Heating device for sample preparation of cryoelectron microscope

Abstract

The utility model relates to the technical field of cryoelectron microscope components, in particular to a heating device for a cryoelectron microscope sample, which comprises a first heating cabin provided with a first liquid carrying space; the first heating cabin comprises an embedded end matched with a sample loading port of the cryoelectron microscope, and a suspension end deviating from the embedded end and integrally formed with the embedded end; a first through hole is formed in the side, away from the embedded end, of the suspension end, and a second through hole is formed in the side, away from the suspension end, of the embedded end. In the process that the sample loading needle penetrates through the first through hole and the second through hole, the sample loading needle penetrates through the liquid with the preset temperature in the first liquid carrying space, the trace sample in the sample loading needle is heated to the preset temperature, and then the trace sample in the sample loading needle is added to a balanced copper net in the cryoelectron microscope; the temperature rising time of the sample is shortened, the temperature fluctuation in the sample transferring process is reduced, it is ensured that the sample can be directly loaded and frozen after being heated, and the state change and molecular activity of the sample caused by heating can be fully fixed.

Description

Technical Field

[0001] The utility model relates to the technical field of cryo-electron microscope components, in particular to a heating device for cryo-electron microscope sample preparation. Background Art

[0002] Cryo-EM sample preparation is a core technology used in cryo-EM sample preparation. This technique involves freezing liquid samples extremely quickly at low temperatures to form a vitreous ice layer. The sample's state is then observed using a cryo-EM microscope (at temperatures down to -185°C) for further research. During the sample preparation process, rapid freezing allows water to be rapidly cooled by liquid ethane, rendering it glassy at low temperatures and minimizing the formation of ice crystals, thus preserving the sample's structure.

[0003] A more established sample preparation method currently utilizes the Vitrobot device, manufactured by Thermo Scientific. This device provides a temperature-controlled (0-60°C) sample chamber. The Vitrobot precisely controls parameters such as the adsorption time between the sample and the copper mesh, the contact strength and duration between the sample and the filter paper, the dwell time before immersion in the coolant after adsorption, and the temperature and humidity of the sample chamber. Furthermore, the Vitrobot system ensures that key cryo-EM sample preparation parameters remain constant, thus ensuring excellent sample quality before imaging single particles or cellular components. This system is also suitable for applications in the food, industrial, pharmaceutical, and nanotechnology sectors.

[0004] However, during the freezing process, while the final freezing temperature remains consistent, the temperature of the sample prior to freezing has a significant impact on the final freezing state. While the Vitrobot maintains the sample's temperature through a temperature-controlled sample chamber, the timescale and efficiency of such heat exchange are far from sufficient for studying the molecular dynamics of biomacromolecules at different temperatures.

[0005] Therefore, the existing technology still needs to be improved and developed. Utility Model Content

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a heating device for sample preparation for cryo-electron microscopy, aiming to solve the problem that the temperature equilibrium time for heating samples in the existing cryo-electron microscopy is too long, making it impossible to fix and explore the molecular dynamic processes of biological macromolecules at different temperatures.

[0007] The technical solution of the utility model is as follows:

[0008] A heating device for preparing samples for cryo-electron microscopy comprises a first heating chamber provided with a first liquid-carrying space; the first heating chamber comprises an embedded end that matches the sample loading port of the cryo-electron microscope, and a suspended end that faces away from the embedded end and is integrally formed with the embedded end; the embedded end and the suspended end are arranged to penetrate to form the first liquid-carrying space; a first through hole is provided on the side of the suspended end facing away from the embedded end, and a second through hole is provided on the side of the embedded end facing away from the suspended end.

[0009] The heating device for preparing samples for cryo-electron microscopy, wherein the heating device for preparing samples for cryo-electron microscopy also includes a first temperature control system; a first heating element and a first temperature sensor are provided in the first liquid-carrying space; the first heating element and the first temperature sensor are electrically connected to the first temperature control system respectively.

[0010] The heating device for preparing samples for cryo-electron microscopy is characterized in that a first liquid injection port is provided on the first heating chamber for injecting liquid into the first liquid-carrying space.

