Rapid freezing sample preparation equipment

Abstract

The utility model discloses rapid freezing sample preparation equipment. The equipment comprises a sample clamping module, a sample liquid absorption module, a freezing sample preparation module, a sample net carrying box and an electric control module, the equipment can automatically control the projection time of tweezers in the sample clamping module, reduce the vibration of the tweezers in the horizontal direction, relieve the rebound pollution problem caused by collision vibration generated after ethane is fed into the tweezers, and control the telescopic distance, telescopic speed, initial position and residence time of the sample liquid suction module through a photoelectric sensor in a high-precision manner, so that the detection accuracy is improved. And the device has the advantages of low manufacturing cost and flexibility in use.

Description

Technical Field

[0001] The utility model discloses a rapid freezing sample preparation device, belonging to the technical field of devices for low-temperature treating samples. Background Art

[0002] The development of electron microscopy can be traced back to the proposal of the "matter wave" hypothesis in 1924, which is the theoretical basis of electron microscopy. With the progress of technology, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) have made remarkable achievements in sample structure analysis and microscopic component characterization, becoming important tools in scientific research and industrial applications. In the field of biological materials, the application of cryo-electron microscopy technology began in 1974. Kenneth A. Taylor and Robert M. Glaeser successfully obtained high-resolution images of cryo-hydrated catalase at -120 °C, revealing the potential of cryo-samples to reduce radiation damage. This discovery laid the foundation for the application of cryo-electron microscopy in biophysics. In 1980, Jacques Dubochet made a breakthrough in the technology of preparing vitrified thin films of pure water and proposed the liquid ethane bath freezing method. With its fast and efficient characteristics, this method has become the standard technology for rapid freezing of samples and has gradually matured. The cryo-technology solution that won the Nobel Prize in 2017 mainly includes spreading the sample solution on a grid to form a thin liquid layer, and then immersing the grid in liquid ethane for rapid freezing at a speed of 10 4 to 10 6 K, thereby forming amorphous ice (glass state). The advantage of this method is that the water in cells and solutions will not form ice crystals that damage the sample structure and generate electron diffraction, avoiding water loss in the high-vacuum environment, and at the same time, the damage caused by electron radiation to the sample at low temperature is smaller, so better data can be obtained.

[0003] A cryo-electron microscopy grid usually consists of two layers: a copper mesh skeleton and a carbon film containing micron-sized pores. After the sample solution to be tested is dropped on the copper mesh, it spreads out, but the liquid layer is usually relatively thick, and it is difficult to reach the glass state directly by freezing. Therefore, the conventional method is to suck off the excess sample liquid with filter paper before rapid freezing to ensure that a liquid layer of 20 um to 50 um remains on the grid.

[0004] Currently, cryo-electron microscopy sample preparation devices are divided into two categories: manual and automated. Manual sample preparation devices rely on manual operation for filter paper pressing, resulting in poor reproducibility of experiments and difficulty in ensuring consistency. After setting parameters and adding samples, automated sample preparation equipment can automatically complete experimental operations with good reproducibility. However, there are problems such as difficult control of filter paper pressing, uncontrollable thickness of the sample liquid layer, few product types, complex operation, large volume, heavy weight, the need for a professional environment for use, low space utilization, and extremely high prices. Although some other cryo-sample preparation equipment for materials solves the problem of high prices, the sample preparation success rate is extremely low. Specifically, the existing rapid freezing sample preparation equipment has the following main defects in terms of accuracy, experimental stability, debugging flexibility, ease of operation, and cost performance:

[0005] 1. Some products hold the filter paper manually for adsorption, unable to ensure the same adsorption time, strength, and angle each time, resulting in poor reproducibility of the experiment.

[0006] 2. The problem of tweezers vibration. When some products put samples into the ethane pool, the tweezers vibrate significantly. This vibration can cause the glassy ice to break and also damage the film on the surface of the grid, resulting in sample preparation failure.

[0007] 3. Although some products have high repeatability, they integrate many unnecessary functions, resulting in a bulky device, low space utilization, inconvenience in dealing with various application scenarios, and difficulty in popularization and application. At the same time, due to market monopoly, the device price is high and the cost performance is low.

[0008] To address these problems, it is necessary to develop a rapid freezing sample preparation device with high experimental accuracy, strong stability, simple operation, and high cost performance to improve the reproducibility of experiments and the efficiency of sample preparation.

