A rapid temperature-varying freezing device and a method for preparing a cryo-em sample by de-glassification

Abstract

The present application relates to a quick temperature changing freezing device and a cryo-EM sample de-glassing preparation method, the quick temperature changing freezing device comprising a freezing frame, a freezing copper cup, a heating device and a temperature detector, the freezing copper cup is tightly connected in the center hole of the freezing frame, the freezing copper cup is used for loading solid-liquid mixed state ethane, the heating device is arranged on the outer cylinder wall of the freezing copper cup and is in thermal conduction connection with the freezing copper cup, and is used for heating the solid-liquid mixed state ethane in the freezing copper cup; the temperature detector is arranged on the freezing copper cup and is used for detecting the temperature of the solid-liquid mixed state ethane in the freezing copper cup. When the cryo-EM sample is prepared, the sample grid loaded with sample solution is put into the freezing copper cup for freezing, after completion, the heating device is controlled to start to heat the solid-liquid mixed state ethane, and the solid-liquid mixed state ethane is heated to the de-glassing temperature, and the cryo-EM sample is de-glassed to eliminate the internal stress of the cryo-EM sample caused by rapid freezing, so as to improve the drift problem in the cryo-EM sample image.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of electron microscopy sample preparation, in particular to a rapid temperature-variable freezing device and a vitrification-free preparation method of cryo-EM samples. BACKGROUND

[0002] Biological samples are radiation-sensitive samples, and sample drift, i.e., beam-induced motion (BIM for short), can be caused by low-dose and short-time irradiation. It is generally believed that the internal stress accumulated in the sample during the rapid freezing process (10 5 ~ 10 6 ℃ / s) and the sample deformation and twisting induced by the irradiation thermal stress during the irradiation process are the main causes of image drift during the data collection process of cryo-EM samples.

[0003] In recent years, with the maturity of multi-frame shooting, recording methods, post-data processing algorithms and software technologies of direct electron detection cameras, a large amount of drift problems generated in the image data collection process of biological cryo-EM samples can be corrected later. However, the initial observation of the cryo-EM sample is the part with the weakest irradiation damage effect and the most high-resolution information of the sample structure, and the drift problem of the initial image shooting or recording (the first few frames of data) cannot be corrected by using the existing technology. Therefore, how to preserve these important structural information is a key problem to be solved for the field of structural biology to move towards the atomic resolution era. SUMMARY

[0004] (I) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a rapid temperature-variable freezing device and a vitrification-free preparation method of cryo-EM samples. By drawing on the principles of material quenching and tempering in material heat treatment, when the sample rapid freezing is completed, the temperature control device is used to heat the cryogen in the copper freezing cup to the vitrification-free temperature, so as to produce a tempering effect, thereby eliminating the internal stress of the sample caused by rapid freezing, and solving the image drift problem in the data of the frozen sample of the prior art.

[0006] (II) Technical solutions

[0007] In order to achieve the above-mentioned purpose, the present application provides a rapid temperature-variable freezing device, and the specific technical solutions are as follows:

[0008] A rapid temperature-variable freezing device, comprising:

[0009] A freezing frame provided with a central hole;

[0010] A freezing copper cup tightly connected in the central hole and used for containing solid-liquid mixed state ethane;

[0011] a heating device arranged on the outer periphery of the frozen copper cup and in thermal conduction connection with the frozen copper cup, for heating the solid-liquid mixed state ethane in the frozen copper cup;

[0012] a temperature detector arranged on the outer wall of the frozen copper cup, for detecting the temperature of the solid-liquid mixed state ethane in the frozen copper cup.

[0013] Further, the heating device comprises:

[0014] a heating cylinder arranged on the outer periphery of the frozen copper cup and connected with the frozen copper cup;

[0015] a plurality of heating ceramic sheets arranged in the heating cylinder at equal intervals and in thermal conduction connection with the frozen copper cup respectively.

[0016] Further, a limiting stop ring is arranged on the cylinder wall of the frozen copper cup;

[0017] the bottom of the heating cylinder is arranged on the limiting stop ring, and the top is connected with the top of the frozen copper cup through the limiting flange.

[0018] Further, a plurality of first grooves are arranged on the inner wall of the heating cylinder, and a plurality of second grooves are arranged on the outer wall of the frozen copper cup correspondingly, the first grooves and the second grooves correspond one by one;

[0019] the heating ceramic sheets are arranged in the first grooves and the second grooves.

