Cryoelectron microscope sample preparation pool and preparation method

Abstract

The invention discloses a cryoelectron microscope sample preparation pool, the cryoelectron microscope sample preparation pool comprises a liquid nitrogen pool, a workbench, a cold conduction piece, a sample freezing device, a cover and a snap ring alignment tool, the workbench comprises a support seat and an objective table, and the sample freezing device comprises an ethane pool and a foaming gasket; the invention also discloses a preparation method of the cryoelectron microscope sample. The cryoelectron microscope sample preparation pool is simple in overall structure, easy to manufacture and low in cost, can prevent infectious samples containing viruses and the like from polluting the outside, has the advantages of being convenient and stable to operate, short in sample preparation period, safe, environmentally friendly and capable of reducing waste, and is suitable for application and popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of transmission electron microscope sample preparation and discloses a cryo-electron microscope sample preparation cell and a preparation method. Background Art

[0002] Cryo-electron microscopy has now developed into an indispensable core technology in the field of structural biology. Cryo-electron microscopy is a technique that uses a transmission electron microscope to image and analyze the structure of samples embedded in cryogenic glassy ice. In the 1980s, Dr. Jacques Dubochet discovered that liquid ethane could be used to embed rapidly inserted ultra-thin biological samples less than 1 micron in cryogenic glassy ice, allowing biological samples to be imaged in a near-physiological environment while reducing the effects of radiation damage on biological samples. Compared with traditional crystallography, cryo-electron microscopy has been widely used in many fields such as biological mechanism research, biopharmaceutical research, and virus research because it can directly observe and calculate the structure of specific proteins or tissues in solutions or cell tissues, demonstrating great scientific and commercial value.

[0003] Samples used for cryo-electron microscopy analysis are usually composed of proteins, protein complexes, viruses or other macromolecules. However, these molecules are usually not resistant to electron beam radiation and will continue to move in room temperature solutions, affecting the imaging effect. For this reason, before observing the sample with an electron microscope, the protein-containing solution is attached to a porous metal grid and then quickly inserted into liquid ethane to cool it into an amorphous solid (abbreviated as: vitrification) state to observe the sample. As the first step in the cryo-electron microscopy experiment, the quality of cryo-electron microscopy sample preparation determines the success of the subsequent experiment. This sample preparation method has the following advantages: (1) High versatility, applicable to samples such as proteins, tissues, viruses and specific materials; (2) During the vitrification process, the sample will form amorphous ice, which maintains the living state of cells or tissues. In this state, the biological sample does not contain ice crystals and will not destroy the structure of the biological sample itself; (3) It avoids the effects of chemical agents on biological samples, reduces damage to the sample's ultrastructure and composition, and is beneficial to protecting the activity of biological macromolecules; (4) The tolerance of glassy biological samples to electron radiation in transmission electron microscopy is greatly improved, and electron diffraction will not interfere with data collection; (5) The operation steps are few and the time is short, which is suitable for rapid research and diagnosis. It avoids the conformational changes that may occur in biological samples during the transfer process, resulting in the loss of some structural information and reducing the accuracy of sample function evaluation.

