An ultra-long endurance sample transport system for cryo-em

Abstract

The present application relates to electron microscope technical field, aims at solving the problems of short low-temperature holding time of sample rod in existing sample transfer support, vibration of sample rod caused by boiling of liquid nitrogen in Dewar jar, and limited sample rod inclination, and provides a kind of super-long endurance sample transmission system for cryo-electron microscopy, comprising Dewar jar and sample rod;Dewar jar includes outer tank and inner tank, inner tank is located in the inside of outer tank, low-temperature fluid one is stored in outer tank, low-temperature fluid two is stored in inner tank, and the boiling point of low-temperature fluid two is higher than the boiling point of low-temperature fluid one;Cold plate is arranged in inner tank, sample rod includes outer rod and cold rod, cold rod is located in the inside of outer rod, one end of cold rod is inserted into inner tank and connected with cold plate, the other end of cold rod is extended out of outer rod and connected with sample holder;Outer rod is connected with connecting assembly one, Dewar jar is connected with connecting assembly two, and connecting assembly one and connecting assembly two are detachably connected;The low-temperature holding time of the present application is long, the drift is small, and the sample rod inclination is large.

Description

Technical Field

[0001] The present invention relates to the field of electron microscopy technology, and in particular to an ultra-long endurance sample transmission system for a cryo-electron microscope. Background Art

[0002] Cryo-electron microscopy preserves the sample's natural state by rapidly freezing it and then observing it at extremely low temperatures (≤-136°C). Cryo-electron microscopy can be used to study the ultrastructure of cells, such as protein complexes, organelles, and cell membranes. It provides scientists with tools to study many important issues in biology, such as cell differentiation, viral infection mechanisms, and cell signaling. When cryo-electron microscopy collects the microscopic structure of samples at low temperatures, it usually requires a liquid nitrogen-cooled freezing sample stage, and the data collection time is relatively long, generally requiring more than 10 hours.

[0003] Currently, low-temperature sample transfer holders are commonly used on the market. The tail of the sample rod is equipped with a vacuum liquid nitrogen tank with a capacity of approximately 200ml. The cold energy is transferred to the sample holder at the front end of the sample rod through the built-in cooling rod, maintaining the sample at a low temperature (≤-150°C). The sample rod is then inserted into the electron microscope to complete data acquisition. Due to the limited capacity of the vacuum liquid nitrogen tank of the sample rod, each time it is filled with liquid nitrogen, the low temperature (≤-150°C) can only be maintained for about 3 hours. When the liquid nitrogen is refilled, the vibration caused by the boiling of the liquid nitrogen will cause the sample to drift and data cannot be collected. Therefore, automatic data acquisition for a long time is not possible.

[0004] In addition, liquid nitrogen needs to be added to the cryo-transfer sample rod before loading the sample to cool the cryo-transfer sample rod and keep it at a low temperature. When the sample rod is inserted into the cryo-electron microscope, the sample rod needs to be rotated 90° for insertion. At this time, the mouth of the liquid nitrogen tank is changed from vertical to horizontal, and the liquid nitrogen in the tank will pour out. After the vacuum is evacuated in the cryo-electron microscope cavity, the sample rod is rotated back to the initial position (the mouth of the liquid nitrogen tank is facing up), and liquid nitrogen is manually added again. This step also has the risk of liquid nitrogen splashing and scalding the operator, and the boiling point of liquid nitrogen is low. The liquid nitrogen poured out of the tank will vaporize instantly, resulting in a waste of liquid nitrogen. For example, in a confined space, there is also a risk of suffocation.

[0005] In summary, the existing sample transfer bracket has the following problems:

[0006] 1. The sample holder can maintain low temperature for a short time, about 3 to 4 hours, which cannot meet the needs of long-term (more than 10 hours) automatic data collection.

[0007] 2. The bubbles of boiling liquid nitrogen in the Dewar tank will vibrate the sample rod, causing the electron microscope image to drift;

[0008] 3. The sample rod has a limited inclination angle. If the rotation angle is too large, the liquid nitrogen will overflow. Due to the limited inclination angle, there is a blind spot in the imaging.

