A method and apparatus for controlled instantaneous freezing of electron microscope samples

Abstract

This invention discloses a controllable instantaneous freezing method and apparatus for electron microscopy sample preparation. The method includes: S10: A TEM copper mesh with the sample is mounted on the sample stage. The control system monitors the sample reaction process. When the sample reaction reaches a trigger threshold, the control system automatically sends a freezing trigger signal; S20: A lead screw drive device drives the lead screw through a high-pressure pneumatic system to immerse the sample stage in the cryogenic liquid in the freezing chamber within 50 ms, thereby obtaining a sample that is instantaneously frozen within 100 ms; S30: The sample stage is separated from the lead screw, and the TEM copper mesh is removed and transferred to a liquid nitrogen freezing transfer rod; S40: The liquid nitrogen freezing transfer rod is inserted into a transmission electron microscope to obtain TEM images of the sample at corresponding moments during liquid crystal phase transitions or electrochemical processes. Through the above settings, when the sample reaction reaches the trigger threshold, the freezing process is automatically triggered, thereby accurately capturing the non-equilibrium structure of the sample at specific reaction states and reaction time points.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of frozen electron microscopy sample preparation technology, in particular to a controllable transient freezing electron microscopy sample preparation method and device. BACKGROUND

[0002] Freeze electron microscopy technology, also known as cryo-EM, can effectively reduce the damage of electron beam to sensitive samples, and has become a key tool for analyzing high-resolution structures of biological macromolecules. One of its core technologies is sample preparation, that is, how to instantaneously freeze water or solution in the sample into amorphous ice, thereby preserving the near-native state of the sample.

[0003] In recent years, cryo-EM technology has been gradually applied to the field of material science, especially in battery systems for observing lithium dendrites, solid electrolyte interphase (SEI) and other metastable structures sensitive to electron beams. Transient structures with short lifetimes and susceptible to environmental disturbances are commonly present during electrochemical reactions, and such structures have a key influence on interface reaction mechanisms and material evolution. Due to limitations in time response, environmental control, and beam dose of existing characterization methods, such transient structures are difficult to effectively capture and stabilize imaging, which has become a technical bottleneck in related research fields. For example, Zhang et al. observed the swelling process of SEI in liquid electrolyte using a cryo-scanning transmission electron microscope, but overall, existing technologies still cannot systematically obtain rapid evolution information of electrochemical interfaces.

[0004] However, the existing mainstream freeze sample preparation technology is mainly designed for samples in thermodynamic equilibrium state. Although the sample environment temperature can be controlled within a certain range, the freezing process is usually manually triggered or triggered by preset time, and cannot be precisely synchronized with external temperature jump or potential step, etc. to trigger rapid dynamic processes such as chemical reactions and phase changes. Although there have been attempts to combine electrochemical operations with freeze sample preparation methods, such as electrochemical tweezers and modified coin cell platforms, these methods still rely heavily on manual operation and are difficult to achieve repeatable automatic triggering and transient freezing at specific millisecond to second reaction time points. Therefore, the existing cryo-EM sample preparation technology still has obvious deficiencies in capturing metastable and non-equilibrium structures generated during chemical reactions or self-assembly processes, and lacks a sample preparation method that can achieve high-precision and repeatable time sequence control. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a controllable transient freezing electron microscopy sample preparation method and device, which can automatically trigger the freezing process when the sample reaction reaches the trigger threshold, thereby accurately capturing the non-equilibrium structure of the sample at a specific reaction state and reaction time point.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] A controllable instant freezing electron microscope sample preparation method, comprising the following steps:

[0008] S10: install the TEM copper mesh with the sample on the sample stage, monitor the reaction process of the sample through the control system, and when the sample reaction reaches the trigger threshold, the control system automatically sends a freezing trigger signal;

[0009] S20: After receiving the freezing trigger signal, the lead screw driving device drives the lead screw through the high-pressure pneumatic system, and uses the lead screw to drive the sample stage to immerse in the freezing liquid in the freezing chamber within 50ms, so as to obtain the sample instantaneously frozen within 100ms;

[0010] S30: Separate the sample stage from the lead screw by operating the clamping mechanism, take out the TEM copper mesh with the frozen sample in the sample stage with the pre-cooled forceps, and then transfer to the liquid nitrogen freezing transfer rod;

[0011] S40: Insert the liquid nitrogen freezing transfer rod into the transmission electron microscope quickly to obtain the TEM imaging of the frozen sample at the corresponding moment of the liquid crystal phase change or the electrochemical process.

