Visual freezing system for observing ultra-fast freezing process of cells

Abstract

A visual freezing system for observing the ultra-fast freezing process of a small amount of cells at low temperature, mainly including a biological sample cabin for placing samples to be tested, a low-temperature freezing table for providing freezing cold, a protective gas subsystem for protecting the system from frost and condensation, an imaging recording subsystem for observing and recording the freezing process, a motion control subsystem for controlling the system movement to realize the system function, and a temperature measurement control subsystem for monitoring the system temperature. The present invention can rapidly and speed-controllably cool down cell samples; can visually image the rapid cooling process in a very short time, which is convenient for studying the formation mechanism of cell ice crystals; can change the atmosphere in the biological sample cabin and change the cabin pressure to a certain extent, which is convenient for studying the freezing process of samples under different atmospheres; can prevent the problem of condensation and frosting at low temperatures by blowing dry protective gas, and the sample changing operation is simple.

Description

Technical Field

[0001] The invention relates to the field of cryobiology and biomedicine, and in particular to a visual observation system for a cell low-temperature ultra-rapid freezing process. Background Art

[0002] Sperm, eggs, embryos, and tiny tissue sections will not die when they are subjected to low temperature under the protection of cryoprotectants, and can be stored at low temperatures for a long time. In the past, a large number of studies on the cryopreservation of biological samples have been carried out at home and abroad, and many preservation schemes have been formed, which are applied to the fields of efficient preservation of cells such as red blood cells, stem cells, and immune cells, assisted reproduction, transplantation of tissues and organs such as corneas and livers, and protection of rare species life resources.

[0003] The main component of biological samples is water. Water molecules may exist in two forms, crystal and glass, at low temperatures (mainly below -80°C), corresponding to two cryopreservation methods: programmed slow cooling and vitrification rapid cooling. The application of programmed slow cooling is relatively mature, but ice crystals are easily formed during the process, causing cryogenic damage, and the operation is mostly based on experience. Vitrification rapid freezing uses an extremely fast cooling rate to allow the water in the biological sample to directly enter a vitrified state, and no ice crystals are formed inside or outside the cells, thereby avoiding cryogenic damage. The study of intracellular ice crystal formation and extracellular cryoprotectant ice crystal formation is a prerequisite for the development of cryopreservation technology that minimizes cell and tissue damage. It is necessary to visualize the physical phenomena that occur during the rapid freezing of biological samples.

[0004] According to the existing patent search technology, the patent with the announcement number CN203985779U discloses a cell tissue vitrification carrier kit, including a metal plug mesh carrier for carrying cell tissue and a supporting member for supporting the carrier. The kit can fix the cells on the mesh carrier and cool them by immersing them in liquid nitrogen. The structure of this invention patent is simple, but it does not have the function of visual observation and high-speed recording of the vitrification process.

[0005] The patent with announcement number CN210809003U discloses a vitrification freezing device, which includes several chambers arranged in sequence along the same direction, the chambers are connected by pipes, and each chamber is filled with different concentrations of cryoprotectants. The invention aims to solve the adverse stress response of cells / embryos caused by the strong change of the protective agent concentration. However, the device is large in size and requires good mechanical coordination, and it is not possible to observe the internal and external morphology of cells during the vitrification process.

[0006] Patent No. CN106153622A discloses a device for observing vitrification, freezing and rethawing, which mainly includes a vertical microscope, a hollow cylindrical isolation box, a base, an observation carrier and a carrier moving instrument. The device can observe the state of different glass liquids during rapid freezing and rethawing, but the device uses liquid nitrogen or nitrogen fumigation, which is prone to frost and fog.

