A sample preparation module, a full-process cryo-EM workstation and a cryo-EM sample preparation method

Abstract

The application discloses a sample preparation module, a whole-process cryo-EM workstation and a cryo-EM sample preparation method. The sample preparation module comprises a sample bearing unit, a thinning component, a second heating component and a cold source. The sample bearing unit has a bearing surface for bearing a sample. The thinning component comprises a first heating component and / or an air supply component, and is used for thinning the sample on the bearing surface. The second heating component is arranged in or connected to the sample bearing unit, and is used for keeping the sample liquid during sample thinning. The cold source is connected to the sample bearing unit and is used for conducting cold to the sample bearing unit. The cold source can freeze the bearing surface when the second heating component stops heating. The sample preparation module can controllably thin the sample, and improves the accuracy of thickness control during sample preparation.

Description

Technical Field

[0001] This application relates to the field of electron microscopy sample preparation technology, and in particular to a sample preparation module, a full-process cryo-electron microscopy workstation, and a cryo-electron microscopy sample preparation method. 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. Summary of the Invention

[0004] To solve at least one of the above-mentioned technical problems, this application provides a sample preparation module that can controllably thin the sample, thereby improving the accuracy of thickness control during sample preparation. The technical solution adopted is as follows.

[0005] The sample preparation module provided in the first aspect of this application includes a sample carrying unit, a thinning component, a second heating component, and a cold source. The sample carrying unit has a carrying surface for carrying a sample. The thinning component includes a first heating component and / or an air supply component, and the thinning component is used to thin the sample on the carrying surface. The second heating component is disposed in or connected to the sample carrying unit and is used to keep the sample in a liquid state during sample thinning. The cold source is connected to the sample carrying unit and is used to conduct cold to the sample carrying unit. The cold source can freeze the carrying surface when the second heating component stops heating.

[0006] In some embodiments of the first aspect of this application, the sample carrier unit is provided with a first film, the surface of the first film is formed as the carrier surface, and the first film is provided with a through structure penetrating both sides of the first film.

[0007] In some embodiments of the first aspect of this application, the air supply component includes a first air supply section and a second air supply section, the first air supply section and the second air supply section are symmetrically disposed on both sides of the first film, and the first air supply section and the second air supply section are used to supply air to both sides of the first film simultaneously.

[0008] In some embodiments of the first aspect of this application, the air pressure of the first air supply section and the second air supply section is the same.

[0009] In some embodiments of the first aspect of this application, the sample carrier unit includes a carrier mesh, the first film is disposed on the carrier mesh, and the second heating component is disposed in the carrier mesh.

[0010] In some embodiments of the first aspect of this application, the sample preparation module includes a fixing member, the second heating member is disposed on the fixing member, and when the sample carrying unit is installed on the fixing member, the second heating member is in contact with the bottom surface of the sample carrying unit.

[0011] In some embodiments of the first aspect of this application, the sample carrier unit is provided with a light-transmitting second film, the surface of the second film is formed as the carrier surface, and the second film is continuously disposed.

[0012] In some embodiments of the first aspect of this application, the sample carrier unit includes a carrier mesh, the second film is disposed on the carrier mesh, and the second heating component is disposed in the carrier mesh.

[0013] In some embodiments of the first aspect of this application, the sample preparation module includes a fixing member, the second heating member is disposed on the fixing member, and when the sample carrying unit is installed on the fixing member, the second heating member is in contact with the bottom surface of the sample carrying unit.

[0014] In some embodiments of the first aspect of this application, the air supply component is used to provide a plasma flow.

[0015] In some embodiments of the first aspect 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.

[0016] In some embodiments of the first aspect of this application, the sample preparation module further includes a flow guiding component, the air supply component is used to supply air to the sample carrying unit in a direction perpendicular to the bearing surface, the flow guiding component is disposed on the outer periphery of the sample carrying unit, 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.

[0017] In some embodiments of the first aspect of this application, the airflow guiding component includes a vacuum device for absorbing the airflow delivered by the air supply component.

[0018] In some embodiments of the first aspect of this application, the sample carrier unit includes a carrier mesh and a first heat sink, at least one end of the carrier mesh is encapsulated in the first heat sink, the carrier surface is exposed from the first heat sink, the first heat sink is used to conduct cooling to the end of the heat sink, and the cold source is connected to the first heat sink and is used to conduct cooling to the first heat sink.