[0020] Further, a third groove is arranged on the inner wall of the heating cylinder, and a fourth groove is arranged on the outer wall of the frozen copper cup correspondingly, and the temperature detector is arranged in the third groove;

[0021] a wire insertion hole is further arranged on the heating cylinder and communicates with the third groove.

[0022] Specifically, the frozen copper cup is connected in the central hole through a fastening device;

[0023] the fastening device comprises a first fastening part and a second fastening part;

[0024] the first fastening part is arranged on the outer wall of the frozen copper cup, and the second fastening part is connected with the frozen frame and can be fastened with the first fastening part.

[0025] Preferably, the first fastening part comprises a plurality of clamping grooves arranged on the outer wall of the frozen copper cup at equal intervals;

[0026] the second fastening part is a semicircular clamping ring which can be clamped with any clamping groove and is connected with the frozen frame through a bolt.

[0027] Specifically, the frozen frame comprises a frozen cylinder and a top cover plate;

[0028] A central hole is arranged on the top cover plate, and the top cover plate is arranged on the top of the freezing cylinder.

[0029] Further, a plurality of first air holes are arranged on the cylinder wall of the freezing cylinder at equal intervals.

[0030] A plurality of second air holes are arranged on the top cover plate.

[0031] A freezing cryo-EM sample de-glassification preparation method is provided, and the method comprises the following steps:

[0032] The de-glassification temperature T of the heating device is preset.

[0033] The sample grid loaded with the sample solution is added into the solid-liquid mixed state ethane in the freezing copper cup for rapid freezing, and the freezing cryo-EM sample preparation is completed.

[0034] The heating device is controlled to start, and the temperature information of the solid-liquid mixed state ethane in the freezing copper cup is detected by the temperature detector.

[0035] The temperature information of the solid-liquid mixed state ethane is received, and when the temperature information reaches the de-glassification temperature T, the heating device is controlled to stop and is kept for t1 time, and the freezing cryo-EM sample de-glassification treatment is completed.

[0036] (III) Beneficial effects

[0037] The rapid temperature change freezing device and the freezing cryo-EM sample preparation method provided by the application solve the problems of the prior art.

[0038] In the application, the rapid temperature change freezing device comprises a freezing frame, a freezing copper cup, a heating device and a temperature

[0039] detector, wherein the freezing copper cup is tightly connected in the central hole of the freezing frame, the solid-liquid mixed state ethane is loaded in the freezing copper cup, the heating device is arranged on the outer cylinder wall of the freezing copper cup and is in thermal conduction connection with the freezing copper cup, and is used for heating the solid-liquid mixed state ethane in the freezing copper cup, and the temperature detector is also arranged on the freezing copper cup and is used for detecting the temperature of the solid-liquid mixed state ethane in the freezing copper cup.

[0040] The application further provides a freezing cryo-EM sample de-glassification preparation method, which comprises the following steps: placing the rapid temperature change freezing device in a freezing box, injecting liquid nitrogen into the freezing box, rapidly putting the sample grid loaded with the sample solution into the freezing copper cup for freezing, controlling the heating device to start to heat the liquid ethane after the freezing is completed, and then heating the frozen sample grid to the de-glassification temperature to produce tempering effect, so as to eliminate the internal stress of the cryo-EM sample caused by rapid freezing, thereby improving the image drift problem in the cryo-EM sample data. BRIEF DESCRIPTION OF DRAWINGS

[0041] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0042] Figure 1 Structure diagram of the first perspective of the rapid temperature change freezing device in the specific embodiment;

[0043] Figure 2 Structure diagram of the second perspective of the rapid temperature change freezing device in the specific embodiment;

[0044] Figure 3 Structure diagram of the freezing copper cup and the heating device in the specific embodiment;

[0045] Figure 4 Structure diagram of the heating device in the specific embodiment;

[0046] Figure 5 Structure diagram of the heating device in the specific embodiment;

[0047] Figure 6 Structure diagram of the freezing copper cup in the specific embodiment;

[0048] Figure 7 Structure diagram of the freezing rack in the specific embodiment;

[0049] Figure 8 Time-temperature curve of the glass-freezing treatment of the cryo-EM sample.

