An automatic electronic staining device for small batches of biological samples

Abstract

The utility model discloses a kind of automatic electronic dyeing devices for small batch of biological sample, including substrate, electronic dyeing mould, circuit control panel and three syringe execution mechanism;Electronic dyeing mould includes runner groove base, rubber plate fixture and upper pressing plate;Runner groove base is equipped with runner groove, one luer external thread adapter is respectively equipped in the side wall of corresponding runner groove inlet, outlet;Inlet luer external thread adapter sequentially connects two luer three-way valves, and the remaining three interfaces of two luer three-way valves are respectively connected with three syringe execution mechanism;Outlet luer external thread adapter is connected with electromagnetic three-way valve one interface, and the other two interfaces of electromagnetic three-way valve are connected with waste liquid collection barrel;Rubber plate fixture is clamped and fixed with copper mesh on the side towards runner groove;Circuit control panel is electrically connected with syringe execution mechanism, electromagnetic three-way valve, luer three-way valve.The utility model has the advantages of simple operation, automatic control, realize light avoidance, air isolation dyeing, reagent consumption is less.

Description

Technical Field

[0001] This invention belongs to the field of electron microscope biological sample preparation technology, and in particular relates to an automated electronic staining device for small batches of biological samples. Background Technology

[0002] In the field of life sciences, electron microscopy is playing an increasingly important role. Electron microscopes can observe the ultrastructure of biological tissues and cells, making them an indispensable tool in basic life science research and clinical medical research. Electron microscopy in life sciences relies heavily on biological sample preparation, including electron staining. This involves immersing ultrathin sections of tissue cells in heavy metal salt solutions, causing varying degrees of adhesion to the fine structures within the cells. This process is essential for revealing the ultrastructure contrast of biological tissues under an electron microscope.

[0003] For example, Chinese patent document CN 217237419 U discloses a device for rapid batch staining of biological tissue sections, including a staining box, a stage, and a sealing cover. The surface of the stage is uniformly provided with a loading groove. The top surface of the sealing cover and the top surface of the staining box are provided with a connecting groove. The bottom surface of the sealing cover is fixedly provided with a connecting post corresponding to the connecting groove. The top surface of the sealing cover is provided with a silicone transparent cover. The bottom surface of the silicone transparent cover is fixedly provided with a connecting post. The bottom surface of the sealing cover is provided with an array of liquid storage cone grooves. The loading groove and the liquid storage cone groove correspond one-to-one. The stage is used to place biological sections. The liquid storage cone grooves on the surface of the sealing cover are filled with staining solution. The top of the sealing cover and the silicone transparent cover are connected. By pressing the silicone transparent cover, the staining solution of multiple sections is injected simultaneously.

[0004] The main steps of staining are as follows: first, immerse the copper mesh containing the ultrathin sections in uranyl acetate for 15-20 minutes, then rinse three times with double-distilled water; next, immerse in lead citrate for 3-10 minutes, and rinse three times with double-distilled water. Traditional electronic staining methods include drop staining, fully automated staining machines, and some self-made staining devices. Drop staining is suitable for staining single or small quantities of ultrathin sections, while fully automated staining machines are suitable for staining large quantities of ultrathin sections. However, for ultrathin sections with a quantity between these two extremes, drop staining becomes very cumbersome, time-consuming, and labor-intensive. Using an automated staining machine consumes a large amount of chemical reagents, significantly increasing costs. Furthermore, uranyl acetate used in staining is easily decomposed by light, and lead citrate solution readily reacts with carbon dioxide in the air, contaminating the ultrastructure of tissue and cell samples. Utility Model Content

[0005] This invention provides an automated electronic staining device for small-batch biological sample production, which has the advantages of simple operation, automated control, light-proof and air-isolated staining, and low reagent consumption.

