Preparation method of cryo-electron microscopy single crystal sample and cryo-electron microscopy single crystal sample
By separating and applying shear force based on the crystal surface characteristics of single crystal samples, and removing irregular grains on the microgate loading network, the problem of uncontrollable sample thickness and orientation in the prior art is solved, and the problem of uncontrollable sample thickness and orientation is achieved High resolution transmission electron microscopy imaging.
Patent Information
- Application Number
- CN202210427566.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-04-22
- Publication Date
- 2024-12-20
- Estimated Expiration
- 2042-04-22
AI Technical Summary
Existing transmission electron microscope sample preparation methods cannot effectively control the thickness and orientation of single crystal samples, resulting in the inability to achieve high-resolution imaging of atomic structure characterization.
By separating the sample according to the crystal surface characteristics of the single crystal sample, applying shear force to break through the crystal, forming refined grains, and removing irregular grains on the microgate loading network, and finally forming a target crystal surface thin by applying the force Layer sample.
Controllable thinning of the thickness and size of single crystal samples is achieved, the controllability and preparation success rate of samples are improved, the randomness of sample orientation is changed, and a thin layer sample of target crystal surface with uniform thickness is obtained.
Smart Images
Figure CN114965527B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cryo-electron microscopy, and in particular to a method for preparing a cryo-electron microscopy single crystal sample and a cryo-electron microscopy single crystal sample. Background Art
[0002] With the development of transmission electron microscopy technology, it has become possible to visualize the atomic-level structure of materials. Conventional transmission electron microscope samples are generally required to be less than 100nm thick. In order to achieve high-resolution transmission electron microscope imaging for atomic-level structural characterization, the sample thickness must be less than 50nm, or even 10nm.
[0003] However, for the transmission electron microscopy characterization of electron beam-sensitive single crystal samples, the existing transmission electron microscopy samples are generally prepared by a direct scraping method. The orientation and thickness of the transmission electron microscopy samples prepared after treatment are uncontrollable, and the sample thickness requirements for high-resolution transmission electron microscopy imaging for atomic-level structural characterization cannot be achieved. Summary of the invention
[0004] The main purpose of the present invention is to provide a method for preparing a cryo-electron microscopy single crystal sample and a cryo-electron microscopy single crystal sample, aiming to solve the technical problem of how to improve the controllability of sample preparation when using cryo-electron microscopy to characterize electron beam sensitive single crystal samples.
[0005] To achieve the above object, the present invention provides a method for preparing a cryo-electron microscopy single crystal sample, the method comprising:
[0006] Separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain broken single crystals;
[0007] Applying shear force to the broken single crystal to cause transcrystalline fracture of the broken single crystal and form refined grains on the first microgrid carrier grid;
[0008] Removing the irregular refined grains from the first micro-grid carrier grid to obtain target refined grains with target oriented crystal planes;
[0009] A force is applied on both sides of the target refined grain by the second microgrid carrier and the first microgrid carrier, so that the surface layer of the target refined grain adheres to the surface of the second microgrid carrier facing the first microgrid carrier, forming a target crystal plane thin layer sample.
[0010] Optionally, before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtaining the broken single crystal, the method further includes:
[0011] By controlling the growth process of the single crystal sample, the single crystal sample having a thickness smaller than the millimeter scale is obtained.
[0012] Optionally, before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtaining the broken single crystal, the method further includes:
[0013] The single crystal sample having a thickness smaller than the millimeter scale is obtained by mechanically processing the single crystal sample.
[0014] Optionally, the step of separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample to obtain a broken single crystal includes:
[0015] Determining a separation direction according to the crystal plane characteristics of the single crystal sample;
[0016] Stress is applied to the single crystal sample in sequence according to the separation direction to separate the single crystal sample, thereby reducing the thickness of the single crystal sample and obtaining the broken single crystal.