[0009] Based on the problems existing in the prior art, the purpose of the present utility model is to provide a rapid freezing sample preparation device with high experimental accuracy, good sample preparation stability, compact structure, simple operation, and higher cost performance. Summary of the Utility Model

[0010] Based on the above purpose, the present utility model provides a rapid freezing sample preparation device, which includes a freezing sample preparation module 400, a grid box 500, and an electric control module 600. The rapid freezing sample preparation device further includes a sample clamping module 200 and a filter paper liquid absorption module 300. The freezing sample preparation module 400 is located below the sample clamping module 200 and is used for freezing sample preparation of the sample grid after liquid absorption treatment. The position of the filter paper liquid absorption module 300 is set corresponding to the position of the sample clamping module 200 to ensure that the filter paper liquid absorption module 300 is used for liquid absorption treatment of the sample liquid film on the sample grid clamped by the sample clamping module 200.

[0011] In a specific embodiment of the present utility model, the filter paper liquid absorption module 300 is located at the lower side of the sample clamping module 200.

[0012] The sample clamping module 200 is used to clamp a sample carrier from a carrier grid box 500, after loading a sample liquid droplet, to make it undergo the liquid absorption treatment of the filter paper liquid absorption module 300, enter the freeze sample preparation module 400 for freeze sample preparation, and enter the workbench. In a preferred embodiment, the sample clamping module 200 includes forceps 270 for clamping the carrier grid, a coupling member 244 connected to the forceps, a flip-up forceps fixing block 240 for accommodating and fixing the forceps 270, an optical axis 220 and a guide rail slider 260 for reducing the vibration of the forceps fixing block 240. The guide rail slider 260 is parallel to the optical axis 220 and has a spacing, and jointly guides the forceps fixing block 240. An electromagnet door lock 210 for fixing the initial state, and symmetrically arranged buffers 230 for buffering the guide rail slider 260; the design of the flip-up clamping mechanism facilitates single-handed operation and reduces the interference of the operation on the experiment. The design of the forceps fixing block increases the stability of the forceps fixing and reduces the vibration during the projection of the forceps. The design of the buffer avoids the rebound of the forceps. The synergistic effect of each component ensures the stability of sample preparation, avoids the diffusion of biological samples, and the damage to the samples to be prepared, ensuring the safety and accuracy of the experiment.

[0013] In a preferred embodiment, the forceps fixing block 240 in the sample clamping module 200 is composed of a fixing block base 241 and a fixing seat flip cover 242, which are connected by a cylindrical pin to form a flip-up clamping mechanism. A fixing groove 245 for placing the forceps 270 connected to the coupling member 244 is provided on the fixing block base 241, and a magnet for fixing the forceps 270 is provided on the back of the fixing seat.

[0014] The filter paper liquid absorption module 300 is used to drive the rotation of the filter paper to perform liquid absorption treatment on the liquid film of the sample on the sample carrier held by the sample clamping module 200. In a preferred embodiment, the filter paper liquid absorption module 300 includes: a rotatable filter paper disk 310, an adjustable photoelectric sensor bracket 320, a photoelectric sensor 321, a push-rod type linear stepper motor 330 for driving the rotatable filter paper disk 310, a horizontal guide rail slider 360 for guiding the rotatable filter paper disk 310, a guide rail slider fixing plate 370, an induction sheet 380, and a lighting strip 390; in the filter paper liquid absorption module 300, the photoelectric sensor 321 and the adjustable photoelectric sensor bracket 320 are respectively installed on both sides of the guide rail slider fixing plate 370 and can be adjusted to any position in the horizontal direction. The induction sheets 380 are respectively installed on both sides of the horizontal guide rail slider 360 and can move along with the horizontal guide rail slider. When the photoelectric sensor 321 senses that the induction sheet 380 passes through, it transmits the position information of the horizontal guide rail slider 360 at this time to the electric control module 600, and the push-rod type linear stepper motor 330 is used to provide stable thrust and pull force.

[0015] More preferably, the filter paper liquid absorption module 300 further includes a filter paper disk angle slide 340 and a Z-axis lifting platform 350. By rotating the filter paper disk angle slide knob 341, the guide rail slider fixing plate 370 can be adjusted for up-and-down angles based on the horizontal direction; by rotating the Z-axis lifting platform knob 351, the Z-axis lifting platform 350 can be raised.

