Fully automatic high-throughput electron microscope grid staining machine

Abstract

The present invention provides a fully automatic high-throughput electron microscopy grid staining machine, comprising a conveyor belt for carrying copper mesh samples, a visual acquisition device located above the conveyor belt, and a dye spraying device. The dye spraying device includes a liquid spraying carriage movable in the X and Y directions. The liquid spraying carriage is located above the conveyor belt and downstream of the visual acquisition device. The liquid spraying carriage is connected to a dye cartridge via a flexible tube. The liquid spraying carriage is used to spray the dye onto the copper mesh sample based on the position of the copper mesh sample captured by the visual acquisition device and the operating speed of the conveyor belt. The present invention enables precise dye spraying, requiring only cleaning of the copper mesh after the first dye application. Through image acquisition and automatic tracking of the liquid spraying carriage, the present invention allows the placement of the copper mesh sample to be unrestricted by positional accuracy. A cross-arm sampling device located at the rear of the conveyor belt can automatically remove the dyed sample into a lifting tray, significantly reducing labor intensity and significantly improving the efficiency of large-scale dyeing operations.

Description

Technical Field

[0001] The invention relates to the technical field of biological sample staining, in particular to a full-automatic high-throughput electron microscope grid staining machine. Background Art

[0002] Transmission electron microscopy (TEM) sample observation and image analysis involve the following steps: 1. Sample collection: Collect appropriate tissue samples; 2. Fixation: Use chemical fixation reagents; 3. Dehydration: Remove moisture from the sample; 4. Infiltration and embedding: The dehydrated sample is placed in a resin for infiltration, which then solidifies to form a solid block; 5. Ultrathin sectioning: Use an ultramicrotome to cut the embedded block into sections approximately 50-100 nanometers thick. 6. Staining: Stain the sections mounted on a copper grid. 7. Microscopic observation. Current staining methods rely on manual operation, requiring a droplet of staining solution and then flipping the copper grid over the droplet to stain the sections. However, some staining solutions require extended soaking times, such as 10 minutes. During this time, sections may fall off, resulting in failed copper grid sample preparation. Furthermore, these methods require high operator skill. This inefficiency is particularly significant for experiments involving large numbers of samples, hindering experimental efficiency. Chinese patent document CN217820089U records an integrated device for clamping and dyeing a copper mesh for electron microscopy, which uses a lead screw as a driving mechanism and uses tweezers to clamp the copper mesh. This solution does not improve the efficiency of dyeing. CN210376190U records an apparatus for making sample staining for electron microscopy, which uses a solution of top dripping to dye the copper mesh sample, but this solution has the problem of high difficulty in accurately controlling the amount of dripping. In addition, the document also records the problem that the copper mesh is small and easily rolls over under the influence of dripping. The solution of this document is to set a fixed protrusion 35 to prevent the copper mesh from rolling over, but this structure increases the difficulty of automated operation. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a fully automatic high-throughput electron microscope grid staining machine, which can conveniently set dyes for copper grid samples and realize automation of staining operations to cope with the substantial increase in experimental samples.

[0004] In order to solve the above-mentioned technical problems, the technical solution of the present invention is: a fully automatic high-throughput electron microscope grid staining machine, comprising a conveyor belt for carrying copper mesh samples, a visual acquisition device is provided above the conveyor belt, and a dye spraying device is also provided. The dye spraying device is provided with a liquid spraying trolley that can move along the X and Y directions. The liquid spraying trolley is located above the conveyor belt and downstream of the visual acquisition device. The liquid spraying trolley is connected to the dye box through a flexible tube. The liquid spraying trolley is used to spray the dye on the copper mesh sample according to the position of the copper mesh sample collected by the visual acquisition device and the operating speed of the conveyor belt.

[0005] In the preferred solution, the copper mesh sample is loaded on a bracket, and the structure of the bracket is as follows: upper pressing plates are provided on both sides above the frame, the upper pressing plates are used to clamp the copper mesh sample, the upper pressing plates are pressed on both sides of the copper mesh sample, and a hollow structure is provided at the position corresponding to the frame and the copper mesh sample.

