Controllable instantaneous freezing electron microscope sample preparation method and device

Abstract

The invention discloses a controllable instantaneous freezing electron microscope sample preparation method and device, and the method comprises the steps: S10, installing a TEM copper mesh with a sample on a sample stage, monitoring the reaction process of the sample by a control system, and automatically sending out a freezing trigger signal by the control system when the reaction of the sample reaches a trigger threshold value; s20, a lead screw driving device drives a lead screw through a high-pressure pneumatic system to drive the sample table to be immersed into the freezing liquid in the freezing bin within 50 ms, so that a sample which is instantly frozen within 100 ms is obtained; s30, separating the sample table from the screw rod, taking out the TEM copper mesh, and transferring the TEM copper mesh to a liquid nitrogen freezing transfer rod; and S40, inserting the liquid nitrogen freezing transfer rod into a transmission electron microscope, and obtaining a TEM image of the sample at a corresponding moment in a liquid crystal phase change or electrochemical process. Through the setting, when the sample reaches the trigger threshold value during reaction, the freezing process is automatically triggered, so that the non-equilibrium state structure of the sample in a specific reaction state and a reaction time point is accurately captured.

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: A controllable instantaneous freezing electron microscope sample preparation method, comprising 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 a trigger threshold, the control system automatically sends a freezing trigger signal; S20: after receiving the freezing trigger signal, the lead screw driving device drives the lead screw through the high-pressure pneumatic system, and the lead screw drives the sample stage to be immersed in the freezing liquid in the freezing chamber within 50 ms to obtain the sample frozen within 100 ms; 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; S40: quickly insert the liquid nitrogen freezing transfer rod into the transmission electron microscope to obtain the TEM imaging of the frozen sample at the corresponding moment of the liquid crystal phase change or the electrochemical process.

[0007] Further, in the S20, the gas pressure value of the high-pressure pneumatic system driving the lead screw is 6-8 bar, and the displacement value of the lead screw driving the sample stage within 50 ms is 12 cm±1 cm.

[0008] Further, in the S40, the freezing liquid is pre-cooled propane selected from liquid nitrogen.

[0009] Further, before the S10, it further comprises: S00: sample preparation: take 1 μL of dichloromethane solution of liquid crystal molecules with a concentration of 0.5 mol / L, drop it on the 400 mesh TEM copper mesh covered with graphene, and after the solvent is completely volatilized, cover it with another piece of TEM copper mesh and seal it to obtain the sample.

[0010] 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 methods are included: the sample undergoes a 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.

[0011] 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 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.

[0012] 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 and 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.

[0013] 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 the external electrochemical workstation, supporting constant potential, constant current, cyclic voltammetry and other test modes.

[0014] 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.

[0015] 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.

[0016] The present application has the following beneficial effects: 1. The present application realizes the accurate and instantaneous capture of non-equilibrium structures: by combining the precise control of sample temperature, potential, current or magnetic field environment with high-speed trigger response mechanism, it can automatically trigger the freezing process at the precise moment when a specific physical or chemical change occurs. This overcomes the defects 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 associated with dynamic processes, realizing high-fidelity freezing and capture of metastable, transient and other non-equilibrium structures.

[0017] 2. The present application establishes a quantifiable and high-time-efficient time correlation from triggering to freezing: using a high-pressure pneumatic system controlled by a single-chip microcomputer system, the response delay can be as low as 50 ms, and the temperature change curve during the freezing process is recorded in real time by an integrated temperature sensor. This makes the time correlation between the trigger signal and the actual freezing time of the sample accurate, providing a reliable time reference for analyzing the evolution of dynamic processes, realizing the leap from approximate time points to precise time points, which is crucial for studying fast reaction kinetics. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The structure diagram of the controllable instantaneous freezing electron microscope sample preparation device of the present application.

[0019] Figure 2 The flowchart of the controllable instantaneous freezing electron microscope sample preparation method of the present application.

[0020] Figure 3 The frozen transmission electron microscope image of the liquid crystal phase transition intermediate state obtained by the method of Example 2.