[0011] The heating device for preparing samples for cryo-electron microscopy, wherein the first through hole and the second through hole are both provided with a diaphragm.

[0012] The heating device for preparing samples for cryo-electron microscopy is characterized in that a first thermal insulation layer is provided on the surface of the first heating chamber.

[0013] The heating device for cryo-electron microscopy sample preparation, wherein the shape of the suspended end includes a cuboid, a cube, a cylinder, and a cone; the suspended end and the embedded end form a funnel shape.

[0014] The heating device for preparing samples for cryo-electron microscopy, wherein the heating device for preparing samples for cryo-electron microscopy also includes a second heating chamber arranged on the side of the cryo-electron microscope loading port away from the first heating chamber.

[0015] The heating device for preparing samples for cryo-electron microscopy, wherein the heating device for preparing samples for cryo-electron microscopy also includes a second temperature control system; the second heating chamber is provided with a second liquid-carrying space; a second heating element and a second temperature sensor are provided in the second liquid-carrying space; the second heating element and the second temperature sensor are electrically connected to the second temperature control system respectively.

[0016] The heating device for preparing samples for cryo-electron microscopy, wherein the second heating chamber is provided with a third through hole corresponding to the second through hole, and a fourth through hole arranged opposite to the third through hole; the first through hole, the second through hole, the third through hole and the fourth through hole are on the same straight line.

[0017] The heating device for cryo-electron microscopy sample preparation is described, wherein the second heating chamber is provided with a second liquid injection port for injecting liquid into the second liquid-carrying space; and the surface of the second heating chamber is provided with a second thermal insulation layer.

[0018] Beneficial effects: The present invention provides a heating device for sample preparation for cryo-electron microscopy, comprising a first heating chamber provided with a first liquid-carrying space; the first heating chamber comprises an embedded end that matches the sample loading port of the cryo-electron microscope, and a suspended end that is away from the embedded end and integrally formed with the embedded end; the embedded end and the suspended end are arranged to penetrate to form the first liquid-carrying space; a first through hole is provided on the side of the suspended end away from the embedded end, and a second through hole is provided on the side of the embedded end away from the suspended end. In the process of the present invention passing the sample loading needle through the first through hole and the second through hole, the sample loading needle passes through the liquid of a predetermined temperature in the first liquid-carrying space, and uses the liquid of the predetermined temperature to heat the trace sample in the sample loading needle to a predetermined temperature, and then adds the trace sample in the sample loading needle to the copper mesh that has been balanced in the cryo-electron microscope, thereby reducing the time for sample heating and the temperature fluctuation during the sample transfer process, and also ensuring that the sample can be directly loaded and frozen after heating, and can fully fix the state changes and molecular activities of the sample caused by heating. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a structural schematic diagram of the first heating chamber;

[0020] Figure 2 This is a structural diagram of the first heating chamber from another perspective;

[0021] Figure 3 is a structural schematic diagram of the second heating chamber;

[0022] Figure 4 This is a schematic structural diagram of the second heating chamber from another perspective;

[0023] Figure 5 A schematic diagram of the first heating chamber and the second heating chamber when used in combination;

[0024] Explanation of the reference numerals: first heating chamber 10, embedded end 11, suspended end 12, first through hole 13, second through hole 14, first liquid injection port 15, first heating element 20, first temperature sensor 30, second heating chamber 40, fixed end 41, third through hole 42, fourth through hole 43, second liquid injection port 44, second sealing cover 441, second heating element 50, second temperature sensor 60, loading needle 100. DETAILED DESCRIPTION

[0025] The present invention provides a heating device for cryo-electron microscopy sample preparation. To make the purpose, technical solution, and effects of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only intended to explain the present invention and are not intended to limit the present invention.

[0026] In the description of this application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "back" and the like indicating orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting this application. In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include one or more of the said features.