[0007] The patent with announcement number CN110057821A discloses a low-temperature microscopic imaging system for observing the rapid freezing and thawing process of human gametes, which mainly includes a gamete freezing and thawing device, a liquid nitrogen circulation cooling system, a microscopic imaging system, a measurement control system and a vacuum system, which can achieve rapid cooling of human gametes and real-time observation and recording of the details of sample changes during the freezing process. The low-temperature components in the system are protected in the form of vacuum. Although the requirements of thermal insulation and prevention of fogging and frost formation of the low-temperature components in the system are solved, the thermal conductivity mechanism and thermal insulation structure design of the system inevitably lead to the sample cabin and the cold source being in the same vacuum environment, making the overall system relatively large, and the sample replacement process requires repeated restoration of normal pressure and vacuuming operations, which is not conducive to research and clinical application. Summary of the invention

[0008] In view of the above-mentioned defects of the prior art, the present invention provides an observation system for the ultra-fast freezing process of cells at low temperatures. The system can cool down cell samples quickly and at a controllable speed; can visualize the rapid cooling process in a very short time, which is convenient for studying the formation mechanism of cell ice crystals; can change the atmosphere in the biological sample chamber and change the pressure in the chamber to a certain extent, which is convenient for studying the freezing process of samples under different atmospheres; can prevent the problem of condensation and frosting at low temperatures by blowing dry protective gas, and the sample changing operation is simple.

[0009] The technical solution of the present invention is as follows:

[0010] A visual freezing system for observing the ultra-fast freezing process of cells at low temperature, characterized in that the system includes a biological sample chamber, a low-temperature freezing table, a protective gas subsystem, an imaging and recording subsystem, a motion control subsystem and a temperature measurement and control subsystem:

[0011] The biological sample cabin is in the shape of a flat pancake as a whole, including a cabin shell, a sample stage and a sample slide. The cabin shell is divided into an upper cover plate and a bottom panel. The upper cover plate and the bottom panel are connected and matched by threads to provide a relatively closed space for the sample. An observation window is provided on the upper cover plate. Three holes are provided on the bottom panel: a central hole located at the center of the bottom panel and a protective gas hose through hole and a lead hole outside the center. The central hole is used to install the sample stage, which is embedded in the bottom panel by threads and low-temperature glue sealing bonding. A sample slide is provided on the sample stage for placing the biological sample to be cooled. The sample stage, the sample slide and the observation window are coaxially distributed.

[0012] The low-temperature freezing table is cylindrical, including a cold bottle, a cold conducting rod and a flexible shield. The cold bottle is a low-temperature liquid container with an opening upward, and the working medium required for freezing is stored inside. The cold conducting rod is a cylinder, which is slightly higher than the cold bottle, and the lower end is fixed to the center of the inner bottom surface of the cold bottle and immersed in the low-temperature working medium. The flexible shield is a retractable thin cylinder, the lower end of which is fixed to the bottle mouth of the cold bottle, and the upper end is a free end, which shields the surrounding of the cold conducting rod exposed outside the cold bottle. The top surface of the cold bottle is provided with a cold bottle protective gas pipe hole and a cold bottle lead hole;

[0013] The protective gas subsystem comprises a cryogenic liquid tank, an electric heating wire, two gas-conducting hoses and a flow regulating valve. The electric heating wire extends into the cryogenic liquid tank to generate dry protective gas by electric heating. The two gas-conducting hoses are respectively provided with the flow regulating valves. The cryogenic liquid tank passes through the two gas-conducting hoses through the protective gas pipe holes of the sample cabin respectively. The cold bottle protective gas pipe holes are in communication with the sample cabin and the upper end surface of the cooling rod.

[0014] The motion control subsystem includes a sample chamber xy-axis fine-tuning displacement stage, a low-temperature freezing stage z-axis moving stage, a motion controller and a computer, and the control end of the low-temperature freezing stage z-axis moving stage is connected to the computer via a wire through the motion controller;

[0015] The biological sample chamber is placed on the sample chamber xy axis fine-tuning displacement platform and fixed in the z direction, and the low-temperature freezing table is placed on the low-temperature freezing table z axis moving platform;

[0016] The imaging and recording subsystem includes a microscope, a high-speed camera, a bracket and a computer. The bracket includes an upper bracket and a lower bracket. The upper bracket fixes the microscope and the high-speed camera directly above the observation window of the biological sample chamber, and the lower bracket is coupled to the sample chamber xy-axis fine-tuning displacement stage and the z-axis moving stage of the low-temperature freezing stage;

