High-throughput zebra fish behavior analysis device and method

Abstract

The invention discloses a high-flux zebra fish behavior analysis device and method, and aims to solve the problem of compatibility of plate edge distortion and a multi-type pore plate, a telescopic mechanism is arranged in a shell of the device, an isolation cavity is formed in the shell when the telescopic mechanism is retracted, a micro-lens array and a light filter plate are sequentially arranged in the isolation cavity from top to bottom, and the micro-lens array and the light filter plate are arranged in the shell. A stimulation module is arranged at the bottom of the light filter plate, a pore plate placement area is arranged between the stimulation module and the light filter plate, and a universal adaptive bracket for accommodating a standard pore plate and a non-standard pore plate is arranged in the pore plate placement area; a camera module comprising at least one high-frame-rate camera with a fixed focal length is correspondingly arranged above the porous plate placing area, the camera module is connected with a control and analysis module, distortionless track diagrams, heat diagrams and behavior parameters are output online, and the optical mechanical integrated distortion correction and porous plate combined universal adaptation structure is used for correcting the distortion of the porous plate. When the same set of light path is switched between standard plates and non-standard plates of different models, the distortion of the edge view field is automatically compensated.

Description

Technical Field

[0001] This invention relates to the field of animal behavior analysis technology, specifically to a high-throughput zebrafish behavior analysis device and method. Background Technology

[0002] In high-throughput behavioral studies of zebrafish, standard multi-well cell culture plates, as well as non-standard plates such as social plates, cross mazes, and linear mazes, are widely used. However, existing imaging systems all employ a fixed-focal-length top-mounted camera with a flat base plate. When the field of view needs to cover the entire plate, the culture wells located at the edges, being in areas of maximum lens curvature and light refraction, experience significant barrel or trapezoidal distortion. This results in zebrafish trajectory position errors >0.5 mm and velocity calculation deviations >10%, directly impacting the reliability of experiments requiring extremely high data accuracy, such as circadian rhythm and drug screening.

[0003] To mitigate distortion, the conventional approach is to perform post-processing pixel coordinate correction in software. However, different plate types have significant differences in aperture spacing and diameter, requiring the creation of separate calibration tables for each type of plate. This process is cumbersome and cannot eliminate aberrations introduced by the optical system itself. For example, Chinese patent CN116171920B, which relates to a high-throughput zebrafish behavior analysis device and method, is insufficient to meet the needs of conventional 6- or 12-well plates, as well as non-standard plates such as linear mazes, cross mazes, and social plate types. Another approach is to reduce the field of view and perform segmented shooting, but this sacrifices the high-throughput advantage of "imaging the entire plate at once." Furthermore, existing equipment brackets are mostly customized for a single plate type. Changing plate types requires manual adjustment of the focal length and recalibration, and sometimes even results in physical installation problems between different plate types, creating a closed "one machine, one plate" system that severely restricts the flexibility of sharing equipment across multiple laboratory scenarios.

[0004] Therefore, how to achieve both "full-field one-time high-definition acquisition" and "plug-and-play multi-orifice plates" in the same imaging optical path, and fundamentally correct edge distortion instead of relying on post-processing software compensation, has become a key technical bottleneck that high-throughput zebrafish behavior analysis devices urgently need to overcome. Summary of the Invention

[0005] To address the compatibility issues between edge distortion and perforated plates, this invention proposes a high-throughput zebrafish behavior analysis device and method. A replaceable microlens array correction module is embedded in the imaging optical path, forming a universal adaptable structure that integrates optical-mechanical distortion correction with perforated plates. This allows for automatic compensation of edge field distortion when switching between different standard and non-standard plates using the same optical path.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a high-throughput zebrafish behavior analysis device, wherein a telescopic mechanism is provided inside the housing, and when the telescopic mechanism is retracted, an isolation cavity is formed inside the housing. A microlens array and a filter plate are arranged sequentially from top to bottom in the isolation cavity. A stimulation module is provided at the bottom of the filter plate. A perforated plate placement area is provided between the stimulation module and the filter plate. The perforated plate placement area is provided with a universal adapter bracket for accommodating standard and non-standard perforated plates. A camera module containing at least one high frame rate camera with a fixed focal length is correspondingly arranged above the perforated plate placement area. The camera module is connected to a control and analysis module, which outputs distortion-free trajectory maps, heat maps, and behavioral parameters online.