[0027] Those skilled in the art will understand that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those skilled in the art in the field to which the present invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art, and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0028] like Figure 1 and Figure 2 As shown, the utility model provides a heating device for sample preparation for cryo-electron microscopy, comprising a first heating chamber 10 provided with a first liquid-carrying space; the first heating chamber 10 comprises an embedded end 11 that matches the sample loading port of the cryo-electron microscope, and a suspended end 12 that is away from the embedded end 11 and is integrally formed with the embedded end 11; the embedded end 11 and the suspended end 12 are arranged to penetrate to form the first liquid-carrying space; a first through hole 13 is provided on the side of the suspended end 12 away from the embedded end 11, and a second through hole 14 is provided on the side of the embedded end 11 away from the suspended end 12.

[0029] In this embodiment, the heating device is installed at the loading port of the cryo-electron microscope, and a liquid with a predetermined temperature is injected into the first liquid-carrying space. Then, in the process of the loading needle 100 with the sample absorbed passing through the first through hole and the second through hole, the loading needle passes through the liquid with a predetermined temperature in the first liquid-carrying space, and the liquid with a predetermined temperature is used to heat the trace sample in the loading needle to a predetermined temperature. Then, the trace sample in the loading needle is added to the copper mesh that has been balanced in the cryo-electron microscope, which reduces the time for sample heating and the temperature fluctuation during the sample transfer process, and also ensures that the sample can be directly loaded and frozen after heating, and can fully fix the state changes and molecular activities of the sample caused by heating.

[0030] Specifically, in the traditional cryo-electron microscopy sample preparation process, the temperature control of the sample before freezing often has the problem of long time and low efficiency, which limits the exploration of the molecular dynamic process of biological macromolecules at different temperatures. The utility model uses the heating device to achieve seamless connection between instant heating and rapid freezing in the cryo-electron microscopy sample preparation process. The temperature of the first heating chamber 10 is controlled by the liquid in the first liquid-carrying space, and the sample loading needle is used to achieve heat conduction with the liquid in the first liquid-carrying space during the process of passing through the first through hole 13 and the second through hole 14, so as to complete the rapid heating of the sample in the sample loading needle, and then quickly transfer it to the copper mesh in the cryo-electron microscope sample chamber for rapid freezing, thereby greatly improving the efficiency of heating and freezing.

[0031] In a preferred embodiment, the heating device for preparing samples for cryo-electron microscopy is suitable for a Vitrobot device.

[0032] In some embodiments, the sample loading needle is made of a metal needle or a polymer plastic needle. Preferably, the metal needle is a stainless steel needle, and the polymer plastic needle is made of PEEK, PPSU, or the like. These polymer plastics have excellent biocompatibility and stability, ensuring that the sample will not be contaminated or damaged during sample transfer. In addition, the metal and polymer plastic needles have a high thermal conductivity.

[0033] In some embodiments, based on the properties of polymer plastics, the hardness and wear resistance of the tip of the loading needle can be increased to improve the comfort of the grip and the loading accuracy, and to enhance its durability and ease of use.

[0034] Specifically, the heating device is used to heat the sample on the sample loading needle, which can make the temperature control accurate and stable; the stainless steel sample loading needle is used to quickly conduct heat, which ensures the uniformity and stability of the sample heating.

[0035] In some embodiments, the heating device for preparing samples for cryo-electron microscopy further comprises a first temperature control system; a first heating element 20 and a first temperature sensor 30 are provided in the first liquid-carrying space; the first heating element 20 and the first temperature sensor 30 are electrically connected to the first temperature control system, respectively. The first temperature sensor 30 can be used to monitor the temperature of the liquid in the first liquid-carrying space in real time and transmit the temperature back to the first temperature control system. The first temperature control system compares the preset temperature value with the temperature value transmitted back to the first temperature control system by the temperature sensor to determine whether the liquid in the first liquid-carrying space needs to be heated. If the preset temperature value is greater than the temperature value transmitted back to the first temperature control system by the temperature sensor, the liquid in the first liquid-carrying space is heated by the first heating element until the temperature value transmitted back to the first temperature control system by the temperature sensor is equal to the preset temperature value; if the preset temperature value is less than the temperature value transmitted back to the first temperature control system by the temperature sensor, an alarm signal is issued to stop the experimental operation. The experimental operation is restarted when the temperature value transmitted back to the first temperature control system by the temperature sensor is equal to the preset temperature value.