[0017] Optionally, applying a shear force on the broken single crystal to cause the broken single crystal to fracture through the crystal and form refined grains on the first microgrid carrier grid includes:
[0018] The broken single crystal is roller-coated on one side surface of the first micro-grid carrier to apply the shear force on the broken single crystal, so that the broken single crystal undergoes transgranular fracture on the first micro-grid carrier, and the refined grains are formed on the first micro-grid carrier.
[0019] Optionally, the step of removing irregular refined grains from the first micro-grid carrier to obtain target refined grains of the target oriented crystal plane includes:
[0020] The first micro-grid carrier grid is shaken to remove the irregular refined grains and obtain the target refined grains of the target oriented crystal plane.
[0021] Optionally, before applying a force on both sides of the target refined grain by the second microgrid and the first microgrid so that the target refined grain adheres to a surface of the second microgrid facing the first microgrid, and forming a target crystal plane thin layer sample, the method further includes:
[0022] The second microgrid carrier is placed on a side surface of the first microgrid carrier carrying the target refined grains, so that the target refined grains contact the second microgrid carrier and the first microgrid carrier.
[0023] Optionally, the step of applying a force on both sides of the target refined grain by the second micro-grid and the first micro-grid so that the target refined grain adheres to a surface of the second micro-grid facing the first micro-grid to form a target crystal plane thin layer sample includes:
[0024] The force is applied on both sides of the target refined grain by the second micro-grid and the first micro-grid within a preset sliding time, so that the second micro-grid and the first micro-grid slide relative to each other, so that the target refined grain adheres to the surface of the second micro-grid facing the first micro-grid, thereby forming the target crystal surface thin layer sample.
[0025] Optionally, before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtaining the broken single crystal, the method further includes:
[0026] The single crystal sample is placed in a vacuum environment to complete the processing of the single crystal sample in the vacuum environment.
[0027] In addition, to achieve the above-mentioned purpose, the present invention also provides a cryo-electron microscopy single crystal sample, which is prepared by the preparation method of the cryo-electron microscopy single crystal sample as described in any of the above items.
[0028] The present invention proposes a method for preparing a cryo-EM single crystal sample and a cryo-EM single crystal sample. The single crystal sample can be separated according to the crystal plane characteristics of the single crystal sample to minimize the thickness of the single crystal sample; a broken single crystal is obtained; a shear force is applied to the broken single crystal to cause the broken single crystal to fracture through the crystal, so that the broken single crystal can be further thinned to form refined grains on a first micro-grid carrier; irregular refined grains are removed from the first micro-grid carrier to obtain refined grains with relatively uniform thickness, and the exposure of the crystal plane of the target orientation can be retained to a large extent on the carrier to obtain target refined grains with the target orientation crystal plane; a force is applied on both sides of the target refined grain by a second micro-grid carrier and the first micro-grid carrier to make the target refined grain adhere to the surface of the second micro-grid carrier facing the first micro-grid carrier, so that the target refined grain is loaded on the second micro-grid carrier to form a target crystal plane thin layer sample. That is, by separating the single crystal samples according to the crystal plane characteristics of the single crystal samples and applying shear force to the broken single crystals, the thinning of the single crystal samples can be controlled. By removing irregular refined grains, refined grains with relatively uniform thickness can be obtained. By applying forces on both sides of the target refined grains with the second microgrid carrier and the first microgrid carrier, a target crystal plane thin layer sample is formed, thereby achieving control over the thinning of the single crystal samples, obtaining samples with relatively uniform thickness, and achieving the loading of the target crystal plane thin layer sample. The phenomenon of completely random sample orientation in the prior art has been changed, the controllability of the prepared samples has been improved, and the success rate of the overall sample preparation has also been improved. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0030] Figure 1 It is a schematic flow chart of the first embodiment of the method for preparing a cryo-electron microscopy single crystal sample of the present invention;
[0031] Figure 2 It is a schematic flow chart of a fourth embodiment of a method for preparing a cryo-electron microscopy single crystal sample of the present invention;
[0032] Figure 3 It is a schematic flow chart of the fifth embodiment of the method for preparing single crystal samples for cryo-electron microscopy of the present invention.
[0033] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION
[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0035] It should be noted that in the embodiments of the present invention, all directional indications (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.