[0050]

Explanation of the reference signs

[0051] 1, freezing rack; 11, cooling cylinder; 12, top cover plate; 13, center hole; 14, first air hole; 15, second air hole; 16, guide hole;

[0052] 2, freezing copper cup; 21, clamping groove; 22, limiting ring; 23, second groove; 24, fourth groove;

[0053] 3, heating device; 31, heating cylinder; 32, first groove; 33, third groove; 34, wire insertion hole; 35, heating ceramic sheet;

[0054] 4, semicircular clamping ring; 5, temperature probe; 6, controller; 7, limiting flange; 8, auxiliary tool pre-cooling cup; 9, sample box support; 10, guide rod. DETAILED DESCRIPTION

[0055] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described below in a more detailed manner with reference to the drawings in the preferred embodiments of the present application. Identical or similar numerals in the drawings represent identical or similar elements or elements with identical or similar functions throughout. The described embodiments are part of the embodiments of the present application, rather than all the embodiments. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation on the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application. The embodiments of the present application will be described in detail below with reference to the drawings.

[0056] In the description of the present embodiment, it should be understood that the terms "center", "longitudinal", "transverse", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present embodiment and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the scope of protection of the present embodiment.

[0057] During the rapid freezing process, the internal stress accumulated in the sample and the sample deformation and twisting induced by the irradiation thermal stress suffered by the sample during the irradiation process cause image deviation during the cryo-EM sample data acquisition process, thereby inhibiting the development of the field of structural biology to the atomic resolution era.

[0058] In the field of materials, after quenching of metal materials, tempering can induce long-range atomic diffusion, induce the metastable structure in the material to transform into a stable structure, and then release the internal stress accumulated during the quenching process, thereby improving the performance of the material.

[0059] As shown in Figures 1 to 7 Based on the causes of image deviation and in combination with the principles of quenching and tempering of materials in material heat treatment, the present application provides a rapid temperature-changing freezing device in the present embodiment, which specifically comprises a freezing rack 1, a freezing copper cup 2, a heating device 3 and a temperature detector 5.

[0060] The freezing rack 1 is provided with a central hole 13, and the freezing copper cup 2 is tightly connected in the central hole 13 and used for containing solid-liquid mixed state ethane. The heating device 3 is arranged on the outer periphery of the freezing copper cup 2 and is in thermal conduction connection with the freezing copper cup 2, and is used for heating the solid-liquid mixed state ethane in the freezing copper cup 2. The temperature detector 5 is arranged on the outer wall of the freezing copper cup 2 and is used for detecting the temperature of the solid-liquid mixed state ethane in the freezing copper cup 2.

[0061] In practical use, the rapid temperature-changing freezing device of this embodiment is placed in a freezing chamber beforehand. Liquid nitrogen is added to the freezing chamber, and after the liquid nitrogen boils twice, a solid-liquid mixture of ethane is added to the freezing copper cup 2. Then, a sample grid of appropriate size is selected and hydrophilic treatment is performed on the sample grid. A small amount of sample solution is taken with a pipette and dropped onto the sample grid. Excess sample on the surface of the sample grid is removed with filter paper. The sample grid is then transferred to the freezing copper cup 2 for rapid freezing. After freezing, the heating device 3 is activated to heat the solid-liquid mixture of ethane in the freezing copper cup 2 to the devitrification temperature to devitrify the frozen sample. Finally, the devitrified frozen sample is stored in the cryo-electron microscope sample box.

[0062] Furthermore, the rapid temperature-changing freezing device in this embodiment also includes a controller 6, which is electrically connected to the temperature detector 5 and the heating device 3 respectively. The temperature detector 5 is used to detect the temperature of the solid-liquid mixed ethane in the freezing copper cup 2 and transmit the temperature information to the controller 6. The controller 6 controls the start and stop of the heating device 3 according to the temperature information.

[0063] When performing devitrification treatment on rapidly frozen samples, the temperature of the solid-liquid mixed ethane is detected in real time by temperature detector 5. When the temperature reaches the devitrification temperature, the heating device 3 is controlled to stop heating, thereby realizing automatic control of the heating device 3 and improving the control accuracy of the heating device 3 and the accuracy of devitrification temperature adjustment.

[0064] In this embodiment, the heating device 3 is an electric heating device 3. After being powered on, it generates heat, which is transferred to the freezing copper cup 2 by thermal conduction. The freezing copper cup 2 is a metal part, and the freezing copper cup 2 then transfers the heat to the solid-liquid mixed ethane by thermal conduction, thereby heating the solid-liquid mixed ethane.