[0036] In some embodiments, as Figure 2 As shown, the first heating chamber 10 is provided with a first liquid injection port 15 for injecting liquid into the first liquid-carrying space. The first liquid injection port 15 facilitates injecting or replacing liquid into the first liquid-carrying space and also facilitates maintenance of the first heating element and first temperature sensor disposed in the first liquid-carrying space.

[0037] In some embodiments, the first liquid injection port 15 is provided with a first sealing cover 151 for sealing the first liquid injection port 15 to prevent the loss of liquid in the first liquid carrying space.

[0038] In some embodiments, the first heating chamber 10 is made of a heat-insulating material and has good heat-insulating performance; the first temperature control system is used to control the temperature of the liquid in the first liquid-carrying space, thereby ultimately achieving control of the sample loading temperature.

[0039] In order to facilitate understanding of the principle of the heating device for preparing samples for cryo-electron microscopy, the following is further described through an operating process, including: filling water of a predetermined temperature into the first heating chamber of the heating device, and using the first temperature control system, the first heating element and the first temperature sensor to control the water temperature; then connecting the loading needle and the 10μl pipette, aspirating 4μl of the sample, and then slowly inserting the loading needle with the sample from the first through hole and staying for 10 seconds until the sample temperature in the needle is consistent with the water temperature, and then continuing to pass the loading needle out of the second through hole to reach the copper mesh in the Vitrobot cabin, adding the sample to the copper mesh with a pipette, and finally performing the Vitrobot Blot operation.

[0040] In some embodiments, the apertures of the first through hole 13 and the second through hole 14 match the diameter of the sample loading needle of the sample loading needle 100, or the apertures of the first through hole 13 and the second through hole 14 are slightly larger than the diameter of the sample loading needle, so as to ensure that the sample loading needle can pass through the first through hole and the second through hole without causing the liquid in the first liquid-carrying space to flow out.

[0041] In some embodiments, a diaphragm is provided at both the first through hole 13 and the second through hole 14. Providing the diaphragms at the first through hole and the second through hole prevents the liquid in the first carrier space from leaking from the first through hole and the second through hole, while allowing the sample loading needle to pass through easily.

[0042] In some embodiments, a first insulation layer is provided on the surface of the first heating chamber 10. By providing the first insulation layer on the surface of the first heating chamber, the insulation effect of the first heating chamber can be improved, heat loss can be reduced, and the temperature variation of the liquid in the first heating chamber can be smaller, thereby achieving precise control of the sample heating temperature.

[0043] In some embodiments, the shape of the overhanging end 12 includes a rectangular parallelepiped, a cube, a cylinder, or a cone; the overhanging end 12 and the embedded end 11 form a funnel shape. Since the size of the cryo-electron microscope loading port is fixed and its diameter cannot be changed, designing the overhanging end and the embedded end into a funnel shape can increase the liquid volume of the first liquid-carrying space to ensure rapid sample heating. The shape of the overhanging end can also be a combination of two of the rectangular parallelepiped, cube, cylinder, or cone, such as a combination of a cylinder and a cone.

[0044] In some embodiments, as Figure 3 and Figure 4As shown, the heating device for cryo-electron microscopy sample preparation also includes a second heating chamber 40 arranged on the side of the cryo-electron microscopy sample loading port away from the first heating chamber 10. The second heating chamber 40 and the first heating chamber 10 can independently control the water temperature, so that the heating device can simultaneously process multiple samples requiring different temperatures, thereby improving experimental efficiency. For example, the temperature of the first heating chamber is higher than that of the second heating chamber, so that the sample can be rapidly cooled when entering the second heating chamber after rapid heating, and then rapidly frozen, completing the multi-stage temperature treatment of the sample.