[0006] An automated electronic staining device for small-batch biological samples includes a substrate and an electronic staining mold, a circuit control board, and three syringe actuators disposed on the substrate. The electronic dyeing mold includes a detachable and fixed flow channel base and an upper pressure plate, with a rubber plate clamping and fixing between the flow channel base and the upper pressure plate; the flow channel base is provided with a flow channel, and the flow channel base is provided with a Luer external thread adapter on the side wall corresponding to the inlet and outlet of the flow channel; wherein, the Luer external thread adapter at the inlet is sequentially connected to two Luer three-way valves, and the remaining three ports of the two Luer three-way valves are respectively connected to three syringe actuators; the Luer external thread adapter at the outlet is connected to one port of an electromagnetic three-way valve, and the other two ports of the electromagnetic three-way valve are used to connect to a waste liquid collection tank; The gel plate clamp holds and fixes a copper mesh on the side facing the flow channel. The copper mesh includes a support membrane for loading the ultrathin section to be stained. The circuit control board is electrically connected to the syringe actuator, the solenoid three-way valve, and the Luer three-way valve.

[0007] Furthermore, the width of the flow channel is 1.5~2mm, the depth is 3~4mm, and it adopts a serpentine arrangement with a rounded transition.

[0008] Furthermore, the surface of the rubber plate clamp is provided with at least one positioning hole, and the upper surface of the flow channel base is provided with a positioning post that mates with the positioning hole.

[0009] Furthermore, the surface of the rubber plate clamp is provided with coordinate symbols and a copper mesh slot matrix, wherein the coordinate symbols are used to identify the position and the copper mesh slot matrix is ​​used to clamp and fix the copper mesh.

[0010] Optionally, the slot shape of the copper mesh slot matrix is ​​rectangular, with a length of 3~3.5mm and a width of 0.03~0.05mm.

[0011] Alternatively, the slot shape of the copper mesh slot matrix is ​​V-shaped.

[0012] Furthermore, the syringe actuator includes a syringe holder and a stepper motor, guide post, lead screw and syringe mounted on the syringe holder; The output shaft of the stepper motor is connected to the lead screw via a coupling; a lead screw nut slider is sleeved on the guide post and the lead screw, and the lead screw nut slider is fixed to the piston rod of the syringe. The injection port of the syringe is connected to the corresponding interface of the Luer three-way valve via an infusion tubing.

[0013] Furthermore, the substrate is also provided with a liquid crystal touch panel, which is electrically connected to the circuit control board.

[0014] Furthermore, the flow channel base and the upper pressure plate are made of brown transparent acrylic material.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This utility model is designed with a flow channel and a gel plate fixture, which can realize the staining of small batches of biological tissue and cell samples with very low chemical reagent consumption, greatly reducing experimental costs.

[0016] 2. This utility model uses brown transparent acrylic material, which can effectively reduce the decomposition of uranium acetate by light; the "sandwich" structure can avoid the reaction of lead citrate with carbon dioxide in the air, thus improving the quality of electron staining.

[0017] 3. This utility model is designed with a positioning hole and positioning post matching structure, which can not only avoid the interference and deformation between the copper mesh and the flow channel caused by mismatch during installation, but also help to keep the flow direction of the dyeing solution perpendicular to the thickness direction of the copper mesh, avoid the dyeing solution directly impacting and damaging the support film, and reduce the dyeing breakage rate of the copper mesh support film.

[0018] 4. The present invention is equipped with a circuit control board, and the overall control method adopts automation, which eliminates the need for manual operation and saves manpower. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this utility model, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the overall structure of an automated electronic staining device for small-batch biological samples according to the present invention.

[0021] Figure 2 This is an exploded view of the electronic dyeing mold in this utility model.

[0022] Figure 3 This is an exploded view of the electronic dyeing mold in this utility model from another angle.

[0023] Figure 4 This is a schematic diagram of a copper mesh containing ultrathin slices in this utility model.

[0024] Figure 5 This is a schematic diagram of the overall structure and cross-sectional structure of the flow channel base of this utility model.

[0025] Figure 6 This is a schematic diagram of the overall structure and cross-sectional structure of the rubber plate clamp in this utility model.

[0026] Figure 7 This is a schematic diagram of the V-shaped opening copper mesh slot matrix in this utility model.

[0027] Figure 8 This is a schematic diagram of the syringe actuator in this utility model.

[0028] Figure 9 This is a schematic diagram of the syringe holder in this utility model. Detailed Implementation

[0029] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0030] It should be noted that, unless otherwise specified, the features in the following embodiments and implementation methods can be combined with each other.