[0065] In some implementations, such as Figure 4 , Figure 5 and Figure 6 As shown, the heating device 3 includes a heating cylinder 31 and a plurality of heating ceramic plates 35. The plurality of heating ceramic plates 35 are equally spaced inside the heating cylinder 31. The heating cylinder 31 is fitted around the outer periphery of the freezing copper cup 2 and is thermally connected to the freezing copper cup 2.

[0066] In this embodiment, a limiting ring 22 is provided on the outer periphery of the freezing copper cup 2. The heating cylinder 31 is sleeved on the outer periphery of the freezing copper cup 2, with its bottom overlapping the upper surface of the limiting ring 22. A stop is provided at the top opening of the heating cylinder 31, and a limiting flange 7 is bolted to the stop of the heating cylinder 31 to fix the limiting flange 7. In this embodiment, the heating cylinder 31 is fixed by the limiting ring 22 and the limiting flange 7, which facilitates installation and disassembly.

[0067] The inner wall of the heating cylinder 31 is further provided with a plurality of first grooves 32, which are equidistantly arranged along the outer periphery of the heating cylinder 31. The outer wall of the frozen copper cup 2 is correspondingly provided with a plurality of second grooves 23, which are equidistantly arranged along the outer periphery of the frozen cylinder cup. The first grooves 32 and the second grooves 23 correspond to each other, and the heating ceramic sheet 35 is arranged in the first grooves 32 and the second grooves 23 to limit the movement of the heating ceramic sheet 35.

[0068] The inner wall of the heating cylinder 31 is further provided with a third groove 33, and the cylinder wall is further provided with a wire insertion hole 34, which is in communication with the third groove 33. The wire insertion hole 34 is used for inserting the communication line of the temperature probe 5. The outer wall of the frozen copper cup 2 is correspondingly provided with a fourth groove 24, and the temperature probe 5 is installed in the third groove 33 and the fourth groove 24.

[0069] Specifically, as shown in Figure 1 、 Figure 3 , the frozen copper cup 2 of the embodiment is connected with the center hole 13 through a fastening device. The fastening device includes a first fastening part and a second fastening part. The first fastening part is arranged on the outer wall of the frozen copper cup 2, and the second fastening part is connected to the cooling rack 1 and can be tightly connected with the first fastening part.

[0070] In the embodiment, the fastening connection mode of the first fastening part and the second fastening part can be any one of hoop connection, threaded connection and clamping connection.

[0071] As a preferred solution, the first fastening part includes a plurality of clamping grooves 21, which are equidistantly arranged on the outer wall of the frozen copper cup 2. Correspondingly, the second fastening part is a semicircular clamping ring 4, which is clamped in any one of the clamping grooves 21. The semicircular clamping ring 4 is connected to the cooling rack by a bolt, and the center of the semicircular clamping ring 4 coincides with the center of the center hole 13.

[0072] The embodiment can adjust the depth of the frozen copper cup 2 inserted into the cooling rack by adjusting the clamping position of the semicircular clamping ring 4, thereby limiting the depth of the frozen copper cup 2 immersed in liquid nitrogen. The clamping type fastening connection mode of the first fastening part and the second fastening part of the embodiment is simple in structure and convenient to disassemble and assemble.

[0073] Specifically, as shown in Figure 1 、 Figure 2 and Figure 7As shown, the cooling rack of the embodiment includes a cooling cylinder 11 and a top cover plate 12 provided at the top of the cooling cylinder 11, and a central hole 13 is provided on the top cover plate 12. A plurality of first air holes 14 are provided on the cylinder wall of the cooling cylinder 11 at equal intervals, and a plurality of second air holes 15 are provided on the top cover plate 12, the diameter of the first air holes 14 is greater than that of the second air holes 15, the first air holes 14 and the second air holes 15 are used for the injection of liquid nitrogen and the overflow of vaporized liquid nitrogen, so that the liquid nitrogen is fully injected into the cooling rack, and the frozen copper cup 2 is fully cooled.

[0074] Further, as shown in Figure 1 and Figure 2 The rapid temperature change freezing device of the embodiment further includes a sample box support 9 and an auxiliary tool pre-cooling cup 8, both of which are connected to the top cover plate 12 through a guide rod 10.

