tunnel (2100) for moving the sample to the observation port (2110). The other transport mechanism is used to send the sample into the inlet (2111) of the transport tunnel (2100) or out from the outlet (2112) of the transport tunnel (2100). The sample can be transferred between the two transport mechanisms.

41. The cryo-electron microscopy workstation according to claim 40, characterized in that, The conveying module (2000) further includes a first buffer chamber (2200) and a second buffer chamber (2400). The first buffer chamber (2200) is connected to the entrance (2111) of the conveying tunnel (2100), and the second buffer chamber (2400) is connected to the exit (2112) of the conveying tunnel (2100). The conveying module (2000) includes three conveying mechanisms, namely a first conveying mechanism (2310), a second conveying mechanism (2320), and a third conveying mechanism. The first conveying mechanism (2310) is movably disposed in the conveying tunnel (2100), the second conveying mechanism (2320) is disposed in the first buffer chamber (2200), the second conveying mechanism (2320) is used to transfer the sample to the first conveying mechanism (2310), and the third conveying mechanism (2330) is disposed in the second buffer chamber (2400), the third conveying mechanism (2330) is used to receive the sample from the first conveying mechanism (2310).

42. The cryo-electron microscopy workstation according to claim 41, characterized in that, When at least one of the second conveying mechanism (2320) or the first conveying mechanism (2310) is working, the third conveying mechanism (2330) performs sample loading; When at least one of the third conveying mechanism (2330) or the first conveying mechanism (2310) is working, the second conveying mechanism (2320) loads the sample.

43. The cryo-electron microscopy workstation according to claim 41, characterized in that, The first buffer chamber (2200) and the transport tunnel (2100) are selectively connected. When the first buffer chamber (2200) is evacuated, the first buffer chamber (2200) and the transport tunnel (2100) are isolated from each other. When the vacuum degree of the first buffer chamber (2200) reaches the vacuum degree of the transport tunnel (2100), the first buffer chamber (2200) and the transport tunnel (2100) are connected. The second conveying mechanism (2320) transfers the sample to the first conveying mechanism (2310) from the entrance (2111) of the transport tunnel (2100). The second buffer chamber (2400) is selectively connected to the transport tunnel (2100). When the second buffer chamber (2400) is evacuated, the second buffer chamber (2400) and the transport tunnel (2100) are isolated from each other. When the vacuum degree of the second buffer chamber (2400) reaches the vacuum degree of the transport tunnel (2100), the second buffer chamber (2400) and the transport tunnel (2100) are connected. The third conveying mechanism (2330) receives the sample from the first conveying mechanism (2310) at the outlet (2112) of the transport tunnel (2100).

44. The cryo-electron microscopy workstation according to any one of claims 40 to 43, characterized in that, The first conveying mechanism (2310) disposed in the conveying tunnel (2100) includes a track (2312) and a first conveying arm (2311). The track (2312) is disposed along the extension direction of the conveying tunnel (2100). The first conveying arm (2311) is slidably connected to the track (2312) and is used to connect to at least one side of the sample.

45. The cryo-electron microscopy workstation according to claim 44, characterized in that, The sample preparation module (1000) includes a first heat sink (120), two tracks (2312) are provided, and two first conveying arms (2311) are provided. The two first conveying arms (2311) are respectively arranged in the two tracks (2312), and the two first conveying arms (2311) are respectively engaged with the first heat sink (120). The two first conveying arms (2311) synchronously drive the first heat sink (120) to move.

46. ​​The cryo-electron microscopy workstation according to claim 45, characterized in that, The first heat sink (120) includes a mounting portion (121) and a side end portion (122). The thickness of the side end portion (122) is greater than the thickness of the mounting portion (121). The mounting portion (121) is recessed relative to the side end portion (122) to form a clearance space (130). The second transmission mechanism (2320) and the third transmission mechanism (2330) both include a claw (2321). The claw (2321) includes at least a pair of spaced-apart teeth (2322). The claw (2321) extends into the clearance space (130). The mounting portion (121) is located between the two teeth (2322).

47. The cryo-electron microscopy workstation according to claim 40, characterized in that, The conveying mechanism includes a clamping structure (2300) with a groove (2301) and a movable part (2302) in the groove (2301). The movable part (2302) extends to abut against and hold the first heat sink (120) when energized, and retracts and moves away from the first heat sink (120) when de-energized to release the first heat sink (120).

48. The cryo-electron microscopy workstation according to claim 38, characterized in that, The conveying module (2000) further includes a first buffer chamber (2200), the first end (2210) of the first buffer chamber (2200) is connected to the entrance (2111) of the conveying tunnel (2100), and the second end (2220) of the first buffer chamber (2200) is used to deliver a sample; The conveying module (2000) includes a second conveying arm, which includes a first sub-arm (2323) and a second sub-arm (2324). The second sub-arm (2324) is sleeved on the outer periphery of the first sub-arm (2323). The first sub-arm (2323) is retractable relative to the second sub-arm (2324) along its own axial direction. When the second conveying arm delivers the sample into the first buffer chamber (2200), the outer periphery of the second sub-arm (2324) abuts against the opening of the second end (2220) of the first buffer chamber (2200) and is sealed to the opening of the second end (2220). The first sub-arm (2323) extends relative to the second sub-arm (2324) and can pass through the opening of the first end (2210) of the first buffer chamber (2200) to enter the conveying tunnel (2100).