[0021] Figure 4 The frozen transmission electron microscope image of the zinc battery solid electrolyte interface film obtained by the method of Example 3.

[0022] Figure 5 The frozen transmission electron microscope image of the liquid crystal phase transition intermediate state obtained by the method of Comparative Example 2.

[0023] In the figure: 1, support; 2, freezing liquid; 3, freezing bin; 4, sample bin; 5, sample stage; 6, screw drive device; 7, screw; 8, control system. DETAILED DESCRIPTION

[0024] The present application will be further described in detail below in conjunction with the drawings and specific examples. It should be emphasized that all the following examples can be carried out in the same way as Figure 1The representative device or its equivalent device is implemented on the device. These embodiments are only used to illustrate the present application and not to limit the scope of the present application. The terms such as "upper", "inner", "middle", "left", "right" and "one" in the specification are only for the convenience of clear description, not to limit the scope of the present application, and the change or adjustment of the relative relationship is also regarded as the implementation of the present application without substantial change of the technical content.

[0025] Embodiment 1 A controllable instantaneous freezing electron microscope sample preparation device, as shown, comprises a support 1, and a freezing bin 3, a sample bin 4, a lead screw driving device 6 and a control system 8 mounted on the support 1. Figure 1

[0026] The lead screw driving device 6 can adopt a pneumatic motor, and the driving end of the lead screw driving device 6 is connected with a lead screw 7 through screw locking. The lead screw driving device 6 drives the lead screw 7 to rotate at high speed through a high-pressure pneumatic system (such as an air compressor or a pneumatic compressor). The lead screw 7 is perpendicular to the horizontal plane. The lead screw 7 is threaded through a sliding block. The sliding block is slidingly connected with the support 1. The sliding block slides along the length direction of the lead screw 7. The sample stage 5 is provided with a clamping mechanism. The clamping mechanism is a conventional clamping function mechanism. The sample stage 5 is detachably connected with the sliding block in a clamping fixed manner through the clamping mechanism, so as to realize the function of quickly loading or taking out the sample stage 5.

[0027] The sample stage 5 is provided with a temperature control device, an electric control device and a magnetic control device, which are used for monitoring and controlling the temperature information, electrical information or magnetic information of the sample on the sample stage 5 and the sample environment. The lead screw driving device 6, the temperature control device, the electric control device and the magnetic control device are electrically connected with the control system 8. The control system 8 adopts a single-chip microcomputer. The system control establishes the precise time correlation between the trigger signal and the freezing time according to the received changes of the temperature information, electrical information or magnetic information of the sample and the sample environment, so as to provide a time reference for data analysis.

[0028] The temperature control system of the temperature control device adopts 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. The temperature change rate of the integrated chip is greater than 30K / s. The temperature control device adopts a conventional heating wire for heating.

[0029] The electric control device is a conventional integrated system in the prior art. It is connected to an external electrochemical workstation through the conductive contact on the sample stage 5. It can monitor or control the potential, current and other parameters of the battery sample in real time, such as supporting constant potential, constant current, cyclic voltammetry and other modes.

[0030] ​The magnetic control device is a conventional magnetic control component, and its core functional device is an electromagnetic coil or a small permanent magnet array integrated near the sample table 5 or the sample chamber 4; it is used to generate a controllable magnetic field in strength and direction in the sample area, and is used to study the phase transition or reaction process under the induction or influence of the magnetic field, such as liquid crystal orientation, evolution of magnetic materials, etc., to monitor the magnetic response of the sample. The magnetic control device is connected with the control system 8, the magnetic field parameters can be set, and it can be set as one of the conditions for triggering instantaneous freezing.

[0031] The sample table 5 is provided with a sample carrier for carrying or mounting a sample; the sample carrier can be selected from a TEM copper mesh, an ultrathin polymer film, or an ultrathin soft-pack battery, etc. micrometer-level ultrathin structure, the thickness of which is less than 50μm, and the ultrathin design is conducive to rapid heat conduction, and can achieve a freezing rate of more than 10000K / s, so that the temperature difference between the sample carrier and the sample is very low. The temperature sensor sensing end of the temperature control device is connected with the sample carrier, so that the temperature control device can quickly and accurately monitor the real-time temperature change of the sample. At the same time, the heating wire of the temperature control device is opposite to the sample carrier, which is used for heating the sample carrier and the sample.

