An ultra-low temperature light-stimulated frozen sample preparation device and a preparation method thereof

Abstract

This invention discloses an ultra-low temperature photostimulated frozen sample preparation device and its preparation method. The device includes an insulated box, an electric magnetic suction insertion rod, and a photostimulation system. The electric magnetic suction insertion rod includes an insertion rod, an electromagnet, a counterweight, a damping module, a guide rail, and tweezers. When the electromagnet is energized, it is fixedly connected to the top of the insertion rod. The counterweight is fixed to the upper middle part of the insertion rod. The guide rail and the damping module are used to control the falling trajectory of the insertion rod and reduce the vibration of the insertion rod during falling, respectively. Tweezers are fixed to the lower part of the insertion rod by screws with handles. The tweezers are used to hold a sample-containing mesh. When the electromagnet is de-energized, the insertion rod falls along the guide rail and drives the tweezers to fall, so that the sample-containing mesh finally falls into the frozen chamber in the insulated box. The photostimulation system is used to photostimulate the sample. This invention can prepare frozen samples of different types, can be used to study the physiological state of samples at the moment of photostimulation, and the thickness of the frozen sample can be flexibly adjusted according to needs.

Description

Technical Field

[0001] This invention relates to the field of ultra-low temperature frozen sample preparation technology, and more particularly to an ultra-low temperature photostimulated frozen sample preparation device and method. This invention can perform ultra-low temperature frozen preparation on different types of samples, ensuring the stability of the preparation process. It also allows for the application of light stimulation of different wavelengths and intensities to the samples before freezing, enabling the study of the physiological state of the samples at the moment of light stimulation, and the investigation of changes in ion concentration, ultrastructural morphology, and material state within cells, tissues, biomolecules, and materials before and after light stimulation. Background Technology

[0002] Cryo-electron microscopy (cryo-EM) is a microscopy technique used to observe samples at extremely low temperatures using a transmission electron microscope. Currently, cryo-EM is primarily used to analyze the structures of large protein complexes and is considered a powerful tool for exploring the mysteries of life, representing a revolutionary and epoch-making technology in life science research. The first step in cryo-EM is sample preparation, which involves spreading the sample in solution onto a hydrophilically treated grid to form a very thin sample liquid layer. The grid is then immersed in liquid ethane for rapid freezing. Rapid freezing transforms the sample into an amorphous state of ice (vitrified sample). Vitrification forms an amorphous solid with minimal structural damage and does not interfere with electron diffraction data collection. Rapidly frozen samples exhibit significantly improved tolerance to electron radiation during transmission electron microscopy, and the frozen sample particles retain their natural morphology. Cryo-EM sample preparation is crucial for cell and structural biology research that requires rapidly lowering the sample temperature to prevent the crystallization of surrounding water molecules. Good vitrification is a key step in structural biology applications such as single-particle analysis (SPA), cryo-electron tomography (cyro-ET), and microcrystalline electron diffraction (MicroED).

[0003] Existing automated sample preparation instruments are mainly designed for the cryogenic preparation of protein solution samples. However, with the development of cryo-electron microscopy (cryo-EM) technology in recent years, especially the maturity of cryo-electron tomography (cryo-EM), the demand for cryo-EM applications in various fields, including cell samples, tissue samples, biomacromolecule samples, materials chemistry, new energy, and geotechnical engineering, has increased significantly, and sample preparation needs have become increasingly diversified. However, existing instruments are difficult to adapt to the diverse needs of these new sample preparation requirements. Furthermore, sample preparation equipment is heavily reliant on imports, resulting in high purchase costs (800,000-1,000,000 RMB per instrument), high costs for accessories and consumables, and limited functionality, hindering the widespread adoption of cryogenic sample preparation technology. There is a pressing need for inexpensive and widely applicable sample preparation equipment that can meet the needs of different disciplines and be used for a range of activation conditions, such as photostimulation and electrical stimulation, to simulate various physiological phenomena and changes. A reliable, universal, and low-cost photostimulated cryogenic sample preparation device and method can significantly improve the applicability of this type of instrument, greatly facilitating the promotion and application of cryo-EM technology across multiple disciplines. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the main objective of this invention is to provide a reliable, universal, and low-cost ultra-low temperature photostimulated frozen sample preparation device and preparation method thereof.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A cryogenic photostimulation freezing sample preparation device includes: a device base and a device back plate fixedly connected to the device base, an insulated box on the device base, a freezing chamber and a sample transfer station placed in the insulated box, an electric magnetic suction insertion rod, an ethane input pipeline and an illumination system on the device back plate, and a photostimulation system.

