Full-automatic immunofluorescence labeling equipment

Abstract

The utility model provides full-automatic immunofluorescence labeling equipment. The full-automatic immunofluorescence labeling equipment comprises a control mechanism, a power mechanism, a reagent suction mechanism and a sample labeling mechanism, the power mechanism is electrically connected with the control mechanism, the reagent suction mechanism and the sample marking mechanism are both connected with the power mechanism, and the power mechanism is used for driving the reagent suction mechanism to suck a reagent required by immunofluorescence labeling and conveying the reagent required by immunofluorescence labeling to the sample marking mechanism; the sample labeling mechanism is used for adding the reagent required by immunofluorescence labeling into the corresponding culture dish to be labeled, so that the step of automatically performing immunofluorescence labeling is realized, and the problems of low immunofluorescence labeling efficiency, poor repeatability and low result reliability caused by manual labeling are avoided.

Description

Technical Field

[0001] The utility model relates to the field of automation equipment, in particular to a full-automatic immunofluorescence labeling device. Background Art

[0002] Immunofluorescence labeling utilizes the specific recognition and efficient binding between antigens and antibodies, and has a wide range of applications in life sciences and biomedicine. Immunofluorescence, combined with fluorescence microscopy equipment, enables visualization and quantitative analysis of biomolecules.

[0003] However, immunofluorescence labeling is tedious, complex, and extremely time-consuming. Currently, organelle immunofluorescence labeling is performed manually, requiring repeated reagent additions and cleanings, which consumes a significant amount of researchers' time. Furthermore, manual operations can result in low immunofluorescence labeling efficiency, poor reproducibility, and low reliability.

[0004] Therefore, the existing technology has defects and needs to be improved and developed. Utility Model Content

[0005] The technical problem to be solved by the present invention is to provide a fully automatic immunofluorescence labeling device in response to the above-mentioned defects of the prior art, aiming to solve the problems in the prior art that immunofluorescence labeling requires manual operation, has low efficiency, poor repeatability and low reliability of the immunofluorescence labeling results.

[0006] The technical solutions adopted by the present invention to solve the technical problems are as follows:

[0007] An embodiment of the present application provides a fully automatic immunofluorescence labeling device, comprising: a control mechanism, a power mechanism, a reagent suction mechanism and a sample labeling mechanism; the power mechanism is electrically connected to the control mechanism, and the reagent suction mechanism and the sample labeling mechanism are both connected to the power mechanism; the power mechanism is used to drive the reagent suction mechanism to absorb the reagent required for immunofluorescence labeling and transport the reagent required for immunofluorescence labeling to the sample labeling mechanism; the sample labeling mechanism is used to add the reagent required for immunofluorescence labeling to the corresponding culture dish to be labeled.

[0008] In one embodiment, the power mechanism comprises:

[0009] A sampling robot arm connected to the reagent suction mechanism and used to position the reagent suction mechanism;

[0010] a sample discharging robot arm connected to the sample marking mechanism and used for positioning the sample marking mechanism;

[0011] and a peristaltic pump, wherein the peristaltic pump has a peristaltic pump hose, and the peristaltic pump hose connects the passage between the reagent aspirating mechanism and the sample marking mechanism.

[0012] In one embodiment, a plurality of peristaltic pumps are provided, and the peristaltic pumps are arranged in parallel.

[0013] In one embodiment, the reagent aspiration mechanism comprises:

[0014] A sample suction rack, arranged on the sample injection robotic arm;

[0015] A plurality of sample aspirating needle clamping slots are provided on the sample aspirating rack;

[0016] A sample aspirating needle, clamped in each of the sample aspirating needle clamping grooves;

[0017] and a sample aspirating pipe, which is arranged at the upper end of the sample aspirating needle;

[0018] A reagent rack is further provided below the sample aspirating rack. A reagent tube holding groove is provided on the reagent rack. The positions of the sample aspirating needle holding groove and the reagent tube holding groove correspond one to one.

[0019] In one embodiment, a cleaning pool is further provided on one side of the reagent rack, and the cleaning pool is used to hold cleaning liquid to clean the sample aspirating needle. The sampling robot arm is used to drive the reagent aspirating mechanism to be positioned above the reagent rack or above the cleaning pool.

[0020] In one embodiment, the sample marking mechanism comprises:

[0021] A clamping member, provided on the sample discharging robot arm;

[0022] and a plurality of clamping parts, which are arranged on the clamping member, and each of the clamping parts is provided with a plurality of sample needle clamping grooves;

[0023] A sample adding needle, clamped in the sample adding needle clamping groove;

[0024] and a sample adding pipe, which is arranged at the upper end of the sample adding needle;

[0025] A culture dish carrying tray is further provided below the clamping member. The culture dish carrying tray is provided with a plurality of culture dish accommodating slots. The positions of the clamping portions correspond one to one with the positions of the culture dish accommodating slots.

[0026] In one embodiment, a waste liquid tank is also provided on one side of the culture dish carrying tray for receiving waste liquid after cleaning the passage between the reagent suction mechanism and the sample marking mechanism, and the sample discharge robot arm is used to drive the sample marking mechanism to be positioned above the culture dish carrying tray or above the waste liquid tank.

