Labeling device for cryo-em automatic grid

Abstract

This application discloses a marking device for an automated screen carrier in cryo-electron microscopy, comprising a support base and a sample platform fixedly mounted on the support base. The sample platform has a circular mounting groove for placing the automated screen carrier, and four opening structures as marking windows are provided on its sidewalls, evenly arranged along the circumference of the mounting groove. Each opening structure extends radially, with its central axis intersecting the central axis of the mounting groove, thus forming a cross-shaped marking channel with the center of the mounting groove as the intersection point. This allows the marker to perform four-point positioning marking of the automated screen carrier through the marking windows. This marking device ensures that the sample is at the correct angle before marking and marks the automated screen carrier through the preset opening structures, thereby guaranteeing the accuracy of the marking point position. Compared with pre-etched automated screen carriers, this device is lower in cost and more flexible.

Description

Technical Field

[0001] This application relates to a screen marking device, and more specifically to a marking device for automated screen loading in cryo-electron microscopy. Background Technology

[0002] Cryo-electron microscopy, a groundbreaking technique in structural biology, has played a crucial role in the analysis of biological macromolecular structures in recent years. By rapidly freezing biological samples, such as proteins, viruses, and organelles, into glassy ice, the native conformation of the samples can be preserved to the greatest extent possible. Subsequently, electron beam imaging of the samples, combined with advanced 3D reconstruction algorithms, can achieve near-atomic resolution, obtaining images of biological macromolecular structures smaller than 3 angstroms.

[0003] In this process, the sample grid is the core tool for supporting and fixing biological samples. Traditional sample grids are typically metal (such as copper or gold) discs about 3 mm in diameter, covered with a porous carbon film to adsorb biological samples. To ensure the accuracy of sample positioning during imaging, marker points need to be set on the grid. This allows the dual-beam electron microscope to thin the sample at a specific angle and rotate it 90 degrees on the transmission electron microscope to align it with the tilt axis, thus ensuring the quality of data collection for tilted samples. Specifically, four marker points are typically used, evenly distributed along the circumference of the automated grid.

[0004] However, current sample netting labeling methods have the following drawbacks.

[0005] (1) Manual marking method: The operator manually adds marking points on the edge of the automatic mesh using a marker. This method is inefficient, inconsistent, and has low marking accuracy. In addition, manual operation may introduce contaminants such as grease and dust, which may interfere with sample distribution or increase background noise, affecting imaging quality.

[0006] (2) Photolithography or electron beam etching: This method can prepare high-precision markers, but it is costly, has a long preparation cycle, and the pre-marked automatic mesh is difficult to align precisely with the sample.

[0007] In view of the above problems, designing an efficient, accurate and low-cost sample marking auxiliary device is of great importance to improving the efficiency of sample marking. Utility Model Content

[0008] To overcome or mitigate at least one deficiency of the prior art, one object of this application is to provide a marking device for an automated screen mounting system in cryo-electron microscopy. This marking device ensures that the sample is at the correct angle before marking and marks the automated screen using a preset opening structure, thereby guaranteeing the accuracy of the marking point positions.

[0009] To achieve the above objectives, the present application may adopt the following technical solutions.

[0010] This application provides a labeling device for automated screen mounting in cryo-electron microscopy, comprising:

[0011] Support base; and

[0012] A sample platform is fixedly mounted on the support base, and the surface of the sample platform is provided with a circular mounting groove for placing an automatic net.

[0013] The sample platform has four opening structures on its side wall as marking windows, and the four opening structures are evenly arranged along the circumference of the mounting groove.

[0014] Each of the opening structures extends radially, with its central axis intersecting the central axis of the mounting groove. Thus, the four opening structures form a cross-shaped marking channel with the center of the mounting groove as the intersection point, allowing the marker to perform four-point positioning marking on the automatic net through the marking window.

[0015] In at least one embodiment, the sample platform is a cubic structure, the mounting slot is located at the center of the sample platform, and the opening structures are respectively located in the central areas of the four side walls of the sample platform.

[0016] In at least one embodiment, the opening structure has a trapezoidal cross-section, having a wide opening facing the outside of the sample platform and a narrow opening facing the center of the mounting groove.