[0006] In a preferred solution, an extension position is further provided on one side of the frame, and a clamping area and an information area are provided on the edge of the extension position.

[0007] In a preferred embodiment, a second dye spraying device is further provided downstream of the first dye spraying device;

[0008] An atomizing spray device is provided between the first dye spray device and the second dye spray device;

[0009] The atomizing spray device cleans the surface of the copper mesh from one side of the copper mesh sample.

[0010] In the preferred solution, the dye spraying device is provided with a transverse guide rail, a transverse beam moving along the transverse guide rail is provided on the transverse guide rail, and a liquid spraying trolley moving along the transverse beam is provided on the transverse beam, and the moving range of the liquid spraying trolley covers the entire width of the conveyor belt.

[0011] In the preferred solution, the visual acquisition device acquires the position (x0, y0) of the copper mesh sample; obtains the current coordinates (x j ,y j ), track the shortest time (t n ) and the intersection position (x n ,y n );

[0012] Output command: Spraying trolley moves (x n -x j ) absolute value; Transverse beam movement (y n -y j ) absolute value, the spraying car passes through time (t n ) Then start spraying the dye.

[0013] In a preferred solution, if the liquid spraying trolley needs to continue spraying for multiple times, an instruction is outputted, and the liquid spraying trolley moves at the same speed as the conveyor belt and continues spraying for a period of time.

[0014] In the preferred solution, if the liquid spraying trolley needs to continue spraying multiple times and cover a preset range, an instruction is output and the liquid spraying trolley generates a motion trajectory of the liquid spraying process so that the liquid spraying trolley follows the copper mesh sample and moves back and forth to cover the copper mesh sample and spray liquid.

[0015] In a preferred embodiment, a cover is provided at the middle of the conveyor belt, the cover covers the first dye spraying device and the second dye spraying device, and openings for the bracket to pass through are provided at both ends of the cover;

[0016] The cover body is a light-shielding structure. The first dye spraying device and the second dye spraying device are separated. Nitrogen is introduced into the cover body space of the second dye spraying device to maintain the nitrogen at a slightly positive pressure to prevent oxygen from entering.

[0017] In the preferred solution, a lifting pallet is also provided on one side of the tail end of the conveyor belt. The structure of the lifting pallet is: a pallet rack is slidably installed on the guide rail bracket, one end of the lifting device is connected to the guide rail bracket, and the other end of the lifting device is connected to the pallet rack so that one layer of the pallet rack is flush with the conveyor belt and the bracket is loaded.

[0018] In a preferred solution, a cross arm sampling device is further provided on one side of the guide rail bracket, and the structure of the cross arm sampling device is as follows: the guide rod is swingably connected to the guide rail bracket via a rotating shaft;

[0019] The rotating shaft is provided with damping to delay the swing of the guide rail bracket;

[0020] The guide rod is slidably connected to the sliding rod. A material taking rod is provided at one end of the sliding rod facing the conveyor belt, and a driving device is also provided to drive the sliding rod to extend and retract.

[0021] A water absorbing strip is provided on the material taking rod at the same height as the side of the copper mesh sample;

[0022] A counterweight is provided at one end of the guide rod away from the conveyor belt;

[0023] When the sliding rod retracts, the pick-up rod is lifted, and when the sliding rod extends, the pick-up rod falls on the surface of the conveyor belt.

[0024] The present invention provides a fully automatic high-throughput electron microscope grid staining machine, which has the following beneficial effects:

[0025] 1. The present invention can realize automatic and accurate dye spraying on a copper mesh with a thickness of only 1 to 2 mm. After the first dye is sprayed, only the copper mesh needs to be cleaned.

[0026] 2. The present invention uses image acquisition and automatic tracking of the liquid spraying trolley to ensure that the placement of the copper mesh sample is not restricted by position accuracy. The cross-arm sampling device set at the tail of the conveyor belt can automatically take the dyed sample into the lifting tray, greatly reducing labor intensity and significantly improving the efficiency of large-scale and large-volume dyeing operations.