[0036] In the present invention, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or system including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or system. In the absence of more restrictions, an element defined by the sentence "includes..." does not exclude the presence of other identical elements in the process, method, article or system including the element.
[0037] In the present invention, if there is a description involving "first", "second", etc., the description of "first", "second", etc. is only used for descriptive purposes and cannot be understood as indicating or implying its relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features.
[0038] For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.
[0039] Since the sample thickness of conventional transmission electron microscope (TEM) samples in the prior art is generally required to be less than 100nm, in order to achieve high-resolution transmission electron microscope (HRTEM) imaging for atomic-level structural characterization, the sample thickness is required to be less than 50nm, or even 10nm. However, many electron beam sensitive samples to be characterized (such as perovskite photoelectric materials MAPbI3 and MAPbBr3, etc.) are single crystals or other bulk materials, and it is necessary to adopt appropriate processing methods to prepare samples to obtain TEM samples with a thickness of <50nm. The processing techniques for making conventional bulk materials into samples for TEM characterization usually include slicing, pre-thinning, electrolytic polishing or ion thinning, and focused ion beam processing, etc., and the thinning process relies on chemical etching, photolithography or high-energy particle irradiation thinning, etc. Electron beam sensitive samples are often unable to withstand such high-energy particle bombardment and interaction, so there are various forms of structural destruction such as melting, phase change, and defects. Many electron beam-sensitive single crystal materials (such as the perovskite photoelectric materials MAPbI3 and MAPbBr3 mentioned above) are environmentally sensitive and cannot be exposed to air for a long period of sample preparation. In addition, the preparation of existing electron beam-sensitive single crystal materials basically adopts the direct scraping method. The orientation and thickness of the TEM samples prepared after treatment are uncontrollable and the success rate is low.
[0040] The present invention provides a solution, which controls the single crystal sample to undergo transcrystalline fracture at a first microgrid carrier grid, thins the single crystal sample, obtains target refined grains, and forms a target crystal surface thin layer sample by applying forces on both sides of the target refined grains.
[0041] Through the above technical scheme, the thinning of single crystal samples can be controlled, samples with relatively uniform thickness are obtained, the loading of thin layer samples on the target crystal surface is achieved, and the controllability of sample preparation is improved.
[0042] In conjunction with the accompanying drawings, a method for preparing a cryo-electron microscopy single crystal sample and a cryo-electron microscopy single crystal sample provided by the present invention are described in detail through specific examples and implementation methods.
[0043] Embodiment 1
[0044] Reference Figure 1 , Figure 1 It is a schematic flow chart of the first embodiment of the method for preparing single crystal samples for cryo-electron microscopy of the present invention.
[0045] In this embodiment, the method includes:
[0046] Step S20: Separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain broken single crystals.
[0047] The crystal plane characteristics of a single crystal sample are the growth characteristics of the single crystal sample, that is, the surface characteristics of the single crystal, including but not limited to the surface shape of the single crystal.
[0048] Specifically, each single crystal sample has different growth characteristics. By separating the single crystal samples according to the crystal plane characteristics of the single crystal samples, cleavage fracture or shear fracture according to the target crystal plane can be achieved, thereby minimizing the crystal size. For example, under an optical microscope, two steel needles can be used to process the single crystal samples by operating with both hands. The steel needles can be controlled to separate the single crystal samples according to the growth characteristics of the single crystal samples, and the single crystal samples can be thinned to obtain broken single crystals.
[0049] It should be noted that cleavage fracture is a fracture surface separation according to a certain crystal plane (i.e., cleavage plane). When a crystalline mineral is subjected to stress, the property of the crystal to split into smooth planes according to a certain crystal direction due to its own structure is called cleavage, and the smooth cracked plane is called a cleavage plane. Slip shear fracture refers to a fracture that occurs along the surface of maximum shear stress. During the entire shear fracture process, a group of inclined tensile cracks first initiate. As the stress increases, these tensile cracks penetrate each other and then form a penetrating shear plane, which leads to the final shear fracture.