[0019] In some embodiments of the first aspect 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, 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.

[0020] In some embodiments of the first aspect of this application, the thickness of the side end portion is greater than the thickness of the mounting portion.

[0021] In some embodiments of the first aspect of this application, the thickness of the side end portion and the mounting portion gradually transitions.

[0022] In some embodiments of the first aspect of this application, the first heat sink has two said side ends, which are symmetrically arranged about the mounting portion.

[0023] In some embodiments of the first aspect of this application, 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 sample carrier unit, and when the air supply component approaches the sample carrier unit, the air supply component extends into the clearance space.

[0024] In some embodiments of the first aspect 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.

[0025] In some embodiments of the first aspect 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.

[0026] In some embodiments of the first aspect of this application, the thickness detection module includes a detection electrode disposed on the bearing surface and used to contact the sample.

[0027] In some embodiments of the first aspect of this application, the bearing surface includes a central region for bearing a 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.

[0028] In some embodiments of the first aspect of this application, the first heating element is disposed around the second heating element.

[0029] In some embodiments of the first aspect of this application, the thinning component includes at least two spaced-apart first heating components, with the second heating component located between the first heating components.

[0030] In some embodiments of the first aspect of this application, the first heating element and the second heating element are integrally formed, and both the first heating element and the second heating element include heating units, wherein the number of heating units in the first heating element is greater than the number in the second heating element.

[0031] Secondly, this application provides a full-process cryo-electron microscopy workstation, including an observation module and a sample preparation module provided in the first aspect, wherein the sample preparation module and the observation module are set up independently.

[0032] Thirdly, this application provides a method for preparing cryo-electron microscopy samples, including...

[0033] Connect the sample carrier unit to a cold source;

[0034] The second heating element heats the bearing surface to a temperature suitable for the sample.

[0035] The sample is added to the sample carrier unit while maintaining the sample in a liquid state;

[0036] The thinning component reduces the sample on the bearing surface to the target thickness;

[0037] The second heating element stops heating the bearing surface, and the cold source freezes the sample.

[0038] In some embodiments of the third aspect of this application, the second heating element heats the bearing surface to a temperature suitable for the sample, including: the second heating element heats the bearing surface to 4°C to 20°C.

[0039] In some embodiments of the third aspect of this application, the thinning component thins the sample on the bearing surface to a target thickness, including: the air supply component supplying air to the bearing surface to blow the sample to thin it to the target thickness.

[0040] In some embodiments of the third aspect of this application, the air supply component supplies air to the bearing surface to blow the sample thinner to the target thickness, including: a thickness detection module detecting the thickness of the sample; and a control device controlling the air supply component to stop supplying air when the sample is thinned to the target thickness.

[0041] In some embodiments of the third aspect of this application, the sample preparation module further includes a flow guiding component disposed on the outer periphery of the sample carrying unit; the air supply component supplies air to the carrying surface to blow the sample thinning to the target thickness, including: the air supply component supplies air to the sample carrying unit in a direction perpendicular to the carrying surface, and the flow guiding component disperses the airflow supplied by the air supply component along the flow guiding surface, the flow guiding surface being parallel or coplanar with the carrying surface.

[0042] In some embodiments of the third aspect of this application, the thinning component thins the sample on the bearing surface to a target thickness, including: a first heating component and a second heating component simultaneously heating the bearing area, and the heating temperature of the first heating component is greater than that of the second heating component.

[0043] The embodiments of this application have at least the following beneficial effects: the cold source has a freezing effect on the sample support unit, maintaining the sample support unit at a low temperature. The continuous heating of the sample by the second heating component prevents the sample from freezing and condensing. While the sample remains liquid, the thinning component thins the sample surface, spreading the liquid sample outwards, thus achieving the liquid thinning effect. Once the sample is thinned to the target thickness, the heating of the second heating component is stopped. At this point, the cooling effect of the cold source is rapidly transferred from the sample support unit to the support surface, quickly freezing and condensing the sample and maintaining it at the target thickness. This prevents the sample from returning to its original thickness due to surface tension after the thinning component stops freezing. The coordinated action of the second heating component, the thinning component, and the cold source enables sample thinning operations, achieving controllable sample thickness preparation, and timely freezing after thinning to maintain the sample in a suitable shape. 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. Attached Figure Description

[0044] 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.