[0009] 4. The commonly used sample transfer support (with a liquid nitrogen tank at the tail), the liquid nitrogen tank has a small capacity and needs to be filled with liquid nitrogen multiple times, which is troublesome and dangerous to some extent. SUMMARY

[0010] The present application aims to provide a long-lasting sample transfer system for cryo-EM to solve the problems of short low-temperature holding time of the sample rod, bubbles in the boiling liquid nitrogen in the dewar causing the sample rod to vibrate and cause the drift of the electron microscope imaging, and limited tilt angle of the sample rod in the existing sample transfer support.

[0011] The present application is implemented by using the following technical solutions:

[0012] The present application provides a long-lasting sample transfer system for cryo-EM, comprising a dewar and a sample rod.

[0013] The dewar comprises an outer tank and an inner tank, the inner tank is located inside the outer tank, the outer tank stores a low-temperature fluid one, and the inner tank stores a low-temperature fluid two, the boiling point of the low-temperature fluid two is higher than that of the low-temperature fluid one.

[0014] A cold plate is arranged in the inner tank, the sample rod comprises an outer rod and a cold rod, the cold rod is located inside the outer rod, one end of the cold rod extends into the inner tank and is connected with the cold plate, and the other end of the cold rod extends out of the outer rod and is connected with a sample holder.

[0015] The outer rod is connected with a connecting assembly one, the dewar is connected with a connecting assembly two, and the connecting assembly one and the connecting assembly two are detachably connected.

[0016] As a preferred technical solution:

[0017] The other side of the dewar is provided with a handle.

[0018] As a preferred technical solution:

[0019] The low-temperature fluid one is liquid nitrogen, and the low-temperature fluid two is liquid oxygen.

[0020] Alternatively, the low-temperature fluid one and the low-temperature fluid two are both liquid nitrogen, and at the same time, the gas pressure in the outer tank is lower than that in the inner tank.

[0021] As a preferred technical solution:

[0022] The outer tank is a vacuum insulation structural member, the outer tank comprises an outer shell and an inner container, the inner container is located inside the outer shell, a vacuum layer is formed between the outer shell and the inner container, and the inner tank is located inside the inner container.

[0023] As a preferred technical solution:

[0024] The connecting assembly two comprises a transition pipe one, a transition pipe two, a transition joint one, a trumpet joint and a vacuum joint one; one end of the transition pipe one is connected to one side of the inner tank, and the transition pipe one is in communication with the inside of the inner tank, and the other end of the transition pipe one is connected to the transition joint one; one end of the trumpet joint is a large end, and the other end is a small end, the large end of the trumpet joint is connected to one side of the outer tank, and the transition pipe two and the transition joint one are located in the trumpet joint; one end of the transition pipe two is connected to the transition joint one, and the other end of the transition pipe two is connected to the inner wall of the small end of the trumpet joint; the outer wall of the small end of the trumpet joint is connected to the vacuum joint one.

[0025] Because the trumpet joint has different opening sizes at two ends, one end has a larger opening, and the other end has a smaller opening, the large end refers to the end of the trumpet joint with a larger opening, and the small end refers to the end of the trumpet joint with a smaller opening.

[0026] As a preferred technical solution:

[0027] The transition pipe two is connected to the transition joint two, and the transition joint two and the transition joint one are provided with a sealing gasket.

[0028] The transition joint two, the transition pipe three and the sealing gasket cooperate as a sealing structure, and the sealing gasket can prevent the fluid in the inner tank from flowing to the vacuum joint one during movement of the system.

[0029] As a preferred technical solution:

[0030] The vacuum joint one and the trumpet joint are connected to a limiting pin and a sealing ring.

[0031] As a preferred technical solution:

[0032] The connecting assembly one comprises a vacuum joint two, and the vacuum joint two and the vacuum joint one are detachably connected.

[0033] As a preferred technical solution:

[0034] The vacuum joint two and the vacuum joint are connected through a hoop.