[0016] Particularly preferably, the angle range for adjusting the rotatable filter paper disk 310 in the horizontal direction by the filter paper disk angle slide knob 341 is -15° to +15°, for example, it can be -15°, -12°, -10°, -8°, -5°, -2°, 0°, 2°, 5°, 8°, 10°, 12°, 15°.

[0017] In another particularly preferred embodiment, the adjustment range for raising the Z-axis lifting platform is 0 - 10 mm, for example, it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mm.

[0018] The cryosampling module 400 is located below the sample clamping module 200 and is used to perform cryosampling treatment on the sample carrier after liquid absorption treatment. It includes a liquid nitrogen pool 460 for providing a cold source, an ethane pool 420 for quickly freezing the sample carrier, a heat conduction sheet 410 for transferring the temperature of the liquid nitrogen pool 460 to the ethane pool 420, a carrier storage box 442 for storing the carrier, a workbench for storing the carrier storage box 442; a workbench base 450 for supporting the workbench and fixing the workbench to the liquid nitrogen pool, a limiting member 470 for fixing the liquid nitrogen pool 460, and a padding member 480 for protecting and raising the equipment.

[0019] The carrier grid box 500 is used to store carrier grids.

[0020] The electric control module 600 includes a module for collecting and processing the position information transmitted back by the photoelectric sensor 321 in the filter paper liquid absorption module 300, a projection control module for controlling the projection time of the tweezers 270 in the sample clamping module 200 based on the position information, a telescopic control module for controlling the telescopic distance, telescopic speed, initial position and residence time of the filter paper in the filter paper liquid absorption module 300 based on the position information, and a control module for controlling the electromagnet door lock 210.

[0021] In a preferred embodiment, the buffer in the sample clamping module 200 is a standard hydraulic buffer.

[0022] In another preferred embodiment, the device is also equipped with a dust cover 100.

[0023] In yet another preferred embodiment, a heat-insulating foam 430 for heat preservation is further arranged on the outer periphery of the ethane pool 420.

[0024] The method for preparing cryo-EM samples using the rapid freezing sample preparation device of the present utility model includes the following steps:

[0025] (1) Fixing and sample addition: Use the tweezers 270 to pick up the hydrophilized carrier grid from the carrier grid box 500, place the tweezers 270 into the coupler 244, and lock it with a screw. Open the fixing seat flip cover of the sample clamping module 200, place the coupler 244 into the fixing groove 245 of the fixing block base 241 of the tweezers fixing block 240, close the fixing seat flip cover 242 and then tighten the fixing seat flip cover knob 243 for fastening. Lift the tweezers fixing block 240 to the electromagnet door lock 210 located above the device and fix it into the initial state;

[0026] (2) Sample addition: Use a pipette to add 3 - 5 μL of sample to the sample carrier grid obtained in step (1) to ensure that there is liquid on both sides of the sample carrier grid;

[0027] (3) Liquid absorption: Operate / control the filter paper liquid absorption module 300 to adsorb the sample on the sample carrier grid obtained in step (2) according to the preset parameters;

[0028] (4) Freezing sample preparation: After step (3) is completed, the sample clamping module 200 automatically clamps the carrier grid and puts it into the freezing sample preparation module 400. When the guide rail slider 260 of the sample clamping module 200 drops to the target position, it contacts the buffers symmetrically distributed on both sides of the guide rail slider 260 and slowly stops descending;

[0029] (5) Unloading: Open the fixing seat flip cover 242 to remove the tweezers 270, and put the sample into the carrier grid storage box 442.

[0030] The rapid freezing sample preparation equipment provided by the utility model has the following technical effects:

[0031] (1) High-precision control: Existing rapid freezing sample preparation equipment requires manual use of filter paper to adsorb liquid droplets, and it is impossible to quantify and control the adsorption time, strength, and angle. The sample preparation results cannot be quantitatively controlled, and there are large random errors, resulting in insufficiently accurate experimental results and consuming additional time and costs. The main structures of the rapid freezing sample preparation equipment provided by the utility model include a tweezer flip clamping mechanism, a filter paper pushing mechanism, a freezing sample preparation component, and an electric control component. The electric control component includes a touch display and a physical button group, which can conveniently control the projection time of the tweezers, control the telescopic distance, telescopic speed, initial position, and residence time of the filter paper pushing mechanism, and realize adjustable experimental parameters through a photoelectric sensor; realize the quantification and repeatability of experimental data, and greatly improve the experimental accuracy.