[0045] Figure 1 This is a schematic diagram of the sample carrier unit provided in Embodiment 2 of this application;

[0046] Figure 2This is a schematic diagram of a first example of the sample preparation module provided in Embodiment 2 of this application;

[0047] Figure 3 This is a schematic diagram of a second example of the sample preparation module provided in Embodiment 2 of this application;

[0048] Figure 4 for Figure 3 A magnified view of part A;

[0049] Figure 5 This is a schematic diagram of a third example of the sample preparation module provided in Embodiment 2 of this application;

[0050] Figure 6 This is a schematic diagram of the fourth example of the sample preparation module provided in Embodiment 2 of this application;

[0051] Figure 7 This is a schematic diagram of the fifth example of the sample preparation module provided in Embodiment 2 of this application;

[0052] Figure 8 This is a schematic diagram of the sixth example of the sample preparation module provided in Embodiment 2 of this application;

[0053] Figure 9 This is a schematic diagram of a first embodiment of the second heating component in the sample preparation module provided in Embodiment 2 of this application;

[0054] Figure 10 This is a schematic diagram of a second embodiment of the second heating component in the sample preparation module provided in Embodiment 2 of this application;

[0055] Figure 11 This is a schematic diagram of the sample preparation module provided in Embodiment 3 of this application;

[0056] Figure 12 This is a schematic diagram showing the layout of a first example of the first heating component and the second heating component in the sample preparation module provided in Embodiment 3 of this application;

[0057] Figure 13 This is a schematic diagram showing the layout of a second example of the first heating component and the second heating component in the sample preparation module provided in Embodiment 3 of this application;

[0058] Figure 14 A flowchart illustrating the cryo-electron microscopy sample preparation method provided in this application embodiment.

[0059] Figure label:

[0060] 1000, Sample Preparation Module;

[0061] 100. Sample carrier unit; 110. Carrier net; 112. Carrier 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; 122. Side end; 130. Clearance space;

[0062] 200. Second heating component; 201. Fixing component;

[0063] 300. Cold source;

[0064] 400. First heating element;

[0065] 600. Air supply component; 610. First air supply section; 620. Second air supply section;

[0066] 700. Airflow guiding components; Detailed Implementation

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Example 1

[0073] Firstly, this application provides a complete cryo-electron microscopy workstation (not shown), which includes a sample preparation module 1000 and an observation module (not shown). The sample preparation module 1000 can prepare samples that meet the observation requirements, and the samples can be stored or immediately sent 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 a storage module can be set up in the workstation, and the sample can be sent to the storage module for storage, or directly sent to the observation module for observation. The independent setting of the sample preparation module 1000 and the observation module can improve the independence of the operation between the two modules, reduce the problem of mutual interference between the two modules, and improve the reliability of the sample preparation module 1000 and the observation module.

[0074] Optionally, the sample preparation module 1000 and the observation module can be connected via a sample delivery channel or other structure.

[0075] The following section will provide a further introduction to the sample preparation module 1000.

[0076] Example 2

[0077] Secondly, please refer to Figures 1 to 4This application provides a sample preparation module 1000, including a sample carrying unit 100, a thinning component, a second heating component 200, and a cold source 300. The sample carrying unit 100 has a carrying surface 112 for carrying a sample. The thinning component is used to thin the sample on the carrying surface 112. The second heating component is disposed in or connected to the sample carrying unit 100 to maintain the sample in a liquid state during sample thinning. The cold source is connected to the sample carrying unit 100 and is used to conduct cooling to the sample carrying unit 100. The cold source can freeze the carrying surface 112 when the second heating component stops heating.

[0078] For example, the thinned component may include an air supply component 600.

[0079] The cold source has a freezing effect on the sample carrier unit 100, maintaining it at a low temperature. Specifically, the cold source can quickly conduct away heat from the sample carrier unit 100 and the sample placed on the carrier surface 112, thus keeping the sample and sample carrier unit 100 at a low temperature. At this time, it is also necessary to keep the sample in a liquid state to avoid freezing and preventing the thinning operation from being impossible. Therefore, the continuous heating of the sample by the second heating component can prevent the sample from freezing and condensing. While the sample remains liquid, the air supply component 600 blows the sample surface, spreading the liquid sample outwards, thereby achieving the effect of thinning the liquid in the blowing area. When the sample is thinned to the target thickness, the heating of the second heating component is stopped. At this time, the cooling effect of the cold source is rapidly transferred from the sample carrier unit 100 to the carrier surface 112, thereby quickly freezing and condensing the sample and maintaining it at the target thickness. This prevents the sample from returning to its original thickness due to liquid surface tension after the air supply component 600 stops blowing. The second heating element typically operates only on the localized area of ​​the sample-bearing surface 112 where the sample is located. Therefore, the heating effect generally does not affect the low-temperature state of other areas of the sample-bearing unit 100. By utilizing the combined action of the second heating element, the air supply element 600, and the cold source, sample thinning can be achieved, enabling controllable sample thickness preparation. Furthermore, the sample can be frozen promptly after thinning to maintain its appropriate shape. 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.