[0045] In some embodiments, a fixed end 41 is provided at one end of the second heating chamber 40 close to the cryo-electron microscope loading port, and the fixed end 41 is embedded in the side of the cryo-electron microscope loading port away from the first heating chamber 10 to achieve the positioning of the second heating chamber 40.

[0046] In some embodiments, the heating device for cryo-electron microscopy sample preparation further includes a second temperature control system; the second heating chamber 40 is provided with a second liquid-carrying space; a second heating element 50 and a second temperature sensor 60 are provided within the second liquid-carrying space; and the second heating element 50 and the second temperature sensor 60 are each electrically connected to the second temperature control system. The second temperature sensor can monitor the temperature of the liquid within the second liquid-carrying space in real time and transmit the temperature back to the second temperature control system. The second temperature control system compares a preset temperature value with the temperature value transmitted back to the second temperature control system by the temperature sensor to determine whether the liquid within the second liquid-carrying space needs to be heated. If the preset temperature value is greater than the temperature value transmitted back to the second temperature control system by the temperature sensor, the second heating element is used to heat the liquid within the second liquid-carrying space until the temperature value transmitted back to the second temperature control system by the temperature sensor equals the preset temperature value. If the preset temperature value is less than the temperature value transmitted back to the second temperature control system by the temperature sensor, an alarm signal is issued, the experimental operation is stopped, and the experimental operation is resumed when the temperature value transmitted back to the second temperature control system by the temperature sensor equals the preset temperature value.

[0047] In some embodiments, the first temperature control system and the second temperature control system further include a temperature selector for selecting the required temperature, and automatically adjusting the water temperature in the corresponding heating chamber by using heating elements and temperature sensors through the first temperature control system and the second temperature control system, thereby simplifying the operation process and reducing the difficulty of operation.

[0048] In some embodiments, the second heating chamber 40 is provided with a third through hole 42 corresponding to the second through hole 14, and a fourth through hole 43 opposite the third through hole 42. The first through hole 13, the second through hole 14, the third through hole 42, and the fourth through hole 43 are aligned on the same straight line. A diaphragm is provided between the third through hole 42 and the fourth through hole 43.

[0049] In some embodiments, the second heating chamber 40 is provided with a second liquid injection port 44 for injecting liquid into the second liquid-carrying space. A second insulation layer is provided on the surface of the second heating chamber 40. This second insulation layer improves the insulation performance of the second heating chamber, reduces heat loss, minimizes temperature fluctuations of the liquid within the second heating chamber, and enables precise control of the sample heating temperature.

[0050] In some embodiments, the second liquid injection port 44 is provided with a second sealing cover 441 for sealing the second liquid injection port to prevent the liquid in the second liquid carrying space from being lost.

[0051] In some embodiments, a safety isolation device is provided between the first heating chamber 10 and the second heating chamber 40 to prevent water of different temperatures from mixing or leaking. Overheat protection and leakage protection are also provided to ensure the safety of the device during use.

[0052] Specifically, if Figure 5 As shown, in the process of passing the loading needle through the first through hole, the second through hole, the third through hole and the fourth through hole, the loading needle passes through the liquid of the first predetermined temperature in the first liquid-carrying space and the liquid of the second predetermined temperature in the second liquid-carrying space, and the trace sample in the loading needle is heated to the first predetermined temperature by using the liquid of the first predetermined temperature and the liquid of the second predetermined temperature and then immediately heated or cooled to the second predetermined temperature, and then the trace sample in the loading needle is added to the copper mesh that has been balanced in the cryo-electron microscope, which reduces the time for sample heating and the temperature fluctuation during the sample transfer process, and also ensures that the sample can be directly loaded and frozen after heating, and can fully fix the state changes and molecular activities of the sample caused by heating.