[0017] The measurement and control subsystem includes an electric heating film, a heating controller, a stress sensor, a micro-thermocouple, a data acquisition instrument and a computer. The electric heating film is placed on the peripheral wall of the biological sample stage in the sample chamber. The electric heating film is connected to the computer through a wire through the lead hole of the biological sample chamber. The stress sensor is placed at the lower end of the cooling rod of the low-temperature freezing table to measure the contact between the cooling rod and the sample stage. The micro-thermocouple is located on the upper surface of the sample stage and the sample slide to monitor the temperature of the sample. The wire of the micro-thermocouple is connected to the computer through the lead hole of the sample chamber and the data acquisition instrument.

[0018] The computer collects the signal of the pressure sensor and drives the motion control subsystem to adjust the height position of the low-temperature freezing table. The stress sensor, cooling rod, low-temperature freezing table, biological sample chamber, biological sample chamber observation window, microscope and high-speed camera are coaxially distributed from bottom to top. The low-temperature freezing table is moved up by the z-axis moving table of the low-temperature freezing table. The computer adjusts the contact degree of the cooling rod of the low-temperature freezing table with the bottom surface of the sample table to achieve on-demand cooling of the sample, and at the same time, the cooling rate of the sample is cooperatively controlled by the electric heating film on the peripheral wall of the sample table.

[0019] The present invention has the following beneficial technical effects:

[0020] 1. The present invention uses a fully pre-cooled heat sink and efficient heat conduction as technical means to achieve ultra-fast cooling of the sample; avoid direct exposure of the sample to liquid nitrogen; and control the sample cooling rate within a certain range by collaboratively controlling the contact degree between the cooling rod and the sample stage and the heating power of the electric heating film.

[0021] 2. The present invention adopts a split structure of sample chamber and cold source, and adopts an open low-temperature component in combination with a dry protective gas atmosphere (blowing dry protective gas to the exposed part of the cooling rod and the biological sample chamber) to achieve freezing operation, simplifying the structure of the entire device, simplifying the sample replacement operation, and eliminating the hidden dangers of frost and condensation. In addition, the atmosphere in the biological sample chamber is controllable, and the pressure is controllable within a certain range, which can achieve rapid freezing of biological samples under different atmospheric conditions.

[0022] 3. It can realize video capture and recording of rapid freezing process in a very short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the structure of the observation system of the low-temperature ultra-rapid freezing process of cells of the present invention.

[0024] Figure 2 This is a schematic diagram of the sample chamber.

[0025] Figure 3 This is a schematic diagram of a low-temperature freezing station. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings to illustrate the invention in more detail.

[0027] See also Figure 1 , Figure 2 , Figure 3 , Figure 1 This is a schematic diagram of the structure of the observation system for the ultra-rapid freezing process of cells at low temperature of the present invention. Figure 2 This is a schematic diagram of the sample chamber. Figure 3 Schematic diagram of a low-temperature freezing table. As can be seen from the figure, the visual freezing system for observing the low-temperature ultra-rapid freezing process of cells of the present invention includes a biological sample cabin 1, a low-temperature freezing table 2, a protective gas subsystem 3, a motion control subsystem 4, an imaging and recording subsystem 5, and a temperature measurement and control subsystem 6:

[0028] The biological sample cabin 1 is in the shape of a flat pancake as a whole, and includes a cabin shell 1-1, a sample stage 1-2 and a sample slide 1-3. The cabin shell 1-1 is divided into an upper cover plate 1-5 and a bottom panel 1-4. The upper cover plate 1-5 and the bottom panel 1-4 are connected and cooperated by threads to provide a relatively closed space for the sample; an observation window 1-6 is provided on the upper cover plate 1-5; three holes are provided on the bottom panel 1-4: a central hole located at the center of the bottom panel 1-4 and a non-central protective gas hose through hole 1-7 and a lead hole 1-8, the central hole is used to install the sample stage 1-2, and the sample stage 1-2 is embedded in the bottom panel 1-4 by threads and low-temperature glue; a sample slide 1-3 is provided on the sample stage 1-2 for placing the biological sample to be cooled, and the sample stage 1-2, the sample slide 1-3 and the observation window 1-6 are coaxially distributed;

