A cryogenic and low-humidity workbench for cryo-EM sample loading

Abstract

The present invention relates to a cryogenic and low-humidity workbench for cryo-EM sample loading, which comprises an airtight chamber, a refrigeration system, a circulating dehumidification system and a control system; an operation port and an operation observation window are provided at the front end of the airtight chamber, and sample loading windows are provided on both sides; the bottom surface of the airtight chamber serves as a workbench surface, and a movable cup holder, a movable tabletop and a groove table are provided on the workbench surface. The refrigeration system includes a temperature and humidity sensor, a refrigeration fin tube and a coolant interface, and is used to adjust the temperature environment in the airtight chamber. The circulating dehumidification system includes a dryer, a first flow equalizer, a second flow equalizer, a first regulating valve, a second regulating valve and a vacuum pump, and is used to form a microcirculation environment of dry air flow in the airtight chamber. The control system is arranged on the airtight chamber and is used to monitor and control parameters such as temperature and humidity in the airtight chamber. The present invention can effectively reduce the ice crystal contamination of samples and improve the sample quality; moreover, it is easy to install and has a smooth operation.

Description

Technical Field

[0001] The present invention relates to a low-temperature and low-humidity workbench, specifically a low-temperature and low-humidity workbench for cryo-EM sample loading. Background Art

[0002] Cryo-electron microscopy technology is one of the main methods for analyzing the high-resolution structural information of biological macromolecules and won the Nobel Prize in Chemistry in 2017. This technology rapidly freezes biological macromolecules and fixes them in a vitreous ice layer, then uses a cryo-EM to collect sample image information, and processes the images through a three-dimensional reconstruction algorithm to analyze the structural information of biological macromolecules. This technology is mainly used to study the working mechanism of biological macromolecules, screen and optimize antibodies and small molecule drugs acting on macromolecules, etc.

[0003] The preparation of cryo-EM samples is mainly achieved by spreading biological samples on a 3-mm-diameter grid, forming a thin-layer solution with a thickness of dozens to hundreds of nm, and then quickly inserting the grid into liquid ethane at -183°C. Liquid ethane has characteristics such as high heat capacity, high thermal conductivity, and high boiling point, and can freeze the sample solution into a vitreous ice layer in an extremely short time, thereby fixing the sample in the vitreous ice layer in a near-native state.

[0004] Before the EM samples prepared by rapid freezing are observed in a cryo-EM, they need to be fixed on a specific device, the Cartridge, and then transferred to the cryo-EM sample stage for observation through the cryo-EM automatic sample loading system. The cryo-EM automatic sample loading system can store up to 12 Cartridges fixed with grids at most. Fixing a 3-mm-diameter grid to the Cartridge is a relatively delicate operation. To prevent the sample ice layer from warming up and melting and then recrystallizing, the whole process is completed in liquid nitrogen at -196°C. During the operation, it is necessary to clearly see the state of the grid in the liquid nitrogen, which requires the liquid nitrogen level to be relatively low, just enough to submerge the moving grid. Qualifying up to 12 sample grids to be fixed on the Cartridge requires an operation time of about 40 minutes to 1 hour (depending on the operator's proficiency). During such a long time, the interface between liquid nitrogen and air and the tools used in the operation, due to the low temperature, will freeze the moisture in the surrounding air into ice crystals and introduce them into the liquid nitrogen or adsorb them on the operation tools, thereby bringing ice crystals to the sample during the grid-fixing process. A large number of ice crystals accumulated on the grid will cover the sample ice layer, making the sample unusable for high-resolution image acquisition. Even if there are only a small number of ice crystals in the sample ice layer, it will also interfere with the normal operation of the data automatic acquisition software and affect the quality of the acquired data. Summary of the Invention

[0005] The present invention aims to provide a low-temperature and low-humidity workbench for cryo-EM sample loading, so as to solve the technical problem of ice crystal contamination of cryo-EM samples during transfer in the prior art.