[0053] In summary, the utility model provides a heating device for cryo-electron microscopy sample preparation, comprising a first heating chamber provided with a first liquid-carrying space; the first heating chamber comprises an embedded end that matches the sample loading port of the cryo-electron microscope, and a suspended end that is away from the embedded end and integrally formed with the embedded end; the embedded end and the suspended end are arranged to penetrate to form the first liquid-carrying space; a first through hole is provided on the side of the suspended end away from the embedded end, and a second through hole is provided on the side of the embedded end away from the suspended end. In the process of the utility model passing the loading needle through the first through hole and the second through hole, the loading needle passes through the liquid of a predetermined temperature in the first liquid-carrying space, and uses the liquid of the predetermined temperature to heat the trace sample in the loading needle to a predetermined temperature, and then adds the trace sample in the loading needle to the copper mesh that has been balanced in the cryo-electron microscope, thereby reducing the time for sample heating and the temperature fluctuation during the sample transfer process, and also ensuring that the sample can be directly loaded and frozen after heating, and can fully fix the state changes and molecular activities of the sample caused by heating. Furthermore, the heating device has a simple structure and is easily integrated with existing Vitrobot equipment, eliminating the need for large-scale modifications to the original equipment. Furthermore, the heating device is highly compatible and can be applied to Vitrobot equipment of different models and specifications.

[0054] Furthermore, the described heating device for cryo-EM sample preparation will enable more precise and stable temperature control, improving experimental accuracy and reliability. Furthermore, the multi-temperature control design will significantly increase experimental efficiency and meet the needs of more complex experiments.

[0055] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, they can make improvements or changes based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A heating device for cryo-electron microscopy sample preparation, characterized in that: It includes a first heating chamber provided with a first liquid-carrying space; the first heating chamber includes an embedded end that matches the sample loading port of the cryo-electron microscope, and a suspended end that is away from the embedded end and is integrally formed with the embedded end; the embedded end and the suspended end are connected to form the first liquid-carrying space; a first through hole is provided on the side of the suspended end away from the embedded end, and a second through hole is provided on the side of the embedded end away from the suspended end.

2. The heating device for cryo-electron microscopy sample preparation according to claim 1, characterized in that: The heating device for preparing samples for cryo-electron microscopy also includes a first temperature control system; a first heating element and a first temperature sensor are provided in the first liquid-carrying space; the first heating element and the first temperature sensor are electrically connected to the first temperature control system respectively.

3. The heating device for cryo-electron microscopy sample preparation according to claim 1, characterized in that: The first heating chamber is provided with a first liquid injection port for injecting liquid into the first liquid carrying space.

4. The heating device for cryo-electron microscopy sample preparation according to claim 1, characterized in that: A diaphragm is provided at each of the first through hole and the second through hole.

5. The heating device for cryo-electron microscopy sample preparation according to claim 1, characterized in that: A first heat-insulating layer is provided on the surface of the first heating chamber.

6. The heating device for cryo-electron microscopy sample preparation according to claim 1, characterized in that: The shape of the suspension end includes a cuboid, a cube, a cylinder, and a cone; the suspension end and the embedded end form a funnel shape.

7. The heating device for cryo-electron microscopy sample preparation according to claim 1, characterized in that: The heating device for preparing samples for cryo-electron microscopy also includes a second heating chamber arranged on the side of the cryo-electron microscopy sample loading port away from the first heating chamber.

8. The heating device for cryo-electron microscopy sample preparation according to claim 7, characterized in that: The heating device for preparing samples for cryo-electron microscopy also includes a second temperature control system; the second heating chamber is provided with a second liquid-carrying space; a second heating element and a second temperature sensor are provided in the second liquid-carrying space; the second heating element and the second temperature sensor are electrically connected to the second temperature control system respectively.

9. The heating device for cryo-electron microscopy sample preparation according to claim 7, characterized in that: The second heating chamber is provided with a third through hole corresponding to the second through hole, and a fourth through hole arranged opposite to the third through hole; the first through hole, the second through hole, the third through hole and the fourth through hole are on the same straight line.

10. The heating device for cryo-electron microscopy sample preparation according to claim 8, characterized in that: The second heating chamber is provided with a second liquid injection port for injecting liquid into the second liquid-carrying space; and the surface of the second heating chamber is provided with a second thermal insulation layer.

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