[0029] The low-temperature freezing table 2 is cylindrical and includes a cold bottle 2-1, a cold conducting rod 2-2 and a flexible shield 2-3. The cold bottle 2-1 is a low-temperature liquid container with an opening upward, and the working medium required for freezing is stored inside. The cold conducting rod 2-2 is a cylinder, which is slightly higher than the cold bottle 2-1, and its lower end is fixed to the center of the inner bottom surface of the cold bottle 2-1 and immersed in the low-temperature working medium; the flexible shield 2-3 is a retractable thin cylinder, the lower end of which is fixed to the bottle mouth of the cold bottle 2-1, and the upper end is a free end, which shields the surrounding of the cold conducting rod 2-2 exposed outside the cold bottle 2-1, and the top surface of the cold bottle 2-1 is provided with a cold bottle protective gas pipe hole 2-4 and a cold bottle lead hole 2-5;

[0030] The protective gas subsystem 3 comprises a cryogenic liquid tank 3-1, an electric heating wire 3-2, two gas-conducting hoses 3-3 and a flow regulating valve 3-4. The electric heating wire 3-2 extends into the cryogenic liquid tank 3-1 to generate dry protective gas by electric heating. The two gas-conducting hoses 3-3 are respectively provided with the flow regulating valves 3-4. The cryogenic liquid tank 3-1 is respectively connected to the sample cabin protective gas pipe hole 1-7 through the two gas-conducting hoses 3-3, and the cold bottle protective gas pipe hole 2-4 is connected to the sample cabin 1 and the upper end surface of the cooling rod 2-2.

[0031] The motion control subsystem 4 includes a sample chamber xy-axis fine-tuning displacement stage 4-1, a low-temperature freezing stage z-axis moving stage 4-2, a motion controller 4-3 and a computer 7. The control end of the low-temperature freezing stage z-axis moving stage 4-2 is connected to the computer 7 via a wire through the motion controller 4-3;

[0032] The biological sample chamber 1 is placed on the sample chamber xy axis fine-tuning displacement stage 4-1 and fixed in its z direction, and the low-temperature freezing stage 2 is placed on the low-temperature freezing stage z axis moving stage 4-2;

[0033] The imaging and recording subsystem 5 includes a microscope 5-1, a high-speed camera 5-2, a bracket 5-3 and a computer 7. The bracket 5-3 includes an upper bracket and a lower bracket. The upper bracket fixes the microscope 5-1 and the high-speed camera 5-2 directly above the biological sample chamber observation window 1-6. The lower bracket is coupled to the sample chamber xy-axis fine-tuning displacement stage 4-1 and the z-axis moving stage 4-2 of the low-temperature freezing stage.

[0034] The measurement and control subsystem 6 includes an electric heating film 6-1, a heating controller 6-2, a stress sensor 6-3, a micro-thermocouple 6-4, a data acquisition instrument 6-5 and a computer 7. The electric heating film 6-1 is placed on the peripheral wall of the biological sample stage 1-2 in the sample chamber. The electric heating film 6-1 is connected to the computer 7 through a wire through the lead hole 1-8 of the biological sample chamber. The stress sensor 6-3 is placed at the lower bottom end of the cooling rod 2-2 of the low-temperature freezing table to measure the contact between the cooling rod 2-2 and the sample stage 1-2; the micro-thermocouple 6-4 is located on the upper surface of the sample stage 1-2 and the sample slide 1-3 to monitor the temperature of the sample. The wire of the micro-thermocouple 6-4 is connected to the computer 7 through the lead hole 1-8 of the sample chamber and the data acquisition instrument 6-5;

[0035] The computer 7 controls the motion control subsystem 4. The low-temperature freezing table 2, biological sample chamber 1, biological sample chamber observation window 1-6, stress sensor 6-3, cooling rod 2-2, microscope 5-1 and high-speed camera 5-2 are coaxially distributed from bottom to top. The low-temperature freezing table 2 is moved up by the low-temperature freezing table z-axis moving table 4-2. The computer 7 adjusts the cooling rod 2-2 of the low-temperature freezing table 2 to contact the lower bottom surface of the sample table 1-2 to achieve rapid cooling of the sample, and the cooling rate of the sample is coordinated by the contact degree between the electric heating film 6-1 on the peripheral wall of the sample table 1-2, the cooling rod 2-2 and the sample table 1-2.