49. The cryo-electron microscopy workstation according to claim 48, characterized in that, The cryo-electron microscopy workstation also includes a storage module (3000) for storing samples. The storage module (3000) is located at the second end (2220) of the first buffer chamber (2200) and is connected to the second end (2220) of the first buffer chamber (2200).

50. A method of using a cryo-electron microscope, characterized in that, include: Add the sample to the sample preparation module (1000); The sample is sent into the delivery module (2000), the delivery module (2000) and the electron microscope tube are evacuated, and the delivery module (2000) delivers the sample to the observation port (2110); Connect the first heat sink (120) to the cold source (300); The second heating element (200) heats the bearing surface (112) to a temperature suitable for the sample, so that the sample remains in a liquid state; The thinning component thins the sample on the bearing surface (112) to the target thickness; The second heating element (200) stops heating the bearing surface (112), and the cold source (300) freezes the sample; The electron microscope tube is used to observe the sample through the observation port (2110).

51. The method of using a cryo-electron microscope according to claim 50, characterized in that, The second heating element (200) heats the bearing surface (112) to a temperature suitable for the sample, keeping the sample in a liquid state, including: The second heating element (200) heats the bearing surface (112) to 4°C to 20°C.

52. The method of using a cryo-electron microscope according to claim 50, characterized in that, The thinning component thins the sample on the bearing surface (112) to the target thickness, including: The air supply component (600) supplies air to the bearing surface (112) to blow the sample thin to the target thickness.

53. The method of using a cryo-electron microscope according to claim 52, characterized in that, The thinning component supplies air to the bearing surface (112) to blow the sample thinner to the target thickness, including: The thickness detection module detects the thickness of the sample; When the sample is thinned to the target thickness, the control device controls the air supply component (600) to stop supplying air.

54. The method of using a cryo-electron microscope according to claim 52, characterized in that, The air supply component (600) supplies air to the bearing surface (112) to blow the sample thinner to the target thickness, including: The air supply component (600) supplies air to the carrier net (110) in a direction perpendicular to the bearing surface (112), and the airflow guide component (700) disperses the airflow supplied from the air supply component (600) along the airflow guide surface. The airflow guide surface is parallel to or coplanar with the bearing surface (112).

55. The method of using a cryo-electron microscope according to claim 50, characterized in that, The thinning component thins the sample on the bearing surface (112) to the target thickness, including: The first heating element (400) and the second heating element (200) heat the bearing area simultaneously, and the heating temperature of the first heating element (400) is greater than that of the second heating element (200).

56. The method of using a cryo-electron microscope according to claim 50, characterized in that, The sample is fed into the transport module (2000), the transport module (2000) and the electron microscope tube are evacuated, and the transport module (2000) transports the sample to the observation port (2110), including: The second transmission mechanism delivers the sample to the first transmission mechanism from the entrance (2111) of the transport tunnel (2100); The first transmission mechanism moves the sample in the transport tunnel (2100) to the observation gap (1003) for observation; The first transfer mechanism delivers the sample from the outlet (2112) of the transport tunnel (2100) to the third transfer mechanism.

57. The method of using a cryo-electron microscope according to claim 56, characterized in that, The sample is fed into the transport module (2000), the transport module (2000) and the electron microscope tube are evacuated, and the transport module (2000) transports the sample to the observation port (2110), including: The third transfer mechanism performs the unloading of the previous sample; and / or, the second transfer mechanism performs the loading of the previous sample.

58. The method of using a cryo-electron microscope according to claim 50, characterized in that, The electron microscope tube observes the sample through the observation port (2110), including: A magnetic field is formed between the objective lens pole pieces (1002); The electron beam is focused by the objective lens pole piece (1002); The sample passes through the observation gap (1003) between the objective lens pole pieces (1002).

59. The method of using a cryo-electron microscope according to claim 58, characterized in that, The sample preparation unit (100) includes a carrier mesh (110) having a slit region (1101) for carrying the sample; the sample passes through the observation gap (1003) between the objective lens pole pieces (1002), including: The carrier net (110) moves in the direction of extension of the slit region (1101); The carrier (110) moves to focus the electron beam at different positions within the slit region (1101).

60. The method of using a cryo-electron microscope according to claim 50, characterized in that, The sample preparation unit (100) includes a carrier wire (110) and a first heat sink (120); a cold source (300) freezes the sample, including: The cold source (300) reduces the temperature of the first heat sink (120) through heat transfer, so that the temperature of the first heat sink (120) is lower than the temperature of the carrier net (110); The first heat sink (120) reduces the temperature of the carrier net (110) through heat transfer.

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