[0017] In at least one embodiment, the opening structure has a rectangular cross-section with a width of 1.5±0.1mm and a height of 2.3±0.1mm, and its corners are rounded with a radius of 0.3mm. The opening structure extends radially to at least partially communicate with the mounting groove.

[0018] In at least one embodiment, the mounting groove is a circular groove or a circular annular groove.

[0019] In at least one embodiment, the mounting groove is a circular groove with a diameter of 3.6±0.2mm and a depth of 0.7±0.1mm.

[0020] In at least one embodiment, the marking device includes a base disposed at the lower part of the support base, which has two symmetrically distributed mounting holes for fixing the marking device.

[0021] In at least one embodiment, the base is provided with a detachable bracket, on which a magnifying glass is mounted to assist in observing and adjusting the position or angle of the automatic net.

[0022] In at least one embodiment, the marking device includes a dust cover that can be mounted from above on the sample platform, and the dust cover has observation notches around its perimeter corresponding to the opening structure.

[0023] In at least one embodiment, the support base and the sample platform are integrally formed.

[0024] By adopting the above technical solution, this application provides a labeling device for an automated screen carrier in cryo-electron microscopy. This device effectively fixes the sample, and during operation, only tweezers are needed to contact the automated screen carrier, thus avoiding contamination caused by manual contact. The pre-set opening structure on the sample platform allows for precise positioning of the labeling points and limits the movement range of the label. Furthermore, since the sample platform has a circular mounting slot for placing the automated screen carrier, the carrier can rotate within the slot. This device allows the operator to fully adjust the angle of the automated screen carrier before labeling to avoid sample alignment problems, thereby improving the success rate of the experiment. Compared with pre-etched automated screen carriers, this device is lower in cost and more flexible. Attached Figure Description

[0025] Figure 1 A schematic diagram of the structure of a marker device for automated grid loading in cryo-electron microscopy according to an embodiment of this application is shown;

[0026] Figure 2 A top view schematic diagram of a marking device for automated netting in cryo-electron microscopy according to an embodiment of this application is shown;

[0027] Figure 3 A side view schematic diagram of a marking device for automated netting in cryo-electron microscopy according to an embodiment of this application is shown.

[0028] Explanation of reference numerals in the attached figures

[0029] 10. Support base;

[0030] 20 Sample Platforms;

[0031] 21 mounting slots;

[0032] 22. Open structure;

[0033] 30 bases;

[0034] 31 mounting holes; Detailed Implementation

[0035] Exemplary embodiments of this application are described below with reference to the accompanying drawings. It should be understood that these specific descriptions are for teaching those skilled in the art how to implement this application only, and are not intended to exhaustively describe all feasible methods of this application, nor to limit the scope of this application.

[0036] Embodiments of this application provide a marking device for automated netting in cryo-electron microscopy, which will hereafter be referred to simply as a "marking device".

[0037] The present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] like Figure 1 As shown, an embodiment of this application provides a labeling device for automatic netting in cryo-electron microscopy, which may include a support base 10 and a sample platform 20 fixedly disposed on the support base 10.

[0039] Furthermore, the sample platform 20 can be in the shape of a cube, cylinder, etc., and the specific shape is not limited here. In this embodiment, the sample platform 20 is a cube structure, and its external dimensions can be designed according to the dimensions of the automatic netting.

[0040] See Figure 1 The upper surface of the sample platform 20 may be provided with a circular mounting groove 21 for placing the automatic net, that is, for fixing and limiting the automatic net.

[0041] Furthermore, the mounting groove 21 can be a circular groove or a circular annular groove, and its specific shape can be adjusted according to the shape of the automatic mesh feeder. Preferably, in this embodiment, the mounting groove 21 can be a circular groove, the size of which is consistent with the size of the commercially available Thermo Fisher automatic mesh feeder (Autogrid). Specifically, the diameter of the circular groove can be 3.6 ± 0.2 mm, and the depth (… Figure 1 The vertical dimension (in the middle) can be 0.7 ± 0.1 mm.