[0007] In this technical solution, a drawer-type isolation cavity structure is combined with Fresnel distortion compensation, multi-stimulus synchronization, online deep learning trajectory segmentation and velocity grading coloring to achieve high-throughput parallel acquisition of zebrafish, distortion-free imaging, and real-time output of trajectory maps and heat maps. It takes into account throughput, accuracy and multimodal stimulation, and completes large-scale behavioral quantification in one go.

[0008] Preferably, the stimulation module includes a light stimulation unit, a vibration unit, an electrical stimulation unit, and a sound stimulation unit. Each stimulation unit is independently controlled and synchronized with the camera module via a hardware synchronization line. The light stimulation unit includes a light stimulation component positioned above the perforated plate placement area and a bottom light source located on the lower layer of the filter plate.

[0009] Preferably, the universal adapter bracket is compatible with 6, 12, 24, 48, and 96-hole standard boards and non-standard boards such as social boards, cross mazes, and straight mazes within the same mechanical interface, and achieves automatic board type recognition and locking through elastic positioning pins.

[0010] Preferably, the bottom light source is an LED side-emitting array that integrates white light and infrared light, which can be switched and provides uniform background light.

[0011] Preferably, the microlens array is composed of Fresnel lenses, the focal length of which is adjustable to eliminate barrel distortion at the edge of the standard orifice plate.

[0012] Preferably, the housing is equipped with a temperature control module, including a heating module, a heat dissipation module and a temperature probe, and the ambient temperature is kept constant at 28℃±0.5℃ through PID control. The housing on the side of the temperature control module is equipped with a temperature control module air duct.

[0013] Preferably, the housing is made of opaque and sound-insulating material, and the side of the housing is provided with air ducts and temperature control module air ducts, so that a closed-loop airflow organization is formed inside the housing, so that the internal noise of the isolation cavity is less than 60dB and the temperature fluctuation range does not exceed 1℃.

[0014] Preferably, the housing is provided with a control platform, which is an interactive screen. The control platform communicates with the main control board, and the housing is provided with status indicator lights.

[0015] The present invention also adopts the following technical solution: a high-throughput zebrafish behavior analysis method, using the above-mentioned high-throughput zebrafish behavior analysis device, comprising the following steps: S1, Place the perforated plate containing zebrafish into the universal adapter bracket, automatically identify the plate type and lock the corresponding microlens array; S2, activate the bottom light source and set the lighting mode, activate the temperature control module to stabilize the ambient temperature at 28℃; S3 acquires images of the entire board, calls distortion compensation algorithms in real time, performs online instance segmentation to obtain the trajectory of each fish, and generates distortion-free trajectory maps and heatmaps in real time. S4, trigger any or a combination of stimuli in the stimulus module as needed, and record the behavioral response simultaneously; S5 performs high-throughput analysis based on the behavioral response data table and trajectory graph.

[0016] Preferably, in step S3, the trajectory is divided into three levels—low, medium, and high—in real time according to a preset speed threshold, and displayed in superimposed colors to form a customizable speed-graded trajectory map.

[0017] The beneficial effects of this invention are: 1) Introducing a microlens array as a pluggable correction unit into the zebrafish behavior imaging system, along with a drawer-type isolation cavity, eliminates edge distortion of the 96-well plate in one go, reduces the overall plate trajectory error, and allows for direct quantification without the need for subsequent software correction; 2) The dual-chip LED side-emitting array of white light and infrared light switches in 0.1 seconds, combined with an 850 nm bandpass filter, to achieve long-term recording of circadian rhythms without disturbing the animal, and to provide continuous and complete 24-hour data; 3) The four modal stimuli of light, sound, vibration and electricity are aligned with the camera frame exposure through the hardware synchronization line, which can accurately measure the latency of fright and the memory recovery time. 4) Online deep learning instance segmentation combined with GPU parallelism enables real-time processing of 1152 tail trajectories at 60 frames per second, completing data acquisition and analysis in 30 minutes, further improving throughput; 5) The velocity graded trajectory map is colored and superimposed in real time, the threshold is adjustable, what you see is what you get, eliminating the need for offline secondary processing and significantly shortening the experimental iteration cycle. Attached Figure Description

[0018] Figure 1 This is a front view of the device in Embodiment 1 of the present invention.