[0080] Understandably, the sample thickness can be controlled by adjusting parameters such as the blowing power and duration of the air supply component 600. The second heating component continuously heats the sample until the target thickness is reached to prevent freezing and solidification. Heating stops only when the target thickness is reached. At this point, due to the disappearance of heating, the small amount of heat remaining in the localized area of ​​the sample can be rapidly conducted and dissipated to the cold source through the sample support unit 100. Since the sample support unit 100 itself has a low temperature and is typically small in size, the freezing and cooling effect of the cold source can be transferred extremely quickly from the sample support unit 100 to the support surface 112. This configuration shortens the freezing action transmission path, increases the rate, and achieves an ultra-fast freezing effect.

[0081] In some embodiments, the sample carrying unit 100 is provided with a first film 113, the surface of which is formed as a carrying 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 carry the sample, allowing the sample carrying unit 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 airflow to the two 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 airflow 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.

[0082] Optionally, the through structure 1131 can be a through hole, through groove, etc.

[0083] In some embodiments, please refer to Figure 5 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.

[0084] 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.

[0085] In some embodiments, please refer to Figure 9 The sample carrier unit 100 includes a carrier mesh 110, a first film 113 disposed on the carrier mesh 110, and a second heating component disposed in the carrier mesh 110. The second heating component can directly heat the carrier mesh 110, and 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.

[0086] Optionally, the second heating element 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.

[0087] 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 element 200 is disposed on the fixing member 201. When the sample carrying unit 100 is installed on the fixing member 201, the second heating element 200 contacts the bottom surface of the sample carrying unit 100. The second heating element 200 is separately disposed on the fixing member 201, other than the sample carrying unit 100, which helps to simplify the structure and reduce the cost of the sample carrying unit 100 itself. When the sample carrying unit 100 is installed on the fixing member 201, the bottom surface of the sample carrying unit 100 can contact the second heating element 200, thus allowing the second heating element 200 to heat the carrying surface 112 of the sample carrying unit 100. When the sample carrying unit 100 is removed from the fixing member 201, the second heating element 200 separates from the sample carrying unit 100. In this way, the second heating element 200 can heat multiple different sample carrying units 100. On the other hand, the fastener 201 can also support and stabilize the sample carrier unit 100, thereby improving the stability of the sample carrier unit 100 during the sample preparation process.

[0088] In some cases, when a first film 113 with a through structure 1131 is used to carry the sample, and a fastener 201 is used to support the sample carrier unit 100, 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 that the sample may stick to the surface of the fastener 201. In this case, removing the sample carrier unit 100 from the fastener 201 may result in the destruction of the sample.

[0089] Therefore, to resolve this issue, please refer to [link / reference]. Figure 6 In some embodiments, the sample carrier unit 100 is provided with a light-transmitting second film 114, the surface of which is formed as a carrier 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 sample carrier unit 100 and the fixing member 201 can be easily separated, avoiding the problem of sample adhesion.

[0090] It should be noted that even if the sample carrier unit 100 uses the first film 113, the sample is not necessarily adhered 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 carrier unit 100 being provided 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.

[0091] In some embodiments, the sample carrier unit 100 includes a mesh 110, a second film 114 disposed on the mesh, and a second heating element 200 disposed in the mesh 110. The second heating element 200 can directly heat the mesh 110, eliminating the need for other external devices to heat the mesh 110, thus simplifying the structure of the sample preparation module 1000.

[0092] 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.