[0050] Step S40: applying a shear force to the broken single crystal to cause the broken single crystal to fracture through the crystal, thereby forming refined grains on the first microgrid carrier grid.
[0051] Shear force or shear force is a force that can cause shear deformation of materials. Shear deformation is a deformation form characterized by relative displacement of the cross section of the rod when a pair of external forces are applied that are parallel to the cross section of the rod, very close to each other, equal in magnitude, and opposite in direction. The cross section where shear deformation occurs is called the shear plane. The key to determining whether "shear" occurs is whether the cross section of the material is relatively displaced.
[0052] Transgranular fracture is a type of crack that follows the edge of the crystal lattice in a granular material, ignoring the grains in each crystal lattice. During transgranular fracture, the crack extends through the interior of the grain. Transgranular fracture can be a macroscopic plastic fracture or a macroscopic brittle fracture. Transgranular fracture can be divided into cleavage fracture and shear fracture according to the fracture mode. The cleavage fracture surface is strictly separated along a certain crystal plane (i.e., cleavage plane), which is usually a brittle fracture, but a brittle fracture is not necessarily a cleavage fracture. Shear fracture occurs under the action of shear stress, and shear fracture can be divided into pure shear fracture and microporous aggregation fracture.
[0053] Specifically, a shear force may be applied to the broken single crystals, so that the broken single crystals undergo transcrystalline fracture under the action of the shear force, thereby reducing the thickness of the broken single crystals, achieving grain refinement, and obtaining refined grains.
[0054] Step S60: removing irregular refined grains from the first micro-grating grid to obtain target refined grains on the target oriented crystal plane.
[0055] Specifically, the residual irregular small grains on the first micro-grating grid can be removed by clamping and refining the grains with tweezers or rotating or shaking the first micro-grating grid, so as to achieve exposure of the crystal plane with the target orientation retained to a large extent on the grid.
[0056] Step S80: applying force on both sides of the target refined grains through the second micro-grid and the first micro-grid, so that the target refined grains adhere to the surface of the second micro-grid facing the first micro-grid, forming a target crystal plane thin layer sample.
[0057] Specifically, by applying force on both sides of the target refined grains through the second micro-grid carrier and the first micro-grid carrier, the exposed upper surface of the target refined grains loaded on the first micro-grid carrier can be "reverse extracted" and fully adhered to the second micro-grid carrier above, thereby achieving an extraction replication effect.
[0058] The present embodiment provides a method for preparing a single crystal sample for cryo-electron microscopy, which separates the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain broken single crystals; applies shear force to the broken single crystal to cause transcrystalline fracture of the broken single crystal to form refined grains on the first microgrid carrier; removes irregular refined grains from the first microgrid carrier to obtain target refined grains with target oriented crystal planes; applies force on both sides of the target refined grains through the second microgrid carrier and the first microgrid carrier to make the target refined grains adhere to the surface of the second microgrid carrier facing the first microgrid carrier to form a thin layer sample of the target crystal plane, thereby achieving control over the thinning of the thickness of the single crystal sample, obtaining a sample with relatively uniform thickness, and achieving the loading of the thin layer sample of the target crystal plane, thereby changing the phenomenon of completely random sample orientation in the prior art, improving the controllability of the prepared samples, and also improving the overall success rate of sample preparation.
[0059] Embodiment 2
[0060] Furthermore, a second embodiment of the method for preparing a single crystal sample for cryo-electron microscopy of the present invention is proposed. The single crystal sample is separated according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample. Before obtaining the broken single crystal, the method further includes:
[0061] Step S15: obtaining the single crystal sample having a thickness smaller than a millimeter scale by controlling the growth process of the single crystal sample.
[0062] Specifically, for a single crystal with a relatively large thickness, such as a single crystal sample with a thickness greater than a millimeter scale, the growth process of the single crystal sample can be controlled to ensure that the thickness of the single crystal sample is below the millimeter scale.