[0019] Figure 2 This is a left view of the device in Embodiment 1 of the present invention.

[0020] Figure 3 This is a partial structural schematic diagram of the device in Embodiment 1 of the present invention.

[0021] Figure 4 This is a front view of the device in Embodiment 1 of the present invention, excluding the housing.

[0022] Figure 5 This is a schematic diagram of the device in Embodiment 1 of the present invention, excluding the housing.

[0023] Figure 6 This is a schematic diagram of the device in Embodiment 1 of the present invention, excluding the housing and telescopic mechanism.

[0024] Figure 7 This is a top view of the device in Embodiment 1 of the present invention, excluding the housing and telescopic mechanism.

[0025] Figure 8 This is a schematic diagram of the structure of the device control area in Embodiment 1 of the present invention.

[0026] Figure 9 This is a left view of the control area of ​​the device in Embodiment 1 of the present invention.

[0027] Figure 10 This is a right view of the device control area in Embodiment 1 of the present invention.

[0028] Figure 11 This is a flowchart of the method in Embodiment 2 of the present invention.

[0029] Reference numerals: 1: Control platform; 2: Status indicator light; 3: Telescopic mechanism; 4: Air duct; 5: Temperature control module air duct; 6: RJ45 interface; 7: USB interface; 8: Power interface; 9: Camera module; 10: Heat dissipation module; 11: Heating module; 12: Photostimulation component; 13: Temperature probe; 14: Fresnel lens; 15: Filter plate; 16: Bottom light source; 17: Vibration unit; 18: Vibration module control board; 19: Relay; 20: Power supply device; 21: Main control board; 22: USB expansion dock. Detailed Implementation

[0030] Example 1 This embodiment provides a high-throughput zebrafish behavior analysis device, such as... Figure 1-10 As shown, it includes a vertical darkroom housing, with an electric drawer-type telescopic mechanism 3 located in the lower center of the front of the housing; when the drawer is fully pushed in and closed, an isolation cavity is formed inside the housing that is isolated from external light and sound.

[0031] In this embodiment, a high-throughput zebrafish behavior analysis device is a vertical integrated machine. The shell is made of opaque and sound-insulating material. The interior is divided into four functional spaces: "imaging area", "stimulation area", "control area" and "environment maintenance area". Each functional space is connected by air duct 4 and temperature control module air duct 5 to form an independent and closed-loop airflow organization, ensuring that the internal noise is <60 dB and the temperature fluctuation is ±1℃.

[0032] The top front of the device features a 15° tilted control platform 1, which integrates a 10.1-inch capacitive touchscreen, an embedded industrial computer, and a physical start / stop button. The control platform 1 communicates bidirectionally with the main control board 21 via an internal USB expansion dock 22, enabling it to send experimental protocols, preview 12 camera feeds in real time, and export data.

[0033] A status indicator light 2, consisting of a three-color LED ring array, is located below the control platform 1. A solid green indicator light indicates that the system is ready; a flashing blue indicator light indicates that data acquisition is in progress; and a solid red indicator light indicates a fault alarm, such as over-temperature, camera disconnection, or short circuit in the stimulation module. The status indicator light 2 is directly connected to the GPIO of the main control board 21, with a response time of <200 ms.

[0034] In this embodiment, the telescopic mechanism adopts a drawer-type sample loading mechanism.

[0035] It adopts linear guide rails and servo motor drive, with a stroke of 250mm and a repeatability of ±0.05mm. The drawer-type sample loading mechanism has an embedded interchangeable plate holder, which is compatible with standard cell culture plates of 6, 12, 24, 48, and 96 wells as well as non-standard plates such as "cross maze" and "social plate"; the four corners of the holder are equipped with spring buckles to achieve quick 5-second replacement.

[0036] After the drawer-type sample loading mechanism is closed, it forms a light-tight seal with the dark box through a magnetic sealing strip, preventing stray light from entering the imaging area.

[0037] The isolation chamber contains a microlens array and a pluggable filter plate arranged horizontally from top to bottom. The microlens array is composed of several threaded Fresnel lenses, and the focal length of each lens can be finely adjusted within the range of 80-120 mm to compensate for the barrel distortion caused by the curvature of the lens at the edge of the 96-aperture plate.