[0006] The insulated box is pre-filled with liquid nitrogen to maintain an ultra-low temperature environment for the samples. The bottom of the insulated box is equipped with a freezing chamber and a sample transfer station. The freezing chamber consists of inner and outer cavities. The inner cavity (i.e., the liquid ethane chamber) is filled with liquid ethane for rapid sample freezing; the outer cavity (i.e., the isolation cavity) is empty and used to maintain the temperature of the inner cavity. The freezing chamber is made of a material with good thermal conductivity, typically copper. The freezing chamber is fixed in position, with the inner cavity and the input rod on the same vertical line. The sample transfer station has multiple sample loading boxes, each capable of holding multiple sample carriers, with samples fixed on the carriers. The sample loading boxes have holes for placing the carriers, which are stably positioned in the holes and not easily moved or detached.

[0007] The electric magnetic insertion rod includes an insertion rod, an electromagnet, a counterweight, a damping module, a guide rail, and tweezers. The electromagnet is fixed to the top of the device's back plate and, when energized, is fixedly connected to the top of the insertion rod, thus securing the insertion rod to the top of the device's back plate. The counterweight is fixed to the upper middle part of the insertion rod. The guide rail and damping module are fixed to the device's back plate, respectively used to control the insertion rod's falling trajectory and reduce vibration during its descent. The lower part of the insertion rod clamps and secures the tweezers with a screw and handle. The tweezers are used to hold a sample-containing mesh, and a rubber ring is fitted at the bottom of the tweezers to secure the mesh after clamping. The insertion rod can rotate 360 ​​degrees, allowing adjustment of the mesh's orientation for easy operation. The insertion rod is aligned vertically with the cryogenic chamber's interior. The initial height of the tweezers holding the mesh is adjustable, ensuring that the tweezers do not touch the inner wall or bottom of the cryogenic chamber after insertion.

[0008] The ethane input pipeline is connected to an ethane cylinder to input ethane into the refrigeration chamber. The ethane input flow rate can be adjusted by an ethane control valve. The height and illumination angle of the lighting system are adjustable, and it is used to provide illumination for the device, facilitating operation during the sample preparation process. The photostimulation system can be synchronously or individually activated via a control switch. The system includes a laser and a controller. The laser wavelength and intensity are adjustable; the controller can adjust parameters such as laser intensity, emission duration, interval duration, and frequency to optimize the photostimulation scheme. The laser spot irradiates the sample or its falling path, providing photostimulation to the sample before cryo-fixation. The spot height can be adjusted via a three-axis lifting platform beneath the laser.

[0009] Before freezing, the sample-containing grid needs to have excess moisture absorbed on the back of the grid using filter paper to ensure that the sample thickness meets the requirements for subsequent imaging.

[0010] This invention also discloses a method for preparing ultra-low temperature photostimulated frozen samples, which is based on the aforementioned ultra-low temperature photostimulated frozen sample preparation device and specifically includes the following steps: 1) Connect the device to the power supply, and the electromagnet will be energized; turn on the lighting, raise the insertion rod, and the insertion rod will be fixed to the back plate of the device by the attraction of the electromagnet. 2) Inject liquid nitrogen into the insulation box, ensuring the liquid nitrogen level is lower than the height of the cryogenic chamber; after the temperature stabilizes, open the ethane control valve and inject ethane into the liquid ethane chamber; 3) Turn on the light stimulation system and set the laser parameters through the laser controller; 4) Use tweezers to hold the sample-loaded grid and fix the tweezers to the lower part of the feed rod with a screw with a handle; 5) Turn on the laser and adjust its position using the three-axis lifting platform so that the optical path is on the path of the falling net and perpendicular to the net; use filter paper to absorb excess liquid, turn off the power of the device, de-energize the electromagnet, and the input rod falls rapidly. The sample is then input into the liquid ethane chamber along with the net. The sample input process passes through the laser optical path. 6) Turn on the power to the device, loosen the screw with the handle, raise the input rod to the highest point, and the input rod is fixed to the back plate of the device by the attraction of the electromagnet; quickly transfer the tweezers together with the sample-loaded net from the liquid ethane chamber to the liquid nitrogen in the insulated box; release the tweezers and transfer the sample-loaded net to the sample loading box in the sample transfer station to complete the photostimulation and freezing preparation of the sample.