[0036] Example

[0037] An observation window 1-6 made of quartz glass is provided on the upper cover plate 1-4. Three holes are provided on the bottom panel 1-5: one hole is located at the center of the bottom panel 1-5, and the hole is used to position and install the sample stage 1-2. The sample stage 1-2 is embedded in the bottom panel 1-5 in the form of threads and low-temperature glue; the other two holes are provided outside the center hole, which are the protective atmosphere control hole 1-7 and the lead hole 1-8. The air duct delivers the protective gas to the biological sample chamber through the protective atmosphere control hole 1-7, thereby controlling the atmosphere in the chamber and controlling the pressure in the chamber to a certain extent. The lead hole 1-8 realizes the connection and communication of the leads inside and outside the sample chamber 1. The sample table 1-2 is made of pure silver material with high thermal conductivity, in the shape of a small cylinder, embedded in the bottom panel 1-5 and slightly protruding outward from the bottom panel 1-5. A sapphire sample carrier 1-3 is provided on the sample table 1-2 for placing the biological sample to be cooled, so as to prevent the biological sample from directly contacting the pure silver sample table. The sample carrier 1-3 and the sample stage 1-2 are mechanically fixed to ensure good heat transfer effect. In terms of spatial position, the observation window 1-6, the sample carrier 1-3 and the sample stage 1-2 are coaxially distributed up and down.

[0038] The low-temperature freezing table 2 is cylindrical in shape as a whole, and is an open system, which is placed directly below the biological sample cabin 1, and includes a cold bottle 2-1, a cold conducting rod 2-2 and a flexible shield 2-3. The cold bottle 2-1 is an open bottle container made of double-layer insulation material with a vacuum interlayer, and stores a certain amount of low-temperature liquid, specifically liquid nitrogen, to provide cold for sample freezing. The cold conducting rod 2-2 is made of a material with a high thermal conductivity, specifically oxygen-free copper, and is cylindrical, with a diameter smaller than the diameter of the sample table, and a height slightly higher than the cold bottle 2-1. The cold conducting rod 2-2 is fixed at the center of the inner bottom surface of the cold bottle 2-1, and the sample table is coaxially distributed: that is, the lower part of the cold conducting rod 2-2 is immersed in the low-temperature liquid, and a small part of the upper end is exposed outside the cold bottle 2-1 and can contact the bottom surface of the sample table 1-2, and the cold of the low-temperature liquid is conducted to the sample table 1-2, and then to the sample on the sample table 1-2, so as to achieve sample freezing and cooling. The flexible shield 2-3 is a thin cylindrical structure that can be retracted and deformed. One end of the flexible shield 2-3 is fixed to the bottle mouth of the cold bottle 2-1, and the other end is a free end. Its extension height is higher than the cold conduction rod 2-2, and it can provide a certain shielding around the cold conduction rod 2-2 exposed outside the cold bottle 2-1. The upper bottom surface of the cold bottle 2-1 is provided with a protective atmosphere control hole 2-4 and a cold bottle lead hole 2-5. The air guide pipe introduces protective gas through the atmosphere control hole 2-4 to blow the upper end surface of the cold conduction rod 2-2 to prevent it from frosting and condensing, thereby causing deterioration of heat transfer. The cold bottle lead hole 2-5 allows the pressure sensor 6-3 lead to pass through the cold bottle and connect to the data acquisition instrument 6-5 to achieve communication.