[0038] The filter plate adopts a pull-out guide rail design, which can quickly replace the visible light cutoff, infrared cutoff or neutral density filter according to experimental needs, so as to achieve spectrum switching without disassembling other components.

[0039] Below the filter plate is the well plate placement area. A universal adapter bracket is embedded in the center of the drawer's bottom plate. The bracket has elastic positioning pins and RFID readers at its four corners, which can complete the mechanical locking and plate type identification of 6, 12, 24, 48, and 96-well standard cell culture plates, as well as non-standard plates such as social plates, cross mazes, and straight mazes within 5 seconds. The identification results are sent to the main control board in real time for automatic retrieval of the corresponding coordinate mapping and distortion compensation parameters.

[0040] Below the bracket is the bottom light source of the stimulation module. In this embodiment, the bottom light source is a side-emitting high-brightness LED array, in which 5600 K white light chips and 850 nm infrared chips are alternately arranged. The high-frequency switching between white light and infrared can be completed in 0.1 seconds through a constant current driving circuit. With the help of a light guide plate and a diffusion film, the uniformity is improved to more than 92%, providing shadowless background illumination for high-speed imaging.

[0041] Specifically, the bottom light source 16 is located 15mm directly below the telescopic mechanism 3. It adopts a side-emitting high-brightness LED array, and its brightness is adjustable from 0 to 100% through PWM dimming. The uniformity is ≥92%, and the lifespan is >50,000 hours. White light and infrared light can be seamlessly switched in 0.1s to meet the requirements of circadian rhythm photoperiod experiments.

[0042] Camera module 9 is located directly above the microlens array, suspended from the top of the imaging area. The module consists of a 2×6 array of twelve 2.2 MP GigE cameras, each equipped with a global shutter and an 850 nm bandpass filter. It is connected to the main control board via a gigabit Ethernet switch, enabling synchronous acquisition at 60 fps with jitter control within 50 microseconds. A 40 mm focusing clearance is provided between the cameras and Fresnel lenses, ensuring that the resolution difference between the center and edge of each aperture on the imaging plane does not exceed 3%, thus guaranteeing subsequent trajectory accuracy.

[0043] A Fresnel lens 14 and a filter plate 15 are arranged sequentially between the camera module 9 and the aperture plate. The Fresnel lens 14 (focal length 120mm, threaded adjustable) is used to eliminate barrel distortion at the edge of the 96-aperture plate, so that the difference in resolution between the edge and the center is <3%. The filter plate 15 is a pluggable structure and can be selected from three specifications: visible light cutoff, infrared cutoff, and neutral density, to adapt to different spectral experiments.

[0044] The bottom light source frame integrates vibration units around its perimeter, consisting of four eccentric wheel motors. Electrode contacts are pre-drilled on the bottom surface of the bracket; when using the matching electrical stimulation plate, the contacts correspond one-to-one with the gold-plated electrode plates on the bottom of the plate, outputting a square wave with a pulse width of 0-5 V and a pulse width of 1-1000 ms. Two full-range speakers are also installed on the inner side of the rear wall of the housing, forming the sound stimulation unit; four linear white LED light strips are arranged on the top, with a maximum output of 8000 lux, for top light stimulation. All four types of stimulation units—light, sound, vibration, and electricity—are connected to the main control board via hardware synchronization lines, ensuring that the TTL error between the stimulation event and the camera exposure frame is less than 50 microseconds.

[0045] Specifically, the light stimulation component 12 has four linear LED light strips arranged at the top four corners of the imaging area, with a maximum of 8000 lux and a color temperature of 6500K. It is connected to the main control board 21 through a constant current drive circuit and can output 1–100Hz flickering or gradient brightness stimulation.

[0046] Vibration unit 17 consists of four eccentric wheel vibration motors evenly arranged below the base plate of telescopic mechanism 3, with a frequency of 1–200Hz and an amplitude of 0.1–1mm, and is independently closed-loop controlled by vibration module control board 18.

[0047] The electrical stimulation unit is a telescopic mechanism with a detachable electrode frame embedded in the bracket. The electrode frame contacts the electrode plate at the bottom of the orifice plate through a gold-plated spring pin, outputting a 0–5V DC square wave with a pulse width of 1–1000ms, which is switched by relay 19 to realize memory or epilepsy experiments.