[0035] As a preferred technical solution:

[0036] The outer rod is provided with a rotary switch, the rotary switch is connected to one side of the vacuum joint two; the rotary switch is connected to a pull rod inside the cold guide rod, a sample cover is slidably arranged on the sample holder, and one end of the pull rod extends out of the cold guide rod and is connected to the sample cover.

[0037] As a preferred technical solution:

[0038] The outer tank and the inner tank are provided with a filling device, the filling device comprises a compensator one connected between the outer shell and the inner tank, the compensator one is internally provided with a filling pipe, the top of the inner tank is connected with a compensator two, and the filling pipe is connected with the compensator two.

[0039] The top of the compensator one is connected with an outer cover, the outer cover is provided with an exhaust port, and an exhaust pipe is installed at the exhaust port.

[0040] As a preferred technical solution:

[0041] The outer shell and the inner tank are connected with a compensator three, the compensator three is internally provided with a liquid level meter, the liquid level meter extends into the inner tank, and the top of the compensator three is connected with an outer cover.

[0042] As a preferred technical solution:

[0043] The system further comprises an adjusting platform, the adjusting platform is provided with a sliding rail, a mounting platform is slidably connected to the sliding rail, and the mounting platform can slide along the sliding rail; the dewar tank is mounted on the mounting platform; and the sample rod is mounted on an electron microscope goniometer stage.

[0044] As described above, due to the adoption of the above technical solutions, the present application has the following advantages:

[0045] 1. In the working state of the sample transmission system, the dewar tank and the sample rod are not in direct contact, and the weight of the dewar tank and the fluid in it will not be transmitted to the electron microscope, so that the loading capacity of the low-temperature fluid in the dewar tank is large, the ultra-long endurance of more than 10h can be realized, the low-temperature holding time of the sample rod is long, and the hidden danger of the operator being frozen by liquid nitrogen due to multiple filling of liquid nitrogen is eliminated.

[0046] 2. In the sample transmission system, the cold guide rod of the sample rod is in contact with the fluid in the inner tank through the cold guide plate in the working state, the fluid in the outer tank continuously transmits cold to the fluid in the inner tank, and then the sample on the sample holder is kept at low temperature through the cold guide plate and the cold guide rod. Due to the heat exchange between the fluid in the outer tank and the fluid in the inner tank and the system heat leakage, the fluid in the outer tank will evaporate and boil, but since the boiling point of the fluid in the outer tank is lower than that of the fluid in the inner tank, the fluid in the inner tank will not boil, and the vibration of the bubbles generated by the boiling of the fluid in the outer tank cannot be directly transmitted to the sample rod, so that smaller imaging drift and higher resolution can be realized.

[0047] 3、The sample transfer system of the application is in working state, the connection assembly one and the connection assembly two are separated, the Dewar and the sample rod are not in direct contact, the cold energy in the inner tank is transferred through the cold guide plate, therefore, the sample rod can be rotated 360° for observation, the observation angle is larger, and the imaging has no dead angle. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The external schematic view of the ultra-long endurance sample transfer system for cryo-EM described in the application.

[0049] Figure 2 The semi-sectional view of the ultra-long endurance sample transfer system for cryo-EM described in the application.

[0050] Figure 3 The schematic view of the ultra-long endurance sample transfer system for cryo-EM after the KF clamp is closed.

[0051] Figure 4 The schematic view of the ultra-long endurance sample transfer system for cryo-EM after the KF clamp is opened.

[0052] Figure 5 The Figure 3 The sectional view in A-A direction.

[0053] Figure 6 The Figure 4 The sectional view in B-B direction.

[0054] Figure 7 The schematic view of the sample transfer support installed on the adjusting platform.