[0006] The present invention realizes the above object through the following technical solutions. A low-temperature and low-humidity workbench for cryo-EM sample loading includes an airtight chamber, a refrigeration system, a circulating dehumidification system, and a control system.

[0007] An operation port and an operation observation window are provided at the front end of the airtight chamber; sample loading windows are provided on both sides of the airtight chamber for liquid nitrogen filling and the entry and exit of sample cups; the bottom surface of the airtight chamber serves as a workbench surface, and a movable cup holder for placing a liquid nitrogen cup or a sample cup, a movable table for placing sample loading tools, and a groove table for sample loading operation are provided on the workbench surface.

[0008] The refrigeration system includes a temperature and humidity sensor, a refrigeration finned tube, and a coolant interface, and is used to adjust the temperature environment in the airtight chamber.

[0009] The circulating dehumidification system includes a dryer, a first flow equalizer, a second flow equalizer, a first regulating valve, a second regulating valve, and a vacuum pump. The first regulating valve is used to connect a rotary dehumidifier; the vacuum pump is used to extract the air in the airtight chamber, and the air flow passes through the first flow equalizer, the dryer, the second regulating valve, the vacuum pump, and the second flow equalizer in sequence in the circulating dehumidification system, so as to form a microcirculation environment of dry air flow in the airtight chamber.

[0010] The control system is arranged on the airtight chamber and is used to monitor the temperature and humidity in the airtight chamber and control the operation of the refrigeration system and the circulating dehumidification system.

[0011] The following technical solutions are preferred technical solutions:

[0012] Preferably, the control system includes a temperature control module for monitoring and controlling temperature, a humidity control module for monitoring and controlling humidity, a pressure monitoring module for monitoring pressure, an air flow monitoring module for monitoring air flow, an illumination module for providing light source, a power supply module for providing power supply, a drying module for heating, and a display screen for displaying temperature, humidity, pressure, and air flow data.

[0013] Preferably, a sleeve for preventing external moisture from entering is provided on the operation port.

[0014] Preferably, the operation observation window is an inclined plane made of a transparent material.

[0015] Preferably, side doors are provided on both sides of the airtight chamber, and the sample loading windows are arranged on the side doors.

[0016] Preferably, the groove table is provided with a corresponding cover plate.

[0017] Preferably, the dryer is provided on the control system, the first flow equalizer and the first regulating valve are provided at the top of the sealed chamber, and the second flow equalizer is provided at the bottom of the sealed chamber.

[0018] Preferably, the workbench further includes a bracket, and the sealed chamber is arranged on the bracket.

[0019] Preferably, feet for adjusting the height of the bracket and universal wheels for facilitating the movement of the bracket are provided at the bottom of the bracket.

[0020] The principle and beneficial effects of the present invention are as follows:

[0021] For the low-temperature and low-humidity workbench of the present invention, except for the operation cuffs, the entire workbench cavity is sealed. During operation, first, use a rotary dehumidifier to reduce the relative humidity in the sealed chamber to below 5%, and at the same time, use the temperature control module of the control system to reduce the air temperature to below 16°C to reduce the air saturation humidity. Then, through the internal circulating dehumidification system, while ensuring the minimum air flow disturbance, keep the humidity below 5%. Within the same operation time, using the low-temperature and low-humidity workbench of the present invention to complete sample loading can greatly reduce sample ice crystal contamination and improve sample quality. In addition, the present invention fully considers ergonomics, and it is very convenient to complete the installation and fixation of the sample carrier grid, and the entire operation steps are relatively smooth. Description of the Drawings

[0022] In order to more clearly illustrate the embodiments of the present invention, the drawings required for describing the embodiments will be briefly introduced below. Obviously, the drawings described below are only partial embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings or embodiments can also be obtained based on these drawings, but they should be covered within the protection scope of the present invention.

[0023] Figure 1 It is the right view of the low-temperature and low-humidity workbench according to the embodiment of the present invention.