[0048] In some other implementations, an auditory stimulation unit may be provided, such as two 8Ω / 5W full-range speakers with a frequency response of 200Hz–20kHz built into the rear wall of the device for auditory fright experiments.

[0049] All stimulation units and camera module 9 are connected in parallel via a hardware synchronization line (BNC) and are powered by power supply unit 20.

[0050] A temperature control module air duct is located on the lower right side of the housing, forming a closed-loop airflow of "bottom in, top out" with the general air duct on the left. The heating module 11 uses two 200 W polyimide film heating films attached to the inner wall of the housing. The heat dissipation module 10 consists of two 120 mm silent fans with a speed of 800-2000 RPM. The PT1000 temperature probe is suspended 20 mm above the orifice plate, with a sampling frequency of 2 Hz. The PID algorithm stabilizes the temperature inside the isolation chamber at 28℃±0.5℃. The closed-loop airflow removes heat while avoiding direct blowing on the liquid surface that could cause evaporation, and also keeps the noise level inside the chamber below 60 dB.

[0051] The control platform is mounted at a 15° angle on the top of the housing, integrating a 10.1-inch capacitive touchscreen, an i7 industrial PC, and a one-button start / stop function. A three-color status indicator light is located on the right side of the platform to show system readiness, data acquisition in progress, or fault alarm status. The back of the housing houses eight power interfaces, seven USB interfaces, six RJ45 interfaces, and a removable dust filter, supporting remote desktop and cluster data upload. The main control board uses an ARM+FPGA architecture, handling camera synchronization triggering, sensor acquisition, stimulation module control, and data caching. It also writes raw images in real-time to a 4 TB NVMe SSD via PCIe x4, meeting the requirements for 24-hour continuous recording.

[0052] During operation, the user places the fish-loading plate into the universal adapter bracket, pushes the drawer closed, and the device automatically completes plate type recognition, temperature and light stabilization, multi-camera synchronous acquisition, online instance segmentation, and behavioral parameter output; trajectory maps, heat maps, and 42 quantitative indicators can be exported with one click through the control platform, achieving truly high-throughput, distortion-free, multimodal zebrafish behavior analysis.

[0053] Example 2 This embodiment provides a high-throughput zebrafish behavior analysis method, which is performed within a high-throughput zebrafish behavior analysis device described in Embodiment 1, with reference to... Figure 11 This includes the following steps.

[0054] Step S1: Before starting the work, simply place the perforated plate containing the zebrafish fry in the perforated plate placement area and close the drawer. The elastic positioning pins at the four corners of the universal adapter bracket will quickly clamp the edge of the plate. At the same time, the RFID reader will read the electronic tag on the bottom of the plate. Based on this, the main control board will determine that the plate type is a 96-hole standard plate and then call the corresponding Fresnel lens focal length and distortion compensation coefficient to complete the mechanical and optical dual locking.

[0055] The system then proceeds to step S2, where the bottom light source first outputs 200 lux white light for 30 seconds to allow the fish to adapt to the environment. The PID temperature control loop, linked to the thin-film heating film and the silent fan, continuously monitors and adjusts the temperature inside the isolation chamber, stabilizing it at 28℃±0.5℃ within 10 seconds. The status indicator light changes from red to green, indicating that data collection can begin.

[0056] In step S3, 12 GigE cameras are simultaneously exposed at 60 fps under the synchronization pulse of the FPGA on the main control board. The original images are directly written to memory via the PCIe channel. The embedded instance segmentation model on the GPU performs frame-by-frame calculations to extract the coordinates of the center point of each fish and corrects the barrel error in real time according to the pre-calibrated distortion lookup table to generate a distortion-free trajectory.

[0057] Meanwhile, the software divides instantaneous velocity into three levels—low, medium, and high—based on user-preset thresholds: trajectory points below 4 mm / s are marked in black, 4–20 mm / s in green, and above 20 mm / s in red. The three-color vectors are superimposed on the video stream in real time to form a velocity-graded trajectory map. The same data stream is also used to calculate 2D kernel density and output the corresponding heatmap. The entire process has a latency of less than 200 ms, achieving true online analysis.