[0039] The protective gas subsystem 3 includes a cryogenic liquid tank 3-1, an electric heating wire 3-2, a gas hose 3-3, and a flow control valve 3-4. The protective gas subsystem is mainly used to generate and transport protective gas. In order to simplify the operation process and reduce the volume of the equipment, the overall system has an open cryogenic component, which is not isolated by vacuum. Instead, dry gas is used to replace the humid atmosphere near the opening component to prevent frost and condensation problems from the source. Specifically, the liquid stored in the cryogenic liquid tank is a certain amount of liquid nitrogen. The electric heating wire 3-2 extends into the cryogenic liquid tank 3-1, and the tank mouth is connected to the gas hose 3-3, and one end of the gas hose is connected to the flow control valve. The liquid nitrogen is vaporized by electric heating to generate protective gas, specifically dry nitrogen. The increase in pressure in the tank causes the protective gas to be discharged outside the tank along the gas hose, and the flow rate of the transported gas is controlled by the flow control valve 3-4. The protective gas is delivered to the sample chamber and the upper section of the cooling rod 2-2 through the protective atmosphere control holes 1-7 and 2-4, changing the ambient atmosphere of the sample chamber and open low-temperature components to prevent frost exposure.

[0040] The motion control subsystem 4 includes a sample chamber xy-axis fine-tuning displacement stage 4-1, a low-temperature freezing stage z-axis moving stage 4-2, a motion controller 4-3 and a computer 17. The biological sample chamber 1 is placed on the sample chamber xy-axis fine-tuning displacement stage 4-1 and fixed in its z direction. The sample chamber xy-axis fine-tuning displacement stage 4-1 can be used to adjust the position in the horizontal plane of the sample chamber so as to perform sample placement operations. The low-temperature freezing stage 2 is placed on the low-temperature freezing stage z-axis moving stage 4-2, and the low-temperature freezing stage z-axis moving stage 4-2 and the computer 17 are connected for communication through the motion controller. The low-temperature freezing stage z-axis moving stage 4-2 can change the position in the vertical plane of the low-temperature freezing stage 2, thereby determining whether the upper end surface of the cooling rod 2-2 is in contact with the lower end surface of the sample stage, thereby controlling the freezing and cooling of the sample.

[0041] The imaging and recording subsystem 5 includes a microscope 5-1, a high-speed camera 5-2, a bracket 5-3 and a computer 7. The upper part of the bracket 5-3 fixes the microscope 5-1 lens and the high-speed camera 5-2 just above the biological sample chamber observation window 1-6, and the lower part of the bracket 5-3 couples and connects the sample chamber xy-axis fine-tuning displacement stage 4-1 and the low-temperature freezing stage z-axis moving stage 4-2.

[0042] The measurement and control subsystem 6 includes an electric heating film 6-1, a heating controller 6-2, a stress sensor 6-3, a micro-thermocouple 6-4, a data acquisition instrument 6-5 and a computer 7. The electric heating film 6-1 is placed on the peripheral wall of the biological sample stage 1-2 in the sample chamber, and is connected to the heating controller 6-2 and the computer 7 through the lead hole 6-1 of the biological sample chamber, and the heating power can be adjusted by the computer 7. The stress sensor 6-3 is placed at the bottom of the cooling rod of the low-temperature freezing table to measure the contact degree between the cooling rod 2-2 and the sample stage 1-2. The micro-thermocouple 6-4 is located on the upper surface of the sample stage 1-2, used to monitor the sample temperature, and is connected to the data acquisition instrument 6-5 and the computer 7 through the lead hole 1-8 of the sample chamber.

[0043] The low-temperature freezing table 2 and the biological sample chamber 1 are coaxially distributed up and down. The low-temperature freezing table 2 is lifted by the low-temperature freezing table z-axis moving table 4-2, so that the upper bottom surface of the cooling rod 2-2 of the low-temperature freezing table contacts the lower bottom surface of the sample table 1-2 of the biological sample chamber. The specific cooling rod 2-2 is made of oxygen-free copper, and the sample table 1-2 is made of pure silver. The rapid cooling of the sample is achieved by heat conduction. During use, an indium sheet can be inserted between the sample table 1-2 and the cooling rod 2-2 to strengthen the contact between the two. The different clamping forces between the two will result in different degrees of contact between the two planes, thereby resulting in different contact thermal resistances between the two, and then cooperate with the electric heating film 6-1 on the peripheral wall of the sample table to coordinately control the cooling rate of the sample.