[0012] Further, in the S20, the gas pressure value of the high-pressure pneumatic system driving the lead screw is 6bar to 8bar, and the displacement value of the lead screw driving the sample stage within 50ms is 12cm±1cm.

[0013] Further, in the S40, the freezing liquid is selected as liquid nitrogen pre-cooled propane.

[0014] Further, before the S10, it further comprises: S00: sample preparation: take 1μL of dichloromethane solution of liquid crystal molecules with a concentration of 0.5mol / L, drop it on the 400 mesh TEM copper mesh covered with graphene, after the solvent is completely volatilized, cover it with another piece of TEM copper mesh and seal it, to obtain the sample.

[0015] In S10, the reaction process of the sample is monitored by the control system, and when the sample reaction reaches the trigger threshold, the following methods are included: the sample undergoes liquid crystal phase change in the sample stage, the heating wire heats the TEM copper mesh, and when the control system monitors that the sample is heated to 130℃ by the temperature control device and maintained for 5 minutes, the trigger threshold of the sample being instantaneously frozen is reached.

[0016] Preferably, before the S10, it further comprises: S00: sample preparation: use a high molecular film as a window piece, use an alumina covered TEM copper mesh as a support, and evaporate zinc interdigital electrodes through a mask plate; drop 1μL of aqueous solution containing 2mol / L zinc sulfate and 10mol / L 1,3-dimethyl-2-imidazolidinone as an electrolyte to assemble the sample into an electrochemical cell.

[0017] In S10, the reaction process of the sample is monitored by the control system, and when the sample reaction reaches a trigger threshold, the following method is included: connecting the electrochemical cell of the sample to the electrochemical workstation, setting the cyclic voltammetry scan parameters to a rate of 1V / s, a voltage range of-1V to 1V, and at the beginning of the 4th scan, the trigger threshold of the sample being instantaneously frozen is reached.

[0018] Further, when the electrochemical cell of the sample is connected to the electrochemical workstation, the following method is used: connecting the electrodes of the sample through elastic contact points on the sample stage, controlled by an external electrochemical workstation, supporting constant potential, constant current, cyclic voltammetry and other test modes.

[0019] A controllable instant freezing electron microscope sample preparation device applied to the controllable instant freezing electron microscope sample preparation method, comprising a support, a freezing bin, a sample bin, a lead screw driving device and a control system arranged on the support; the driving end of the lead screw driving device is connected with a lead screw, the lead screw is perpendicular to the horizontal plane, the lead screw is threaded through a sliding block, the sliding block is slidingly connected with the support, the sliding block slides along the length direction of the lead screw, the sample stage is detachably connected with the sliding block through a clamping mechanism, the sample stage is provided with a copper mesh clamping seat for detachably installing a sample carrier to realize rapid installation and disassembly of the sample carrier and the sample; the sample stage is provided with a temperature control device, an electric control device and a magnetic control device for monitoring and controlling the temperature information, electrical information or magnetic information of the sample on the sample stage; the lead screw driving device, the temperature control device, the electric control device and the magnetic control device are electrically connected with the control system; the freezing bin is located below the sample bin, the freezing bin is provided with a freezing liquid, the bottom surface of the sample bin is provided with an opening opposite to the freezing bin, one end of the lead screw is inserted into the sample bin from top to bottom and placed in the freezing bin through the opening, and the sample stage is located in the sample bin in the initial state.

[0020] Further, the temperature control device uses a heating wire for heating; the temperature control system of the temperature control device uses an integrated chip of an integrated temperature sensor and a heating control circuit, the temperature control range of the integrated chip is 277K to 473K, and the temperature change rate of the integrated chip is greater than 30K / s.

[0021] The present application has the following beneficial effects:

[0022] 1. This invention achieves precise and instantaneous capture of non-equilibrium structures: By combining precise control of environmental factors such as sample temperature, potential, current, or magnetic field with a high-speed trigger response mechanism, the freezing process can be automatically triggered at the precise moment of a specific physical or chemical change. This overcomes the shortcomings of existing immersion freezing technology, which can only handle equilibrium samples, and existing electrochemical freezing technology, which relies on manual control and cannot be precisely correlated with dynamic processes. It achieves high-fidelity freezing and capture of metastable, transient, and other non-equilibrium structures.