[0004] However, the formation of vitrified biological samples has very strict environmental requirements. One of the conditions for forming the vitrified state during the preparation process is that the sample is frozen by liquid ethane fast enough, and the sample needs to be stored at liquid nitrogen temperature in real time to ensure the stability of the amorphous ice properties. The cycle from inactivation to freezing of biological samples is long, and conformational changes may occur during the transfer process, resulting in the loss of some structural information and reducing the accuracy of the functional evaluation of biological samples. Currently, there are few manufacturers in the world capable of producing rapid freezing preparation cells, and they are expensive. At the same time, they lack convenience and safety. The defects of the existing rapid freezing preparation cells in terms of convenience are mainly reflected in the following aspects: the number of grid storage slots is very small, and it may be necessary to frequently replace the grid storage box when there are more biological sample preparation requirements; the shapes and angles of the preparation cell and the grid storage slot do not follow ergonomics, which is not convenient for biological sample preparation operations; the structural stability is poor, and it does not support the operation of the retaining ring. An additional instrument is required for the retaining ring; the structure easily causes the sample to be attached by ice, resulting in inability to image. The defects of the existing rapid freezing preparation cells in terms of safety are mainly reflected in the following aspects: the preparation cell is open. During the preparation operation, due to a large amount of liquid nitrogen volatilization, it is necessary to frequently pour liquid nitrogen, increasing the risk of liquid nitrogen contaminating the ethane pool; in the field of virus structure research, cryo-electron microscopes are often used because viruses often have the characteristics of high pathogenicity and high infectivity. During the process of quickly putting the grid into the freezing preparation cell, it is necessary to block the ways in which the sample and liquid ethane cause harm to the experimenters through sputtering.

[0005] Based on the problems existing in the prior art, the purpose of the present invention is to provide a cryo-electron microscope sample preparation cell and a preparation method that reduce costs, shorten the biological sample preparation cycle, improve the convenience of biological sample preparation and the accuracy of functional evaluation, and ensure safety during the preparation process. Summary of the Invention

[0006] Based on the above purpose, the present invention first provides a cryo-electron microscope sample preparation cell that can prevent external contamination by infectious samples such as viruses, and has low cost, convenient operation, high safety, and high accuracy in the functional evaluation of biological samples. The cryo-electron microscope sample preparation cell includes: a liquid nitrogen pool, a workbench, a heat conduction member, a sample freezing device, as well as a lid and a retaining ring alignment tool. The workbench includes a support seat and a loading platform. The center of the loading platform is a hollow structure and cooperates with the central convex structure of the inserted support seat to form a central circular groove. The sample freezing device includes an ethane pool and a foaming gasket. The workbench is located in the cavity of the liquid nitrogen pool. The retaining ring alignment tool is located in the grid storage slot provided on the loading platform; the ethane pool is located in the foaming gasket; the foaming gasket is located in the central circular groove of the loading platform; the heat conduction member is located at the top of the ethane pool, and the bottom of the heat conduction member is in contact with the surface of the loading platform; the lid is placed on the upper surface of the liquid nitrogen pool.

[0007] In a preferred embodiment, a handle is provided on the liquid nitrogen pool.

[0008] In a specific embodiment of the present invention, the support base includes four positioning columns. The positioning columns are integrally formed with the support base.

[0009] In a preferred embodiment, the included angle between two adjacent positioning columns among the four positioning columns is 90°.

[0010] In a specific embodiment of the present invention, the stage is connected to the support base by a rotating shaft.

[0011] In another specific embodiment of the present invention, the stage is fixed to the support base by bolts to enhance the structural stability.

[0012] In a preferred embodiment, digital markings, a grid storage groove, an O-ring storage groove, and a central circular groove are provided on the stage. The digital markings are located between the grid storage groove and the central circular groove, are in one-to-one correspondence with the grid storage groove, and are evenly distributed in the circumferential direction. The digital markings are used to mark the position of the grid storage groove; the grid storage groove is used to store the snap ring alignment tool; the O-ring storage groove is located between the grid storage grooves and is used to store O-rings; the central circular groove is located at the center and is used to place the foaming gasket.

[0013] In a specific embodiment of the present invention, an O-ring placement groove is provided in the middle of the snap ring alignment tool, and both ends of the O-ring placement groove are open to allow the passage of liquid nitrogen.

[0014] In a specific embodiment of the present invention, the lid is composed of a first lid body and a second lid body. The first lid body and the second lid body are respectively provided with two positioning structures that cooperate with the liquid nitrogen pool. The positioning structure is in a stepped shape and fits the outer contour of the liquid nitrogen pool. The positioning structure aligns the center of the lid with the center of the liquid nitrogen pool; the lid is directly placed on the upper surface of the liquid nitrogen pool.