[0027] In one embodiment, the sample injection robot arm includes: a first driving source, a first X-axis screw slide connected to the first driving source, a second driving source, and a first Y-axis screw slide connected to the second driving source; the first Y-axis screw slide is arranged on the first X-axis screw slide, and the reagent aspiration mechanism is arranged on the first Y-axis screw slide;

[0028] The sample discharging robot arm includes: a third driving source, a second X-axis screw slide connected to the third driving source, a fourth driving source, and a second Y-axis screw slide connected to the fourth driving source; the second Y-axis screw slide is arranged on the second X-axis screw slide, and the sample marking mechanism is arranged on the second Y-axis screw slide.

[0029] In one embodiment, the control mechanism includes: a host computer, and a drive controller and a relay connected to the host computer for communication; four drive controllers are provided, which are respectively connected to the first drive source, the second drive source, the third drive source and the fourth drive source; multiple relays are provided, and the multiple relays are used to control the sample addition paths in different experimental steps.

[0030] The utility model discloses a fully automatic immunofluorescence labeling device, which comprises: a control mechanism, a power mechanism, a reagent suction mechanism and a sample labeling mechanism; the power mechanism is electrically connected to the control mechanism, and the reagent suction mechanism and the sample labeling mechanism are both connected to the power mechanism; the power mechanism is used to drive the reagent suction mechanism to absorb the reagent required for immunofluorescence labeling and transport the reagent required for immunofluorescence labeling to the sample labeling mechanism; the sample labeling mechanism is used to add the reagent required for immunofluorescence labeling to the corresponding culture dish to be labeled, thereby realizing the step of automatically performing immunofluorescence labeling and avoiding the problems of low immunofluorescence labeling efficiency, poor repeatability and low result reliability caused by manual labeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a structural schematic diagram of a preferred embodiment of a fully automatic immunofluorescence labeling device in the present utility model.

[0032] Figure 2 is the cell immunofluorescence labeling step and the corresponding time.

[0033] Figure 3 This is a hardware collaboration flow chart of the fully automatic immunofluorescence labeling system in the utility model.

[0034] Figure 4 It is a structural diagram of the reagent aspirating mechanism in the present invention.

[0035] Figure 5 It is a structural diagram of the sample marking mechanism in the utility model.

[0036] Figure 6 It is an exploded view of the sample marking mechanism in the utility model.

[0037] Figure 7 This is the wiring diagram of the relay and peristaltic pump motor of the fully automatic immunofluorescence labeling system in this utility model.

[0038] Figure 8 This is the software interface diagram of the fully automatic immunofluorescence labeling system in the present invention (standard mode).

[0039] Figure 9 This is a flow chart of the software initialization process of the fully automatic immunofluorescence labeling system in the present invention.

[0040] Figure 10 This is a diagram of the custom step mode interface of the fully automatic immunofluorescence labeling system in the present invention.

[0041] Figure 11 This is a software flow chart of the fully automatic immunofluorescence labeling system in the utility model.

[0042] Description of reference numerals:

[0043] 100. Control mechanism; 200. Reagent aspiration mechanism; 210. Sample aspiration rack; 211. Sample aspiration needle; 220. Reagent rack; 230. Reagent tube clamping groove; 240. Cleaning tank; 300. Sample marking mechanism; 310. Clamping member; 311. Clamping portion; 312. Sample injection needle clamping groove; 313. Clamping buckle; 320. Culture dish carrying tray; 321. Culture dish receiving tank; 400. Sample injection robot arm; 410. First driving source; 420. First X-axis screw slide; 430. Second driving source; 440. First Y-axis screw slide; 500. Sample discharging robot arm; 510. Third driving source; 520. Second X-axis screw slide; 530. Fourth driving source; 540. Second Y-axis screw slide. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clear and distinct, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] Current commercially available pathology slide labeling systems are generally relatively expensive, making them unaffordable for ordinary laboratories. This results in manual labeling of tissue sections. Furthermore, pathology slide labeling systems are primarily designed for clinical tissue slide samples in hospitals and are not suitable for subcellular immunofluorescence labeling related to life sciences. The automated instrument provided in the embodiments of this application can be used for both tissue slide and subcellular immunofluorescence labeling applications.

[0046] like Figure 1 As shown, the present invention provides a fully automatic immunofluorescence labeling device, comprising: a control mechanism 100, a power mechanism, a reagent aspirating mechanism 200, and a sample labeling mechanism 300. The power mechanism is electrically connected to the control mechanism 100, and the reagent aspirating mechanism 200 and the sample labeling mechanism 300 are both connected to the power mechanism. The power mechanism is used to drive the reagent aspirating mechanism 200 to aspirate the reagent required for immunofluorescence labeling and transfer the reagent required for immunofluorescence labeling to the sample labeling mechanism 300; the sample labeling mechanism 300 is used to add the reagent required for immunofluorescence labeling to the corresponding culture dish to be labeled.

[0047] Specifically, the immunofluorescence labeling of frozen sections and cells is mainly achieved by the specific binding of corresponding antigens and antibodies. Generally, the whole process is divided into seven independent operation steps, and each operation step is corresponding to three washing processes. Figure 2 shown.