[0032] Regarding the structure of the sample carrier mounted on the sample table 5: the inside of the sample table 5 is provided with a copper mesh clamping seat, which has a spring buckle type mechanical clamp for stably clamping the TEM copper mesh to prevent displacement during high-speed movement or transfer.

[0033] The sample table 5 is also provided with an elastic contact point, which is electrically connected with the electric control device and the magnetic control device, and is used for electrically connecting the electrode tab of the battery sample to monitor and control the electrical information or magnetic information of the battery sample. At the same time, the elastic contact point is used for connecting with the electrode prepared in advance on the TEM copper mesh, so as to realize the access of the external electrochemical workstation.

[0034] The freezing chamber 3 is located below the sample chamber 4, and the distance between the sample chamber 4 and the freezing chamber 3 is 8cm-10cm. The bottom surface of the sample chamber 4 is provided with an opening opposite to the freezing chamber 3, and one end of the lead screw 7 is inserted into the sample chamber 4 from top to bottom and placed in the freezing chamber 3 through the opening. In the initial state, the sample table 5 is located in the sample chamber 4, and the sample chamber 4 is used to ensure that the sample is at a suitable temperature and humidity before freezing. The freezing chamber 3 is provided with a freezing liquid 2, and the freezing liquid 2 adopts liquid nitrogen pre-cooled propane, which has better cooling effect than pure liquid nitrogen or liquid nitrogen pre-cooled ethane, and can instantaneously freeze the sample within 100ms.

[0035] Therefore, in operation, the temperature control device, the electric control device and the magnetic control device monitor the sample and the sample environment in real time, and when the sample and the sample environment reach the instantaneous freezing trigger threshold, the lead screw driving device 6 drives the lead screw 7 through the high-pressure pneumatic system to make the sample table 5 and the sample thereof fall into the freezing bin 3 within 50 ms, and the sample is instantaneously frozen by the freezing liquid 2 within 100 ms, thereby facilitating the capture of the sample morphology at the moment when the trigger threshold is reached.

[0036] Based on this, the controllable instantaneous freezing electron microscope sample preparation device of the present application realizes accurate and instantaneous capture of non-equilibrium state structures. By providing the temperature control device, the electric control device and the magnetic control device on the sample table 5, in combination with the lead screw driving device 6 and the control system 8, the accurate control of the temperature, electric potential, current or magnetic field information of the sample environment is combined with a high trigger response mechanism, so that the freezing process can be automatically triggered at a critical moment of a specific phase change or chemical reaction. This overcomes the defects of existing investment freezing technology which can only handle equilibrium state samples and existing electrochemical freezing technology which relies on manual control and cannot be accurately associated with dynamics, and realizes high-fidelity freezing and capture of metastable state, transient state and other non-equilibrium state structures.

[0037] Based on the above introduction to the structure of the controllable instantaneous freezing electron microscope sample preparation device, the controllable instantaneous freezing electron microscope sample preparation method will be further introduced below.

[0038] Example 2 As shown in Figure 1 and Figure 2 , a controllable instantaneous freezing electron microscope sample preparation method, which is a method for capturing liquid crystal phase change intermediate state structures, mainly uses the controllable instantaneous freezing electron microscope sample preparation device of Example 1, and includes the following steps: S00: sample preparation: take 1 μL of liquid crystal molecule dichloromethane solution with a concentration of 0.5 mol / L, and drop it on a 400-mesh TEM copper mesh covered with graphene; after the solvent is completely volatilized, cover and seal it with another 400-mesh TEM copper mesh covered with graphene to obtain the sample.