[0058] Step S4: If the experimental design requires external stimulation, the main control system first sends a pre-trigger to the camera via a hardware synchronization line, and then immediately starts the corresponding stimulation unit: for example, the top light bar outputs an 8000 lux white light pulse lasting 2 seconds, or a vibration motor provides mechanical stimulation with a 100 Hz amplitude of 0.5 mm; the stimulation start and end times are written to the image metadata in the form of TTL pulses to ensure that the event corresponds to the frame number with zero error. After the stimulation ends, the system continues to collect recovery behavior for no less than 5 minutes to calculate the latency and recovery rate.

[0059] Step S5: The software automatically summarizes 42 parameters for each hole, including total swimming distance, low-speed percentage, high-speed bursts, peak coordinates of the heat map, stimulus response latency, etc., along with the speed-graded trajectory. Figure 1 The data is then written into CSV and PDF reports; users can export the data with one click through the control platform, or upload it to a local area network database via RJ45, completing a high-throughput behavioral analysis of 1152 zebrafish on the entire plate.

[0060] This invention deeply integrates mechanical, optical, electronic, and software technologies with biological experimental procedures, resulting in a significant improvement in efficiency. Traditional manual recording or low-throughput equipment can often only observe a few dozen zebrafish simultaneously, requiring researchers to time, count, export videos, and then analyze them frame by frame using third-party software. A complete dose-response curve often takes several weeks.

[0061] This invention employs a drawer-type automatic loading system and a twelve-camera array, capable of accommodating twelve 96-well plates at a time, with a theoretical peak of 1152 individuals recorded simultaneously. Combined with FPGA hardware synchronous triggering and GPU online instance segmentation, it can complete image acquisition, trajectory calculation, heatmap generation, and parameter output for the entire plate within 30 minutes, truly achieving an analytical throughput of thousands of individuals per hour. For new drug screening or toxicity evaluation, this speed means that effective concentration ranges can be quickly identified early on, reducing the number of ineffective compounds entering subsequent validation and significantly lowering R&D costs.

[0062] Image quality is the foundation of the credibility of behavioral quantification and is also the area where traditional devices are most prone to compromise. Common USB 2.0 cameras on the market can only maintain 15 frames per second at 640×480 resolution, and their fixed lenses require the entire tripod to be moved if the working distance changes, easily introducing mechanical errors. This invention uses a GigE interface camera, which can continuously output 60 frames per second at full resolution of 1920×1080, with ample bandwidth margin to ensure that even high-speed escapes or subtle twitches of zebrafish can be clearly captured.

[0063] More importantly, the GigE protocol supports 100-meter cabling and Power over Ethernet, with multi-camera synchronization jitter of less than 50 microseconds, laying the hardware foundation for subsequent frame-level data fusion. The camera front end is also equipped with a replaceable 850nm bandpass filter, allowing only infrared light to enter during nighttime or dark periods of recording, which meets the requirements of circadian rhythm experiments and avoids additional interference from visible light on animal behavior.

[0064] Long-term operation poses challenges to heat dissipation and stability. This invention directly attaches the camera housing to the thermal pad on the top of the darkroom, and creates negative pressure to dissipate heat through the air duct. After 24 hours of continuous operation, the body temperature rises by less than 8 degrees Celsius, and the image noise remains at the original level, ensuring data consistency.

[0065] Optical distortion often leads to deviations in the trajectory of edge apertures, resulting in systematic errors in speed and rotation angle calculations. This invention inserts a Fresnel lens array between the camera and the aperture plate. The lens surfaces provide reverse compensation for the field of view at the edge of the 96-aperture plate, reducing the actual resolution difference between the center and outermost apertures within the same plate to less than 3%.

[0066] Meanwhile, the lens frame and filter share a sliding rail, allowing experimenters to switch between visible light cutoff, infrared cutoff, or neutral density filters within 30 seconds without refocusing or calibration, greatly expanding the flexibility of spectral experiments. The bottom light source uses a side-emitting method, with the LED chip closely attached to the side surface of the light guide plate. After multiple internal reflections, the light evenly overflows from the top surface, forming a background light field with a brightness difference of less than 8%. Even when shooting high-contrast silhouettes, there will be no local overexposure, providing high signal-to-noise ratio images for subsequent contour recognition.

[0067] The richness and compatibility of the stimulation modules are another significant advantage of this invention compared to single-function monitors. Different models, such as optogenetics, startle response, and learning / memory, have varying requirements for stimulation morphology. This invention integrates five methods—top light stimulation, bottom light switching, vibration, sound, and electrical stimulation—into a single cavity, each with its own independent closed-loop drive. Taking electrical stimulation as an example, traditional methods require inserting large electrodes into the fish tank, which easily creates dead zones in the electric field.