[0048] The extraction process primarily involves extracting free microtubules from the cell using an extraction reagent to prevent artifacts during subsequent imaging. The fixation process involves fixing the cell structure with a paraformaldehyde solution to facilitate labeling. The permeabilization process involves using chemical reagents such as Triton X-ray to permeabilize the cell membrane, allowing antibodies and fluorescent probes to more efficiently enter the cell structure. The blocking process involves using reagents such as serum proteins to block nonspecific adsorption of materials within the cell. The antibody incubation process depends primarily on the experimental design, and the secondary or tertiary antibody steps may be omitted. For example, a primary antibody conjugated to a fluorescent tag can be used for direct labeling. This approach is convenient but also carries the highest cost. Alternatively, a primary antibody conjugated to biotin and a fluorescent tag linked to streptavidin can be used as a secondary antibody. For some unusual antibody combinations, a secondary antibody may be used as an intermediate to connect the primary antibody and the tertiary antibody fluorescent probe. Incubation times and conditions vary depending on the experimental requirements. Therefore, it takes 6-40 hours to complete a cell immunofluorescence labeling experiment, and if it is a frozen section labeling experiment, it basically takes more than 48 hours.

[0049] Antigen-antibody binding is currently the most mature immunofluorescence labeling method. However, the biggest challenge with this experiment is that both research institutes and testing companies currently rely on manual labeling. Besides wasting significant time for researchers, manual immunofluorescence labeling also suffers from poor reproducibility and reliability, issues that urgently need to be addressed.

[0050] Key elements for successful immunofluorescence labeling include strict control of sample volume, incubation time at each step, and timely aspiration and washing. These critical factors are easily affected by external factors and can be altered during manual labeling. However, instrument-based labeling ensures that all of these elements are met.

[0051] The fully automatic immunofluorescence labeling equipment of the embodiment of the present application is provided with a control mechanism 100, a power mechanism, a reagent aspirating mechanism 200 and a sample labeling mechanism 300. The control mechanism 100 can control the power mechanism to drive the reagent aspirating mechanism 200 to absorb the reagents required for immunofluorescence labeling, and transport the reagents required for immunofluorescence labeling to the sample labeling mechanism 300; the control mechanism 100 controls the sample labeling mechanism 300 to add the reagents required for immunofluorescence labeling to the corresponding culture dish to be labeled, thereby realizing the automatic immunofluorescence labeling step and avoiding the problems of low efficiency, poor repeatability and low reliability of immunofluorescence labeling caused by manual labeling.

[0052] like Figure 3 As shown, the system corresponding to this device can realize an automated labeling process through the coordinated operation of five modules: the reagent end, the hardware control unit, the software control unit, the power unit, and the biological sample end. Two-dimensional robotic arms are equipped on the reagent end and the biological sample end to automatically switch the sample aspiration and sample injection needles between cleaning and adding reagents and samples. The hardware control unit and the power unit are placed inside the machine housing; the software control unit is implemented by a touch screen computer embedded in the housing.

[0053] In one specific embodiment, the immunofluorescence labeling system utilizes a horizontally distributed structure, consisting of three sections: a reagent section, a main unit, and a biological sample section. The entire device occupies a space of 55 cm in length, 58 cm in width, and 30 cm in height, allowing it to be placed directly on a standard horizontal tabletop. The mechanical structure of the system is constructed entirely of 6061 aluminum alloy, which is milled, cut, and surface-treated to create the complete device.

[0054] In one embodiment of the present application, the power mechanism includes:

[0055] The sample injection robot 400 is connected to the reagent suction mechanism 200 and is used to position the reagent suction mechanism 200;

[0056] A sample discharging robot 500, connected to the sample marking mechanism 300, for positioning the sample marking mechanism 300;

[0057] and a peristaltic pump, wherein the peristaltic pump has a peristaltic pump hose, and the peristaltic pump hose connects the passage between the reagent aspirating mechanism 200 and the sample marking mechanism 300 .

[0058] Specifically, the sampling robot arm 400, the sampling robot arm 500 and the peristaltic pump all belong to the power unit of this system. The sampling robot arm 400 is the two-dimensional robot arm of the reagent end, and the sampling robot arm 500 is the two-dimensional robot arm of the biological sample end. The reagent end is mainly responsible for the storage of the cleaning fluid and biological reagents of the system. In addition, the sampling robot arm 400 can ensure that the sampling needle is controlled to be in the correct position of the cleaning tank or reagent rack in different experimental steps. When the system performs the pipeline cleaning operation, the sampling robot arm 400 drives the sampling needle to the cleaning tank filled with cleaning fluid. When performing the marking task, the sampling robot arm 400 drives the sampling needle to be positioned in the reagent tube.

[0059] In a specific embodiment, the peristaltic pump is a Baines BPH-DK-B10-A micro peristaltic pump. The peristaltic pump has the characteristics of simple structure and low failure rate, which improves the stability of the system.

[0060] In one embodiment of the present application, a plurality of peristaltic pumps are provided, and the peristaltic pumps are arranged in parallel.