[0063] In this embodiment, the single crystal sample having a thickness smaller than the millimeter scale is obtained by controlling the growth process of the single crystal sample, thereby ensuring that the size of the single crystal sample is below the millimeter scale, and facilitating further processing of the single crystal sample.
[0064] Embodiment 3
[0065] Furthermore, a third embodiment of the method for preparing a single crystal sample for cryo-electron microscopy of the present invention is proposed. Before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtaining the broken single crystal, the method further includes:
[0066] Step S16: Obtain the single crystal sample having a thickness smaller than the millimeter scale by mechanically processing the single crystal sample.
[0067] Specifically, for single crystals with relatively large thickness dimensions, such as single crystal samples with a thickness dimension greater than the millimeter scale, the thickness dimension of the single crystal sample can be reduced by simple mechanical processing. For example, the single crystal sample can be directly processed by using tweezers to crush the single crystal sample, a steel needle to poke the single crystal sample, a blade to cut the single crystal sample, etc., to reduce the thickness dimension of the single crystal sample and ensure that the thickness dimension of the single crystal sample is below the millimeter scale.
[0068] In this embodiment, the single crystal sample is mechanically processed to obtain the single crystal sample having a thickness smaller than the millimeter scale, thereby ensuring that the size of the single crystal sample is below the millimeter scale, and facilitating further processing of the single crystal sample.
[0069] Embodiment 4
[0070] Further, see Figure 2 , Figure 2 Schematic diagram of the fourth embodiment of the method for preparing a cryo-EM single crystal sample according to the present invention. Figure 1 In the embodiment shown, the step of separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain a broken single crystal includes:
[0071] Step S200: determining a separation direction according to the crystal plane characteristics of the single crystal sample.
[0072] Specifically, each single crystal sample has different growth characteristics. By determining the separation direction according to the growth characteristics of the single crystal sample, the single crystal sample can be separated more quickly according to the separation direction.
[0073] Step S202: applying stress to the single crystal sample in sequence according to the separation direction to separate the single crystal sample, thereby reducing the thickness of the single crystal sample and obtaining the broken single crystal.
[0074] Specifically, tensile stress and shear stress can be applied to the single crystal sample in sequence according to the separation direction, so that the single crystal sample can achieve transcrystalline fracture according to the target crystal plane under the action of tensile stress and shear stress, further reduce the thickness of the single crystal sample, and obtain a broken single crystal. For example, under an optical microscope, two steel needles can be used to process the single crystal sample by two hands, and the operation direction of the steel needles can be controlled according to the growth characteristics of the single crystal sample, and tensile stress and shear stress can be applied to the sample in sequence to achieve transcrystalline fracture according to cleavage or slip-shear of the target crystal plane, thereby minimizing the crystal size.
[0075] In this embodiment, the separation direction is determined according to the crystal plane characteristics of the single crystal sample; stress is applied to the single crystal sample according to the separation direction, so that the single crystal sample is transcrystalline fractured according to the target crystal plane to reduce the thickness of the single crystal sample, thereby obtaining the broken single crystal. Transcrystalline fracture according to cleavage or slip shear of the target crystal plane is achieved, the crystal size is minimized, and the thinning of the single crystal sample is controlled.
[0076] Embodiment 5
[0077] Further, see Figure 3 , Figure 3 Schematic diagram of the fifth embodiment of the method for preparing a cryo-EM single crystal sample according to the present invention. Figure 2 In the embodiment shown, the shear force is applied to the broken single crystal to cause the broken single crystal to fracture through the crystal and form refined grains on the first microgrid carrier, including:
[0078] Step S400: Roll-coating the broken single crystal on one side surface of the first microgrid carrier to apply the shear force on the broken single crystal, so that the broken single crystal undergoes transgranular fracture on the first microgrid carrier to form the refined grains on the first microgrid carrier.
[0079] Roller coating uses a roller as a carrier of the coating. The coating forms a wet film of a certain thickness on the surface of the roller, and then the coating is applied to the surface of the coating by means of the roller contacting the coating object during the rotation process. Roller coating is suitable for flat coating objects and is widely used in the coating of metal plates, plywood, cloth and paper, especially for the coating of metal coils.