[0039] S10: setting and triggering: the prepared sample is detachably installed on the sample table 5 through the sample carrier (i.e. the TEM copper mesh), and 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, i.e. the sample undergoes liquid crystal phase change in the sample table 5, the sample is heated to 130℃ by the temperature control device. Since 130℃ is the critical temperature of the liquid crystal molecule phase change, the trigger threshold of the instantaneous freezing of the sample can be reached when it is heated to 130℃ for 5 minutes, at which time the control system 8 automatically sends a freezing trigger signal.

[0040] S20: Instantaneous freezing: Upon receiving the freezing trigger signal, the lead screw drive 6 drives the lead screw 7 to rotate at a pressure of 6-8 bar through a high-pressure pneumatic system. According to the principle of relative motion, the lead screw 7 drives the sample stage 5 to move 12 cm ± 1 cm in 50 ms to immerse the sample stage 5 and the sample into the freezing liquid 2 in the freezing chamber 3, so as to obtain the sample that is instantaneously frozen in 100 ms.

[0041] S30: Sample transfer: The clamping mechanism is operated to separate the sample stage 5 from the lead screw 7. After the TEM copper mesh with the sample in the sample stage 5 is taken out by the pre-cooled tweezers in the freezing chamber 3, it is then transferred to the liquid nitrogen freezing transfer rod.

[0042] S40: Electron microscope observation: The liquid nitrogen freezing transfer rod is quickly inserted into the transmission electron microscope to obtain the TEM imaging of the sample in the TEM copper mesh at the corresponding moment of the liquid crystal phase change or the electrochemical process. The representative images obtained are shown in Figure 3 , which successfully capture the metastable structure.

[0043] Example 3 As shown in Figure 1 and Figure 2 , an electron microscope sample preparation method with controllable instantaneous freezing is provided. The method is a method for capturing the SEI film formation process of a zinc battery. The electron microscope sample preparation device with controllable instantaneous freezing of Example 1 is used, and the method comprises the following steps: S00: Sample preparation: An ultrathin polymer film is used as a window sheet, and an aluminum oxide covered TEM copper mesh is used as a support. Zinc interdigital electrodes are evaporated through a mask plate. 1 μL of an aqueous solution containing 2 mol / L zinc sulfate and 10 mol / L 1,3-dimethyl-2-imidazolidinone is added as an electrolyte to assemble the sample into an electrochemical cell. The addition of 1,3-dimethyl-2-imidazolidinone can improve the stability of the electrolyte.

[0044] S10: Setting and triggering: The prepared sample is detachably mounted on the sample stage 5 through the sample carrier (i.e., the supporting TEM copper mesh). During installation, the electrode tabs of the sample are connected through the elastic contact points on the sample stage 5. An external electrochemical workstation is controlled to support constant potential, constant current, and cyclic voltammetry test modes. During the process, the reaction process of the sample is monitored through the electric control device and the 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 1 V / s and a voltage range of -1 V to 1 V at the start of the 4th scan. When the sample is instantaneously frozen, the control system 8 automatically sends a freezing trigger signal.

[0045] S20: Instantaneous freezing: The same as step S20 of Example 2.

[0046] S30: Sample transfer: same as step S30 of Example 2.

[0047] S40: Electron microscope observation: the liquid nitrogen-cooled transfer rod was quickly inserted into the transmission electron microscope to obtain TEM imaging of the sample in the TEM copper mesh at the corresponding time during the liquid crystal phase transition or electrochemical process. The representative images obtained are shown in Figure 4 The SEI film structure of the zinc battery was successfully captured.

[0048] Comparative Example 1 The method for artificially capturing the intermediate state structure during the liquid crystal phase transition includes the following steps: S00: Sample preparation: 1 μL of a dichloromethane solution of liquid crystal molecules with a concentration of 0.5 mol / L was dropped onto a sample carrier, which can be a TEM copper mesh; after the solvent completely evaporated, another sample carrier was used to cover and seal the sample.

[0049] S10: Setting and triggering: the sample carrier with the sample was placed on the heater, and the temperature of the sample was monitored by the temperature sensor; when the sample was heated to 130°C and maintained for 5 minutes, the sample was in the intermediate state structure during the liquid crystal phase transition.