[0055] Figure legend: 1-Dewar, 2-sample rod, 3-handle, 4-outer tank, 5-inner tank, 6-outer shell, 7-inner liner, 8-vacuum layer, 9-liquid nitrogen, 10-liquid oxygen, 11-cold guide plate, 12-cold guide rod, 13-outer rod, 14-transition pipe one, 15-transition joint one, 16-transition pipe two, 17-transition joint two, 18-sealing gasket, 19-transition pipe three, 20-horn joint, 21-KF vacuum joint one, 22-limiting pin, 23-sealing ring, 24-KF vacuum joint two, 25-center support, 26-KF clamp, 27-sample holder, 28-sample cover, 29-knob switch, 30-pull rod, 31-compensator one, 32-filling pipe, 33-compensator two, 34-outer cover, 35-exhaust pipe, 36-compensator three, 37-liquid level meter, 38-vacuum valve, 39-navigation plug joint, 40-adjusting platform, 41-sliding rail, 42-installation platform, 43-sample transfer support. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0057] Example 1

[0058] like Figures 1-7 As shown, this embodiment proposes an ultra-long endurance sample transmission system for a cryo-electron microscope, comprising a dewar 1 and a sample rod 2, one end of the sample rod 2 extends into the dewar 1 from one side of the dewar 1, and a handle 3 is provided on the other side of the dewar 1.

[0059] The Dewar tank 1 includes an outer tank 4 and an inner tank 5 , and the inner tank 5 is located inside the outer tank 4 .

[0060] The outer tank 4 is a vacuum-insulated structure that reduces heat transfer and system heat leakage. Specifically, the outer tank 4 includes an outer shell 6 and an inner liner 7. The inner liner 7 is located inside the outer shell 6. A vacuum layer 8 is formed between the outer shell 6 and the inner liner 7. The inner tank 5 is located inside the inner liner 7.

[0061] The outer tank 4 stores a low-temperature fluid 1. Specifically, the low-temperature fluid 1 is located in the inner tank 7. The inner tank 5 stores a low-temperature fluid 2. The boiling point of the low-temperature fluid 2 is higher than that of the low-temperature fluid 1. The temperatures of the low-temperature fluid 1 in the outer tank 4 and the low-temperature fluid 2 in the inner tank 5 can be transferred to each other. The cold energy of the low-temperature fluid 1 can be transferred to the low-temperature fluid 2, so that the temperature of the low-temperature fluid 2 is reduced to the same as the temperature of the low-temperature fluid 1, while the volume remains unchanged. The low-temperature fluid 1 will partially evaporate due to heat exchange with the low-temperature fluid 2. Since the boiling point of the low-temperature fluid 1 is lower, all heat leakage in the system will first cause the low-temperature fluid 1 to boil and evaporate. The low-temperature fluid 2 will continue to remain in liquid form and will not evaporate because its boiling point has not been reached. Naturally, the volume of the low-temperature fluid 2 remains unchanged.

[0062] In this embodiment, the inner tank 5 is a metal thin-walled tank, which is beneficial to the temperature transfer between the fluid in the outer tank 4 and the inner tank 5.

[0063] In the embodiment, the inner container 7 stores liquid nitrogen 9, which has a melting point of -210℃ and a boiling point of -195.8℃ at standard atmospheric pressure (101.325kPa); the inner tank 5 stores liquid oxygen 10, which has a melting point of -218℃ and a boiling point of -183℃ at standard atmospheric pressure; below the melting point, it is in a solid state; between the melting point and the boiling point, it is in a liquid state; above the boiling point, it is in a gaseous state. After heat exchange through the wall of the inner tank 5, the liquid nitrogen 9 and the liquid oxygen 10 are both at -195.8℃. Since the boiling point of the liquid oxygen 10 is higher than that of the liquid nitrogen 9, the liquid nitrogen 9 will evaporate due to heat exchange with the liquid oxygen 10 and system heat leakage, but the liquid oxygen 10 will not evaporate because its boiling point has not been reached, and its volume remains unchanged.

[0064] The inner tank 5 is provided with a cold guide plate 11, the sample rod 2 includes an outer rod 13 and a cold guide rod 12, the cold guide rod 12 is located inside the outer rod 13, one end of the cold guide rod 12 is connected to one side of the cold guide plate 11, and the cold guide plate 11 is partially immersed in the low-temperature fluid two, so as to transfer cold to the sample rod 2.