[0061] Specifically, during the immunofluorescence labeling process, reagents between different steps, especially different antibodies, must be kept pure and free of contamination. Using parallel peristaltic pumps as reagent addition units effectively eliminates the problem of reagent crosstalk, laying the foundation for accurate, reliable, and efficient fluorescent labeling. Compared to using a single pump as the sample injection unit, parallel addition eliminates the need to wait for the sample aspiration probe 211 and tubing to be cleaned, reducing waiting time between steps and improving labeling efficiency.

[0062] In one embodiment of the present application, Figure 4 As shown, the reagent aspirating mechanism 200 includes:

[0063] The sample suction rack 210 is provided on the sample injection robot arm 400;

[0064] A plurality of sample aspirating needle 211 holding slots are provided on the sample aspirating rack 210;

[0065] The sample aspirating needle 211 is clamped in each of the sample aspirating needle 211 clamping grooves;

[0066] and a sample aspirating pipe, which is provided at the upper end of the sample aspirating needle 211;

[0067] A reagent rack 220 is further provided below the sample aspirating rack 210 . A reagent tube holding groove 230 is provided on the reagent rack 220 . The holding groove of the sample aspirating needle 211 corresponds to the position of the reagent tube holding groove 230 .

[0068] Specifically, a device base can be provided on which the reagent rack 220 is fixed. Driven by the sample injection robot 400, the clamping groove of the sample aspirating needle 211 can be accurately aligned with the reagent tube clamping groove 230, thereby realizing mechanical manipulation of the sample aspirating and further realizing the automation of the device.

[0069] In one embodiment of the present application, a cleaning pool 240 is further provided on one side of the reagent rack 220. The cleaning pool 240 is used to hold cleaning liquid to clean the sample aspiration needle 211. The sampling robot 400 is used to drive the reagent aspiration mechanism 200 to be positioned above the reagent rack 220 or above the cleaning pool 240.

[0070] Specifically, the embodiment of the present application is provided with a cleaning pool 240. Driven by the sampling robot 400, the reagent suction mechanism 200 can be positioned above the cleaning pool 240, thereby realizing mechanically controlled cleaning and further realizing the automation of the equipment.

[0071] In one embodiment of the present application, Figure 5 and Figure 6 As shown, the sample marking mechanism 300 includes:

[0072] The clamping member 310 is provided on the sample discharging robot arm 500;

[0073] and a plurality of clamping portions 311 disposed on the clamping member 310 , each of the clamping portions 311 being provided with a plurality of sample needle clamping grooves 312 ;

[0074] A sample injection needle, clamped in the sample injection needle clamping groove 312;

[0075] and a sample adding pipe, which is arranged at the upper end of the sample adding needle;

[0076] A culture dish carrying tray 320 is further provided below the clamping member 310 . The culture dish carrying tray 320 is provided with a plurality of culture dish receiving slots 321 . The positions of the clamping portions 311 and the culture dish receiving slots 321 correspond one to one.

[0077] In one specific embodiment, the biological sample handling system features four culture dish slots 321 on a culture dish tray 320, along with a sample needle, its positioning arm, and a rocker module. The culture dish tray allows for flexible interchangeability based on the type of culture dish used in the experiment. The sample needle is secured with a clamp 310 that matches the tray, ensuring consistent placement in each culture dish.

[0078] In one embodiment, each clamping portion 311 is provided with a U-shaped retaining buckle 313, which defines a receiving space within which the clamping portion 311 is received. Both end surfaces of the retaining buckle 313 have pinholes corresponding to the sample needle retaining grooves 312. The provision of the retaining buckle 313 improves the stability of retaining the sample needle.

[0079] The sample addition pipeline in the embodiment of the present application is connected to the peristaltic pump, and the reagent is then added to the culture dish, thereby realizing automated sample addition and thus realizing automation of the equipment.

[0080] In one embodiment of the present application, a waste liquid tank is also provided on one side of the culture dish carrying tray 320 for receiving waste liquid after cleaning the passage between the reagent aspiration mechanism 200 and the sample marking mechanism 300, and the sample discharge robot arm 500 is used to drive the sample marking mechanism 300 to be positioned above the culture dish carrying tray 320 or above the waste liquid tank.

[0081] Specifically, the sample discharging robot arm 500 can drive the sample adding needle to accurately position at the culture dish and the waste liquid tank according to the current operation steps.

[0082] The biological sample end of this system can also be equipped with a shaker module, and the swing amplitude of the shaker can be set according to the expected experimental requirements to ensure sufficient contact and reaction between the reagent and the biological sample.

[0083] In this way, the system-integrated mechanical control of the embodiment of the present application can realize the steps of adding, removing, and cleaning reagents automatically, accurately control the incubation time and reagent dosage, realize the standardization of the entire labeling process, effectively reduce the time investment of scientific researchers, and improve the quality of cell labeling and the repeatability of fluorescence imaging.

[0084] In one embodiment of the present application, the sampling robot 400 includes: a first driving source 410, a first X-axis screw slide 420 connected to the first driving source 410, a second driving source 430, and a first Y-axis screw slide 440 connected to the second driving source 430; the first Y-axis screw slide 440 is arranged on the first X-axis screw slide 420, and the reagent suction mechanism 200 is arranged on the first Y-axis screw slide 440; the sampling robot 500 includes: a third driving source 510, a second X-axis screw slide 520 connected to the third driving source 510, a fourth driving source 530, and a second Y-axis screw slide 540 connected to the fourth driving source 530; the second Y-axis screw slide 540 is arranged on the second X-axis screw slide 520, and the sample marking mechanism 300 is arranged on the second Y-axis screw slide 540.