[0031] like Figure 1 As shown, an automated electronic staining device for small-batch biological sample production includes an electromagnetic three-way valve 1, an electronic staining mold 2, a substrate 3, a syringe actuator 4, a liquid crystal touch panel 5, and a circuit control board 6. In this embodiment, the number of syringe actuators 4 is three.

[0032] like Figures 2-4 As shown, the electronic dyeing mold 2 consists of a flow channel base 21, a rubber plate clamp 22, a Luer external thread adapter 23, a Luer three-way valve 24, an upper pressure plate 25, nylon bolts 26, and a copper mesh assembly 27 carrying ultrathin slices (including a copper mesh 71 with a support film and ultrathin slices 72). The copper mesh 71 with the support film is generally 3mm in diameter and 0.01~0.03mm thick.

[0033] The flow channel base 21 and the upper pressure plate 25 are made of brown transparent acrylic material, which can effectively reduce the decomposition of the chemical reagent uranyl acetate by light and improve the quality of electron staining; the flow channel base 21 includes a flow channel 211 and a positioning post 212, such as Figure 5 As shown; the flow channel 211 has a width of 1.5~2mm and a depth of 3~4mm, and its shape adopts a serpentine arrangement with a rounded transition, which is conducive to the slow and smooth flow of the dyeing solution.

[0034] like Figure 6As shown, the adhesive plate clamp 22 includes positioning holes 221, coordinate symbols 222, and a copper mesh slot matrix 223. The copper mesh slot matrix 223 is rectangular in shape, with a length of 3~3.5mm and a width of 0.03~0.05mm. A slit is made in the adhesive plate using a sharp scalpel, and the adhesive plate clamp 22 is bent to open the copper mesh slot matrix 223, using the gap to hold the solid edge of the copper mesh 71 of the supporting membrane.

[0035] Furthermore, the shape of the copper mesh slot matrix 223 can also be processed into a V-shape. In practice, tweezers can be used to sequentially insert the copper mesh 71 containing the support film into different copper mesh slot matrices 223 with the V-shaped opening direction, making the loading of the copper mesh more convenient. (See...) Figure 7 As shown. Coordinate symbol 222 is used to identify a unique position, which can avoid sample confusion when electronically staining copper meshes of different sample numbers in small batches. The positioning hole 221 cooperates with the positioning post 212 to avoid misfitting during installation, which would cause interference and deformation between the copper mesh and the flow channel 211. It also helps to keep the flow direction of the dyeing solution perpendicular to the thickness direction of the copper mesh, avoiding direct impact of the dyeing solution on the support film and reducing the dyeing failure rate of the copper mesh support film.

[0036] As described above, the flow channel base 21 and the upper pressure plate 25 press the rubber plate clamp 22 in the middle with four nylon bolts 26 to form a tight "sandwich" structure. This structure helps to isolate air and prevent the lead citrate staining solution from reacting with carbon dioxide in the air to form a precipitate that contaminates the sample, thereby further improving the staining quality.

[0037] The Luer external thread adapter 23 connects to the threaded holes at both ends of the flow channel base 21, with one inlet and one outlet. Before connection, the threads of the Luer external thread adapter 23 need to be wrapped clockwise with 3-5 turns of PTFE tape to increase liquid sealing. Two Luer three-way valves 24 are connected in series and then connected to the Luer external thread adapter 23 at the inlet to realize the function of a four-way valve. The electromagnetic three-way valve 1 is connected to the Luer external thread adapter 23 at the outlet. The two hoses of the electromagnetic three-way valve 1 are respectively introduced into the inorganic heavy metal waste solution collection tank and the ordinary waste water tank.

[0038] The syringe actuator 4 can push and pull the syringe at a constant rate to perform injection and air release functions. For example... Figure 8 As shown, the syringe actuator 4 includes a syringe holder 41, a stepper motor 42, a coupling 43, a guide post 44, a lead screw nut slider 45, a lead screw 46, a syringe 47, an infusion tubing 48, and a deep groove ball bearing 49.