[0044] The working process of the device is as follows:

[0045] First, connect each subsystem of the system correctly.

[0046] Then check whether the protective gas subsystem 3 can output the protective gas normally and whether the gas hose 3-3 has any leakage; check whether the motion control subsystem 4 can work normally; and check whether the reading of the measurement control subsystem 6 is correct.

[0047] After checking that there are no system problems in the experimental system, lower the cryogenic freezing table 1 to the lowest end, turn on the protective gas subsystem 3, open the protective gas valve 3-4 of the cryogenic freezing table, wait for a while until the cooling rod 2-2 is in the protective gas atmosphere, and pour a suitable volume of liquid nitrogen into the cold bottle 2-2. Then open the upper cover plate 1-4 of the biological sample chamber to place the sample, then tighten the upper cover plate 1-4, and open the protective gas valve 3-4 of the biological sample chamber. Adjust the sample chamber xy axis fine-tuning displacement stage 4-1 and the microscope 5-1 so that the biological sample appears in the center of the eyepiece field of view, and the image is clear.

[0048] Turn on the high-speed camera 5-2 to prepare for recording, control the low-temperature freezing stage z-axis moving stage 4-2 to lift the low-temperature freezing stage 2, until the cooling rod 2-2 contacts the sample stage 1-2 at an appropriate contact level, input the appropriate power of the electric heating film 6-1, and record the sample cooling process. After the cooling and freezing process is completed, lower the low-temperature freezing stage 2 to separate the cooling rod 2-2 from the sample stage 1-2, wait for the sample to reheat, check whether the liquid nitrogen in the cold bottle 2-1 has a suitable volume, if the amount of liquid nitrogen is too little, add it, and then re-set the sample to start the next low-temperature freezing experiment.

[0049] Experiments show that the present invention can cool down cell samples quickly and at a controllable speed; can visually image the rapid cooling process in a very short time, which is convenient for studying the formation mechanism of ice crystals inside and outside cells; can change the atmosphere in the biological sample chamber and change the pressure in the chamber to a certain extent, which is convenient for studying the freezing process of samples under different atmospheres; can prevent the problem of condensation and frosting at low temperatures by blowing dry protective gas, and the sample changing operation is simple.