[0093] In some embodiments, 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 sample carrying unit 100 is mounted on the fixing member 201, the second heating member 200 contacts the bottom surface of the sample carrying unit 100. The second heating member 200 is separately disposed on the fixing member 201 other than the sample carrying unit 100, which helps to simplify the structure and reduce the cost of the sample carrying unit 100 itself. When the sample carrying unit 100 is mounted on the fixing member 201, the bottom surface of the sample carrying unit 100 can contact the second heating member 200, thus allowing the second heating member 200 to heat the carrying surface 112 of the sample carrying unit 100. When the sample carrying unit 100 is removed from the fixing member 201, the second heating member 200 separates from the sample carrying unit 100. In this way, the second heating member 200 can heat multiple different sample carrying units 100. On the other hand, the fastener 201 can also support and stabilize the sample carrier unit 100, thereby improving the stability of the sample carrier unit 100 during the sample preparation process.

[0094] 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.

[0095] 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.

[0096] In some embodiments, please refer to Figure 8 The sample preparation module 1000 also includes a flow guiding component 700. The air supply component 600 supplies air to the sample carrying unit 100 in a direction perpendicular to the bearing surface 112. The flow guiding component 700 is disposed on the outer periphery of the sample carrying unit and is used to disperse 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.

[0097] 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.

[0098] In some embodiments, the sample carrier unit 100 includes a carrier mesh 110 and a first heat sink 120. At least one end of the carrier mesh 110 is encapsulated in the first heat sink 120, and the carrier 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. A cold source 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 heat conduction effect of the first heat sink 120 on the carrier mesh 110, and enabling the cold source 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, 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 conducts heat to the carrier mesh 110, achieving a freezing effect on both the carrier mesh 110 and the sample.

[0099] In some embodiments, 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 enclose 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 is 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 to the bearing area, freezing samples, and observing samples.

[0100] 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.

[0101] In some embodiments, the thickness of the side end portion 122 and the mounting portion 121 gradually changes. Since the thicknesses of the side end portion 122 and the mounting portion 121 are different, by providing a gradual change in thickness between them, 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.

[0102] 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 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, thereby improving the cooling effect on the carrier mesh 110 and improving the efficiency of cooling and freezing the carrier mesh 110.

[0103] 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, and the mounting portion 121 is suspended relative to the second heat sink. 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, and improve the reliability of the first heat sink 120.

[0104] In some embodiments, 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 sample support unit 100. When the air supply component 600 approaches the sample support unit 100, the air supply component 600 extends into the clearance space 130. The clearance space 130 formed by the recess of the mounting portion 121 relative to the side end portion 122 allows the air supply component 600 to enter the clearance space 130, thereby further shortening the distance between the air supply component 600 and the support surface 112, enabling the air supply component 600 to get closer to the sample and improving the effect of blowing and thinning the sample.

[0105] 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.

[0106] 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.

[0107] Alternatively, the thickness of the sample can be measured using optical principles or electrical principles, which will be explained below.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Example 3

[0112] The difference between Example 3 and Example 2 is that the sample can be thinned using a temperature difference. The remaining undeveloped portions can be found in Example 2. Please refer to [link / reference]. 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] Example 4

[0118] In this embodiment, the thinning component can simultaneously include a first heating component 400 and an air supply component 600. While thinning by blowing air, the temperature difference between the first heating component 400 and the second heating component 200 is used to spread the sample liquid, thereby achieving a better sample thinning effect. The implementation methods and effects of the first heating component 400 and the air supply component 600 can be found in Embodiments 2 and 3, respectively, and will not be repeated here.

[0119] Example 5

[0120] Please see Figure 14 This application provides a method for preparing cryo-electron microscopy samples, comprising the following steps:

[0121] S100. Connect the sample carrier unit 100 to the cold source 300;

[0122] S200. The second heating element 200 heats the bearing surface 112 to a temperature suitable for the sample; wherein, the temperature suitable for the sample means that the temperature is not too high, which would cause the sample to become inactive when added to the bearing surface 112, and the temperature is not too low, which would cause the sample to freeze and solidify and lose its fluidity. Therefore, the temperature suitable for the sample can be from 4°C to 20°C.

[0123] S300. Add the sample to the sample carrier unit 100 to keep the sample in a liquid state;

[0124] S400. The thinning component reduces the thickness of the material on the bearing surface 112 to the target thickness;

[0125] S500. The second heating element 200 stops heating the bearing surface 112, and the cold source freezes the sample.

[0126] 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 level, the second heating component 200 can stop heating the bearing area, allowing the sample to be quickly frozen solidified and maintained at the reduced thickness, achieving a sample preparation effect with controllable thickness.