[0075] The sample box support 9 is used to store sample boxes, and the frozen samples after the glass transition treatment are directly transferred to the sample boxes for storage, which shortens the transfer distance of the frozen samples and avoids contamination during the transfer process. The auxiliary tool pre-cooling cup 8 is used to store the tools required during the test, and is used to pre-cool the tools required for the test.

[0076] In the embodiment, a guide hole 16 is provided on the top cover plate 12, and the guide rod 10 is inserted into the guide hole 16. The height of the sample box support 9 and the auxiliary tool pre-cooling cup 8 can be adjusted by adjusting the connection position of the guide rod 10 and the top cover plate 12.

[0077] The guide rod 10 of the embodiment is a metal piece, and the bottom of the guide rod 10 is immersed in liquid nitrogen. The temperature of the liquid nitrogen can be conducted to the sample box support 9 and the auxiliary tool pre-cooling cup 8, so that the sample box support 9 and the auxiliary tool pre-cooling cup 8 are always in a cooling state, and the tools in the sample box support 9 and the auxiliary tool pre-cooling cup 8 are always in a cooling state.

[0078] The above is the specific structure of the rapid temperature change freezing device of the embodiment. By providing the heating device 3, the temperature detector 5 and the controller 6, after the sample is rapidly frozen, the temperature information fed back by the temperature detector 5 is used to control the heating device 3 to heat the solid-liquid mixed state ethane in the frozen copper cup 2, and then the rapid heating of the solid-liquid mixed state ethane is realized, so as to perform the glass transition treatment on the rapidly frozen sample, eliminate the internal stress of the sample caused by rapid freezing, and thus improve the image offset problem in the data acquisition process of the frozen sample, which is conducive to the development of the field of structural biology to the atomic resolution era.

[0079] Based on the above rapid temperature change freezing device, the embodiment further provides a cryo-EM sample glass transition preparation method, which includes the following steps:

[0080] 1) The devitrification temperature T of the preset heating device 3;

[0081] The temperature T for the glass transition is in the range of -135 to -100℃, but the specific value needs to be determined based on the application requirements.

[0082] 2) The grid containing the sample solution is added to the solid-liquid mixture of ethane in the freezing copper cup 2 for rapid freezing. When the freezing reaches the set time t1, the cryo-electron microscopy sample preparation is complete.

[0083] It should be noted that the sample grid needs to be treated with glow discharge before use. The sample solution is dropped onto the glow-treated side of the sample grid using a pipette, and then the excess sample solution on the surface of the grid is removed by filter paper or non-contact blowing and suction methods to form a sample film of uniform thickness on the surface of the grid.

[0084] In this embodiment, before preparing the cryo-electron microscopy sample, a rapid temperature-changing freezing device is placed in a freezing chamber, and liquid nitrogen is added to the freezing chamber. The temperature range of the liquid nitrogen is -184±1℃. The liquid nitrogen precools the freezing copper cup 2 and the solid-liquid mixed ethane inside the freezing copper cup 2 through heat conduction. Then, the carrier mesh carrying the sample solution is added to the freezing copper cup 2 through a transfer device for freezing to prepare the cryo-electron microscopy sample.

[0085] 3) The heating device 3 is started, and the temperature information of the solid-liquid mixture of ethane in the frozen copper cup 2 is detected by the temperature detector 5;

[0086] 4) Receive the temperature information of the solid-liquid mixed ethane. When the temperature information reaches the devitrification temperature, control the heating device 3 to stop and maintain it until time t1. The devitrification process of the cryo-electron microscopy sample is completed.

[0087] In this embodiment, as Figure 8 The figure shows the temperature-time curve of the devitrification process for a cryo-electron microscopy (cryo-EM) sample. The devitrification temperature T is reached within 2 to 3 minutes of starting the heating device 3, achieving rapid temperature change. In this embodiment, the temperature of the solid-liquid mixture of ethane inside the cryo-copper cup 2 is monitored in real time by a temperature detector 5, and the heating device 3 is repeatedly started to maintain a constant temperature within the range of devitrification temperature T ± 1°C. The cryo-EM sample is then placed in the cryo-copper cup and held for time t1 to completely devitrify the sample.

[0088] After devitrification, the sample grid is transferred to a cryogenic sample box and then to liquid nitrogen for storage in preparation for cryo-electron microscopy observation.