[0023] 2. This invention establishes a quantifiable and highly time-sensitive time correlation from triggering to freezing: It employs a high-pressure pneumatic system controlled by a microcontroller, achieving a response delay as low as 50ms. An integrated temperature sensor records the temperature change curve during the freezing process in real time. This allows for precise calibration of the time correlation between the trigger signal and the actual freezing moment of the sample, providing a reliable time reference for analyzing the evolution of dynamic processes. It achieves a leap from approximate time points to precise time points, which is crucial for studying rapid reaction kinetics. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the controllable instantaneous freezing electron microscope sample preparation device of the present invention.

[0025] Figure 2 This is a flowchart of the electron microscope sample preparation method with controllable instantaneous freezing according to the present invention.

[0026] Figure 3 This is a cryo-transmission electron microscope image of the liquid crystal phase transition intermediate state obtained by the method in Example 2.

[0027] Figure 4 This is a cryo-transmission electron microscope image of the solid electrolyte interface film of the zinc battery obtained by the method in Example 3.

[0028] Figure 5 This is a cryo-transmission electron microscope image of the liquid crystal phase transition intermediate obtained by the method in Comparative Example 2.

[0029] In the diagram: 1. Support; 2. Coolant; 3. Freezing chamber; 4. Sample chamber; 5. Sample stage; 6. Lead screw drive device; 7. Lead screw; 8. Control system. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. It should be emphasized that all the following embodiments can be implemented in accordance with... Figure 1The embodiments are implemented on representative devices or equivalent devices shown. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Terms such as "upper," "inner," "middle," "left," "right," and "one" used in this specification are for clarity of description only and are not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0031] Example 1

[0032] An electron microscope sample preparation device with controllable instantaneous freezing, such as Figure 1 As shown, it includes a support 1, and a freezing chamber 3, a sample chamber 4, a lead screw drive device 6, and a control system 8 installed on the support 1.

[0033] The lead screw drive device 6 can be a pneumatic motor. The drive end of the lead screw drive device 6 is connected to the lead screw 7 by screws. The lead screw drive device 6 drives the lead screw 7 to rotate at high speed through a high-pressure pneumatic system (such as an air compressor or pneumatic compressor). The lead screw 7 is perpendicular to the horizontal plane. The lead screw 7 has a threaded slider that is slidably connected to the bracket 1. The slider slides along the length of the lead screw 7. The sample stage 5 is equipped with a clamping mechanism. The clamping mechanism is a conventional clamping mechanism. The sample stage 5 is detachably connected to the slider by clamping and fixing it, so as to realize the function of quickly installing or removing the sample stage 5.

[0034] The sample stage 5 is equipped with a temperature control device, an electrical control device, and a magnetic control device to monitor and control the temperature, electrical, or magnetic information of the sample and the sample environment on the sample stage 5. The lead screw drive device 6, the temperature control device, the electrical control device, and the magnetic control device are all electrically connected to the control system 8. The control system 8 uses a microcontroller. The system control establishes a precise time correlation between the trigger signal and the freezing time based on the received temperature, electrical, or magnetic information of the sample and the sample environment, thus providing a time reference for data analysis.

[0035] The temperature control system of the temperature control device uses an integrated chip that combines a temperature sensor and a heating control circuit. The integrated chip has a temperature control range of 277K to 473K and a temperature change rate greater than 30K / s. The temperature control device uses a conventional heating wire for heating.

[0036] The electronic control device is a conventional integrated system in existing technology. It is mainly connected to an external electrochemical workstation through conductive contacts on the sample stage 5. It can monitor or control parameters such as potential and current of the battery sample in real time, and supports modes such as constant potential, constant current, and cyclic voltammetry.

[0037] The magnetron sputtering device is a conventional magnetron sputtering component. Its core functional device is an electromagnetic coil or a small permanent magnet array integrated near the sample stage 5 or sample chamber 4. It is used to generate a magnetic field with controllable strength and direction in the sample area to study phase transitions or reaction processes such as liquid crystal orientation and magnetic material evolution under the influence of the magnetic field, and to monitor the magnetic response of the sample. The magnetron sputtering device is connected to the control system 8, and the magnetic field parameters can be set. It can also be set as one of the conditions to trigger instantaneous freezing.