[0011] The beneficial effects of this invention are as follows: 1) It can perform cryopreservation preparation of different types of samples, such as cells, tissues, biomolecules, and materials, ensuring the stability of the preparation process; 2) Samples can be stimulated with light of different wavelengths, intensities, and durations before freezing preparation; 3) It can be used to study the physiological state of a sample at the moment of light stimulation, and to study the changes in ion concentration, ultrastructure morphology, etc. in cells, tissues and other samples before and after light stimulation; 4) It can be used to study the ultrastructural changes of biological macromolecules and materials under light stimulation; 5) The thickness of frozen samples is controllable and can be flexibly adjusted according to experimental needs. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the structural composition of the device of the present invention; Figure 2 This is a top view of the insulation box in the device of this scheme; The components include: 1. Electromagnet; 2. Counterweight; 3. Damping module; 4. Guide rail; 5. Lighting system; 6. Screw with handle; 7. Tweezers; 8. Ethane input terminal; 8-1. Ethane output terminal; 9. Ethane control valve; 10. Sample-containing mesh; 11. Liquid ethane chamber; 12. Isolation chamber; 13. Liquid nitrogen; 14. Insulation box; 15. Control switch; 16. Laser and controller; 17. Three-axis lifting platform; 18. Sample transfer station; 19. Sample loading box. Detailed Implementation

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0014] like Figure 1The image shows an ultra-low temperature photostimulated cryogenic sample preparation device according to the present invention. The device specifically includes a device base and a device back plate fixedly connected to the device base, an insulated box 14 disposed on the device base, a cryogenic chamber and a sample transfer station 18 placed in the insulated box 14, an electric magnetic suction insertion rod, an ethane input pipeline and an illumination system 5 disposed on the device back plate, and a photostimulation system.

[0015] Liquid nitrogen is pre-filled into the insulated box to maintain an ultra-low temperature environment for the sample. Figure 2 As shown, the bottom of the insulated box 14 is equipped with a freezing chamber and a sample transfer station 18. The freezing chamber consists of an inner and an outer cavity. The inner cavity (i.e., the liquid ethane chamber 11) is filled with liquid ethane for rapid freezing of samples. The outer cavity (i.e., the isolation cavity 12) is used to maintain the temperature of the inner cavity. The isolation cavity 12 is a hollow cavity to prevent external liquid nitrogen from directly contacting the inner cavity and causing the inner cavity temperature to drop too low. The freezing chamber is made of a material with good thermal conductivity, typically copper. The freezing chamber is fixed in position, and the inner cavity is on the same vertical line as the input rod. The sample transfer station 18 is equipped with multiple sample loading boxes 19, each of which can hold multiple carrier nets, on which samples are fixed. The sample loading boxes 19 have holes for placing the carrier nets, and the carrier nets are stably placed in the holes and are not easily moved or detached.

[0016] The electric magnetic insertion rod includes an insertion rod, an electromagnet 1, a counterweight 2, a damping module 3, a guide rail 4, and tweezers 7. The electromagnet 1 is fixed to the top of the device's back plate, and its energization is controlled by a control switch 15. When energized, the electromagnet 1 is fixedly connected to the top of the insertion rod, thus securing the insertion rod to the top of the device's back plate. The counterweight 2 is fixed to the upper middle part of the insertion rod. The guide rail 4 and damping module 3 are fixed to the device's back plate, respectively controlling the insertion rod's falling trajectory and reducing vibration during its descent. The damping module can be made of a low-temperature resistant and shock-absorbing material, such as polytetrafluoroethylene (PTFE), to prevent the insertion rod from rebounding after falling into liquid ethane, thus preventing the sample from rebounding above the liquid surface and causing the sample to heat up. The lower part of the insertion rod is clamped and fixed with a screw 6 with a handle; the tweezers 7 are used to clamp the sample-containing mesh 10, and the lower part of the tweezers 7 is equipped with a rubber ring to fix the mesh after clamping; the insertion rod can rotate 360 ​​degrees to adjust the orientation of the mesh for easy operation; the insertion rod is on the same vertical line as the inner cavity of the freezing chamber, the initial height of the tweezers clamping the mesh is adjustable, and the tweezers will not touch the inner wall and bottom of the inner cavity of the freezing chamber after being inserted into the freezing chamber.