[0015] In a specific embodiment of the present invention, a through hole is provided in the middle of the lid, and the through hole is used for the tweezer to pass through.

[0016] The present invention also provides a method for preparing a cryo-EM sample using the above-mentioned cryo-EM sample preparation pool, and the method includes the following steps: Step 1. After assembling the support base, the foaming gasket, the stage, and the ethane pool, place them in the liquid nitrogen pool; Step 2. Pour liquid nitrogen to lower the temperature in the liquid nitrogen pool; Step 3. Introduce ethane gas into the ethane pool and liquefy it into ethane liquid until an appropriate amount; Step 4. Use tweezers to hold the grid and drop the biological sample onto the grid; Step 5. Cover the upper surface with two lids having through holes in the middle, align the through holes with the bottom of the tweezers, and quickly drop the grid into the ethane pool through the sample projection device to complete the rapid freezing and fixation of the sample. In a specific embodiment of the present invention, the sample projection device is Vitrobot of Thermo Fisher; Step 6. Remove the tweezers, quickly move the grid to the liquid nitrogen pool, and then transfer it to the snap ring alignment tool. Use the C-type snap ring insertion tool to fix the grid to the O-ring.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The cryo-EM sample preparation pool of the present invention is provided with a support base to provide stable structural support and support the removal of ice contamination by rotation. The stage is provided with a grid storage groove and a snap ring placement groove, and can be fixed to the support base using bolts; the liquid nitrogen pool is provided with a handle to facilitate movement, provide an operation fulcrum, and ensure stable operation; the technical solution of the present invention is provided with a lid, which plays a role in heat preservation, reducing volatilization, and preventing splashing, and prevents the external contamination of infectious samples such as viruses. The overall structure of the technical solution of the present invention is simple, easy to manufacture, and low in cost. At the same time, it has the characteristics of convenient and stable operation, short sample preparation cycle, good effect, safe and environmentally friendly, and reducing waste, and is suitable for popularization and application. Description of the Drawings

[0018] Figure 1 is a schematic diagram of the overall structure of the cryo-EM sample preparation pool of the present invention; Figure 2 is a schematic diagram of the workbench structure of an embodiment of the present invention; Figure 3 is a schematic diagram of the cooperation structure of the workbench, the heat conduction member and the sample freezing device of an embodiment of the present invention; Figure 4 is a schematic diagram of the snap ring alignment tool structure of an embodiment of the present invention; Figure 5 is the spatial arrangement of the O-ring, the grid and the C-type ring during the placement of the C-type ring of an embodiment of the present invention; Figure 6 is a schematic diagram of the structure after the placement of the C-type ring of an embodiment of the present invention; Figure 7 is a photo of the ice layer of the biological sample prepared in an embodiment of the present invention.

[0019] In the figure: 1 - Liquid nitrogen pool; 2 - Workbench; 21 - Support base; 22 - Stage; 211 - Positioning post; 221 - Digital marking; 222 - Central circular groove; 223 - Grid storage groove; 224 - O-ring storage groove; 225 - Rotating shaft; 3 - Snap ring alignment tool; 31 - Open end of O-ring placement groove; 32 - O-ring placement groove; 33 - Protrusion; 4 - Sample freezing device; 41 - Ethane pool; 42 - Foam gasket; 5 - Heat conduction member; 51 - Heat conduction member handle; 6 - Lid; 61 - First cover body; 62 - Second cover body; 63 - Through hole; 64 - Matching positioning structure; 7 - Liquid nitrogen pool handle; 8 - O-ring; 9 - Grid; 10 - C-ring. Detailed implementation manners

[0020] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the protection scope defined by the claims of the present invention.