[0032] (2) Stability: The design of the existing sample preparation equipment at the tweezer fixing part is relatively simple. When the tweezers are put into the ethane pool, there is a large vibration phenomenon. If the carrier grid is not firmly clamped, it may fall due to vibration, resulting in experimental failure; the carrier grid may touch the ethane pool or solidified ethane, causing bending deformation, damaging the carrier grid, and causing waste; the vibration of the tweezers in the ethane pool may cause liquid ethane to splash out. If the sample has a biosafety hazard (such as a virus), it will cause environmental pollution and various safety problems; the existing sample preparation equipment does not consider the vibration buffering problem, and it is difficult to conduct the vibration force out, easily causing the vibration of the tweezers to intensify when suddenly stopped, thus damaging the film structure on the sample surface. In the tweezer flip clamping mechanism of the rapid freezing sample preparation equipment provided by the utility model, there is a buffer. The buffer absorbs energy through symmetric arrangement to prevent rebound; the use of an optical axis and a guide rail slider ensures the structural parallelism during sliding and reduces the vibration of the tweezers in the horizontal direction. The use of an optical axis and a guide rail slider ensures the movement accuracy and reduces the probability of problems caused by the collision vibration after the tweezers are put into ethane; the use of symmetrically arranged buffers avoids the rebound of the tweezer flip clamping mechanism. A buffer pad is added to the bottom of the equipment to ensure that the overall vibration of the equipment is reduced after the carrier grid enters the liquid ethane, and the stability of the equipment is significantly improved. The combination of the filter paper disk angle slide and the Z-axis lifting platform provides more adjustable filter paper adsorption angles; the overall structure of the equipment is compact, occupying a small space, being light in weight, and convenient for handling; each component can be independently adjusted in position; the filter paper disk is marked with numbers and is convenient to move on the structure; different types of tweezers can be replaced for the tweezer fixing part; in the freezing sample preparation component, the liquid nitrogen pool and the workbench can be replaced according to requirements; there is a special position for placing the sample box.

[0033] (3)Flexibility and ease of use: The existing sample preparation equipment has a complex and large structure, occupies a large space, and cannot cope with special environments (e.g., inside an anaerobic workstation). Moreover, the existing sample preparation equipment has complex functions and is difficult to operate. The rapid freezing sample preparation equipment provided by the present utility model has a compact structure, with a volume approximately 1 / 3 of that of similar products, 348 mm in length, 253 mm in width, 506 mm in height, and is light in weight, about 10.2 kg, making it easy to carry. Each component is relatively independent, and its relative position is adjustable in the x, y, and z-axis directions. In the freezing sample preparation module, the position-limiting piece and the heightening piece are fixed to the electronic control module with long slotted holes. The relative position between the freezing and sample preparation module and other components in the x and y axes can be adjusted by adjusting the position of the fastening screws in the long slotted holes, and the elevation angle of the filter paper tray can also be adjusted by the filter paper tray angle slide. A precision displacement stage is adopted. The filter paper tray angle slide is equipped with an inclination angle scale, and the filter paper tray angle slide knob is equipped with a height scale, which is clear and quantifiable. The tweezer fixing block can be used with different models of tweezers without additional drilling, and the replacement is convenient. In the freezing sample preparation component, the workbench is detachable and replaceable, and different liquid nitrogen pools, workbenches, and their accessories can be selected according to needs. The sample addition position is in the front, which is convenient for both left and right hands to operate and also convenient for observation. The filter paper tray is marked with numbers, which is convenient for recording whether it is used, and it is easy to toggle and rotate in terms of structure. The placement directions of the grid storage boxes on the workbench are all in convenient directions (attached Figure 8 ), which is convenient for single-handed operation, and both left and right hands can operate conveniently, and there are replaceable different workbenches; Quick clamps are used, and a tweezer flip clamping mechanism can be operated with one hand; There is a special sample box placement groove inside the equipment; No additional gas source is required, and it can be used after being powered on.