[0065] One side of the inner tank 5 is connected with a transition pipe one 14, the transition pipe one 14 is in communication with the inside of the inner tank 5, one end of the transition pipe one 14 is fixedly connected to the side of the inner tank 5, the other end of the transition pipe one 14 penetrates the inner container 7, the outer wall of the transition pipe one 14 is connected and sealed with the contact part of the inner container 7, and the liquid level of the low-temperature fluid two in the inner tank 5 is lower than the bottom height of the transition pipe one 14. The inner container 7 is filled with the low-temperature fluid one, and the low-temperature fluid one surrounds the inner tank 5 and the transition pipe one 14.

[0066] The end of the transition pipe one 14 protruding out of the inner container 7 is located in the vacuum layer 8, and the end of the transition pipe one 14 is connected with a transition joint one 15, one end of the transition joint one 15 is inserted into the transition pipe one 14 and fixedly connected with the transition pipe one 14, and the other end of the transition joint one 15 penetrates the outer shell 6, and the outer wall of the transition joint one 15 is connected and sealed with the contact part of the outer shell 6. The end of the transition joint one 15 protruding out of the outer shell 6 is connected with a transition pipe two 16.

[0067] The outer part of the cold guide rod 12 is connected with a transition joint two 17, the transition joint two 17 is located in the transition joint one 15, and a sealing gasket 18 is arranged between the transition joint two 17 and the transition joint one 15. One end of the transition joint two 17 is connected with a transition pipe three 19, and the transition pipe three 19 is located inside the transition pipe two 16.

[0068] The outer side of the shell 6 is connected with a horn joint 20, one end of which is large in opening and the other end is small in opening, and the large opening end of the horn joint 20 is fixed on the shell 6. The transition joint one 15, the transition joint two 17, the transition tube two 16 and the transition tube three 19 are located in the horn joint 20, one end of the transition tube two 16 is connected with the transition joint one 15, the other end of the transition tube two 16 is connected with the inner wall of the small opening end of the horn joint 20, so as to fix the transition tube two 16. The outer wall of the small opening end of the horn joint 20 is connected with a KF vacuum joint one 21, and the KF vacuum joint one 21 can rotate 360° around the axis direction of the horn joint 20.

[0069] A limiting pin 22 is connected between the KF vacuum joint one 21 and the horn joint 20, and the KF vacuum joint one 21 is limited to move along the axial direction by the limiting pin 22. Two sealing rings 23 are arranged between the KF vacuum joint one 21 and the horn joint 20 to seal them.

[0070] One end of the KF vacuum joint one 21 is connected with the horn joint 20, and the other end of the KF vacuum joint one 21 is connected with a KF vacuum joint two 24. A center frame 25 is arranged between the KF vacuum joint one 21 and the KF vacuum joint two 24, and the KF vacuum joint one 21 and the KF vacuum joint two 24 are positioned and sealed by the center frame 25, and the KF vacuum joint one 21 and the KF vacuum joint two 24 are connected and fixed by a KF clamp 26.

[0071] One end of the cold lead 12 penetrates from the outer rod 13, then penetrates through the KF vacuum joint two 24, the KF vacuum joint one 21, the transition tube three 19, the transition tube one 14 in sequence, and is connected to the center of the cold plate 11. The cold lead 12 is in a suspended state and does not directly contact the dewar jar 1, but only contacts the fluid in the inner jar 5 through the cold plate 11. Since the cold lead 12 does not directly contact the fluid in the outer jar 4, the vibration generated by the boiling of the fluid in the outer jar 4 cannot be directly transmitted to the sample rod 2, so the drift caused by the electron microscope imaging is smaller than that of the prior art with direct contact, which is very beneficial to imaging. The other end of the cold lead 12 extends out of the outer rod 13 and is connected with a sample holder 27, a sample groove is arranged on the sample holder 27, and the sample groove is used to place a sample. The cold plate 11 transmits the cold energy in the inner jar 5 to the sample holder 27 in a solid conduction manner, and keeps the sample in a low temperature state. A sample cover 28 is slidably arranged on the sample holder 27, and the sample cover 28 can close the sample groove, so as to protect the sample from being contaminated during sample transfer.