[0017] The ethane input pipeline connects to an ethane cylinder, supplying ethane into the refrigeration chamber. The ethane input flow rate can be adjusted via an ethane control valve 9. The ethane input pipeline includes an ethane input end 8 and an ethane output end 8-1. The ethane input end 8 is connected to the ethane cylinder and is used to introduce ethane into the ethane input pipeline; the ethane output end 8-1 is used to inject ethane into the liquid ethane chamber 11. Correspondingly, a rack can be installed on the back panel of the device so that the ethane output end 8-1 can be placed on the rack after ethane injection is complete.

[0018] As ethane flows from the ethane cylinder into the liquid ethane chamber 11 through the ethane inlet pipeline, it undergoes the following changes: Initially, the ethane stored in the high-pressure cylinder is liquid and under high pressure. Next, the ethane is released from the high-pressure cylinder and undergoes violent vaporization; during this process, the flow rate can be controlled using the ethane control valve 9. Finally, after the ethane enters the cryogenic chamber, the liquid nitrogen 13 in the insulation box 14 provides a low temperature to the liquid ethane chamber 11, causing the ethane to rapidly cool and become liquid again. If there are many frozen samples, resulting in a long processing time, the ethane may solidify. In this case, it can be stirred using a material with good thermal conductivity, such as a copper rod, to maintain its liquid state.

[0019] The height and illumination angle of the lighting system 5 are adjustable, and it is used to provide illumination for the device, facilitating operation during the sample preparation process.

[0020] The photostimulation system can be synchronously or individually activated via a control switch. The system includes a laser and a controller 16. The laser wavelength and intensity are adjustable; the controller can adjust parameters such as laser intensity, emission duration, interval duration, and frequency to optimize the photostimulation scheme. The laser spot irradiates the sample or its falling path, providing photostimulation to the sample before freeze-fixation. The spot height can be adjusted via a three-axis lifting platform below the laser.

[0021] Before freezing, the sample-containing grid 10 needs to have excess moisture absorbed on the back of the grid using filter paper to ensure that the sample thickness meets the requirements for subsequent imaging.

[0022] Using the cryopreservation of cultured light-sensitive channel cells as a specific example, the process of preparing ultra-low temperature photostimulated frozen samples is explained. The sample preparation process is realized based on the above-mentioned ultra-low temperature photostimulated frozen sample preparation device.

[0023] First, the cell culture medium was pretreated: the cell culture medium (model: R2 / 1 Au 200, Quantifoil) was placed in a glass-bottomed culture dish, hydrophilized using a glow discharge apparatus (model: PELCO easiGlow, TED PELLA), then soaked in 75% ethanol and irradiated with ultraviolet light for 10 minutes, washed 6 times with ultrapure water, coated with Fibronectin (model: FC010, Millipore) at 37°C for 30 minutes, washed 6 times with ultrapure water, and then added to cell culture medium for later use.

[0024] Next, cell preparation was performed: Newborn mice with light-sensitive channel transgenic structures (P0) were sprayed with disinfectant alcohol. The mice were decapitated to obtain their heads. In a clean bench, the scalp and skull were cut along the midline of the skull using ophthalmic scissors. The skull was then carefully removed using ophthalmic forceps, and the brain was quickly transferred to a 35-mm culture dish containing pre-chilled HBSS (placed on an ice box). Under a dissecting microscope, the left and right hemispheres were separated using tethering forceps. The meninges, residual blood sinuses, and vascular tissue were removed, and the hippocampus was harvested. The hippocampus was digested for 30 minutes in a 37°C water bath using papain (200 μL, for one pair of cortical tissues or three pairs of hippocampal tissues; 2 μL DNase was added before use). The tissue block was gently removed, and the sample was mechanically ground 15-20 times with 1 mL of grinding buffer using a 1 mL grinding head (avoiding air bubbles). The sample was washed twice. The cells were centrifuged at 1,000 rpm for 5 minutes at 4°C. After washing the separated cells, they were then cultured in a 1 mL DNase-containing solution. Resuspend cells in ml of homogenate and count them using a cell counter. Dilute the cell suspension with culture medium (DMEM / F12 / 10% fetal bovine serum / fetal bovine serum albumin) and seed at a low density of 50,000–100,000 cells / dish in culture dishes containing the pretreated grids. After culturing for 3 hours (or overnight), replace the seeding medium with Neurobasal medium containing 2% B27, 0.5 mM L-glutamine, and antibiotics. One or two days after seeding, add cytarabine (Ara-C) to prevent excessive proliferation of glial cells. On days 4, 7, and 14, replace one-third of the culture volume with fresh culture medium. Check cell confluence and grid integrity every other day using an optical microscope to ensure that the cells are in a suitable state for subsequent experiments, such as cryo-electron microscopy.