[0068] In addition, the present invention patterns the electrodes and plates them with gold on the bottom shell of the perforated plate. Each hole forms a ring-shaped microelectrode. When the drawer is pushed in, the spring pin automatically presses it in place. The electric field covers the entire liquid surface. Only 3-5 volts are needed to generate a uniform current density in the hole, which ensures the intensity of stimulation and avoids the precipitation of bubbles and metal ions.

[0069] All stimulation units are connected in parallel with the camera trigger signal via a hardware synchronization line, ensuring that the time difference between the stimulation start edge and the first frame exposure edge is less than 1 millisecond. Researchers can use this to accurately calculate the response latency without having to align the timeline afterward.

[0070] The temperature control module may seem conventional, but it is the key to recording success over a long period of time.

[0071] Zebrafish exhibit significant changes in metabolic rate within the temperature range of 26-30 degrees Celsius; a temperature drift of 1 degree Celsius can cause a difference in swimming distance of more than 10%.

[0072] This invention suspends the PT1000 platinum resistance probe 20 mm directly above the orifice plate. This position can represent the surface temperature of the water without being bumped by fish. The PID algorithm samples twice per second, and the proportional-integral-derivative parameters have been calibrated through a large number of long-term experiments. It can restore the temperature to the set value within 180 seconds after the door is opened to take or put in the sample, with an overshoot of less than 0.2 degrees Celsius.

[0073] The heating film is made of polyimide material with a thickness of only 0.15 mm. It can be bent and attached to the inner wall to achieve surface heating instead of point heating, thus avoiding local hot spots. The cooling fan adopts PWM linear speed regulation, and the speed change is proportional to the temperature error, reducing start-up and shutdown noise.

[0074] After the system ran continuously for 7 days, the temperature standard deviation remained within 0.3 degrees Celsius, providing reliable environmental consistency for longitudinal comparison experiments.

[0075] Traditional threshold segmentation is prone to missing detections when fish overlap or lighting is uneven. This invention employs a deep learning instance segmentation network, trained separately for a 96-well plate scene. Data augmentation includes random halos, water ripples, reflections, and particle noise, making the model robust to changes in the real aquatic environment. Inference on a GPU takes only 18 milliseconds per frame, and parallel streaming processing with 12 cameras can meet the real-time requirement of 60 frames per second. After the network outputs a fish mask, the major axis of an ellipse is further fitted to obtain the head orientation, providing angular parameters for studying social interaction or phototaxis.

[0076] The velocity grading module allows users to adjust thresholds online, and the system instantly redraws trajectory colors without needing to rerun offline scripts. This WYSIWYG approach significantly reduces trial-and-error time. All trajectories, heatmaps, and indicators are stored in HDF5 format, supporting append writing and ensuring data continuation even after power outages. It also outputs CSV and PDF reports, which can be directly used for submission to high-impact journals, reducing the post-processing burden on researchers.

[0077] Long-term reliability and consistency are important dimensions for evaluating the investment value of instruments. All moving parts in this invention adopt a stepper motor and linear guide rail scheme. After 50,000 opening and closing tests, the positioning accuracy of the drawer is still better than 0.05 mm. A shock-absorbing gasket is added between the camera and the lens to prevent image blurring caused by motor vibration. The electronic components are selected to operate in a temperature range of -25 to 85 degrees Celsius, and the gold plating thickness of key connectors reaches 30 microinches to ensure no oxidation in high humidity environments.

[0078] The entire device underwent 72 hours of continuous high-temperature and high-humidity aging, followed by a 12-hour random vibration test, and then returned to room temperature for functional testing. The trajectory error and noise level remained consistent with those before aging. For longitudinal experiments requiring repeated measurements, such as chronic drug administration or aging studies, this stability means that data from different batches can be directly compared without additional correction factors, thereby improving the reliability of the conclusions.

[0079] In summary, this invention achieves a comprehensive breakthrough in four dimensions—through deep coupling of engineering and biology—in terms of throughput, it enables simultaneous analysis of over a thousand tails; in terms of accuracy, it controls the marginal error to within 3%; in terms of flexibility, it allows for arbitrary combinations of five stimulus methods; and in terms of stability, it guarantees 24 / 7 unattended operation.