[0039] like Figure 9As shown, the syringe holder 41 is made of transparent acrylic or aluminum alloy and includes a flange hole 411, a bearing groove 412, a guide post hole 413, and a retaining groove 414. A stepper motor 42 is fixed in the flange hole 411; a pair of guide posts 44 pass through the guide post hole 413; a set of deep groove ball bearings 44 is fixed in the bearing groove 412, and the two ends of the lead screw 46 are coaxially fitted with these deep groove ball bearings 44. A coupling 43 connects the output shaft of the stepper motor 42 to the lead screw 46 to achieve power transmission; the lead screw nut slider 45 passes through the two guide posts 44 and is threadedly fitted with the lead screw 46. The syringe 47 and the infusion tubing 48 are made of brown transparent material. The syringe 47 has a volume of 50ml. The plunger of the syringe 47 is fixed in the slot of the lead screw nut slider 45, and the syringe barrel of the syringe 47 is fixed in the slot 414. The output shaft of the stepper motor 42 rotates clockwise, which drives the lead screw 46 to rotate, thereby pushing the lead screw nut slider 45 to move forward and squeeze the plunger of the syringe 47 to realize the injection function of the syringe. If the output shaft of the stepper motor 42 rotates counterclockwise, the plunger of the syringe 47 is pulled back.

[0040] The circuit control board 6 is connected to three syringe actuators 4, which can drive stepper motors 42, control electromagnetic three-way valves 1, and contain a timer module. The electromagnetic three-way valve 1 is a normally open type with one inlet and two outlets, and is controlled separately according to the chemical reagent and aqueous solution being injected, so as to realize the classification, collection and treatment of inorganic heavy metal waste solutions.

[0041] The LCD touch panel 5 has human-machine interaction functions and is electrically connected to the circuit control board 6. It can define the number and duration of timers, the syringe injection rate, whether to emit a buzzer, and display the current status. According to the actual electronic dyeing process requirements, an appropriate time and injection rate are set. After the injection action is completed, the current status is maintained for a period of time, while displaying the current running steps and status. After the electronic dyeing is completed, a prompt sound is emitted through the buzzer.

[0042] The following example uses the electronic dyeing process table in Table 1 as an illustration.

[0043] Table 1 Select a V-shaped adhesive plate clamp 22, and according to the coordinate symbol 222, use tweezers to insert the copper mesh 71 containing the support film into different copper mesh slot matrices 223 in the direction of the V-shaped opening.

[0044] Nylon bolts 26 secure the flow channel base 21, upper pressure plate 25 and rubber plate clamp 22 together in a diagonal assembly manner.

[0045] Referring to Table 1, each of the three syringes draws 45 ml of double-distilled water, 5 ml of uranium acetate dye solution, and 5 ml of lead citrate dye solution, respectively. The plungers of the three syringes 47 are fixed in the slots of the lead screw nut slider 45, and the syringe barrels of the syringes 47 are fixed in the slots 414. One end of the tubing 48 connected to the syringes 47 is connected to a Luer three-way valve 24. It should be noted that the infusion tubing connected to the three syringes should be filled with liquid to avoid excess air being trapped in the flow channels.

[0046] Set the following steps: Inject the solution using a 45ml double-distilled water syringe 47 at a rate of 0.5ml / s for 10 seconds, then let it stand for 0.5 minutes to clean the flow channel 211 and remove air. Inject the solution using a 5ml uranium acetate dye syringe 47 at a rate of 0.2ml / s for 25 seconds, then let it stand for 15 minutes. Inject the solution using a 45ml double-distilled water syringe 47 at a rate of 0.5ml / s for 40 seconds, then let it stand for 0.5 minutes to clean the previous staining solution. Inject the solution using a 5ml lead citrate dye syringe 47 at a rate of 0.2ml / s for 25 seconds, then let it stand for 20 minutes. Inject the solution using a 45ml double-distilled water syringe 47 at a rate of 0.5ml / s for 40 seconds, then let it stand for 0.5 minutes to clean the previous staining solution. After completing these settings, click the "Start Staining" button.