[0024] Figure 2 It is the front view of the low-temperature and low-humidity workbench according to the embodiment of the present invention.

[0025] Figure 3 It is the left view of the low-temperature and low-humidity workbench according to the embodiment of the present invention.

[0026] Figure 4 It is the top view of the workbench surface according to the embodiment of the present invention.

[0027] Figure 5 It is the rear perspective view of the low-temperature and low-humidity workbench according to the embodiment of the present invention.

[0028] Figure 6This is a block diagram of the working principle of the cyclic dehumidification system described in the embodiments of the present invention.

[0029] Reference numerals: 1, operation port; 2, operation observation window; 3, control system; 4, dryer; 5, first flow equalizer; 6, first regulating valve; 7, sample loading window; 8, temperature and humidity sensor; 9, coolant interface; 10, universal wheel; 11, floor footing; 12, bracket; 13, side door; 14, second regulating valve; 15, movable cup holder; 16, refrigeration system; 17, movable tabletop; 18, second flow equalizer; 19, groove table; 20, power socket. Detailed implementation manners

[0030] In order to make the objectives, features, and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the embodiments described below are only some embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] In the description of the present invention, it should be understood that the orientation relationships and position relationships indicated by terms such as "upper", "top", "bottom", etc. are based on the orientations or position relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or component referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0032] Please refer to Figures 1-6 As shown, a low-temperature and low-humidity workbench for cryo-electron microscopy sample loading includes an airtight chamber, a refrigeration system 16, a cyclic dehumidification system, and a control system 3.

[0033] The front end of the airtight chamber is provided with an operation port 1 and an operation observation window 2. A sleeve for preventing external moisture from entering is provided on the operation port 1; the operation observation window 2 is an inclined surface made of a transparent material; side doors 13 are provided on both sides of the airtight chamber, and a sample loading window 7 is provided on the side door 13 for liquid nitrogen filling and the entry and exit of sample cups; the bottom surface of the airtight chamber serves as a workbench surface, and a movable cup holder 15 for placing a liquid nitrogen cup or a sample cup, a movable tabletop 17 for placing sample loading tools, and a groove table 19 for operating sample loading are provided on the workbench surface; a corresponding cover plate is provided on the groove table 19. The airtight chamber is fixed on a bracket 12, and floor footings 11 for adjusting the height of the bracket 12 and universal wheels 10 for facilitating the movement of the bracket 12 are provided at the bottom of the bracket 12.

[0034] The refrigeration system 16 includes a temperature and humidity sensor 8, refrigeration fin tubes, and a coolant interface 9. The refrigeration system 16 is used to adjust the temperature environment in the airtight chamber.

[0035] The cyclic dehumidification system includes a dryer 4, a first flow equalizer 5, a second flow equalizer 18, a first regulating valve 6, a second regulating valve 14, and a vacuum pump. The dryer 4 and the second regulating valve 14 are arranged on a control system 3. The first flow equalizer 5 and the first regulating valve 6 are arranged at the top of a sealed chamber, and the second flow equalizer 18 is arranged at the bottom of the sealed chamber. The first regulating valve 6 is used to connect a rotary wheel dehumidifier. The vacuum pump is used to extract the air in the sealed chamber. The air flow sequentially passes through the first flow equalizer 5, the dryer 4, the second regulating valve 14, the vacuum pump, and the second flow equalizer 18 in the cyclic dehumidification system, so as to form a microcirculation environment of dry air flow in the sealed chamber.

[0036] The control system 3 includes a temperature control module for monitoring and controlling temperature, a humidity control module for monitoring and controlling humidity, a pressure monitoring module for monitoring pressure, an air flow monitoring module for monitoring air flow, an illumination module for providing light source, a power supply module for providing power supply, a drying module for heating, and a touch screen display for displaying and setting parameters such as temperature, humidity, pressure, and air flow.