[0050] S20: Sample freezing: the sample carrier was manually clamped and placed in the freezing liquid 2 for instantaneous freezing; the sample carrier transfer process took 10-30 seconds.

[0051] S30: Sample transfer and electron microscope observation: the sample carrier was taken out of the freezing liquid 2 with a pre-cooled forceps, and then placed on the transmission electron microscope to capture the imaging of the sample during the liquid crystal phase transition on the sample carrier; the imaging result was difficult to achieve the goal of capturing the metastable state.

[0052] Comparative Example 2 The method for capturing the intermediate state structure during the liquid crystal phase transition by replacing the driving system includes the following steps: The method of this comparative example 2 was operated in the same way as the method of Example 2, but the driving system used a high-speed servo motor to drive the rack instead of the high-pressure pneumatic drive screw 7 system. Under this configuration, the time delay from triggering to sample immersion in the freezing liquid 2 was 100-200 ms, which was significantly longer than the 50 ms of the preferred scheme of the present application, proving the advantage of pneumatic driving in response speed. As in Example 2, it was used to capture the intermediate state of the liquid crystal, and the result is shown in Figure 5 The final state structure with high crystallinity after thermodynamic equilibrium was captured, and it was difficult to achieve the goal of capturing the metastable state.

[0053] In summary, based on Example 2 and Example 3, it can be seen that when the controllable transient freezing electron microscope sample preparation device of the application is used to implement the controllable transient freezing electron microscope sample preparation method, the sample bin 4 can provide a suitable temperature and humidity environment for the sample, and can meet the precise capture of the liquid crystal phase transition intermediate state structure and the SEI film structure of the zinc battery; wherein the control system 8 can precisely monitor the temperature information, electrical information and magnetic information of the sample through the temperature control device, the electric control device or the magnetic control device, and can accurately time, receive monitoring information and automatically send a freezing trigger signal by using the control system 8 of the single-chip microcomputer, so as to precisely capture the non-equilibrium state structure of the sample at a specific reaction state and a reaction time point.

[0054] Based on Example 2 and Comparative Example 1, it can be seen that the manual method for capturing the liquid crystal phase transition intermediate state structure cannot achieve the purpose of transient freezing of the sample at the freezing time, compared with the method of Example 2 using the screw drive device 6 driven by the high-pressure pneumatic system, so it is difficult to capture the liquid crystal phase transition intermediate state structure.

[0055] Based on Example 2 and Comparative Example 2, it can be seen that the method of Example 2 using the screw drive device 6 to drive the sample stage 5 to move rapidly by using the high-pressure pneumatic system is more rapid than the method of Comparative Example 2 using a high-speed servo motor to drive a rack instead of a high-pressure pneumatic screw 7 to drive the sample stage 5 to move, so the method of Comparative Example 2 is still difficult to achieve precise capture of the liquid crystal phase transition intermediate state structure, so it is difficult to precisely capture the non-equilibrium state structure of the sample at a specific reaction state and a reaction time point.

[0056] The embodiments of the application are not limited thereto, and according to the above content of the application, using the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or combinations can be made without departing from the above basic technical idea of the application, which all fall within the protection scope of the application.

Claims

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

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

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 step is further included. S00: sample preparation: 1 μL of a dichloromethane solution of liquid crystal molecules with a concentration of 0.5 mol / L is dropped on a 400-mesh TEM copper mesh covered with graphene, after the solvent is completely volatilized, another TEM copper mesh is used to cover and seal, so that the sample is obtained.

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 a trigger threshold, the following method is adopted: The sample is subjected to liquid crystal phase change in the sample table, the TEM copper mesh is heated through the heating wire of the temperature control device, and when the control system monitors that the sample is heated to 130 DEG C and maintained for 5 minutes by the temperature control device, the trigger threshold of the sample being transiently 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 alumina-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 electrode tabs of the sample are connected through elastic contact points on the sample stage, and are controlled by an 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