[0047] The buzzer in the LCD touch panel 5 sounds, indicating that the dyeing process is over. The nylon bolts 26 are used to separate the flow channel base 21, the upper pressure plate 25 and the glue plate clamp 22 in a diagonal assembly manner. The copper mesh 71 containing the support film in the glue plate clamp 22 is removed with tweezers.

[0048] The embodiments described above provide a detailed explanation of the technical solution and beneficial effects of this utility model. It should be understood that the above descriptions are only specific embodiments of this utility model and are not intended to limit this utility model. Any modifications, additions, and equivalent substitutions made within the scope of the principles of this utility model should be included within the protection scope of this utility model.

Claims

1. An automated electronic staining device for small-batch biological sample processing, characterized in that, It includes a substrate (3) and an electronic dyeing mold (2), a circuit control board (6) and three syringe actuators (4) disposed on the substrate (3); The electronic dyeing mold (2) includes a detachable and fixed flow channel base (21) and an upper pressure plate (25). A rubber plate clamp (22) is clamped and fixed between the flow channel base (21) and the upper pressure plate (25). The flow channel base (21) is provided with a flow channel (211). The flow channel base (21) is provided with a Luer external thread adapter (23) on the side wall of the corresponding flow channel (211) inlet and outlet. The Luer external thread adapter (23) at the inlet is connected to two Luer three-way valves (24) in sequence. The remaining three ports of the two Luer three-way valves (24) are connected to three syringe actuators (4). The Luer external thread adapter (23) at the outlet is connected to one port of the electromagnetic three-way valve (1). The other two ports of the electromagnetic three-way valve (1) are used to connect to the waste liquid collection tank. The adhesive plate clamp (22) clamps and fixes a copper mesh (71) on the side facing the flow channel (211). The copper mesh (71) includes a support membrane for loading the ultrathin section (72) to be stained. The circuit control board (6) is electrically connected to the syringe actuator (4), the electromagnetic three-way valve (1), and the Luer three-way valve (24).

2. The automated electronic staining device for small-batch biological sample processing according to claim 1, characterized in that, The width of the flow channel (211) is 1.5~2mm and the depth is 3~4mm. It adopts a serpentine arrangement and a rounded transition.

3. The automated electronic staining device for small-batch biological sample processing according to claim 1, characterized in that, The surface of the rubber plate clamp (22) is provided with at least one positioning hole (221), and the upper surface of the flow channel base (21) is provided with a positioning post (212) that cooperates with the positioning hole (221).

4. The automated electronic staining device for small-batch biological sample processing according to claim 1, characterized in that, The surface of the adhesive plate clamp (22) is provided with coordinate symbols (222) and copper mesh slot matrix (223), wherein the coordinate symbols (222) are used to mark the position, and the copper mesh slot matrix (223) is used to clamp and fix the copper mesh (71).

5. The automated electronic staining device for small-batch biological sample processing according to claim 4, characterized in that, The copper mesh slot matrix (223) has a rectangular slot shape with a length of 3~3.5mm and a width of 0.03~0.05mm.

6. The automated electronic staining device for small-batch biological sample processing according to claim 4, characterized in that, The slot shape of the copper mesh slot matrix (223) is V-shaped.

7. The automated electronic staining device for small-batch biological sample processing according to claim 1, characterized in that, The syringe actuator (4) includes a syringe holder (41) and a stepper motor (42), a guide post (44), a lead screw (46) and a syringe (47) mounted on the syringe holder (41). The output shaft of the stepper motor (42) is connected to the lead screw (46) via a coupling (43); a lead screw nut slider (45) is fitted on the guide post (44) and the lead screw (46), and the lead screw nut slider (45) is fixed to the piston rod of the syringe (47). The injection port of the syringe (47) is connected to the corresponding interface of the Luer three-way valve (24) via an infusion tubing.

8. The automated electronic staining device for small-batch biological sample processing according to claim 1, characterized in that, The substrate (3) is also provided with a liquid crystal touch panel (5), which is electrically connected to the circuit control board (6).

9. The automated electronic staining device for small-batch biological sample processing according to claim 1, characterized in that, The flow channel base (21) and the upper pressure plate (25) are made of brown transparent acrylic material.

Images