[0089] The de-glassified frozen electron microscope sample obtained by the above method produces a similar tempering effect, eliminates the internal stress of the electron microscope sample caused by rapid freezing, and solves the image offset problem caused by rapid freezing.

[0090] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes within the technical scope disclosed by the present application and according to the technical solutions and inventive concepts of the present application, which are all covered within the protection scope of the present application.

Claims

1. A rapid temperature-changing freezing device, characterized in that, include: Freezer rack (1) with a central hole (13); A freezing copper cup (2) is tightly fitted into the central hole (13) and is used to hold a solid-liquid mixture of ethane. A heating device (3) is located on the outer periphery of the frozen copper cup (2) and is thermally connected to the frozen copper cup (2). It is used to heat the solid-liquid mixed ethane in the frozen copper cup (2) to the devitrification temperature so as to devitrify the frozen sample located in the frozen copper cup. A temperature detector (5) is installed on the outer wall of the frozen copper cup (2) to detect the temperature of the solid-liquid mixed ethane inside the frozen copper cup (2).

2. The rapid temperature-changing refrigeration device according to claim 1, characterized in that, The heating device (3) includes: A heating cylinder (31) is fitted around the outer periphery of the freezing copper cup (2) and connected to the freezing copper cup (2); Multiple heating ceramic plates (35) are equally spaced inside the heating cylinder (31) and are thermally connected to the freezing copper cup (2).

3. The rapid temperature-changing refrigeration device according to claim 2, characterized in that, A limit ring (22) is provided on the cylinder wall of the freezing copper cup (2); The bottom of the heating cylinder (31) is placed on the limiting ring (22), and the top is connected to the top of the freezing copper cup (2) through the limiting flange (7).

4. The rapid temperature-changing refrigeration device according to claim 3, characterized in that, The inner wall of the heating cylinder (31) is provided with a plurality of first grooves (32), and the outer wall of the freezing copper cup (2) is provided with a plurality of second grooves (23), with the first grooves (32) and the second grooves (23) corresponding one to one; The heating ceramic plate (35) is placed in the first groove (32) and the second groove (23).

5. The rapid temperature-changing refrigeration device according to claim 4, characterized in that, The inner wall of the heating cylinder (31) is also provided with a third groove (33), and the outer wall of the freezing copper cup (2) is provided with a corresponding fourth groove (24). The temperature detector (5) is installed in the third groove (33). The heating cylinder (31) is also provided with a wire insertion hole (34), which is connected to the third groove (33).

6. The rapid temperature-changing refrigeration device according to claim 1, characterized in that, The freezing copper cup (2) is connected to the central hole (13) by a fastening device; The fastening device includes a first fastening part and a second fastening part; The first fastening part is provided on the outer wall of the freezing copper cup (2), and the second fastening part is connected to the freezing rack (1) and can be fastened to the first fastening part.

7. The rapid temperature-changing refrigeration device according to claim 6, characterized in that, The first fastening part includes a plurality of slots (21), and the plurality of slots (21) are equally spaced on the outer wall of the freezing copper cup (2); The second fastening part is a semi-circular retaining ring (4), which can be engaged with any of the retaining slots (21) and connected to the freezer rack (1) by bolts.

8. The rapid temperature-changing refrigeration device according to claim 1, characterized in that, The freezer rack (1) includes a freezer cylinder (11) and a top cover plate (12); The central hole (13) is located on the top cover plate (12), which is located on the top of the freezing cylinder (11).

9. The rapid temperature-changing refrigeration device according to claim 8, characterized in that, The freezing cylinder (11) has a plurality of first vent holes (14) evenly spaced on its cylinder wall; The top cover plate (12) is provided with a plurality of second vent holes (15).

10. A method for preparing cryo-electron microscopy samples by devitrification, characterized in that, The rapid temperature-changing freezing apparatus according to any one of claims 1 to 9 comprises the following steps: The devitrification temperature T of the preset heating device; The sample solution-containing grid is added to the solid-liquid mixture of ethane in a freezing copper cup for rapid freezing, thus completing the preparation of the cryo-electron microscopy sample; The heating device is activated, and the temperature information of the solid-liquid mixture of ethane inside the frozen copper cup is detected by a temperature detector. The temperature information of the solid-liquid mixed ethane is received. When the temperature information reaches the devitrification temperature T, the heating device is controlled to stop and held for a time t1. The devitrification process of the cryo-electron microscopy sample is then completed.

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