[0027] 3. The bracket provided in the present invention can prevent the copper mesh sample from turning over during the dyeing process and prevent it from falling due to its small size. In particular, the information area is provided on the bracket, which can carry the information of the sample, thereby ensuring that the information of the sample is connected with the real object. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below with reference to the accompanying drawings and examples:

[0029] Figure 1 It is a top view of the overall structure of the present invention.

[0030] Figure 2 It is a schematic diagram of the operation of the cross arm sampling device of the present invention.

[0031] Figure 3 It is a structural schematic diagram of the lifting tray of the present invention.

[0032] Figure 4 This is a schematic structural diagram of the bracket of the present invention when loading a copper mesh sample.

[0033] In the figure: bracket 1, clamping area 101, information area 102, upper pressing plate 103, frame 104, conveyor belt 2, cover body 3, dye box 4, flexible tube 5, liquid spraying trolley 6, transverse beam 7, transverse guide rail 8, cross arm sampling device 9, feeding rod 91, sliding rod 92, counterweight 93, guide rod 94, feeding motor 95, feeding belt 96, rotating shaft 97, water absorption strip 98, copper mesh sample 10, supporting platform 11, lifting tray 12, lifting device 121, tray rack 122, guide rail bracket 123, visual acquisition device 13, second dye spraying device 14, atomizing spray device 15. DETAILED DESCRIPTION

[0034] like Figure 1 In the invention, a fully automatic high-throughput electron microscope grid staining machine includes a conveyor belt 2 for carrying a copper mesh sample 10. A visual acquisition device 13 is provided above the conveyor belt 2. A dye spraying device is also provided. The dye spraying device is provided with a liquid spraying trolley 6 that can move along the X and Y directions. The liquid spraying trolley 6 is located above the conveyor belt 2 and downstream of the visual acquisition device 13. The liquid spraying trolley 6 is connected to the dye box 4 through a flexible tube 5. The liquid spraying trolley 6 is used to spray the dye on the copper mesh sample 10 according to the position of the copper mesh sample 10 collected by the visual acquisition device 13 and the operating speed of the conveyor belt 2.

[0035] The preferred solution is Figure 4 In the figure, the copper mesh sample 10 is loaded on the bracket 1, and the structure of the bracket is as follows: upper pressing plates 103 are provided on both sides above the frame 104, and the upper pressing plates 103 are used to clamp the copper mesh sample 10. The upper pressing plates 103 are pressed on both sides of the copper mesh sample 10, and a hollow structure is provided at the position corresponding to the frame 104 and the copper mesh sample 10.

[0036] The preferred solution is Figure 4 In the embodiment, an extension position is provided on one side of the frame 104, and a clamping area 101 and an information area 102 are provided at the edge of the extension position.

[0037] The preferred solution is Figure 1 In the embodiment, a second dye spraying device is further provided downstream of the first dye spraying device;

[0038] An atomizing spray device 14 is provided between the first dye spray device and the second dye spray device;

[0039] The first dye spray device uses uranyl acetate, a dye that binds to nucleic acids within cells, increasing their electron density and making images clearer under electron microscopy, facilitating observation of the distribution and structure of nucleic acids within cells. It also binds to most molecules within cells, enhancing the contrast between proteins and connective tissue fibers. The second dye spray device uses lead citrate, a heavy metal salt containing lead. Lead ions specifically bind to certain components in biological samples. Due to the heavy metal's strong electron scattering ability, this dye enhances image contrast and improves observation clarity.

[0040] The atomizing spray device 14 cleans the surface of the copper mesh sample 10 from one side. This ensures that the dye is completely removed while preventing sample slices from falling off. Specifically, when the copper mesh sample 10 is approximately 10 to 5 cm from the atomizing spray device 14, the atomizing spray device 14 begins spraying to clean the dye from the copper mesh sample 10. The atomizing spray device 14 is precisely controlled to prevent large amounts of liquid droplets from falling onto the conveyor belt 2.