[0072] The outer rod 13 is provided with a knob switch 29 connected to one side of the KF vacuum joint two 24. The inside of the knob switch 29 is connected with a pull rod 30 in the inside of the cold guide rod 12, one end of the pull rod 30 is connected with the knob switch 29, the other end extends out of the cold guide rod 12 and is connected with the sample cover 28, by rotating the knob switch 29 to move the pull rod 30 forward and backward, so as to open and close the sample cover 28, to protect the sample during sample transfer to the cryo-EM.

[0073] Further, in order to realize the filling of the inner tank 5 and the outer tank 4, the outer tank 4 and the inner tank 5 are provided with a filling device, the filling device includes a compensator one 31 connected between the outer shell 6 and the inner container 7, the inside of the compensator one 31 is provided with a filling pipe 32, the top of the inner tank 5 is connected with a compensator two 33, and the filling pipe 32 is connected with the compensator two 33. The inner tank 5 is filled through the filling pipe 32, and the outer tank 4 is filled through the gap between the compensator one 31 and the filling pipe 32. The top of the compensator one 31 is threadedly connected with an outer cover 34, an exhaust port is formed in the outer cover 34, and an exhaust pipe 35 is installed at the exhaust port.

[0074] Further, the outer shell 6 and the inner container 7 are connected with a compensator three 36, the inside of the compensator three 36 is provided with a liquid level meter 37, and the liquid level meter 37 extends into the inner container 7. The top of the compensator three 36 is also connected with an outer cover 34.

[0075] Further, the two sides of the KF vacuum joint two 24 are respectively connected with a vacuum valve 38 and a plug-in connector 39, as shown in Figure 3

[0076] In this embodiment, the KF vacuum joint one 21 and the KF vacuum joint two 24 adopt KF vacuum flanges.

[0077] As shown in Figure 7 The system further includes an adjusting platform 40, a pair of sliding rails 41 are arranged on the adjusting platform 40, an installation platform 42 is slidably connected on the sliding rails 41, and the installation platform 42 can slide along the sliding rails 41. The Dewar flask 1 and the sample rod 2 form a sample transfer support 43, and the sample transfer support 43 can be fixedly installed on the installation platform 42, and moves linearly forward and backward together with the installation platform 42.

[0078] ​In the working state, the Dewar flask 1 is directly installed on the installation platform 42, and the sample rod 2 is installed on the electron microscope goniometer; since the Dewar flask 1 is not in direct contact with the sample rod 2, the weight of the Dewar flask 1 and the fluid in the Dewar flask 1 cannot be transmitted to the sample rod 2 and the electron microscope, so that the fluid capacity in the Dewar flask 1 can be made larger without damaging the electron microscope goniometer, and more liquid nitrogen 9 can realize super-long temperature endurance, and the super-long endurance eliminates the hidden danger that the operator is frozen by liquid nitrogen during multiple liquid nitrogen filling operations. Moreover, since the Dewar flask 1 is not in direct contact with the sample rod 2, the cold energy of the inner tank 5 is transmitted through the cold guide plate 11, so that the sample rod 2 can realize 360° rotation observation, the observation angle is larger, and the imaging has no dead angle.

[0079] In the working state, the cold guide plate 11 is only in contact with the fluid in the inner tank 5, since the boiling point of the fluid in the outer tank 4 is higher than that of the fluid in the inner tank 5, the fluid in the inner tank 5 will not boil, and the vibration of the bubbles generated by the boiling of the fluid in the outer tank 4 cannot be directly transmitted to the sample rod 2, so that the application can realize smaller imaging drift and higher resolution.