[0085] Specifically, the sample aspirator 210 is mounted on the first Y-axis lead screw slide 440, and the clamp 310 is mounted on the second Y-axis lead screw slide 540. In one embodiment, both the sample injection robot 400 and the sample discharge robot 500 utilize a 35-type two-phase four-wire stepper motor to drive a "T"-shaped lead screw slide to precisely position the reagent-end sample aspirator 211. In one embodiment, the lead screw has an 8mm diameter and a 2mm pitch, and the lead screw slide has a positioning accuracy of 0.05mm.

[0086] The screw slide used in the embodiments of this application combines a base made of aluminum alloy profiles with linear guides, achieving both lightweight and high rigidity. Lightweighting results in a compact and lightweight device, while high rigidity translates to high precision. The low cost of aluminum alloy further reduces costs. The screw slide offers higher repeatability than other linear slides, making it particularly suitable for high-precision machining and positioning applications.

[0087] In one embodiment of the present application, the control mechanism 100 includes: a host computer, and a drive controller and relay connected to the host computer for communication; the drive controllers are provided in four numbers, which are respectively connected to the first drive source 410, the second drive source 430, the third drive source 510 and the fourth drive source 530; the relays are provided in plurality, and the plurality of relays are used to control the sample addition paths in different experimental steps.

[0088] Specifically, in the system of the embodiment of the present application, the hardware control unit is divided into a stepper motor drive control system for controlling the robotic arm and a relay control module for controlling the start and stop of the peristaltic pump. In one embodiment, the stepper motor drive controller communicates with the host computer through RS485; the relay module communicates with the host computer through the RS232 protocol. According to the design of this system, the relay uses a 16-way serial port relay, and relays 1 to 7 respectively control the extraction, fixation, punching, sealing, primary antibody, secondary antibody and tertiary antibody addition paths in the experimental steps. Relay No. 8 is responsible for the control of the addition of phosphate buffer solution (PBS); Relay No. 9 is used to control the suction of waste liquid. In order to ensure that there is no excess waste liquid in the biological tissue culture dish after each step, this system is equipped with waste liquid suction needles in the center and side of the culture dish. Relays No. 10 to No. 12 are idle ends, reserved for expansion and upgrading. Relays No. 13 to No. 16 have special functions and are not used to control the start and stop of the sample injection peristaltic pump, but are used to control the four sample channels of the system. Among them, the peristaltic pump motor uses a PWM speed regulator for flow control. The specific wiring method is as follows Figure 7 shown.

[0089] The host computer is the software control unit of this system, and the host computer software controls the hardware control unit and the power unit through serial communication. Figure 8 As shown. After the system is powered on and the control software is opened, it first performs a hardware communication self-test. Once the self-test passes, the hardware system is initialized and the software buttons are switched. During system initialization, four dishes A, B, C, and D are set to operate simultaneously, with the corresponding buttons enabled. The default operation procedure is "Standard," with the corresponding buttons enabled. The standard operation sequence is: extraction - fixation - fixative washes (3 times, 5 minutes each) - perforation - blocking - primary antibody incubation - primary antibody waste washes (3 times, 5 minutes each) - secondary antibody / dye incubation - secondary antibody / dye waste washes (3 times, 5 minutes each) - PBS buffer added to the dish - automatic tubing cleans - step completion indication - reset the robotic arm. Each step begins with the corresponding sample pump (peristaltic pump) pumping the corresponding reagent. At the end, two waste pumps (peristaltic pumps) aspirate the solution into the waste bottle. When the "Standard" procedure is selected, the operation time for each step is internally defined in the software and cannot be modified by the user.

[0090] To provide users with more flexible operation, simply turn off the "Standard" button after initialization to enter the user-defined "Custom" mode. The software is set to work from left to right, and users only need to click the required experimental steps based on actual experimental conditions. In addition, in user-defined mode, the operation time of all steps can be modified by the user. In addition, to prevent the operator from accidentally touching the operation time of each step after the experiment begins, the software has a "Parameter Lock" button to ensure the security of each step parameter in custom mode.

[0091] The software also features control buttons for the sample injection arm 400 and sample discharge arm 500, facilitating pre-experimental operations such as cleaning the lines and loading samples. Upon completion of an experiment, the system software presets the sample injection arm 400 to "up" and "before" positions, and the sample discharge arm 500 to "up" and "before" positions. Unless there are unexpected events during the experiment, such as a forced power shutdown or power outage, the arms automatically adjust to the correct position. The software was designed to prevent unexpected situations during the labeling process. Therefore, the buttons controlling the arms are essential. The arm's movement is intelligent, ensuring that the arm-driven injection needle does not collide with the cleaning reservoir 240 or reagent tubes on the reagent side. Similarly, the arm-driven injection needle does not collide with the walls of the culture dish or waste container on the biological sample side. Each step and start time of the software will be displayed in the "Current Step" text box, and the progress of the current execution step will be intuitively and accurately prompted by the progress bar.