[0025] Next, the preparation of ultra-low temperature photostimulated frozen samples can be carried out, which includes the following steps: 1) Turn on the control switch 15 to connect the device power supply; turn on the lighting and raise the input rod, which is fixed to the back plate of the device by the attraction of the electromagnet 1. 2) Inject liquid nitrogen into the insulation box 14, being careful not to submerge the isolation chamber. After the temperature stabilizes, open the ethane control valve 9 and inject liquid ethane into the liquid ethane chamber 11. 3) Turn on the laser and set the laser parameters; 4) Take the above-cultured mature cells, rinse with buffer, label with specific live cell dye, such as calcium dye, rinse with buffer after labeling, carefully grasp the edge of the sample-containing grid 10 with tweezers 7, pay attention to the front and back of the sample-containing grid 10, adjust the rubber ring on the tweezers 7, clamp and fix the sample-containing grid 10, fix the tweezers 7 to the lower part of the input rod with the screw 6 with the handle, rotate the input rod so that the back of the sample-containing grid 10 faces outward. 5) Turn on the laser, and adjust the optical path using the three-axis lifting platform 17 to be positioned on the path of the sample-containing mesh 10 as it falls, and perpendicular to the mesh. Use filter paper to absorb excess liquid, paying attention to controlling the liquid thickness. Quickly turn off the control switch 15, de-energize the electromagnet 1, and the insertion rod will fall rapidly. The sample-containing mesh 10 will be inserted into the liquid ethane chamber 11. During the insertion process, the sample passes through the laser optical path, and the photosensitive channel is opened after a momentary light stimulation. Optionally, the height of the laser optical path can be adjusted using the three-axis lifting platform 17 to ensure that the laser optical path passes through the sample-containing mesh 10. The laser parameters can be adjusted using the controller to control the laser intensity, frequency, and duration, thereby adjusting the light stimulation scheme and achieving different modes of light stimulation. 6) Turn on the control switch 15, loosen the screw 6 with the handle, raise the input rod to the highest point, and fix the input rod to the back plate of the device by the attraction of the electromagnet 1; quickly transfer the tweezers 7 together with the sample-containing mesh 10 from the liquid ethane chamber 11 to the liquid nitrogen 13, move the rubber ring of the tweezers 7 upward, release the tweezers 7, and carefully transfer the sample-containing mesh 10 to the sample loading box 19 located in the sample transfer station 18 to complete the photostimulation and freezing of the cell sample.

[0026] Using the sample loading box 19, the grid is transferred to a cryo-optical microscope or cryo-electron microscope for observation, recording, and analysis of changes in intracellular calcium ions and other signals before and after light stimulation.

[0027] In another embodiment, a method for preparing ultra-low temperature photostimulated frozen samples using biological macromolecules as samples is provided. The preparation process is similar to that for cell samples, with the only differences being the sample preparation and the loading of the sample onto a grid.

[0028] During sample preparation, it is necessary to prepare a solution of biological macromolecules that meets the requirements for electron microscopy observation. For example, if used for cryo-electron microscopy structure analysis, the purity is generally required to be greater than 80% and the concentration is higher than 1 mg / ml.

[0029] The method for loading the sample onto the carrier is to use a pipette to take 3-5 microliters of sample solution and add it directly to the carrier.

[0030] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A device for preparing ultra-low temperature photostimulated frozen samples, characterized in that, Includes a device base and a device back plate fixedly connected to the device base, an insulation box on the device base, an electric magnetic insertion rod, an ethane input pipeline and a lighting system on the device back plate, and a photostimulation system; The insulated box is used to maintain an ultra-low temperature environment for the sample, and the insulated box is equipped with a freezing chamber; The electric magnetic insertion rod includes an insertion rod, an electromagnet, a counterweight, a damping module, a guide rail, and tweezers. The electromagnet, damping module, and guide rail are all fixedly connected to the back plate of the device. When the electromagnet is energized, it is fixedly connected to the top of the insertion rod. The counterweight is fixed to the upper middle part of the insertion rod. The guide rail and damping module are used to control the falling trajectory of the insertion rod and reduce the vibration of the insertion rod during its fall, respectively. The tweezers are fixed to the lower part of the insertion rod by screws with handles. The tweezers are used to hold a sample-containing mesh. When the electromagnet is de-energized, the insertion rod falls along the guide rail and drives the tweezers to fall. The sample-containing mesh held by the tweezers finally falls into the cryogenic chamber. The ethane input pipeline is used to input ethane into the freezing chamber and form liquid ethane, thereby rapidly freezing the sample; The lighting system is used to provide illumination for the device; The photostimulation system is used to stimulate the sample before it falls into the cryogenic chamber.