[0042] like Figure 1 and Figure 2 As shown, in this embodiment, four opening structures 22 can be provided on the side wall of the sample platform 20 as marking windows, and the four opening structures 22 can be evenly arranged along the circumference of the mounting groove 21.

[0043] Specifically, see Figure 2 Each opening structure 22 extends radially. Here, if the sample platform 20 is cylindrical, this radial direction is the radial direction of the cylinder; if the sample platform 20 is a cuboid (including a cube), this radial direction is the radial direction of the circumscribed cylindrical surface of the cuboid. Figure 2 From the perspective of (the radial direction of the circumcircle of the sample platform 20).

[0044] The central axis of each opening structure 22 intersects the central axis of the mounting groove 21. This forms a cross-shaped marking channel with the center of the mounting groove 21 as the intersection point, allowing the marker to perform four-point positioning marking on the automatic netting through the marking window.

[0045] The pre-set opening structure 22 allows for precise four-point positioning marking of the automated net. Furthermore, the pre-set opening structure 22 effectively limits the movement range of the markers, achieving higher accuracy than manual marking. See also... Figure 3 This design also makes it easy to mark the sides of the automatic loading net, which is beneficial for adjusting the loading angle when the sample is placed vertically in the cryo-electron microscope sample chamber.

[0046] Furthermore, during operation, operators only need to use tweezers to contact the automatic mesh carrier, thus avoiding contamination caused by manual contact. In addition, using this marking device, operators can mark the automatic mesh carrier multiple times, making the operation simple and convenient, and its cost is far lower than that of automatic mesh carriers with pre-etched grooves.

[0047] In addition, the opening structure 22 not only allows for precise marking of the automatic netting, but also allows the operator to fully adjust the angle of the automatic netting before marking, that is, to allow the automatic netting to rotate in the circular mounting groove 21, thereby avoiding the problem of sample misalignment caused by pre-marking.

[0048] It should be noted that the marking material can be a marking pen, a wire, or a carving knife, as long as it can create scratches or marks on the automatic netting.

[0049] Furthermore, the cross-sectional shape of the opening structure 22 can be rectangular, and the corners can be designed as rounded corners. Specifically, the width of the opening structure 22 (i.e., in the cross-section, along the direction of the sidewall of the sample platform 20) can be 1.5 ± 0.1 mm, and the height ( Figure 1 The vertical dimension (in the middle) is 2.3 ± 0.1 mm, its corners are rounded with a radius of 0.3 mm, and the opening structure 22 can extend radially to at least partially communicate with the mounting groove 21.

[0050] The opening structure 22 is a through groove with a trapezoidal cross-section, having a wide opening facing the outside of the sample platform 20 and a narrow opening facing the center of the mounting groove 21.

[0051] Preferably, in this embodiment, see Figure 1 , Figure 2 The opening structure 22 can be a through groove with an isosceles trapezoidal cross-section, and the angle between the two sides of the through groove and the central axis of the opening structure 22 can be 5 to 10 degrees.

[0052] like Figure 2As shown, the marking device may also include a base 30, which can be disposed below the support base 10 to provide stable support. Specifically, the base 30 can be rectangular, and its size can be flexibly adjusted. In addition, the base 30 may also be provided with a plurality of symmetrically distributed mounting holes 31 for fixing and mounting the marking device.

[0053] Furthermore, the side wall of the base 30 may be provided with a detachable bracket, on which a magnifying glass may be provided to assist in observing and adjusting the position or angle of the automatic net carrier. For example, the magnification of the magnifying glass may be 5x.

[0054] In this embodiment, the marking device may further include a dust cover, which is transparent and can be installed on the sample platform 20 from above. This dust cover serves to limit the vertical movement of the automatic net and prevent dust accumulation. It also ensures that the automatic net does not warp when marking with markers. The dust cover may also have observation notches corresponding to the opening structure 22 along its perimeter.

[0055] In this embodiment, the sample platform 20, support base 10, and base 30 can be made of the same material and integrally formed. For example, metal can be integrally formed by machining, or plastic can be integrally formed by 3D printing.

[0056] The marking device includes a support base 10 and a sample platform 20, both made of plastic and manufactured as a single piece using 3D printing. The following further describes the method of using the marking device provided in this embodiment.