[0038] Sample stage 5 is equipped with a sample carrier for supporting or mounting samples. The sample carrier can be made of micron-level ultrathin structures such as TEM copper mesh, ultrathin polymer film, or ultrathin soft-pack battery, with a thickness of less than 50 μm. This ultrathin design facilitates rapid heat conduction, enabling freezing rates exceeding 10,000 K / s, resulting in an extremely low temperature difference between the sample carrier and the sample. The temperature sensor of the temperature control device is connected to the sample carrier, allowing the temperature control device to quickly and accurately monitor real-time temperature changes of the sample. Simultaneously, the heating wire of the temperature control device is pressed against the sample carrier to heat both the sample carrier and the sample.

[0039] Regarding the structure of the sample stage 5 for mounting the sample carrier: The sample stage 5 is equipped with a copper mesh clamping seat. The copper mesh clamping seat has a spring-loaded mechanical clamp, which is used to firmly clamp the TEM copper mesh to prevent displacement during high-speed movement or transfer.

[0040] The sample stage 5 is also equipped with elastic contact points, which are electrically connected to the electronic and magnetic control devices for connecting the electrode tabs of battery samples. This allows for monitoring and control of the electrical or magnetic information of the battery samples. Simultaneously, the elastic contact points are used to connect to pre-prepared electrodes on the TEM copper grid, thus enabling access to an external electrochemical workstation.

[0041] The freezing chamber 3 is located below the sample chamber 4, with a distance of 8-10 cm between them. The bottom surface of the sample chamber 4 has an opening directly opposite the freezing chamber 3. One end of the lead screw 7 is inserted into the sample chamber 4 from top to bottom and then placed into the freezing chamber 3 through the opening. Initially, the sample stage 5 is located inside the sample chamber 4, which is used to ensure the sample is at a suitable temperature and humidity before freezing. The freezing chamber 3 contains a cryogenic liquid 2, which uses liquid nitrogen to pre-cool propane. Its cooling effect is superior to pure liquid nitrogen or liquid nitrogen-pre-cooled ethane, enabling instantaneous freezing of the sample within 100 ms.

[0042] Therefore, during operation, the temperature control device, the electrical control device, and the magnetic control device monitor the sample and the sample environment in real time. When the sample and the sample environment reach the instantaneous freezing trigger threshold, the lead screw drive device 6 drives the lead screw 7 through the high-pressure pneumatic system so that the sample stage 5 and its sample fall into the freezing chamber 3 within 50ms and are instantly frozen by the freezing liquid 2 within 100ms, thereby facilitating the capture of the sample morphology at the moment the trigger threshold is reached.

[0043] Based on this, the controllable instantaneous freezing electron microscope sample preparation device of the present invention achieves precise and instantaneous capture of non-equilibrium structures. By setting a temperature control device, an electrical control device, and a magnetic control device in the sample stage 5, combined with a lead screw drive device 6 and a control system 8, the precise control of the temperature, potential, current, or magnetic field information of the sample environment is combined with a highly triggered response mechanism, which can automatically trigger the freezing process at critical moments of specific phase transitions and chemical reactions. This overcomes the shortcomings of existing immersion freezing technology, which can only handle equilibrium samples, and existing electrochemical freezing technology, which relies on manual control and cannot be accurately correlated with dynamics, achieving high-fidelity freezing and capture of metastable, transient, and other non-equilibrium structures.

[0044] Based on the above description of the structure of the electron microscope sample preparation device with controllable instantaneous freezing, the following section further introduces the electron microscope sample preparation method with controllable instantaneous freezing.

[0045] Example 2

[0046] like Figure 1 and Figure 2 As shown, a method for preparing electron microscope samples by controlled instantaneous freezing is described. This method is for capturing the intermediate state structure of liquid crystal phase transition. It mainly uses the controlled instantaneous freezing electron microscope sample preparation device of Example 1 and includes the following steps:

[0047] S00: Sample preparation: Take 1 μL of a 0.5 mol / L liquid crystal molecular dichloromethane solution and drop it onto a 400-mesh TEM copper mesh covered with graphene; after the solvent has completely evaporated, cover and seal it with another 400-mesh TEM copper mesh covered with graphene to obtain the sample.