[0021] In combination with the drawings of the present invention, as Figures 1 to 4 shown, in a cryo-EM sample preparation cell provided by the present invention, it includes: a liquid nitrogen pool 1, a workbench 2, a heat conduction member 5, and a sample freezing device 4, and further includes: a lid 6 and a snap ring alignment tool 3. The workbench 2 includes a support base 21 and a stage 22 located thereon. The sample freezing device 4 includes an ethane pool 41 and a foam gasket 42. The liquid nitrogen pool 1 is placed on the tabletop; the workbench 2 is placed in the cavity of the liquid nitrogen pool 1. A plurality of grid storage grooves 223 are sequentially arranged along the circular outer edge of the stage 22. Spaces are left between the grid storage grooves 223 for arranging O-ring storage grooves 224. A plurality of grid storage grooves 223 surround to form a central circular hollow structure, and a central circular groove 222 is formed by sleeving the central cylindrical protrusion structure of the support base 21. The support base 21 and the stage 22 of the workbench 2 are connected by bolts and self-locking nuts at the center as a rotating shaft 225, not completely locked, so that the support base 21 and the stage 22 can rotate relative to each other. The stage 22 extends radially outward to form a plurality of positioning posts 211 that catch the periphery of the liquid nitrogen pool cavity; each grid storage groove 223 is provided with a digital marking 221 for convenient recording and querying. In the attached Figure 2In a specific embodiment, there are 9 sample cells and 223 grid storage slots; the ethane cell 41 is installed in the foam gasket 42; the foam gasket 42 is placed in the central circular groove 222 in the middle of the workbench 2; the heat conduction member 5 is a hollow triangular structure and can be placed on the top of the ethane cell 41. The hollow structure cooperates with the raised step at the top of the ethane cell 41. There are 3 hexagon studs at the bottom of the heat conduction member 5 in contact with the corresponding O-ring storage slots 224 on the surface of the stage 22. When not in use, the heat conduction member 5 can be removed as a whole. A heat conduction member handle 51 for installing and removing the heat conduction member itself is provided at the top of the heat conduction member 5; the lid 6 is placed on the upper surface of the liquid nitrogen cell 1. The fitting positioning structure 64 on the lid 6 can fasten the outer edge of the liquid nitrogen cell 1 so that there is no gap after the lid 6 and the liquid nitrogen cell 1 are combined. The central through hole 63 is on the same axis as the ethane cell 41.

[0022] In a preferred embodiment, the liquid nitrogen cell 1 is provided with a liquid nitrogen cell handle 7, which, like the foam gasket 42 and the lid 6, plays a role in insulating temperature.

[0023] In a specific embodiment of the present invention, the support base 21 includes four positioning posts 211, which are integrally formed with the support base 21 and are used to support components such as the stage 22, the snap ring alignment tool 3, the ethane cell 41, and the foam gasket 42. Placed in the liquid nitrogen cell 1, the ethane cell 41 can be positioned at a specific position. Only four positioning posts 211 are retained, reducing the surface area to the minimum and reducing the contact area with liquid nitrogen to avoid generating bubbles.

[0024] In a preferred embodiment, the included angle between two adjacent positioning posts 211 among the four positioning posts 211 is 90°.

[0025] In a specific embodiment of the present invention, the stage 22 is connected to the support base 21 by a rotating shaft 225. The stage 22 is directly placed on the support base 21 and rotates around the axis.

[0026] In another specific embodiment of the present invention, the stage 22 is fixed to the support base 21 by bolts to enhance the structural stability.

[0027] In a preferred embodiment, the outer contour of the stage 22 closely adheres to the grid storage groove 223, and there is a space between the grid storage grooves 223. The digital markings 221 are located between the grid storage grooves 223 and the central circular groove 222, corresponding to the grid storage grooves 223 one by one and evenly distributed circumferentially. The digital markings 221 are used to mark the positions of the grid storage grooves 223. The grid storage grooves 223 in the present invention can be used to store grid storage boxes or the snap ring alignment tool 3. The grid storage box has a three-dimensional structure that fits the outer contour of the grid storage groove 223 and is used to store multiple grids. The O-ring storage groove 224 is located between the grid storage grooves 223 and is used to store O-rings and to place the three hexagon studs provided at the bottom of the heat conduction member 5. The central circular groove 222 is located at the center and is used to place the foam gasket 42.