[0034] (4)Cost: Compared with the benchmark product, the rapid freezing sample preparation equipment provided by the present utility model has a relatively simple structure, is easy to mass-produce, is convenient for production and manufacturing, has a relatively low price, and has excellent cost performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a three-dimensional structure schematic diagram of the rapid freezing sample preparation equipment according to an embodiment of the present utility model;

[0036] Figure 2 is a front view and a side view of the dust cover according to an embodiment of the present utility model;

[0037] Figure 3 is a front view and a side view of the rapid freezing sample preparation equipment according to an embodiment of the present utility model;

[0038] Figure 4 is a front view and a side view of the structure of the tweezer flip clamping mechanism according to an embodiment of the present utility model;

[0039] Figure 5 is a front view and a side view of the filter paper liquid absorption module according to an embodiment of the present utility model;

[0040] Figure 6 Stereo exploded view of the freezing sample preparation module structure according to an embodiment of the present utility model;

[0041] Figure 7 Open view and back view of the tweezer fixing block according to an embodiment of the present utility model;

[0042] Figure 8 Top view of the first replaceable workbench according to an embodiment of the present utility model;

[0043] Figure 9 Schematic diagram of the working condition when the rotatable filter paper disk rotates to the No. ① position according to an embodiment of the present utility model;

[0044] Figure 10 Schematic diagram of the working condition when the rotatable filter paper disk rotates to the No. ③ position according to an embodiment of the present utility model;

[0045] Figure 11 Schematic diagram of the working condition when the angle slide table is at +15° according to an embodiment of the present utility model;

[0046] Figure 12 Schematic diagram of the working condition when the angle slide table is at -15° according to an embodiment of the present utility model;

[0047] Figure 13 Schematic diagram of the working condition when the angle slide table is at 0° according to an embodiment of the present utility model.

[0048] The reference numerals in the figure are respectively:

[0049] 100 - dust cover, 200 - sample clamping module, 210 - electromagnetic lock, 220 - optical axis, 230 - buffer, 240 - tweezer fixing block, 241 - fixing block base, 242 - fixing seat flip cover, 243 - fixing seat flip cover knob, 244 - coupling member, 245 - fixing groove, 250 - magnet, 260 - guide rail slider, 270 - tweezer, 280 - specimen grid, 300 - filter paper liquid absorption module, 310 - rotatable filter paper disk, 311 - filter paper, 320 - adjustable photoelectric sensor bracket, 321 - photoelectric sensor, 330 - push rod type linear stepping motor, 340 - filter paper disk angle slide table, 341 - filter paper disk angle slide table knob, 350 - Z-axis lifting platform, 351 - Z-axis lifting platform knob, 360 - horizontal guide rail slider, 370 - guide rail slider fixing plate, 380 - induction piece, 390 - lighting lamp strip, 400 - freezing sample preparation module, 410 - cold conduction sheet, 420 - ethane pool, 430 - heat insulation foam, 440 - first replaceable workbench, 441 - second replaceable workbench, 442 - specimen grid storage box, 450 - workbench base, 460 - liquid nitrogen pool, 470 - limiting member, 480 - heightening member, 500 - specimen grid box, 600 - electric control module. Specific Embodiments

[0050] The following specific embodiments are used to further describe the present utility model, and the advantages and features of the present utility model will become clearer as the description progresses. However, these embodiments are exemplary only and do not constitute any limitation to the protection scope defined by the claims of the present utility model.

[0051] Structural Embodiment

[0052] Figure 1 FIG. is a schematic three-dimensional structure diagram of a rapid freezing sample preparation device according to an embodiment of the present utility model. Attached Figure 1 shows the dust-proof cover and the main body of the rapid freezing sample preparation device. Above the main body structure of the rapid freezing sample preparation device is the sample clamping module 200, behind it is the filter paper liquid absorption module 300, below it is the freezing sample preparation module 400, and below the freezing sample preparation module 400 is the electric control module 600.

[0053] Figure 2 FIGS. are the front view and side view of the dust-proof cover according to an embodiment of the present utility model. Handles are provided at the lower sides of the front and back of the dust-proof cover for easy taking and placing.

[0054] Figure 3 FIGS. are the front view and side view of the rapid freezing sample preparation device according to an embodiment of the present utility model. Figure 3 shows the relative positions of the sample clamping module 200, the filter paper liquid absorption module 300, the freezing sample preparation module 400, the grid holder 500, and the electric control module 600. The left button is the switch of the lighting lamp strip 390, and the right button is the emergency stop switch.