[0048] S10: Setting and Triggering: The prepared sample is detachably mounted on the sample stage 5 via the sample carrier (i.e., TEM copper mesh). The reaction process of the sample is monitored by the temperature control device of the control system 8. When the sample reaction reaches the trigger threshold, that is, when the sample undergoes liquid crystal phase transition in the sample stage 5, the sample is heated to 130°C by the temperature control device. Since 130°C is the critical temperature for liquid crystal molecule phase transition, the trigger threshold for instantaneous freezing of the sample can be reached when the temperature is maintained at 130°C for 5 minutes. At this time, the control system 8 automatically sends a freezing trigger signal.

[0049] S20: Instantaneous freezing: After receiving the freezing trigger signal, the lead screw drive device 6 drives the lead screw 7 to rotate at a pressure of 6 bar to 8 bar through the high-pressure pneumatic system. Utilizing the principle of relative motion, the lead screw 7 drives the sample stage 5 to move 12 cm ± 1 cm towards the freezing chamber 3 within 50 ms, so as to immerse the sample stage 5 and the sample in the freezing liquid 2 in the freezing chamber 3, so as to obtain the sample that is instantaneously frozen in 100 ms.

[0050] S30: Sample transfer: The clamping mechanism is used to separate the sample stage 5 from the lead screw 7. The TEM copper mesh with the sample is taken out of the sample stage 5 with pre-cooled tweezers in the freezing chamber 3 and then transferred to the liquid nitrogen freezing transfer rod.

[0051] S40: Electron Microscopy Observation: The liquid nitrogen cryogenic transfer rod is rapidly inserted into the transmission electron microscope to obtain TEM images of the sample in the TEM copper grid at corresponding moments during the liquid crystal phase transition or electrochemical process. Representative images obtained are shown below. Figure 3 As shown, metastable structures were successfully captured.

[0052] Example 3

[0053] like Figure 1 and Figure 2 As shown, a controllable instantaneous freezing method for electron microscopy sample preparation is described. This method captures the formation process of the SEI film in zinc batteries and employs the controllable instantaneous freezing electron microscopy sample preparation apparatus of Example 1. The method includes the following steps:

[0054] S00: Sample preparation: Using an ultrathin polymer membrane as a window and an alumina-covered TEM copper mesh as a support, zinc interdigitated electrodes were deposited through a mask; 1 μL of an aqueous solution containing 2 mol / L zinc sulfate and 10 mol / L 1,3-dimethyl-2-imidazolinone was added as an electrolyte to assemble the sample into an electrochemical cell; the added 1,3-dimethyl-2-imidazolinone can improve the stability of the electrolyte.

[0055] S10: Setting and Triggering: The prepared sample is detachably mounted on the sample stage 5 via the sample carrier (i.e., the TEM copper mesh as support). During installation, it is connected to the sample electrode tabs through the elastic contact points on the sample stage 5. It is controlled by an external electrochemical workstation and supports multiple test modes such as constant potential, constant current, and cyclic voltammetry. During the process, the reaction process of the sample is monitored by the electronic control device and magnetic control device of the control system 8. When the sample reaction reaches the trigger threshold, the control system 8 sets the cyclic voltammetry scan parameters to a rate of 1V / s and a voltage range of -1V to 1V through the electronic control device. At the beginning of the 4th scan, the trigger threshold for instantaneous freezing of the sample is reached. At this time, the control system 8 automatically sends out a freezing trigger signal.

[0056] S20: Instant freezing: Same as step S20 in Example 2.

[0057] S30: Sample transfer: Same as step S30 in Example 2.

[0058] S40: Electron Microscopy Observation: The liquid nitrogen cryogenic transfer rod is rapidly inserted into the transmission electron microscope to obtain TEM images of the sample in the TEM copper grid at corresponding moments during the liquid crystal phase transition or electrochemical process. Representative images obtained are shown below. Figure 4 As shown, the SEI film structure of zinc batteries was successfully captured.

[0059] Comparative Example 1

[0060] A method for artificially capturing the intermediate state structure of liquid crystal phase transition includes the following steps:

[0061] S00: Sample preparation: Take 1 μL of a 0.5 mol / L liquid crystal molecular dichloromethane solution and drop it onto the sample carrier, which can be a TEM copper mesh; after the solvent has completely evaporated, cover and seal it with another sample carrier to obtain the sample.

[0062] S10: Setting and triggering: Place the sample carrier with the sample on the heater, and monitor the temperature of the sample through a temperature sensor. When the sample is heated to 130°C and maintained for 5 minutes, the sample is in the intermediate state of liquid crystal phase transition.