[0080] For basic neural mechanism research, researchers can set up multiple gene mutation groups and drug treatment groups on the same plate, and obtain sufficient statistical power in a single experiment; for the pharmaceutical industry, the early toxicity screening cycle is reduced from several months to several days, significantly reducing the risk of failure in the later stages; for environmental toxicology, large-scale experiments with multiple concentrations, multiple time points, and multiple repetitions can be completed in one device, reducing the space occupied and the number of operators.

Claims

1. A high-throughput zebrafish behavioral analysis apparatus, characterized by, The shell is provided with a telescopic mechanism. When the telescopic mechanism is retracted, an isolated cavity is formed in the shell. A microlens array and a filter plate are sequentially arranged in the isolated cavity from top to bottom. The bottom of the filter plate is provided with a stimulation module. A hole plate placement area is arranged between the stimulation module and the filter plate. The hole plate placement area is provided with a universal adapter cradle for accommodating standard hole plates and non-standard hole plates. A camera module comprising at least one high-frame-rate camera with a fixed focal length is arranged above the hole plate placement area. The camera module is connected with a control and analysis module, and outputs online distortion-free trajectory maps, heat maps and behavior parameters.

2. The high-throughput zebrafish behavior analysis device according to claim 1, wherein, The stimulation module comprises a light stimulation unit, a vibration unit, an electric stimulation unit and a sound stimulation unit, which are independently controlled and synchronized with the camera module through a hardware synchronization line. The light stimulation unit comprises a light stimulation assembly arranged at a corresponding position above the hole plate placement area and a bottom light source arranged at the lower layer of the filter plate.

3. The high-throughput zebrafish behavior analysis apparatus according to claim 1, wherein, The universal adapter cradle is compatible with 6, 12, 24, 48 and 96 standard plates and social plates, cross mazes and non-standard plates in the same mechanical interface, and realizes automatic identification and locking of the plate type through elastic positioning pins.

4. The high-throughput zebrafish behavior analysis apparatus according to claim 2, characterized in that, The bottom light source is a LED side light array integrating white light and infrared light, which can switch and provide uniform background light.

5. The high-throughput zebrafish behavior analysis apparatus according to claim 1, wherein, The microlens array is composed of a Fresnel lens, and the focal length of the Fresnel lens is adjustable to eliminate the barrel distortion of the edge of the standard hole plate.

6. The high-throughput zebrafish behavior analysis apparatus according to claim 1, wherein, The shell is provided with a temperature control module comprising a heating module, a heat dissipation module and a temperature probe. The environmental temperature is kept constant at 28℃±0.5℃ through PID control. A temperature control module air duct is arranged on the side of the shell of the temperature control module.

7. The high-throughput zebrafish behavior analysis apparatus according to claim 1 or 6, characterized in that, The shell is made of light-tight and sound-proof material. The shell is provided with an air duct and a temperature control module air duct, so that a closed-loop air flow is formed in the shell, the internal noise of the isolated cavity is lower than 60dB, and the temperature fluctuation range is not more than 1℃.

8. The high-throughput zebrafish behavior analysis apparatus according to claim 1, wherein, A control platform is arranged on the shell. The control platform is an interactive screen. The control platform communicates with a main control board. The shell is provided with a state indicator light.

9. A high-throughput zebrafish behavior analysis method, using the high-throughput zebrafish behavior analysis device according to any one of claims 1-8, characterized in that, The method comprises the following steps: S1. Put the hole plate containing zebrafish into the universal adapter cradle, automatically identify the plate type and lock the corresponding microlens array; S2. Start the bottom light source and set the light mode. Start the temperature control module to stabilize the environmental temperature at 28℃; S3. Collect the whole plate image, call the distortion compensation algorithm in real time, obtain the trajectory of each fish through online instance segmentation, and generate distortion-free trajectory maps and heat maps in real time; S4. Trigger any or combined stimulation in the stimulation module as needed, and record the behavior response synchronously; S5. Perform high-throughput analysis according to the data table and trajectory map of the behavior response.

10. The high-throughput zebrafish behavior analysis method according to claim 9, wherein, In step S3, the trajectory is divided into three grades of low, medium and high according to a preset speed threshold, and is displayed in different colors, forming a self-definable speed grading trajectory map.

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