[0092] The system in this application embodiment is equipped with easy-to-use host computer operating software and a high-resolution touch screen. The various functional modules are rationally arranged, which can significantly reduce the initial learning costs of researchers and thus improve overall scientific research efficiency. At the same time, the system has an efficient mechanical structure, making it a desktop scientific research instrument suitable for various experimental environments, with stable operation and high space utilization.

[0093] The working principle of this utility model includes:

[0094] The control mechanism 100 controls the power mechanism to drive the reagent aspirating mechanism 200 to aspirate the reagents required for immunofluorescence labeling based on a preset standard experimental procedure, and delivers the reagents required for immunofluorescence labeling to the sample labeling mechanism 300;

[0095] The control mechanism 100 controls the sample labeling mechanism 300 to add the reagents required for immunofluorescence labeling into the corresponding culture dish to be labeled, so as to complete the immunofluorescence labeling.

[0096] The utility model is a fully automatic cell immunofluorescence labeling system for life science research experiments and cell-level detection applications. By introducing a parallel sampling system and writing touch-screen control software, immunofluorescence labeling experiments of frozen sections or cells are realized. The system is equipped with seven sampling channels, which is sufficient to ensure the number of sampling channels in the commonly used immunofluorescence labeling steps. It is equipped with a buffer solution addition channel and two waste liquid suction channels to ensure that there is no excess waste liquid residue at the bottom of the culture dish. The system is also equipped with an automatic pipeline cleaning function to ensure that there is no risk of reagent contamination and crosstalk during different experimental processes. The sampling robot 400 equipped with the system can be flexibly operated and controlled, and has the function of automatically finding a position. The shaker module equipped with the system can control the swing amplitude and adapt to a variety of labeling situations. The entire system realizes fully automatic operation of frozen sections and cell immunofluorescence labeling.

[0097] The software program corresponding to the method of the embodiment of the present application is written in a state machine mode. When the system hardware is powered on and the control software is opened, the software is first initialized to set the state of each button in the software interface and the corresponding parameters.

[0098] like Figure 9 As shown, during the initialization process, the software first assigns values and changes the status of each button on the interface, modifying the properties of the interface controls. Next, the software performs a self-test. This self-test includes checking the communication between the serial relay and the stepper motor controller. The serial relay is controlled using the RS-232 protocol. First, an initialization command is sent to the serial relay. After the relay responds, it returns a handshake command. The software then compares the returned command with the preset correct handshake command. A successful comparison indicates that communication between the software and the serial relay has been established, allowing the next step of checking the communication with the stepper motor controller. In this system, the reagent side and the biological sample side are each equipped with a two-axis robotic arm, totaling four stepper motors. The stepper motor controllers communicate using the RS-485 protocol. To conserve serial ports, the four controllers are linked using a serial cascade configuration. This allows the four stepper motors to be effectively controlled using a single serial channel. During the self-test process, the software also sends a handshake command to the designated serial port. Once communication is successfully established, a command is sent to reset the robotic arms on each side. The reagent end adjusts the injection needle to the top of the cleaning pool 240, and the biological sample end adjusts the injection needle to the top of the waste liquid tank to prevent affecting the sample addition and biological sample process and wait for subsequent operations to be executed.

[0099] After initialization, the software executes according to user instructions. The underlying control software uses a state machine model, treating each action on the software interface as a corresponding event. When the state of a control changes, the program jumps directly to the corresponding event state and performs the corresponding operation. If no instruction is executed, the program refreshes the state at a fixed interval. At this time, all buttons related to the operation steps on the software interface are active.

[0100] When the default "Standard Steps" button is selected, the buttons related to the experimental operation steps in the software cannot be clicked, and the program only executes the built-in standard operation steps.

[0101] Deselecting the "Standard Steps" button allows the program to enter a self-selected state. The user can now select any immunofluorescence labeling steps. For each injection step, such as "Extraction" or "Fixation," the user can choose whether to select the appropriate process based on their needs.

[0102] Software interface such as Figure 10 As shown. Figure 10 In Step 1, Standard Step / Custom Step: Displays the instrument's current operating mode. The Standard Step uses the same parameters as the cytoskeleton alpha-tubulin labeling, with all parameters locked except for the channel selection. In the Custom Step, you can select channels and change parameters as needed.

[0103] Step 2, Channel A / B / C / D: Different channels correspond to different cell culture dishes, and the channel is selected when the green light is on.

[0104] Step 3: Wash and add PBS indicator light: Status display, the green light flashes to indicate that it is being executed.

[0105] Step 4, One-Click Cleaning: Before the experiment begins, clean the pipes. Pure water in cleaning tank 240 flows through the pipes and then to the waste tank. The default pipe cleaning time is 60 seconds. Ensure there is sufficient pure water in cleaning tank 240 and that no water remains in the pipes after cleaning.