[0127] In some embodiments, step S400, thinning the sample on the bearing surface 112 to the target thickness by the thinning component, includes:

[0128] The S410 air supply component delivers air to the bearing surface 600 times, blowing the sample to reduce its thickness to the target thickness.

[0129] In some embodiments, step S410, where the air supply component 600 supplies air to the bearing surface 112 to thin the sample to the target thickness, includes:

[0130] S411. The thickness detection module detects the thickness of the sample;

[0131] S412. When the sample is thinned to the target thickness, the control device controls the air supply component 600 to stop supplying air.

[0132] 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.

[0133] In some embodiments, the sample preparation module 1000 further includes a flow guiding component 700 disposed on the outer periphery of the sample carrying unit 100. Step S400, where the air supply component 600 supplies air to the carrying surface 112 to thin the sample to the target thickness, includes:

[0134] The air supply component 600 supplies air to the sample support unit 100 in a direction perpendicular to the support surface 112. The flow guiding component 700 disperses the airflow supplied by the air supply component 600 along the flow guiding surface, which is parallel to or coplanar with the support 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 support 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 out from the center, thereby achieving the effect of sample thinning.

[0135] In some embodiments, step S400, thinning the sample on the bearing surface 112 to the target thickness by the thinning component, includes:

[0136] S420. 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.

[0137] 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.

[0138] 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 sample preparation module, characterized in that: include A sample carrying unit has a carrying surface for carrying a sample; A thinning component, including a first heating component, is used to thin a sample on the bearing surface; The second heating element is disposed in or connected to the sample carrying unit and is used to keep the sample liquid during sample thinning. The carrying surface includes a central area for carrying the sample and an outer edge area of ​​the central area. The second heating element is disposed corresponding to the central area, and the first heating element is disposed corresponding to the edge area. The heating temperature of the first heating element is greater than that of the second heating element. A cold source is connected to the sample support unit and is used to conduct cold to the sample support unit. The cold source can freeze the support surface when the heating element stops heating.

2. The sample preparation module according to claim 1, characterized in that: The sample carrier unit is provided with a first film, the surface of which is formed as the carrier surface, and the first film is provided with a through structure that penetrates both sides of the first film.

3. The sample preparation module according to claim 2, characterized in that: The thinning component further includes an air supply component, which includes a first air supply section and a second air supply section. The first air supply section and the second air supply section are symmetrically arranged on both sides of the first film. The first air supply section and the second air supply section are used to supply air to both sides of the first film simultaneously.

4. The sample preparation module according to claim 3, characterized in that: The air pressure supplied by the first air supply section and the second air supply section is the same.

5. The sample preparation module according to claim 2, characterized in that: The sample carrier unit includes a carrier mesh, the first film is disposed on the carrier mesh, and the second heating component is disposed in the carrier mesh.

6. The sample preparation module according to claim 2, characterized in that: The sample preparation module includes a fixing component, and the second heating component is disposed on the fixing component. When the sample carrying unit is installed on the fixing component, the second heating component is in contact with the bottom surface of the sample carrying unit.

7. The sample preparation module according to claim 1, characterized in that: The sample carrier unit is provided with a light-transmitting second film, the surface of which is formed as the carrier surface, and the second film is continuously arranged.

8. The sample preparation module according to claim 7, characterized in that: The sample carrier unit includes a carrier mesh, the second film is disposed on the carrier mesh, and the second heating component is disposed in the carrier mesh.

9. The sample preparation module according to claim 7, characterized in that: The sample preparation module includes a fixing component, and the second heating component is disposed on the fixing component. When the sample carrying unit is installed on the fixing component, the second heating component is in contact with the bottom surface of the sample carrying unit.

10. The sample preparation module according to claim 3 or 4, characterized in that: The air supply component is used to provide plasma airflow.

11. The sample preparation module according to claim 10, characterized in that: The polarity of the plasma gas flow provided by the air supply component is the same as that of the sample.

12. The sample preparation module according to claim 3 or 4, characterized in that: The sample preparation module further includes a flow guiding component. The air supply component is used to supply air to the sample carrying unit in a direction perpendicular to the bearing surface. The flow guiding component is disposed on the outer periphery of the sample carrying unit. The flow guiding component is used to disperse the airflow supplied by the air supply component along the flow guiding surface. The flow guiding surface is parallel to or coplanar with the bearing surface.