[0080] Specifically, the operator can set the number of roller coating times, and apply shear force on the broken single crystal by roller coating, so that the broken single crystal is transcrystalline fractured on the first microgrid carrier under the action of the shear force, and the thickness of the broken single crystal is reduced to obtain refined grains. For example, the operator can process the broken single crystal under an optical microscope, use a steel needle to poke the broken single crystal, and roller-coat the broken single crystal on the first microgrid carrier, and roll-coat it back and forth 5 times, and further transcrystalline fracture the broken single crystal under the action of the shear force, so as to achieve grain refinement, obtain refined grains, and evenly coat them on the carrier.
[0081] It should be noted that the operator needs to reasonably set the number of roller coating times. If the number of roller coating times is too few, the broken single crystal cannot be thinned, the refined grains cannot be obtained, and the broken single crystal cannot be evenly coated on the carrier grid, and the thinning effect of the broken single crystal cannot be achieved; if the number of roller coating times is too many, it is easy to cause the thickness of the broken single crystal to be too small, affecting the subsequent operation of the broken single crystal.
[0082] Furthermore, the step of removing irregular refined grains from the first micro-grid carrier to obtain target refined grains of target oriented crystal planes includes:
[0083] Step S600: shaking the first micro-grid carrier grid to remove irregular refined grains and obtain target refined grains of target oriented crystal planes.
[0084] Specifically, when the broken single crystal is transcrystalline fractured on the first microgrid carrier to form refined grains, there will be a part of the residual irregular small grains that are not loaded on the first microgrid carrier or the load is not reliable. Therefore, the operator can set the number of shakes, and shake the first microgrid carrier according to the number of shakes to remove this part of the residual irregular small grains, so that the refined grains on the first microgrid carrier can achieve exposure of the crystal plane with a target orientation to a large extent. For example, after the operator rolls the broken single crystal on one side of the first microgrid carrier, the operator can clamp the first microgrid carrier with tweezers, and lightly shake the first microgrid carrier with the load surface facing downward for 3 times, so that the residual irregular small grains fall off, and the exposure of the crystal plane with a target orientation can be achieved on the carrier.
[0085] It should be noted that the operator needs to set the number of jitters reasonably. If the number of jitters is too small, it is easy to cause the remaining broken and irregular small grains on the first micro-grating carrier grid, affecting the acquisition of the target crystal surface thin layer sample; if the number of jitters is too many, it is easy to cause the target refined grains to fall off, and the target crystal surface thin layer sample cannot be obtained.
[0086] In this embodiment, the broken single crystal is roll-coated on one side surface of the first microgrid carrier according to a preset roll-coating number of times, so as to apply the shear force on the broken single crystal, so that the broken single crystal is transcrystalline fractured on the first microgrid carrier, and the refined grains are formed on the first microgrid carrier. The first microgrid carrier is shaken according to a preset shaking number of times to remove the irregular refined grains and obtain the target refined grains of the target oriented crystal plane. The thinning of the broken single crystal is controlled, so that the thickness of the broken single crystal remains relatively uniform, and the exposure of the target oriented crystal plane is retained to a large extent on the carrier, so as to obtain the target refined grains of the target oriented crystal plane.
[0087] Embodiment 6
[0088] Furthermore, a sixth embodiment of the method for preparing a cryo-electron microscopy single crystal sample of the present invention is proposed, wherein a force is applied on both sides of the target refined grain by the second microgrid carrier and the first microgrid carrier so that the target refined grain adheres to a surface of the second microgrid carrier facing the first microgrid carrier, and before forming a target crystal plane thin layer sample, the method further comprises:
[0089] Step S602: placing the second micro-grid carrier on the surface of the first micro-grid carrier on one side of which the target refined grains are supported, so that the target refined grains contact the second micro-grid carrier and the first micro-grid carrier.
[0090] Specifically, the target refined grains need to contact the second micro-grid carrier to achieve loading. Therefore, the second micro-grid carrier can completely cover the micro-grid surface of the loaded sample, so that the upper surface and the lower surface of the sample are in contact with the second micro-grid carrier and the first micro-grid carrier respectively.