[0063] S20: Sample freezing: The sample carrier is manually clamped and placed into the freezing liquid 2 for instant freezing. The sample carrier transfer process takes 10-30 seconds.

[0064] S30: Sample transfer and electron microscopy observation: The sample carrier is removed from the cryo-liquid 2 with pre-cooled tweezers and then placed on a transmission electron microscope to capture the liquid crystal phase transition process of the sample on the sample carrier. The imaging results are difficult to achieve the goal of capturing metastable states.

[0065] Comparative Example 2

[0066] A method for replacing the driving system to capture the intermediate state of liquid crystal phase transition includes the following steps:

[0067] The method in Comparative Example 2 is the same as that in Example 2, but the drive system uses a high-speed servo motor-driven rack and pinion instead of the high-pressure pneumatic drive screw 7 system. With this configuration, the time delay from triggering to sample immersion in the cryogenic liquid 2 is 100ms-200ms, significantly longer than the 50ms of the preferred embodiment of this invention, demonstrating the advantage of pneumatic drive in response speed. As in Example 2, it was used to capture the liquid crystal intermediate state, and the results are as follows... Figure 5 As shown, it captures the final state structure with high crystallinity after thermodynamic equilibrium, making it difficult to capture the metastable state.

[0068] In summary, based on Examples 2 and 3, it can be seen that when using the controllable instantaneous freezing electron microscope sample preparation device of the present invention to implement the controllable instantaneous freezing electron microscope sample preparation method, the sample chamber 4 can provide a suitable temperature and humidity environment for the sample, and at the same time can meet the requirements for accurate capture of liquid crystal phase transition intermediate structure and zinc battery SEI film structure. This is due to the fact that the control system 8 can accurately monitor the temperature, electrical and magnetic information of the sample through temperature control device, electrical control device or magnetic control device, and can use the microcontroller control system 8 to accurately time, receive monitoring information and automatically send freezing trigger signal, thereby accurately capturing the non-equilibrium structure of the sample at a specific reaction state and reaction time point.

[0069] Based on Example 2 and Comparative Example 1, it can be seen that, compared with the method of using the screw drive device 6 to drive the liquid crystal phase transition intermediate structure through a high-pressure pneumatic system in Example 2, the method of capturing the liquid crystal phase transition intermediate structure by means of artificial means cannot achieve the purpose of instantaneous freezing of the sample at the time of freezing, and therefore it is difficult to capture the liquid crystal phase transition intermediate structure.

[0070] Based on Example 2 and Comparative Example 2, it can be seen that Example 2 uses a lead screw drive device 6 to drive the lead screw 7 to move the sample stage 5 quickly using a high-pressure pneumatic system. Compared with the method in Comparative Example 2, which uses a high-speed servo motor to drive a rack instead of a high-pressure pneumatic drive to move the lead screw 7 to move the sample stage 5, it is faster. Therefore, the method in Comparative Example 2 is still difficult to accurately capture the intermediate state structure of liquid crystal phase transition, and thus it is difficult to accurately capture the non-equilibrium state structure of the sample at a specific reaction state and reaction time point.

[0071] The embodiments of the present invention are not limited thereto. Based on the above description of the present invention, and using common technical knowledge and conventional means in the field, the present invention can be modified, replaced or combined in various other forms without departing from the basic technical idea of ​​the present invention, and all such modifications, replacements or combinations fall within the scope of protection of the present invention.