[0050] The specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many changes based on the concept of the present invention without creative work. Therefore, any modifications, substitutions, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A visual freezing system for observing the ultra-fast freezing process of cells at low temperature, characterized in that the system comprises a biological sample chamber (1), a low temperature freezing table (2), a protective gas subsystem (3), a motion control subsystem (4), an imaging recording subsystem (5) and a temperature measurement control subsystem (6): The biological sample chamber (1) is in the shape of a flat pancake as a whole, and comprises a chamber shell (1-1), a sample stage (1-2) and a sample slide (1-3). The chamber shell (1-1) is divided into an upper cover plate (1-5) and a bottom panel (1-4). The upper cover plate (1-5) and the bottom panel (1-4) are connected and matched by threads to provide a relatively closed space for the sample. The upper cover plate (1-5) is provided with an observation window (1-6). The bottom panel (1-4) is provided with three holes: one located at the upper cover plate (1-5) and the other located at the bottom panel (1-4). The bottom panel (1-4) comprises a central hole in the center and a non-central protective gas hose through hole (1-7) and a lead hole (1-8), wherein the central hole is used to install the sample stage (1-2), and the sample stage (1-2) is embedded in the bottom panel (1-4) by means of threads and low-temperature adhesive sealing bonding; a sample carrier (1-3) is provided on the sample stage (1-2) for placing biological samples to be cooled, and the sample stage (1-2), the sample carrier (1-3) and the observation window (1-6) are coaxially distributed; The low-temperature freezing table (2) is cylindrical and includes a cold bottle (2-1), a cold conducting rod (2-2) and a flexible shield (2-3). The cold bottle (2-1) is a low-temperature liquid container with an opening facing upward, and stores the working medium required for freezing inside. The cold conducting rod (2-2) is a cylinder, which is slightly higher than the cold bottle (2-1). Its lower end is fixed to the center of the inner bottom surface of the cold bottle (2-1) and immersed in the low-temperature working medium. The flexible shield (2-3) is a retractable thin cylinder, the lower end of which is fixed to the bottle mouth of the cold bottle (2-1) and the upper end is a free end, which shields the surrounding of the cold conducting rod (2-2) exposed outside the cold bottle (2-1). The top surface of the cold bottle (2-1) is provided with a cold bottle protective gas pipe hole (2-4) and a cold bottle lead hole (2-5). The protective gas subsystem (3) comprises a cryogenic liquid tank (3-1), an electric heating wire (3-2), two gas-conducting hoses (3-3) and a flow regulating valve (3-4); the electric heating wire (3-2) extends into the cryogenic liquid tank (3-1) to generate dry protective gas by electric heating; the two gas-conducting hoses (3-3) are respectively provided with the flow regulating valves (3-4); the cryogenic liquid tank (3-1) is respectively connected to the sample cabin protective gas pipe hole (1-7) through the two gas-conducting hoses (3-3); the cold bottle protective gas pipe hole (2-4) is connected to the biological sample cabin (1) and the upper end surface of the cooling rod (2-2); The motion control subsystem (4) comprises a sample chamber xy-axis fine-tuning displacement stage (4-1), a low-temperature freezing stage z-axis moving stage (4-2), a motion controller (4-3) and a computer (7), wherein the control end of the low-temperature freezing stage z-axis moving stage (4-2) is connected to the computer (7) via a wire through the motion controller (4-3); The biological sample chamber (1) is placed on the sample chamber xy-axis fine-tuning displacement stage (4-1) and fixed in its z-direction, and the low-temperature freezing stage (2) is placed on the low-temperature freezing stage z-axis moving stage (4-2); The imaging and recording subsystem (5) comprises a microscope (5-1), a high-speed camera (5-2), a bracket (5-3) and a computer (7); the bracket (5-3) comprises an upper bracket and a lower bracket; the upper bracket fixes the microscope (5-1) and the high-speed camera (5-2) directly above the biological sample chamber observation window (1-6); the lower bracket is coupled to the sample chamber xy-axis fine-tuning displacement stage (4-1) and the z-axis moving stage (4-2) of the low-temperature freezing stage; The measurement and control subsystem (6) comprises an electric heating film (6-1), a heating controller (6-2), a stress sensor (6-3), a micro-thermocouple (6-4), a data acquisition instrument (6-5) and a computer (7). The electric heating film (6-1) is placed on the peripheral wall of the biological sample stage (1-2) in the biological sample chamber. The electric heating film (6-1) is connected to the computer (7) through a wire through the wire hole (1-8) of the biological sample chamber. The stress sensor (6-3) is placed at the lower end of the cooling rod (2-2) of the low-temperature freezing table and is used to measure the contact between the cooling rod (2-2) and the sample stage (1-2). The micro-thermocouple (6-4) is located on the upper surface of the sample stage (1-2) and the sample slide (1-3) and is used to monitor the temperature of the sample. The wire of the micro-thermocouple (6-4) is connected to the computer (7) through the wire hole (1-8) of the biological sample chamber and the data acquisition instrument (6-5). The computer (7) collects the signal of the stress sensor (6-3) and drives the motion control subsystem (4) to adjust the height position of the low-temperature freezing table (2). The stress sensor (6-3), the cooling rod (2-2), the low-temperature freezing table (2), the biological sample chamber (1), the biological sample chamber observation window (1-6), the microscope (5-1) and the high-speed camera (5-2) are coaxially distributed from bottom to top. The low-temperature freezing table (2) is moved up by the z-axis moving table (4-2) of the low-temperature freezing table. The computer (7) adjusts the contact degree of the cooling rod (2-2) of the low-temperature freezing table (2) with the bottom surface of the sample table (1-2) to achieve on-demand cooling of the sample, and at the same time, the cooling rate of the sample is cooperatively controlled by the electric heating film (6-1) on the peripheral wall of the sample table (1-2).

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