[0041] The preferred solution is Figure 1 In the figure, the dye spraying device is provided with a transverse guide rail 8, on which a transverse beam 7 is provided that moves along the transverse guide rail 8, and on which a liquid spraying trolley 6 is provided that moves along the transverse beam 7. The moving range of the liquid spraying trolley 6 covers the entire width of the conveyor belt 2.

[0042] The preferred solution is Figure 1In the process, the visual acquisition device 13 acquires the position (x0, y0) of the copper mesh sample 10; obtains the current coordinate (x j ,y j ), tracking the shortest time (t n ) and the intersection position (x n ,y n );

[0043] Output command: Spraying car 6 moves (x n -x j ) absolute value; the transverse beam 7 moves (y n -y j ) absolute value, the spraying car 6 passes the time (t n ) Then start spraying the dye.

[0044] In a preferred solution, if the liquid spraying trolley 6 needs to continue spraying for multiple times, an instruction is outputted, and the liquid spraying trolley 6 moves at the same speed as the conveyor belt 2 and continues spraying for a period of time.

[0045] In a preferred embodiment, if the spraying trolley 6 needs to continue spraying multiple times and cover a preset range, an instruction is output, and the spraying trolley 6 generates a motion trajectory for the spraying process and superimposes the motion trajectory with the movement direction of the conveyor belt 2, so that the spraying trolley 6 follows the copper mesh sample 10 and reciprocates to cover the copper mesh sample 10. The spraying process involves an "S"-shaped movement of the spraying trolley 6 to ensure that the dye is evenly sprayed on the surface of the copper mesh sample 10. The motion trajectory of the spraying trolley 6 needs to be superimposed on the motion trajectory of the conveyor belt 2.

[0046] The preferred solution is Figure 1 In the embodiment, a cover 3 is provided in the middle of the conveyor belt 2, enclosing the first and second dye spraying devices. Openings are provided at both ends of the cover 3 for the carriage 1 to pass through. The cover 3 is a light-shielding structure, and the first and second dye spraying devices are separated. Nitrogen is introduced into the space within the cover 3 of the second dye spraying device, maintaining a slightly positive pressure to prevent oxygen from entering.

[0047] The preferred solution is Figure 1 、 3 In the figure, a lifting pallet 12 is also provided on one side of the tail end of the conveyor belt 2. The structure of the lifting pallet 12 is as follows: a pallet rack 122 is slidably installed on a guide rail bracket 123, one end of the lifting device 121 is connected to the guide rail bracket 123, and the other end of the lifting device 121 is connected to the pallet rack 122, so that one layer of the pallet rack 122 is flush with the conveyor belt 2 and the bracket 1 is loaded.

[0048] The preferred solution is Figure 1 、2 In the embodiment, a cross arm sampling device 9 is further provided on one side of the guide rail bracket 123. The cross arm sampling device 9 has the following structure: a guide rod 94 is swingably connected to the guide rail bracket 123 via a rotating shaft 97;

[0049] The rotating shaft is provided with damping to delay the swing of the guide rail bracket 123;

[0050] The guide rod 94 is slidably connected to the sliding rod 92. A material picking rod 91 is provided at one end of the sliding rod 92 facing the conveyor belt 2. A driving device is also provided to drive the sliding rod 92 to extend and retract.

[0051] A counterweight 93 is provided at one end of the guide rod 94 away from the conveyor belt 2;

[0052] A water wick 98 is installed on the retrieval rod 91 at the same height as the side of the copper mesh sample 10. With this structure, when the retrieval rod 91 approaches the copper mesh sample 10, the water wick 98 absorbs excess liquid from the side of the copper mesh sample 10. After a period of use, the water wick 98 should be replaced.

[0053] When the sliding rod 92 retracts, the material picking rod 91 is lifted, and when the sliding rod 92 extends, the material picking rod 91 falls on the surface of the conveyor belt 2.