2. The ultra-low temperature photostimulated cryogenic sample preparation device according to claim 1, characterized in that, The cryogenic chamber consists of an inner and an outer cavity. Ethane is injected into the inner cavity through an ethane inlet pipe to form liquid ethane for rapid freezing of samples. The outer cavity is used to maintain the temperature of the inner cavity. The center of the inner cavity of the cryogenic chamber is on the same vertical line as the injection rod.

3. The ultra-low temperature photostimulated cryogenic sample preparation apparatus according to claim 1, characterized in that, The tweezers are equipped with a rubber ring at the bottom to secure the carrier net after clamping.

4. The ultra-low temperature photostimulated cryogenic sample preparation apparatus according to claim 1, characterized in that, The initial height of the tweezers is adjustable, ensuring that the tweezers do not touch the inner wall and bottom of the cryogenic chamber after being inserted into it.

5. The ultra-low temperature photostimulated cryogenic sample preparation apparatus according to claim 1, characterized in that, The ethane input pipeline is connected to an ethane cylinder, and the ethane input flow rate is regulated by an ethane control valve.

6. The ultra-low temperature photostimulated cryogenic sample preparation apparatus according to claim 1, characterized in that, The photostimulation system includes a laser and a controller. The laser intensity, emission duration, interval duration, and frequency are adjusted by the controller to adjust the photostimulation scheme. The laser is used in conjunction with a three-axis lifting platform. The laser spot height is adjusted by adjusting the position of the laser through the three-axis lifting platform.

7. The ultra-low temperature photostimulated cryogenic sample preparation apparatus according to claim 1, characterized in that, The insulated box is also equipped with a sample transfer station; the sample transfer station is equipped with multiple sample loading boxes, and each sample loading box can load multiple carrier nets.

8. The ultra-low temperature photostimulated cryogenic sample preparation apparatus according to claim 1, characterized in that, The sample loading box has holes for placing a carrier net, and the carrier net is fixed inside the sample loading box through the holes.

9. The ultra-low temperature photostimulated cryogenic sample preparation apparatus according to claim 8, characterized in that, Before freezing, excess moisture in the sample-containing grid is absorbed by wiping the back of the grid with filter paper.

10. A method for preparing ultra-low temperature photostimulated frozen samples, characterized in that, The preparation of ultra-low temperature photostimulated cryogenic samples based on any one of claims 1-9 specifically includes the following steps: 1) Connect the device to the power supply, and the electromagnet will be energized; turn on the lighting, raise the insertion rod, and the insertion rod will be fixed to the back plate of the device by the attraction of the electromagnet. 2) Inject liquid nitrogen into the insulation box, ensuring the liquid nitrogen level is lower than the height of the cryogenic chamber; after the temperature stabilizes, open the ethane control valve and inject ethane into the liquid ethane chamber; 3) Turn on the light stimulation system and set the laser parameters through the laser controller; 4) Use tweezers to hold the sample-loaded grid and fix the tweezers to the lower part of the feed rod with a screw with a handle; 5) Turn on the laser and adjust its position using the three-axis lifting platform so that the optical path is on the path of the falling net and perpendicular to the net; use filter paper to absorb excess liquid, turn off the power of the device, de-energize the electromagnet, and the input rod falls rapidly. The sample is then input into the liquid ethane chamber along with the net. The sample input process passes through the laser optical path. 6) Turn on the power to the device, loosen the screw with the handle, raise the input rod to the highest point, and the input rod is fixed to the back plate of the device by the attraction of the electromagnet; quickly transfer the tweezers together with the sample-loaded net from the liquid ethane chamber to the liquid nitrogen in the insulated box; release the tweezers and transfer the sample-loaded net to the sample loading box in the sample transfer station to complete the photostimulation and freezing preparation of the sample.

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