[0080] In use, first, the KF vacuum joint one 21 and the KF vacuum joint two 24 are manually clamped and fixed by the KF clamp 26, so that the sample rod 2 is integrated with the dewar 1, and at the same time, since the transition joint one 15 and the transition joint two 17 are sealed by the sealing gasket 18, the fluid in the inner tank 5 can be prevented from flowing to the KF vacuum joint one 21 during movement; then, the outer cover 34 on the top of the compensator one 31 is unscrewed, and the outer tank 4 is filled with fluid medium (such as liquid nitrogen 9) from the gap between the compensator one 31 and the filling tube 32; after the temperature of each component is balanced, the outer tank 4 is supplemented with liquid nitrogen 9 to the full liquid level, and the outer cover 34 is screwed on; then, the inner tank 5 is quantitatively supplemented with fluid (such as liquid oxygen 10), and the cold energy of the liquid oxygen 10 will be transmitted to the sample holder 27 at the front end through the cold lead rod 12, and the cold energy of the liquid nitrogen 9 in the outer tank 4 will be continuously transmitted to the liquid oxygen 10 through the thin-walled inner tank 5, and the actual consumption in this system is the cold energy of the liquid nitrogen 9 in the outer tank 4; at this time, the sample can be loaded onto the sample holder 27 by the sample stage, and the sample cover 28 is closed. The whole system is moved to the adjustment platform 40, and the sample rod 2, the KF vacuum joint two 24, the KF clamp 26 and other components are rotated by 90° along the axis direction of the horn joint 20, at this time, the outer tank 4 and the inner tank 5 remain stationary, the cold lead rod 12 and the cold lead plate 11 are synchronously rotated by 90°, and the sample rod 2 is aligned with the electron microscope port, then the sample transfer bracket 43 and the mounting platform 42 are synchronously pushed forward, after the electron microscope is evacuated, the KF vacuum joint two 24 and other components are manually rotated to rotate back to the original position, the sample transfer bracket 43 is continuously pushed to the bottom, and the KF clamp 26 is manually opened, the outer tank 4 is moved backward by about 10-15 mm as a whole, and the system is installed. As shown in Figure 4 and Figure 6 At this time, the sample rod 2 and the dewar 1 on the electron microscope are disconnected from the KF vacuum joint two 24, without any hard connection, and the cold energy continues to be transmitted from the cold lead rod 12 to the sample holder 27 at the front end. This system can completely avoid the vibration caused by the boiling of liquid nitrogen 9, and reduce the sample drift rate. Since the dewar 1 is supported by the mounting platform 42, and is completely separated from the sample rod 2 on the electron microscope, the capacity of the dewar 1 can be larger, the amount of liquid nitrogen 9 filled at one time is more, and the low-temperature holding time is longer (more than 10 h), thereby prolonging the effective observation time of the sample.

[0081] Example 2

[0082] The difference between this embodiment and example 1 is that:

[0083] The outer tank 4 and the inner tank 5 are filled with liquid nitrogen 9, and after the outer tank 4 is filled with liquid nitrogen 9, the outer tank 4 is sealed by covering the outer cover 34, and the exhaust pipe 35 connected to the outer cover 34 is connected to a vacuum pump, the vacuum pump is started, the air pressure in the outer tank 4 is lower than the standard atmospheric pressure, and the air pressure in the inner tank 5 is unchanged, at this time, the temperature and the phase change point of the liquid nitrogen 9 in the outer tank 4 will also decrease; only the temperature in the outer tank 4 needs to be maintained in the interval of -195.8~ -210℃, which can ensure that the liquid nitrogen 9 in the inner tank 5 remains liquid and does not boil; if the air pressure in the outer tank 4 is controlled at an absolute pressure of 45KPa, the temperature and the boiling point of the liquid nitrogen 9 are -202℃, at this time, the liquid nitrogen 9 in the inner tank 5 and the liquid nitrogen 9 in the outer tank 4 are heated through the wall of the inner tank 5 and are jointly at -202℃, and the boiling point of the liquid nitrogen 9 in the inner tank 5 is higher than that of the liquid nitrogen 9 in the outer tank 4. By changing the air pressure of the outer tank 4 and the inner tank 5, the air pressure in the outer tank 4 is lower than that in the inner tank 5, so that the boiling points of the fluids in the two tanks change.