Claims

1. A controllable flash freezing method for electron microscopy sample preparation, characterized in that, An electron microscope sample preparation device with controllable transient freezing is adopted; The electron microscope sample preparation device with controllable transient freezing comprises a support and a sample stage, and a freezing bin, a sample bin, a screw rod driving device and a control system arranged on the support; The driving end of the screw rod driving device is connected with a screw rod, the screw rod is perpendicular to the horizontal plane, the screw rod is threaded through a sliding block, the sliding block is slidingly connected with the support, the sliding block slides along the length direction of the screw rod, the sample stage is detachably connected with the sliding block through a clamping mechanism, the sample stage is provided with a copper mesh clamping seat for detachably mounting a sample carrier, the sample carrier is selected from TEM copper mesh, so that the sample carrier and the sample can be quickly mounted and detached; The sample stage is provided with a temperature control device, an electric control device and a magnetic control device for monitoring and controlling the temperature information, electrical information or magnetic information of the sample on the sample stage; the screw rod driving device, the temperature control device, the electric control device and the magnetic control device are electrically connected with the control system; The freezing bin is located below the sample bin, the freezing bin is provided with a freezing liquid, the bottom surface of the sample bin is provided with an opening opposite to the freezing bin, one end of the screw rod is inserted into the sample bin from top to bottom and placed in the freezing bin through the opening, and the sample stage is located in the sample bin in the initial state; The electron microscope sample preparation method with controllable transient freezing comprises the following steps: S10: install the TEM copper mesh with the sample on the sample stage, monitor the reaction process of the sample through the control system, and when the sample reaction reaches the trigger threshold, the control system automatically sends a freezing trigger signal; S20: after the screw rod driving device receives the freezing trigger signal, the screw rod driving device drives the screw rod through a high-pressure pneumatic system, and the screw rod drives the sample stage to be immersed in the freezing liquid in the freezing bin within 50 ms to obtain the sample which is transiently frozen within 100 ms; S30: separate the sample stage from the screw rod by operating the clamping mechanism, take out the TEM copper mesh with the frozen sample in the sample stage by using a pre-cooled forceps, and then transfer the TEM copper mesh to a liquid nitrogen freezing transfer rod; S40: insert the liquid nitrogen freezing transfer rod into a transmission electron microscope to obtain the TEM imaging of the frozen sample at the corresponding moment in the liquid crystal phase change or electrochemical process.

2. The controllable flash frozen electron microscopy sample preparation method of claim 1, wherein, In the S20, the gas pressure value of the high-pressure pneumatic system when driving the screw rod is 6bar to 8bar, and the displacement value of the screw rod driving the sample stage is 12cm±1cm.

3. The controllable flash frozen electron microscopy sample preparation method of claim 1, wherein, In the S40, the freezing liquid is selected from liquid nitrogen pre-cooled propane.

4. The controllable flash frozen electron microscopy sample preparation method of claim 1, wherein, Before the S10, the following steps are further included: S00: sample preparation: take 1μL of dichloromethane solution of liquid crystal molecules with a concentration of 0.5mol / L, drop it on a 400-mesh TEM copper mesh covered with graphene, cover another piece of TEM copper mesh after the solvent is completely volatilized, and seal it to obtain the sample.

5. The controllable flash frozen electron microscopy sample preparation method of claim 4, wherein, In the S10, the reaction process of the sample is monitored through the control system, and when the sample reaction reaches the trigger threshold, the following methods are included: The sample is subjected to liquid crystal phase transition in the sample stage, and the temperature control device heats the TEM copper mesh through the heating wire. When the control system monitors that the sample is heated to 130℃ by the temperature control device and maintained for 5 minutes, the trigger threshold of the sample being instantaneously frozen is reached.

6. The controllable flash frozen electron microscopy sample preparation method of claim 1, wherein, Before the S10, further comprising: S00: sample preparation: using a polymer film as a window sheet, using an aluminum oxide covered TEM copper mesh as a support, and evaporating zinc interdigital electrodes through a mask plate; adding 1 μL of an aqueous solution containing 2 mol / L zinc sulfate and 10 mol / L 1,3-dimethyl-2-imidazolidinone as an electrolyte to assemble the sample into an electrochemical cell.

7. The controllable flash frozen electron microscopy sample preparation method of claim 6, wherein, In S10, the reaction process of the sample is monitored by the control system, and when the sample reaction reaches the trigger threshold, the following methods are included: The electrochemical cell of the sample is connected to the electrochemical workstation, and the cyclic voltammetry scanning parameters are set to a rate of 1 V / s and a voltage range of -1 V to 1 V. When the 4th scanning starts, the trigger threshold of the sample being instantaneously frozen is reached.

8. The controllable flash frozen electron microscopy sample preparation method of claim 7, wherein, When the electrochemical cell of the sample is connected to the electrochemical workstation, the following methods are used: the elastic contact points on the sample stage are connected to the electrode tabs of the sample, which are controlled by the external electrochemical workstation, supporting constant potential, constant current, and cyclic voltammetry test modes.

9. The controllable flash frozen electron microscopy sample preparation method of claim 1, wherein, The temperature control device uses a heating wire for heating; the temperature control system of the temperature control device uses an integrated chip of an integrated temperature sensor and a heating control circuit, the temperature control range of the integrated chip is 277 K to 473 K, and the temperature change rate of the integrated chip is greater than 30 K / s.

Images