[0037] The working principle of the present invention is as follows:

[0038] During operation, the sealed cavity can isolate the external environment of the cavity, and the operation is carried out through the operation port 1 with a sleeve. In order to achieve a low-temperature environment inside the sealed cavity, the present invention is designed with a refrigeration system 16. The refrigeration system 16 passes through refrigeration fin tubes and a cold chain circulating medium, and through the weak internal air flow circulation, ensures that the inside of the cavity is in a low-temperature environment. In order to achieve a low-humidity environment inside the cavity, two-stage dehumidification is designed: first, the relative humidity inside the cavity is reduced to less than 5% by using a rotary wheel dehumidifier; then, the cyclic dehumidification system is used to maintain a low-humidity and micro-air flow environment inside the cavity. For the convenience of operation and real-time monitoring of various parameters, a control system 3 is designed. The system realizes the monitoring and control of parameters such as temperature, humidity, pressure, air flow, illumination, power supply, and heating through the touch screen display and each control module.

[0039] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0040] In summary, the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A cryogenic and low-humidity workbench for cryo-electron microscopy sample loading, characterized in that, It includes an airtight chamber, a refrigeration system, a circulating dehumidification system and a control system; An operation port and an operation observation window are provided at the front end of the airtight chamber; sample addition windows are provided on both sides of the airtight chamber for liquid nitrogen filling and the access of sample cups; the bottom surface of the airtight chamber serves as a workbench surface, on which there are movable cup holders for placing liquid nitrogen cups or sample cups, a movable table surface for placing sample loading tools, and a groove table for sample loading operation; The refrigeration system includes a temperature and humidity sensor, refrigeration fin tubes and a coolant interface, and is used to adjust the temperature environment in the airtight chamber; The circulating dehumidification system includes a dryer, a first flow equalizer, a second flow equalizer, a first regulating valve, a second regulating valve and a vacuum pump. The first regulating valve is used to connect a rotary dehumidifier; the vacuum pump is used to extract the air in the airtight chamber, and the air flow passes through the first flow equalizer, the dryer, the second regulating valve, the vacuum pump and the second flow equalizer in sequence in the circulating dehumidification system, so as to form a microcirculation environment of dry air flow in the airtight chamber; The control system is arranged on the airtight chamber and is used to monitor the temperature and humidity in the airtight chamber and control the operation of the refrigeration system and the circulating dehumidification system; The control system includes a temperature control module for monitoring and controlling temperature, a humidity control module for monitoring and controlling humidity, a pressure monitoring module for monitoring pressure, an air flow monitoring module for monitoring air flow, an illumination module for providing light source, a power supply module for providing power supply, a drying module for heating, and a display screen for displaying temperature, humidity, pressure and air flow data; The dryer is arranged on the control system, the first flow equalizer and the first regulating valve are arranged at the top of the airtight chamber, and the second flow equalizer is arranged at the bottom of the airtight chamber.

2. The cryogenic and low-humidity workbench for cryo-EM sample loading according to claim 1, wherein, A sleeve for preventing external moisture from entering is provided on the operation port.

3. The cryogenic and low-humidity workbench for cryo-EM sample loading according to claim 1, wherein The operation observation window is an inclined plane made of a transparent material.

4. The cryogenic and low-humidity workbench for cryo-EM sample loading according to claim 1, wherein, Side doors are provided on both sides of the airtight chamber, and the sample addition windows are arranged on the side doors.

5. The cryogenic and low-humidity workbench for cryo-EM sample loading according to claim 1, characterized in that, The groove table is provided with a corresponding cover plate.

6. The cryogenic and low-humidity workbench for cryo-EM sample loading according to claim 1, characterized in that, The workbench further includes a bracket, and the airtight chamber is arranged on the bracket.

7. The cryogenic and low-humidity workbench for cryo-EM sample loading according to claim 6, wherein, The bottom of the bracket is provided with feet for adjusting the height of the bracket and universal wheels for facilitating the movement of the bracket.

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