[0091] Furthermore, the step of applying a force on both sides of the target refined grain by the second micro-grid and the first micro-grid so that the target refined grain adheres to a surface of the second micro-grid facing the first micro-grid to form a target crystal plane thin layer sample includes:
[0092] Step S800: within a preset sliding time, the force is applied on both sides of the target refined grain by the second micro-grid and the first micro-grid, so that the second micro-grid and the first micro-grid slide relative to each other, so that the target refined grain adheres to the surface of the second micro-grid facing the first micro-grid, thereby forming the target crystal surface thin layer sample.
[0093] Specifically, the operator can set the sliding time between the second microgrid and the first microgrid. For example, the operator can set the sliding time to 30s, and lightly press the second microgrid and the first microgrid with the fingertips to make the second microgrid and the first microgrid slide left and right, so that the target refined grains produce friction interaction on the surface of the second microgrid and the first microgrid, so that the exposed upper surface of the target refined grains loaded on the first microgrid is "reversely extracted" through friction, and fully adheres to the second microgrid above, achieving the extraction replica effect. Collect the second microgrid as a sample for cryo-EM characterization to achieve the loading of the target crystal surface thin layer sample.
[0094] In this embodiment, the second microgrid carrier is placed on the side surface of the first microgrid carrier loaded with the target refined grains, so that the target refined grains contact the second microgrid carrier and the first microgrid carrier. Within the preset sliding time, the second microgrid carrier and the first microgrid carrier apply the force on both sides of the target refined grains, so that the second microgrid carrier and the first microgrid carrier slide relative to each other, so that the target refined grains adhere to the side surface of the second microgrid carrier facing the first microgrid carrier, forming the target crystal plane thin layer sample. The exposed upper surface of the target refined grain loaded on the first microgrid carrier is "reversely extracted" and fully adhered to the second microgrid carrier above, that is, the target crystal plane thin layer sample is loaded, and the target crystal plane thin layer sample is obtained, which changes the phenomenon of completely random sample orientation existing in the prior art, improves the controllability of the prepared sample, and also improves the overall success rate of sample preparation.
[0095] Embodiment 7
[0096] Furthermore, a seventh embodiment of the method for preparing a single crystal sample for cryo-electron microscopy of the present invention is proposed. Before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain the broken single crystal, the method further includes:
[0097] Step S10: placing the single crystal sample in a vacuum environment to complete the processing of the single crystal sample in the vacuum environment.
[0098] Specifically, a vacuum environment is conducive to the processing of single crystal samples that are sensitive to the environment. Therefore, for special single crystal samples that are sensitive to the atmospheric environment, the single crystal samples can be processed in a vacuum environment to obtain target crystal surface thin layer samples. For example, special single crystal samples that are sensitive to the atmospheric environment can be placed in a vacuum glove box and processed in the vacuum glove box.
[0099] In this embodiment, the single crystal sample is placed in a vacuum environment to complete the processing of the single crystal sample in a vacuum environment, thereby avoiding the influence of the atmospheric environment on sensitive single crystal samples, achieving the thinning of the single crystal sample without exposure to the atmospheric environment, obtaining a target crystal surface thin layer sample, changing the phenomenon of completely random sample orientation in the prior art, improving the controllability of the prepared sample, and also improving the overall success rate of sample preparation.
[0100] In addition, an embodiment of the present invention proposes a cryo-electron microscopy single crystal sample, and the cryo-electron microscopy sample is prepared using the preparation method of the cryo-electron microscopy single crystal sample as described in any of the above items.
[0101] Specifically, the cryo-EM single crystal sample is obtained by the cryo-EM single crystal sample preparation method as described above. Therefore, the cryo-EM single crystal sample preparation method will not be described in detail here. In addition, the description of the beneficial effects of using the same method will not be described in detail.
[0102] It should be noted that the serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.
[0103] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structures or equivalent process changes made using the contents of the present invention's specification and drawings, or directly or indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.