[0055] Figure 4 FIGS. are the front view and side view of the sample clamping module according to an embodiment of the present utility model. Figure 4 As can be seen, the tweezers 270 for clamping the grid are fixed to the tweezers fixing block 240. The front part of the tweezers fixing block 240 is the fixing seat flip cover 242, and the back part is the fixing seat base 241. The fixing seat flip cover knob 243 is provided on the face of the fixing seat flip cover 242, and a magnet 250 is provided on the back of the fixing seat base 241 for magnetically strengthening the tweezers to ensure its stability. The optical axis 220 is arranged in parallel with the guide rail slider 260 with a spacing. The guide rail slider 260 is connected to the tweezers fixing block 240. The optical axis 220 and the guide rail slider 260 jointly realize the guiding of the tweezers fixing block 240. Standard oil pressure buffers 230 are symmetrically arranged at the lower part of the running track of the guide rail slider 260. When the guide rail slider 260 descends to a specified position, it contacts the buffer 230 to reduce the vibration of the fixing block, avoid splashing of the sample liquid, ensure biological safety and avoid damage to the sample.

[0056] Figure 5The front view and side view of the filter paper pushing mechanism structure according to an embodiment of the present utility model. Figure 5 Among them, the rotatable filter paper disk 310 is installed on the horizontal guide rail slider 360. Digital serial numbers ①-⑧ are laser engraved on the filter paper disk, which can record the experimental serial numbers; the horizontal guide rail slider 360 is fixed on the guide rail slider fixing plate 370. The rotatable filter paper disk 310 is connected to the push rod type linear stepper motor 330, is driven by its power and moves through the guidance of the horizontal guide rail slider 360. The photoelectric sensor 321 and the adjustable photoelectric sensor bracket 320 are respectively installed on both sides of the guide rail slider fixing plate 370 and can be adjusted to any position along the horizontal direction. The induction sheets 380 are respectively installed on both sides of the horizontal guide rail slider 360 and can move along with the horizontal guide rail slider. When the photoelectric sensor 321 senses that the induction sheet 380 passes through, the position information of the horizontal guide rail slider 360 at this time is transmitted to the electric control module 600. The filter paper disk angle slide 340 is installed under the push rod type linear stepper motor 330, and the Z-axis lifting platform 350 is installed under the filter paper disk angle slide 340. A filter paper disk angle slide knob 341 is provided on the filter paper disk angle slide 340, and the guide rail slider fixing plate 370 can be adjusted by ±15°; a Z-axis lifting platform knob 351 is provided on the Z-axis lifting platform 350, and the Z-axis lifting platform can be raised, and the raising range is 0-10 mm.

[0057] Figure 6 The three-dimensional exploded view of the freezing sample preparation module structure according to an embodiment of the present utility model. The freezing sample preparation module 400 includes a liquid nitrogen pool 460 that provides a cold source, an ethane pool 420 that quickly freezes the sample carrier grid. An insulating foam 430 for heat preservation is also arranged on the outer periphery of the ethane pool 420. A heat conduction sheet 410 that transfers the temperature of the liquid nitrogen pool 460 to the ethane pool 412, a carrier grid storage box 442 for storing the carrier grid, and a workbench for storing the carrier grid storage box 442; a workbench base 415 that supports the workbench and fixes the workbench to the liquid nitrogen pool, a limiting member 470 that fixes the liquid nitrogen pool 460, and a padding member 480 that protects and pads the equipment. Different heat conduction sheets and liquid nitrogen pools can be selected according to different application requirements. In the freezing sample preparation module provided by the present utility model, the workbench is optional. Figure 6 Two optional workbenches are given: the first replaceable workbench 440, the second replaceable workbench 441, and the workbench base 450. Figure 8 The top view of a first replaceable workbench 440 is given. The first replaceable workbench 440 can place 2 carrier grid storage boxes 442 and 8 carrier grids 280, and the second replaceable workbench 441 can place 3 carrier grid storage boxes 442.

[0058] Application embodiment

[0059] 1. Place the rapid freezing sample preparation equipment on a horizontal tabletop. After turning on the power, turn on the main switch of the equipment. When on the working interface and the equipment status is in the READY state, you can enter the configuration interface from the touch screen for settings. The main adjustable parameters are as follows:

[0060] (1) The time for the filter paper to extend for liquid absorption: This time will also be displayed in the countdown information on the working interface. The parameter can be adjusted according to user needs;

[0061] (2) Initial position: The initial position where the filter paper retracts after the equipment reset is completed. The parameter can be adjusted according to user needs;

[0062] (3) Working position: The position where the filter paper extends during operation. The parameter can be adjusted according to user needs;

[0063] (4) Quick retraction position: The position where the filter paper quickly retracts after adsorption is completed. To prevent the forceps from colliding with the filter paper when falling, the forceps will only be released and fall when the second sensor senses that the filter paper has retracted. The parameter can be adjusted according to user needs;

[0064] (5) Quick retraction speed: The speed at which the filter paper retracts to the quick retraction position after adsorption is completed. The parameter can be adjusted according to user needs;

[0065] (6) Equipment reset speed: The slower this speed is, the higher the accuracy of the equipment reset. The parameter can be adjusted according to user needs.