[0084] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An ultra-long-duration sample transport system for cryo-electron microscopy, characterized by: Includes Dewar and sample rod; The Dewar tank includes an outer tank and an inner tank, wherein the inner tank is located inside the outer tank, the outer tank stores a first cryogenic fluid, and the inner tank stores a second cryogenic fluid, wherein the boiling point of the second cryogenic fluid is higher than that of the first cryogenic fluid; A cooling plate is provided in the inner tank, and the sample holder includes an outer rod and a cooling rod. The cooling rod is located inside the outer rod, one end of the cooling rod extends into the inner tank and is connected to the cooling plate, and the other end of the cooling rod extends out of the outer rod and is connected to the sample holder; The outer rod is connected to a connecting component 1, the Dewar tank is connected to a connecting component 2, and the connecting component 1 and the connecting component 2 are detachably connected.

2. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 1, characterized in that: The first cryogenic fluid is liquid nitrogen, and the second cryogenic fluid is liquid oxygen; Alternatively, the cryogenic fluid 1 and the cryogenic fluid 2 are both liquid nitrogen, and the gas pressure in the outer tank is lower than the gas pressure in the inner tank.

3. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 1, characterized in that: The outer tank is a vacuum insulation structural component, comprising an outer shell and an inner liner. The inner liner is located inside the outer shell, a vacuum layer is formed between the outer shell and the inner liner, and the inner tank is located inside the inner liner.

4. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 1, characterized in that: The connecting component 2 includes a transition pipe 1, a transition pipe 2, a transition joint 1, a horn joint and a vacuum joint 1; one end of the transition pipe 1 is connected to one side of the inner tank, and the transition pipe 1 is communicated with the interior of the inner tank, and the other end of the transition pipe 1 is connected to the transition joint 1; one end of the horn joint is a large head end, and the other end is a small head end, the large head end of the horn joint is connected to one side of the outer tank, and the transition pipe 2 and the transition joint 1 are both located in the horn joint; one end of the transition pipe 2 is connected to the transition joint 1, and the other end of the transition pipe 2 is connected to the inner wall of the small head end of the horn joint; the outer wall of the small head end of the horn joint is connected to the vacuum joint 1.

5. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 4, characterized in that: The outside of the cooling rod is connected to a transition joint 2, the transition joint 2 is connected to a transition pipe 3, and a sealing gasket is provided between the transition joint 2 and the transition joint 1.

6. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 4, characterized in that: The connecting component 1 includes a vacuum connector 2, and the vacuum connector 2 is detachably connected to the vacuum connector 1.

7. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 6, characterized in that: A knob switch is provided on the outer rod, and the knob switch is connected to one side of the second vacuum joint; the knob switch is connected to the pull rod inside the cooling rod, and a sample cover is slidably provided on the sample rack, and one end of the pull rod extends out of the cooling rod and is connected to the sample cover.

8. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 3, characterized in that: A filling device is provided between the outer tank and the inner tank, the filling device comprising a compensator 1 connected between the outer shell and the inner tank, a filling pipe being provided inside the compensator 1, a compensator 2 being connected to the top of the inner tank, the filling pipe being connected to the compensator 2; The top of the compensator 1 is connected with an outer cover, the outer cover is provided with an exhaust port, and an exhaust pipe is installed at the exhaust port.

9. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 3, characterized in that: A compensator three is connected between the outer shell and the inner tank. A liquid level gauge is provided inside the compensator three. The liquid level gauge extends into the inner tank. An outer cover is connected to the top of the compensator three.

10. The ultra-long endurance sample transport system for cryo-electron microscopy according to claim 1, characterized in that: The system also includes an adjustment platform, which is provided with a slide rail, and a mounting platform is slidably connected to the slide rail, and the mounting platform can slide along the slide rail; the Dewar tank is mounted on the mounting platform; and the sample rod is mounted on the electron microscope goniometer.

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