Claims
1. A method for preparing single crystal samples for cryo-electron microscopy, It is characterized in that The method comprises: Separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain broken single crystals; Applying shear force to the broken single crystal to cause transcrystalline fracture of the broken single crystal and form refined grains on the first microgrid carrier grid; Removing the irregular refined grains from the first micro-grid carrier grid to obtain target refined grains with target oriented crystal planes; The second microgrid and the first microgrid apply forces on both sides of the target refined grains so that the target refined grains adhere to the surface of the second microgrid facing the first microgrid, forming a target crystal plane thin layer sample.
2. The method for preparing a cryo-electron microscopy single crystal sample according to claim 1, It is characterized in that Before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain the broken single crystal, the method further includes: By controlling the growth process of the single crystal sample, the single crystal sample having a thickness smaller than the millimeter scale is obtained.
3. The method for preparing a cryo-electron microscopy single crystal sample according to claim 1, It is characterized in that Before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain the broken single crystal, the method further includes: The single crystal sample having a thickness smaller than the millimeter scale is obtained by mechanically processing the single crystal sample.
4. The method for preparing a cryo-electron microscopy single crystal sample according to claim 1, It is characterized in that The step of separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample to obtain a broken single crystal comprises: Determining a separation direction according to the crystal plane characteristics of the single crystal sample; Stress is applied to the single crystal sample according to the separation direction to separate the single crystal sample, thereby reducing the thickness of the single crystal sample and obtaining the broken single crystal.
5. The method for preparing a cryo-electron microscopy single crystal sample according to claim 1, It is characterized in that The step of applying a shear force on the broken single crystal to cause the broken single crystal to fracture through the crystal and form refined grains on the first microgrid carrier grid comprises: The broken single crystal is roller-coated on one side surface of the first micro-grid carrier to apply the shear force on the broken single crystal, so that the broken single crystal undergoes transgranular fracture on the first micro-grid carrier, and the refined grains are formed on the first micro-grid carrier.
6. The method for preparing a cryo-electron microscopy single crystal sample according to claim 5, It is characterized in that The step of removing irregular refined grains from the first micro-grid carrier to obtain target refined grains of target oriented crystal planes comprises: The first micro-grid carrier grid is shaken to remove the irregular refined grains and obtain the target refined grains of the target oriented crystal plane.
7. The method for preparing a cryo-electron microscopy single crystal sample according to claim 1, It is characterized in that Before applying a force on both sides of the target refined grain by the second microgrid carrier and the first microgrid carrier, so that the target refined grain adheres to a surface of the second microgrid carrier facing the first microgrid carrier, and forming a target crystal plane thin layer sample, the method further includes: The second microgrid carrier is placed on a side surface of the first microgrid carrier carrying the target refined grains, so that the target refined grains contact the second microgrid carrier and the first microgrid carrier.
8. The method for preparing a cryo-electron microscopy single crystal sample according to claim 7, It is characterized in that The step of applying a force on both sides of the target refined grain by the second microgrid carrier and the first microgrid carrier so that the target refined grain adheres to a surface of the second microgrid carrier facing the first microgrid carrier to form a target crystal plane thin layer sample comprises: The force is applied on both sides of the target refined grain by the second micro-grid and the first micro-grid within a preset sliding time, so that the second micro-grid and the first micro-grid slide relative to each other, so that the target refined grain adheres to the surface of the second micro-grid facing the first micro-grid, thereby forming the target crystal surface thin layer sample.
9. The method for preparing a cryo-electron microscopy single crystal sample according to any one of claims 1 to 8, It is characterized in that Before separating the single crystal sample according to the crystal plane characteristics of the single crystal sample to reduce the thickness of the single crystal sample and obtain the broken single crystal, the method further includes: The single crystal sample is placed in a vacuum environment to complete the processing of the single crystal sample in the vacuum environment.
10. A cryo-electron microscopy single crystal sample, prepared by the method for preparing a cryo-electron microscopy single crystal sample as described in any one of claims 1 to 9.