[0066] 2. Operating steps

[0067] Attachment Figure 7 The three-dimensional exploded view of the forceps fixing block of an embodiment of the present utility model is given, showing the specific structure after the sample clamping module's flip cover. The actual operation process in the working state is as follows:

[0068] (1) Fixing and sample addition:

[0069] 1) Use forceps 270 to pick up the hydrophilized carrier grid from the carrier grid box 500;

[0070] 2) Put the tweezers 270 into the coupling 244 and tighten it with screws. After tightening, put the coupling 244 into the fixing groove of 241. The magnet 250 will produce a magnetic attraction on the tweezers 270 to ensure that the tweezers 270 are fixed and will not fall. After closing the fixing seat flap 242, you can tighten it by hand by screwing the fixing seat flap knob 243 to ensure that the tweezers will not shake. This process can be operated with one hand. The prior art uses a dovetail groove structure to fix the tweezers. This operation requires both hands to align the slots to install the tweezers. The buffer 230 used to prevent the vibration of the tweezers is a standard oil pressure buffer. When the guide rail slider 260 falls to the target position, it will contact the buffer to play a buffering and limiting role. The two buffers are symmetrically distributed on both sides of the guide rail slider 260, which can effectively absorb the vibration of the guide rail slider 260 during the falling process. Finally, the tweezers fixing block 240 is lifted to the electromagnet door lock 210 located above the device and fixed into the initial state.

[0071] (2) Sample addition: Use a pipette to add 3 to 5 μl of sample to the sample grid obtained in step (1), ensuring that there is liquid on both sides of the sample grid.

[0072] (3) Aspiration: Replace the filter paper of the filter paper aspiration module 300 and fix it to the center of the silicone pad of the filter paper disc with a round magnet. The filter paper disc is laser engraved with digital numbers ①-⑧, which can be used to record the experimental sequence; use your fingers to move the filter paper disc so that the number is at the top. Figure 9 This is a schematic diagram of the working condition of rotating the rotatable filter paper disc to position ①; Figure 10 The figure shows the working condition of rotating the rotatable filter paper disc to position ③. Manually fine-tune the parameters of the filter paper disc angle slide and the Z-axis lifting platform (the parameters can be adjusted according to user needs). Figure 11 The filter paper disc angle slide knob 341 is used to adjust the angle of the rotatable filter paper disc 310 to a working state of +15°; Figure 12 The working state is -15°. Figure 13 The working state is 0°. Press the start button, and the filter paper liquid absorption module 300 absorbs the sample according to the preset parameters (the parameters can be adjusted according to user needs).

[0073] (4) Frozen sample preparation:

[0074] After assembling and precooling the freezing sample preparation component, inject ethane and install the freezing sample preparation component on the limiter to ensure that the limiter matches the outer contour of the freezing component. After step (3) is completed, the sample clamping module 200 automatically clamps the carrier net and puts it into the freezing sample preparation module. When the guide rail slider 260 of the sample clamping module 200 falls to the target position, it contacts the buffers symmetrically distributed on both sides of the guide rail slider 260 and slowly stops descending;

[0075] (5)Unloading: Open the fixed base flip cover 242, remove the forceps 270, and place the sample into the grid storage box 442. Transfer the grid storage box to a liquid nitrogen container for storage. Turn off the main switch of the rapid freezing sample preparation equipment and cover it with a dust cover.

Claims

1. A rapid freezing sample preparation device, characterized in that, The device includes a freezing sample preparation module (400), a grid cassette (500), and an electric control module (600). The rapid freezing sample preparation device further includes a sample clamping module (200) and a filter paper liquid absorption module (300). The freezing sample preparation module (400) is located below the sample clamping module (200) and is used to perform freezing sample preparation on the sample grid after liquid absorption treatment. The position of the filter paper liquid absorption module (300) is set corresponding to the position of the sample clamping module (200) to ensure that the filter paper liquid absorption module (300) is used to perform liquid absorption treatment on the sample liquid film on the sample grid clamped by the sample clamping module (200).