[0054] like Figure 1In the present invention, when in use, the copper mesh loaded with ultra-thin slices is loaded on the bracket 1, and the bracket 1 is transferred to the conveyor belt 2 in turn by a tray. When the bracket 1 passes under the visual acquisition device 13, the position below the visual acquisition device 13 is set as the coordinate zero point. The visual acquisition device 13 identifies the copper mesh sample 10 on the bracket 1, and sets the center point of the copper mesh sample 10 as the current bracket 1 coordinate, and sets a speed value for the bracket 1. The first dye spraying device calculates the motion trajectory of the spray trolley 6 according to the coordinate and speed value of the bracket 1. The spray trolley 6 follows the copper mesh sample 10 according to the motion trajectory and sprays the dye on the surface of the copper mesh sample 10. When passing through the spray atomization device 15, the spray atomization device 15 sprays droplets from the upper side to dye the copper mesh sample 10. Excess dye is removed from the surface of the web sample 10, and then the second dye is sprayed at the second dye spraying device 14. After spraying is completed, the multiple trays 1 are transported to the end of the conveyor belt 2. At this time, the lifting device 121, preferably a cylinder or electric push rod, is activated to raise or lower the tray rack 122 to align an empty tray with the height of the conveyor belt 2. The material picking motor 95 is activated, driving the material picking belt 96 to rotate the driven wheel. The driven wheel is provided with teeth, and the bottom of the sliding rod 92 is provided with teeth that mesh with the driven wheel, thereby driving the sliding rod 92 to extend from the guide rod 94. In the initial state, the sliding rod 92 is in a retracted state. Under the action of the counterweight 93, the entire sliding rod 92 is in a tilted state, and the material picking rod 91 leaves the surface of the conveyor belt 2. As the sliding rod 92 extends, the material picking rod 91 falls to a position near the edge of the conveyor belt 2. Then, the retrieving motor 95 reverses, retracting the sliding rod 92 via the retrieving belt 96 and the driven wheel, and the multiple dyed brackets 1 are taken to the empty tray. During the process of returning the brackets 1, the water absorbing strip 98 absorbs the excess dye from the surface of the copper mesh sample 10. The lifting tray 12 then rises or lowers one level, leaving the empty tray on standby, thus completing the fully automatic dyeing process of the copper mesh sample 10. As an important part of the fully automatic production process of ultrathin sample slices, the present invention lays the foundation for the full automation of ultrathin sample preparation.

[0055] The above embodiments are merely preferred technical solutions of the present invention and should not be construed as limiting the present invention. The embodiments and features in the embodiments of this application may be arbitrarily combined with each other unless they conflict. The scope of protection of the present invention shall be the technical solutions described in the claims, including equivalent alternatives to the technical features of the technical solutions described in the claims. Equivalent alternatives and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A fully automatic high-throughput electron microscope grid staining machine, characterized by: The invention comprises a conveyor belt (2) for carrying a copper mesh sample (10), a visual acquisition device (13) being provided above the conveyor belt (2), and a first dye spraying device being provided, wherein the dye spraying device is provided with a liquid spraying trolley (6) movable in the X and Y directions, the liquid spraying trolley (6) being located above the conveyor belt (2), the liquid spraying trolley (6) being located downstream of the visual acquisition device (13), the liquid spraying trolley (6) being connected to a dye box (4) via a flexible tube (5), and the liquid spraying trolley (6) being used to spray the dye on the copper mesh sample (10) according to the position of the copper mesh sample (10) acquired by the visual acquisition device (13) and the running speed of the conveyor belt (2); A lifting tray (12) is also provided on one side of the tail of the conveyor belt (2). The lifting tray (12) has the following structure: a tray rack (122) is slidably mounted on a guide rail bracket (123), one end of a lifting device (121) is connected to the guide rail bracket (123), and the other end of the lifting device (121) is connected to the tray rack (122), so that one layer of the tray rack (122) is flush with the conveyor belt (2) and the bracket (1) is loaded; A cross arm sampling device (9) is further provided on one side of the guide rail bracket (123). The cross arm sampling device (9) has the following structure: a guide rod (94) is swingably connected to the guide rail bracket (123) via a rotating shaft (97); The rotating shaft is provided with damping so that the swing of the guide rail bracket (123) has a time delay; The guide rod (94) is slidably connected to the sliding rod (92), and a material taking rod (91) is provided at one end of the sliding rod (92) facing the conveyor belt (2), and a driving device is also provided to drive the sliding rod (92) to extend and retract; A water absorbing strip (98) is provided on the material taking rod (91) at a position equal to the height of the side of the copper mesh sample (10); A counterweight (93) is provided at one end of the guide rod (94) away from the conveyor belt (2); When the sliding rod (92) is retracted, the material picking rod (91) is lifted, and when the sliding rod (92) is extended, the material picking rod (91) falls on the surface of the conveyor belt (2).