13. The sample preparation module according to claim 12, characterized in that: The airflow guiding component includes a vacuum device, which is used to absorb the airflow delivered by the air supply component.

14. The sample preparation module according to claim 3 or 4, characterized in that: The sample carrier unit includes a carrier mesh and a first heat sink. At least one end of the carrier mesh is encapsulated in the first heat sink, and the carrier surface is exposed from the first heat sink. The first heat sink is used to conduct heat to the end of the heat sink, and the cold source is connected to the first heat sink and is used to conduct heat to the first heat sink.

15. The sample preparation module according to claim 14, characterized in that: The first heat sink includes a mounting part and a side end connected in sequence. The mounting part is used to enclose the carrier mesh. The mounting part is provided with an observation window. The bearing surface is exposed from the observation window. The side end is used to connect to a cold source.

16. The sample preparation module according to claim 15, characterized in that: The thickness of the side end is greater than the thickness of the mounting portion.

17. The sample preparation module according to claim 16, characterized in that: The thickness of the side end and the mounting part gradually transitions.

18. The sample preparation module according to claim 16, characterized in that: The first heat sink has two side ends, which are symmetrically arranged about the mounting portion.

19. The sample preparation module according to any one of claims 16 to 18, characterized in that: 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 sample carrying unit. When the air supply component approaches the sample carrying unit, the air supply component extends into the clearance space.

20. The sample preparation module according to claim 3 or 4, characterized in that: The sample preparation module also 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. 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.

21. The sample preparation module according to claim 20, characterized in that: The thickness detection module includes a light generating structure and a light receiving structure. The light generating structure is disposed facing the bearing surface, and the light receiving structure is disposed in the optical path of the reflected light from the sample.

22. The sample preparation module according to claim 20, characterized in that: The thickness detection module includes a detection electrode, which is disposed on the bearing surface and is used to contact the sample.

23. The sample preparation module according to claim 1, characterized in that: The temperature of the edge region is higher than that of the center region, and the sample can diffuse and thin from the center region to the edge region under the influence of the temperature difference between the center region and the edge region.

24. The sample preparation module according to claim 23, characterized in that: The first heating element is arranged around the second heating element.

25. The sample preparation module according to claim 23, characterized in that: The thinning component includes at least two spaced-apart first heating components, with the second heating component located between the first heating components.

26. The sample preparation module according to claim 23, characterized in that: The first heating element and the second heating element are integrally formed. Both the first heating element and the second heating element include heating units, and the number of heating units in the first heating element is greater than the number in the second heating element.

27. A complete cryo-electron microscopy workstation, characterized in that: It includes an observation module and a sample preparation module as described in any one of claims 1 to 26, wherein the sample preparation module and the observation module are set independently.

28. A method for preparing cryo-electron microscopy samples, applied to the sample preparation module as described in claim 1, characterized in that: include Connect the sample carrier unit to a cold source; The second heating element heats the bearing surface to a temperature suitable for the sample. The sample is added to the sample carrier unit while maintaining the sample in a liquid state; The thinning component reduces the sample on the bearing surface to the target thickness; The second heating element stops heating the bearing surface, and the cold source freezes the sample.

29. The method for preparing cryo-electron microscopy samples according to claim 28, characterized in that: The second heating element heats the bearing surface to a temperature suitable for the sample, including: The second heating element heats the bearing surface to 4°C to 20°C.

30. The method for preparing cryo-electron microscopy samples according to claim 28, characterized in that: The thinning component thins the sample on the bearing surface to the target thickness, including: The air supply component delivers air to the bearing surface, causing the sample to thin to the target thickness.

31. The method for preparing cryo-electron microscopy samples according to claim 30, characterized in that: The thinning component supplies air to the bearing surface, blowing the sample to thin it 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 to stop supplying air.

32. The method for preparing cryo-electron microscopy samples according to claim 30, characterized in that: The air supply component delivers air to the bearing surface, blowing the sample to thin it to the target thickness, including: The air supply component supplies air to the sample support unit in a direction perpendicular to the support surface, and the airflow guide component disperses the airflow supplied by the air supply component along the airflow guide surface, which is parallel or coplanar with the support surface.

33. The method for preparing cryo-electron microscopy samples according to claim 28, characterized in that: The thinning component thins the sample on the bearing surface to the target thickness, including: 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.

Images