[0028] In a specific embodiment of the present invention, an O-ring placement groove 32 is provided in the middle of the snap ring alignment tool 3, and it has a guiding function for the C-type snap ring insertion tool. Both ends 31 of the O-ring placement groove 32 are open ends for connecting liquid nitrogen. The shapes of the two protrusions 33 on the snap ring alignment tool 3 match the outer contour of the grid storage groove 223 on the stage 22, aiming to prevent the snap ring alignment tool 3 from rotating in the grid storage groove during the operation. During work, the grid is placed in the groove of the O-ring, then horizontally placed in the O-ring placement groove 32, and then the C-type snap ring 10 is snapped into the O-ring along the O-ring placement groove 32 in the vertical direction by the C-type snap ring insertion tool, which functions to protect the grid (the grid is very thin and easily bent and damaged). The C-type snap ring insertion tool is a ballpoint pen-shaped tool, and the C-type snap ring 10 is placed into the hole at the front end of the ballpoint pen-shaped tool by tweezers instead of the pen core. The diameter of the pen barrel structure at the front end of the tool matches the O-ring placement groove 32. In a specific placement process, after inserting the ballpoint pen-shaped tool into the O-ring placement groove 32, press the button at the rear end of the ballpoint pen-shaped tool to push the C-type snap ring 10 out of the pen barrel and snap it onto the O-ring 8 and the grid 9. The spatial arrangement during the placement of the C-type snap ring is as Figure 5 shown, Figure 6 showing the structural schematic diagram after the C-type ring is placed.

[0029] In a specific embodiment of the present invention, the lid 6 is composed of two parts, a first lid body 61 and a second lid body 62. The first lid body 61 and the second lid body 62 are respectively provided with a fitting and positioning structure 64 for cooperating with the liquid nitrogen pool 1. The fitting and positioning structure 64 is stepped and matches the outer contour of the liquid nitrogen pool. Each lid is provided with two positioning structures 64 to align the center of the lid with the center of the liquid nitrogen pool. The lid is directly placed on the upper surface of the liquid nitrogen pool 1 for convenient taking and placing.

[0030] In a specific embodiment of the present invention, a through hole 63 is provided in the middle of the lid 6, and the through hole 63 is used for the tweezers to pass through.

[0031] The present invention also provides a method for preparing a novel cryo-EM sample, comprising the following steps: Step 1: After assembling the support base 21, the foam gasket 42, the stage 22, and the ethane pool 41, place them in the liquid nitrogen pool 1. Step 2: Pour liquid nitrogen to lower the temperature in the liquid nitrogen pool 1. Step 3: Introduce ethane gas into the ethane pool 41 to liquefy it into ethane liquid until an appropriate amount. Step 4: Use forceps to hold the grid and drop the biological sample onto the grid. Step 5: Cover the upper surface with two lids 6 having through holes in the middle, align the through holes with the bottom of the forceps, and quickly drop the grid into the ethane pool 41 through the sample projection device of the Vitrobot of Thermo Fisher to complete rapid freezing and fixation of the sample. Step 6: Remove the forceps, quickly move the grid to the liquid nitrogen pool 1, and then transfer it to the snap ring alignment tool 3, and use the C-type snap ring insertion tool to fix the grid to the O-ring.

[0032] Using hemocyanin, imaging tests and data collection were carried out under a 300 keV electron microscope by using the sample preparation method of the present invention. Under the condition of defoucs = -2um, Figure 7 Very clear particles can be seen.