2. The fully automatic high-throughput electron microscope grid staining machine according to claim 1, characterized in that: The copper mesh sample (10) is loaded on a bracket (1), and the bracket has the following structure: upper pressing plates (103) are provided on both sides above the frame (104), the upper pressing plates (103) are used to clamp the copper mesh sample (10), the upper pressing plates (103) are pressed on the positions on both sides of the copper mesh sample (10), and a hollow structure is provided at the position corresponding to the frame (104) and the copper mesh sample (10); An extended position is also provided on one side of the frame (104), and a clamping area (101) and an information area (102) are provided at the edge of the extended position.

3. The fully automatic high-throughput electron microscope grid staining machine according to claim 2, characterized in that: A second dye spraying device is further provided downstream of the first dye spraying device; An atomizing spray device (14) is provided between the first dye spray device and the second dye spray device; The atomizing spray device (14) cleans the surface of the copper mesh sample (10) from one side thereof.

4. The fully automatic high-throughput electron microscope grid staining machine according to claim 2 or 3, characterized in that: The dye spraying device is provided with a transverse guide rail (8), a transverse beam (7) that moves along the transverse guide rail (8) is provided on the transverse guide rail (8), and a liquid spraying trolley (6) that moves along the transverse beam (7) is provided on the transverse beam (7), and the moving range of the liquid spraying trolley (6) covers the entire width of the conveyor belt (2).

5. The fully automatic high-throughput electron microscope grid staining machine according to claim 4, characterized in that: The visual acquisition device (13) acquires the position (x0, y0) of the copper mesh sample (10); obtains the current coordinate (x j ,y j ), track the shortest time (t n ) and the intersection position (x n ,y n ); Output command: Spraying trolley (6) moves (x n -x j ) absolute value; the transverse beam (7) moves (y n -y j ) absolute value, the spraying car (6) after the time (t n ) Then start spraying the dye.

6. The fully automatic high-throughput electron microscope grid staining machine according to claim 5, characterized in that: If the liquid spraying trolley (6) needs to continue spraying for several times, an instruction is outputted, and the liquid spraying trolley (6) moves at the same speed as the conveyor belt (2) and continues spraying for a period of time.

7. The fully automatic high-throughput electron microscope grid staining machine according to claim 5, characterized in that: If the liquid spraying trolley (6) needs to continue spraying multiple times and cover a preset range, an instruction is output, and the liquid spraying trolley (6) generates a motion trajectory of the liquid spraying process, so that the liquid spraying trolley (6) follows the copper mesh sample (10) and reciprocates to cover the copper mesh sample (10) to spray liquid.

8. The fully automatic high-throughput electron microscope grid staining machine according to claim 1, characterized in that: A cover body (3) is provided at the middle section of the conveyor belt (2), the cover body (3) covers the first dye spraying device and the second dye spraying device, and openings for the bracket (1) to pass through are provided at both ends of the cover body (3); The cover (3) is a light-shielding structure. The first dye spraying device and the second dye spraying device are separated. Nitrogen is introduced into the cover (3) space of the second dye spraying device to maintain the nitrogen at a slightly positive pressure to prevent oxygen from entering.

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