[0106] Step 5. Labeling experimental steps: The immunofluorescence labeling experimental process includes extraction (cytoskeleton protein labeling is optional), fixation, perforation, blocking, primary antibody, secondary antibody / dye incubation and other steps; if the indicator light is dark, the step is not selected and will not be executed; if the indicator light is selected and turns yellow, waiting for execution; if the indicator light turns green and flashes, the step is being executed; if the indicator light is always green, the step has been completed. When washing, the PBS washing step can be selected. If the indicator light is dark, washing will not be performed after the above steps are completed; if the indicator light is selected and turns green, the washing step will be performed after the above steps are executed. When setting the time, the default is the preset time of the standard step. In the custom mode, the parameters can be modified according to needs.

[0107] Step 6. Current number of washes: Displays the number of PBS washes currently being performed, with a default range of 0-3.

[0108] Step 7. Pipeline cleaning time (s): In the standard step, the pipeline self-cleaning is carried out according to the system preset time; in the custom mode, the pipeline cleaning time can be modified according to actual needs.

[0109] Step 8. Sample addition and waste liquid aspiration time (s): The time to aspirate the sample from the reagent end to the culture dish, and the time to aspirate the liquid in the culture dish to the waste liquid bottle. The default waste liquid aspiration time is 5s longer than the sample addition time. In the custom mode, the parameters can be modified according to actual needs.

[0110] Step 9. Control buttons for the sample injection robot 400 and the sample discharging robot 500: Control the robot arms by clicking the direction keys (the movement of the robot arms is a two-point displacement and will not stay in the middle position); do not operate the buttons during the initialization process.

[0111] Step 10. Current step: Real-time broadcast of the step being executed and the start time of the marked experiment.

[0112] Step 11. Cleaning time (min) and number of times: The default cleaning time is 5 minutes and the number of times of cleaning is 3 times. In the custom step mode, the parameters can be freely set according to actual needs.

[0113] Step 12. Current execution step progress: Display the progress (percentage) of the current execution step in real time.

[0114] Step 13. Parameter lock: The yellow light will turn on to remind you that the current screen parameters have been locked and cannot be changed.

[0115] Step 14: Self-cleaning of pipelines: When the yellow light is on, the pipelines will be automatically cleaned after the experiment is completed. If you check the self-cleaning option, please ensure that there is enough pure water in the cleaning pool.

[0116] Step 15. Start marking: Click this button to start the marking experiment; a pop-up window will pop up to confirm the start of marking again.

[0117] Step 16. Stop. Click the Stop button to terminate the current marking experiment step, start the initialization process, and then stop. If you find that the experimental parameters are set incorrectly after marking begins, press the Stop button immediately and re-mark the experiment. Do not press the emergency switch unless necessary.

[0118] If the user manually selects, that is, the user edits the marking steps by himself, first cancel the "Standard Step" and the button will be displayed as "Custom Step". At this time, after the user selects to start marking, the program adjusts the robot arm to the corresponding position in the same way as the standard step. Then, each operation step and the corresponding cleaning process are judged one by one to determine whether they need to be executed. Figure 11As shown, the program first determines whether the "Extraction" step is selected. If so, it executes the extraction process. After the extraction process is complete, it determines whether the user has selected the corresponding "Wash" button. If so, it executes the wash process according to the user-set "Number of Washes" and "Wash Time." After the extraction and wash process is complete, it determines whether the remaining steps are selected. If "Extraction" is not selected in the first step, the program skips the "Wash" step and directly determines whether the "Fix" step is selected. This process continues after each step until it reaches the "Wash" step for the triple antibody. Approximately 3 mL of buffer solution is then added to the culture dish to prevent the sample from drying out and crystallizing. The program then determines whether the "Self-Clean Pipeline" function is selected. If selected, the program jumps to the Self-Clean Pipeline module, performs the appropriate robotic arm adjustments and cleaning, and returns the robotic arm to its initial position. If "Self-Clean Pipeline" is not selected, the program automatically resets the robotic arms on the "Reagent End" and "Biological Sample End" to their initial positions. A dialog box pops up to prompt the user to confirm that the step is completed.

[0119] The system in this application embodiment is equipped with multiple modes of software control. Through the "custom mode", it can realize diverse labeling processes, realize the independent setting and parallel control of multiple channels, and meet the diverse needs of researchers with different backgrounds. The high-throughput system allows for multi-channel parallel labeling, greatly improving labeling efficiency.

[0120] The present invention overcomes the drawbacks of manual immunofluorescence labeling by automating the immunofluorescence labeling steps and developing a fully automated immunofluorescence labeling system. Compared to manual operation by experimenters, the automated labeling system overcomes the following problems:

[0121] (1) Strict control of operating steps: The machine can ensure that the operation is carried out in full accordance with the preset experimental steps and in a strict order.

[0122] (2) Strictly control the incubation time of each step: The software of the labeling system can accurately control the time, and the time of each operation step is strictly executed according to the preset time.

[0123] (3) Timely removal of waste liquid generated in each step: The marking system has smooth connections between adjacent steps, and waste liquid can be collected promptly and effectively after each step. All pipelines are cleaned promptly after each experiment to ensure the normal progress of the next round of experiments and to extend the service life of the entire system.

[0124] (4) Completely free up the experimenter's operating time: The experimenter only needs to prepare the required reagents and corresponding biological samples before starting. The use of the labeling system can greatly save the experimenter's time and integrate the fragmented time during manual operation, leaving more time for scientific research and experimenters.