2. The rapid freezing sample preparation device according to claim 1, characterized in that, The sample clamping module (200) includes tweezers (270) for clamping the grid, a coupling member (244) connected to the tweezers, a flip-up tweezers fixing block (240) for accommodating and fixing the tweezers (270), an optical axis (220) and a guide rail slider (260) for reducing the vibration of the tweezers fixing block (240). The guide rail slider (260) is parallel to the optical axis (220) and has a spacing therebetween to jointly guide the tweezers fixing block (240). There is an electromagnet door lock (210) for fixing the initial state, and symmetrically arranged buffers (230) for buffering the guide rail slider (260).

3. The rapid freezing sample preparation device according to claim 2, characterized in that, The tweezers fixing block (240) in the sample clamping module (200) is composed of a fixing block base (241) and a fixing seat flip cover (242), which are connected by a cylindrical pin to form a flip-up clamping mechanism. A fixing groove (245) for placing the tweezers (270) connected with the coupling member (244) is provided on the fixing block base (241), and a magnet (250) for fixing the tweezers (270) is provided on the back of the fixing seat.

4. The rapid freezing sample preparation device according to claim 2, wherein The buffer (230) in the sample clamping module (200) is an oil pressure buffer.

5. The rapid freezing sample preparation device according to claim 1, characterized in that, The filter paper liquid absorption module (300) includes a rotatable filter paper disk (310), an adjustable photoelectric sensor bracket (320), a photoelectric sensor (321), a push rod type linear stepper motor (330) for driving the rotatable filter paper disk (310), a horizontal guide rail slider (360) for guiding the rotatable filter paper disk (310), a guide rail slider fixing plate (370), and an induction piece (380). In the filter paper liquid absorption module (300), the photoelectric sensor (321) and the adjustable photoelectric sensor bracket (320) are respectively installed on both sides of the guide rail slider fixing plate (370) and can be adjusted to any position in the horizontal direction. The induction pieces (380) are respectively installed on both sides of the horizontal guide rail slider (360) and can move along with the horizontal guide rail slider (360). When the photoelectric sensor (321) senses that the induction piece (380) passes through, the position information of the horizontal guide rail slider (360) at this time is transmitted to the electric control module (600).

6. The rapid freezing sample preparation device according to claim 5, wherein, The filter paper liquid absorption module (300) further includes a filter paper disk angle slide table (340) and a Z-axis lifting platform (350). By rotating the filter paper disk angle slide table knob (341), the angle of the rotatable filter paper disk (310) can be adjusted for up-and-down angle adjustment based on the horizontal direction; by rotating the Z-axis lifting platform knob (351), the Z-axis lifting platform (350) can be raised.

7. The rapid freezing sample preparation device according to claim 1, wherein, The freezing sample preparation module (400) includes a liquid nitrogen pool (460) that provides a cold source, an ethane pool (420) that quickly freezes the sample carrier grid, a heat conduction sheet (410) that transfers the temperature of the liquid nitrogen pool (460) to the ethane pool (420), a carrier grid storage box (442) for storing the carrier grid, a workbench (440) for storing the carrier grid storage box (442); a workbench base (450) that supports the workbench and fixes the workbench to the liquid nitrogen pool, a limiting member (470) that fixes the liquid nitrogen pool (460), and a padding member (480) that protects and raises the quick-freezing sample preparation device.

8. The rapid freezing sample preparation device according to claim 1, characterized in that, The electric control module (600) includes a module that collects and processes the position information of the horizontal guide rail slider (360) transmitted back by the photoelectric sensor (321) in the filter paper liquid absorption module (300), a projection control module that controls the projection time of the tweezers (270) in the sample clamping module (200) based on the position information, a telescopic control module that controls the telescopic distance, telescopic speed, initial position, and residence time of the filter paper in the filter paper liquid absorption module (300) based on the position information, and a control module that controls the electromagnet door lock (210).

9. The rapid freezing sample preparation device according to claim 1, wherein The device is also equipped with a dust cover (100).

10. The rapid freezing sample preparation device according to claim 7, characterized in that, An insulating foam (430) for heat preservation is further arranged on the outer periphery of the ethane pool (420).

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