[0057] In use, the operator first uses tweezers to place the automatic netting into the mounting slot 21 of the marking device and adjusts the placement angle of the automatic netting to align it with one of the opening structures 22. Then, using markers, the operator sequentially marks the angles of the automatic netting surface through the opening structures 22. After marking is complete, the operator can use tweezers to pick up the automatic netting along one of the opening structures 22 to complete the entire marking process.

[0058] This marking method is very helpful in the sample chambers of cryo-electron microscopes where samples are placed vertically, such as the Autoloader equipped with the Titan Krios (a model of cryo-transmission electron microscope), to determine and adjust the angle of the automatic mesh during sample loading.

[0059] The automated screen marking device for cryo-electron microscopy provided in this application can effectively fix the sample, and during operation, the operator can use tweezers to contact the automated screen throughout the process, thus avoiding contamination caused by manual contact. The pre-set opening structure 22 on the sample platform 20 can limit the movement range of the marker. Furthermore, this device allows the operator to fully adjust the screen angle before marking to avoid sample alignment problems, thereby improving the success rate of the experiment. Compared with pre-etched automated screens, this device is lower in cost and more flexible.

[0060] It should be understood that the above implementation methods, embodiments, or examples are merely exemplary and are not intended to limit this application. Those skilled in the art can make various modifications and changes to the above implementation methods, embodiments, or examples under the teachings of this application without departing from the scope of this application.

Claims

1. A marking device for automatic grid loading in cryo-electron microscopy, characterized in that, include: Support base; as well as A sample platform is fixedly mounted on the support base, and the surface of the sample platform is provided with a circular mounting groove for placing an automatic net. The sample platform has four opening structures on its side wall as marking windows, and the four opening structures are evenly arranged along the circumference of the mounting groove. Each of the opening structures extends radially, with its central axis intersecting the central axis of the mounting groove. Thus, the four opening structures form a cross-shaped marking channel with the center of the mounting groove as the intersection point, allowing the marker to perform four-point positioning marking on the automatic net through the marking window.

2. The marking device for automatic grid loading in cryo-electron microscopy according to claim 1, characterized in that, The sample platform has a cubic structure, the mounting slot is located at the center of the sample platform, and the opening structures are respectively located in the central areas of the four side walls of the sample platform.

3. The marking device for automatic grid loading in cryo-electron microscopy according to claim 2, characterized in that, The opening structure has a trapezoidal cross-section, with a wide opening facing the outside of the sample platform and a narrow opening facing the center of the mounting groove.

4. The marking device for automatic grid loading in cryo-electron microscopy according to claim 2, characterized in that, The opening structure has a rectangular cross-section with a width of 1.5±0.1mm and a height of 2.3±0.1mm. Its corners are rounded with a radius of 0.3mm, and the opening structure extends radially to at least partially communicate with the mounting groove.

5. The marking device for automatic grid loading in cryo-electron microscopy according to claim 1, characterized in that, The mounting groove is a circular groove or a circular annular groove.

6. The marking device for automatic grid loading in cryo-electron microscopy according to claim 5, characterized in that, The mounting groove is a circular groove with a diameter of 3.6±0.2mm and a depth of 0.7±0.1mm.

7. The marking device for automatic grid loading in cryo-electron microscopy according to any one of claims 1 to 6, characterized in that, The marking device includes a base disposed at the lower part of the support base, which has two symmetrically distributed mounting holes for fixing the marking device.

8. The marking device for automatic grid loading in cryo-electron microscopy according to claim 7, characterized in that, The base is equipped with a detachable bracket, on which a magnifying glass is mounted to assist in observing and adjusting the position or angle of the automatic net carrier.

9. The marking device for automatic grid loading in cryo-electron microscopy according to claim 1, characterized in that, The marking device includes a dust cover that can be installed from above on the sample platform, and its four edges are provided with observation notches corresponding to the opening structure.

10. The marking device for automatic grid loading in cryo-electron microscopy according to any one of claims 1 to 6, 8, and 9, characterized in that, The support base and the sample platform are integrally machined and formed.

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