[0125] (5) Good repeatability of experimental results and high marking efficiency: The experimental results of machine marking are stable and the marking efficiency is high.

[0126] The utility model provides a fully automatic immunofluorescence labeling device, which comprises: a control mechanism, a power mechanism, a reagent suction mechanism and a sample labeling mechanism; the power mechanism is electrically connected to the control mechanism, and the reagent suction mechanism and the sample labeling mechanism are both connected to the power mechanism; the power mechanism is used to drive the reagent suction mechanism to absorb the reagent required for immunofluorescence labeling and transport the reagent required for immunofluorescence labeling to the sample labeling mechanism; the sample labeling mechanism is used to add the reagent required for immunofluorescence labeling to the corresponding culture dish to be labeled, thereby realizing the step of automatically performing immunofluorescence labeling and avoiding the problems of low immunofluorescence labeling efficiency, poor repeatability and low result reliability caused by manual labeling.

[0127] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, they can make improvements or changes based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A fully automatic immunofluorescence labeling device, characterized in that: include: A control mechanism, a power mechanism, a reagent suction mechanism and a sample marking mechanism; the power mechanism is electrically connected to the control mechanism, and the reagent suction mechanism and the sample marking mechanism are both connected to the power mechanism. The power mechanism is used to drive the reagent suction mechanism to absorb the reagent required for immunofluorescence marking and transport the reagent required for immunofluorescence marking to the sample marking mechanism; the sample marking mechanism is used to add the reagent required for immunofluorescence marking to the corresponding culture dish to be marked.

2. The fully automatic immunofluorescence labeling device according to claim 1, characterized in that: The power mechanism comprises: A sampling robot arm connected to the reagent suction mechanism and used to position the reagent suction mechanism; a sample discharging robot arm connected to the sample marking mechanism and used for positioning the sample marking mechanism; and a peristaltic pump, wherein the peristaltic pump has a peristaltic pump hose, and the peristaltic pump hose connects the passage between the reagent aspirating mechanism and the sample marking mechanism.

3. The fully automatic immunofluorescence labeling device according to claim 2, characterized in that: There are multiple peristaltic pumps, and the peristaltic pumps are arranged in parallel.

4. The fully automatic immunofluorescence labeling device according to claim 2, characterized in that: The reagent aspirating mechanism comprises: A sample suction rack, arranged on the sample injection robotic arm; A plurality of sample aspirating needle clamping slots are provided on the sample aspirating rack; A sample aspirating needle, clamped in each of the sample aspirating needle clamping grooves; and a sample aspirating pipe, which is arranged at the upper end of the sample aspirating needle; A reagent rack is further provided below the sample aspirating rack. A reagent tube holding groove is provided on the reagent rack. The positions of the sample aspirating needle holding groove and the reagent tube holding groove correspond one to one.

5. The fully automatic immunofluorescence labeling device according to claim 4, characterized in that: A cleaning pool is further provided on one side of the reagent rack, and the cleaning pool is used to contain cleaning liquid to clean the sample aspirating needle. The sampling robot arm is used to drive the reagent aspirating mechanism to be positioned above the reagent rack or above the cleaning pool.

6. The fully automatic immunofluorescence labeling device according to claim 2, characterized in that: The sample marking mechanism comprises: A clamping member, provided on the sample discharging robot arm; and a plurality of clamping parts, which are arranged on the clamping member, and each of the clamping parts is provided with a plurality of sample needle clamping grooves; A sample adding needle, clamped in the sample adding needle clamping groove; and a sample adding pipeline, which is arranged at the upper end of the sample adding needle; A culture dish carrying tray is further provided below the clamping member. The culture dish carrying tray is provided with a plurality of culture dish accommodating slots. The positions of the clamping portions correspond one to one with the positions of the culture dish accommodating slots.

7. The fully automatic immunofluorescence labeling device according to claim 6, characterized in that: A waste liquid tank is also provided on one side of the culture dish carrying tray for receiving waste liquid after cleaning the passage between the reagent suction mechanism and the sample marking mechanism. The sample discharging robot arm is used to drive the sample marking mechanism to be positioned above the culture dish carrying tray or above the waste liquid tank.

8. The fully automatic immunofluorescence labeling device according to claim 2, characterized in that: The sample injection robot arm includes: a first driving source, a first X-axis screw slide connected to the first driving source, a second driving source, and a first Y-axis screw slide connected to the second driving source; the first Y-axis screw slide is arranged on the first X-axis screw slide, and the reagent aspiration mechanism is arranged on the first Y-axis screw slide; The sample discharging robot arm includes: a third driving source, a second X-axis screw slide connected to the third driving source, a fourth driving source, and a second Y-axis screw slide connected to the fourth driving source; the second Y-axis screw slide is arranged on the second X-axis screw slide, and the sample marking mechanism is arranged on the second Y-axis screw slide.

9. The fully automatic immunofluorescence labeling device according to claim 8, characterized in that: The control mechanism includes: a host computer, and a drive controller and relay connected to the host computer for communication; four drive controllers are provided, which are respectively connected to the first drive source, the second drive source, the third drive source and the fourth drive source; multiple relays are provided, and the multiple relays are used to control the sample addition paths in different experimental steps.

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