[0033] In the present invention, unless otherwise clearly specified and defined, terms such as "installation", "connection", "connection", "fixation", etc. should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral body; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium; it can be the communication inside two components or the interaction relationship between two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0034] It should be understood that the above specific embodiments of the present invention are only used for exemplary illustration or explanation of the principle of the present invention, and do not constitute a limitation to the present invention. Therefore, any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modification examples falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A cryo-electron microscopy sample preparation cell, characterized in that, The cryo-EM sample preparation cell includes: a liquid nitrogen pool, a workbench, a heat conduction member, a sample freezing device, as well as a lid and a clamp alignment tool. The workbench includes a support base and a stage. The center of the stage is a hollow structure and cooperates with the central convex structure of the inserted support base to form a central circular groove. The sample freezing device includes an ethane pool and a foaming gasket. The workbench is located in the cavity of the liquid nitrogen pool, and the clamp alignment tool is located in the grid storage groove provided on the stage. The ethane pool is located within the foaming gasket. The foaming gasket is located in the central circular groove of the stage. The heat conduction member is located at the top of the ethane pool, and the bottom of the heat conduction member contacts the surface of the stage. The lid is placed on the upper surface of the liquid nitrogen pool.

2. The cryo-EM sample preparation cell according to claim 1, wherein, A handle is provided on the liquid nitrogen pool.

3. The cryo-EM sample preparation cell according to claim 1, wherein The support base includes four positioning posts.

4. The cryo-EM sample preparation cell according to claim 3, wherein, The included angle between two adjacent positioning posts among the four positioning posts is 90°.

5. The cryo-electron microscopy sample preparation cell according to claim 1, wherein The stage and the support base are connected by a rotating shaft.

6. The cryo-electron microscopy sample preparation cell according to claim 1, wherein The stage is fixed to the support base by bolts.

7. The cryo-EM sample preparation cell according to claim 1, wherein, The stage is further provided with digital markings, a grid storage groove, and an O-ring storage groove. The digital markings are located between the grid storage groove and the central circular groove, correspond to the grid storage groove one by one, and are evenly distributed along the circumferential direction. The digital markings are used to mark the positions of the grid storage grooves. The grid storage groove is used to store the clamp alignment tool. The O-ring storage groove is located between the grid storage grooves and is used to store O-rings.

8. The cryo-electron microscopy sample preparation cell according to claim 1, wherein An O-ring placement groove is provided in the middle of the clamp alignment tool, and both ends of the O-ring placement groove are open for connecting liquid nitrogen.

9. The cryo-EM sample preparation cell according to claim 1, wherein, The lid is composed of two parts, a first lid body and a second lid body. The first lid body and the second lid body are respectively provided with two positioning structures that cooperate with the liquid nitrogen pool. The positioning structures are step-shaped and match the outer contour of the liquid nitrogen pool. The positioning structures align the center of the lid with the center of the liquid nitrogen pool. The lid is directly placed on the upper surface of the liquid nitrogen pool.

10. A method for preparing a cryo-EM sample using the cryo-EM sample preparation cell according to any one of claims 1-9, the method comprising the following steps: Step 1. After assembling the support base, the foaming gasket, the stage, the ethane pool, and the heat conduction member, place them in the liquid nitrogen pool. Step 2. Pour liquid nitrogen to lower the temperatures in the liquid nitrogen pool and the ethane pool. Step 3. Introduce ethane gas into the ethane pool to liquefy it into ethane liquid until an appropriate amount, and then remove the heat conduction member. Step 4. Use forceps to hold the grid and drop the biological sample onto the grid. Step 5. Cover the upper surface with the lid, align the through hole with the bottom of the forceps, and quickly insert the grid into the ethane pool through the sample projection device to complete rapid freezing and fixation of the sample. Step 6. Remove the forceps and the lid, quickly transfer the grid to the liquid nitrogen pool, and then transfer it to the clamp alignment tool. Use a C-type clamp insertion tool to fix the grid to the O-ring.

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