Intelligent nematode regulating and monitoring system

Abstract

The invention discloses an intelligent nematode regulating and monitoring system, which mainly covers a physical induced movement system and a nematode movement detection system, and the physical induced movement system and the nematode movement detection system realize collaborative operation by means of the same set of matched software. The set of matched software has two core functions of regulation and monitoring, a physical induction movement system and a nematode movement monitoring system are enabled respectively, and efficient management and control and monitoring of the whole system are achieved. On one hand, analysis of an influence mechanism of physical stimulation on nematodes is facilitated; on the other hand, due to the important value of the nematodes in the fields of medicine research and development, neuroscience and the like, the application range of the device can be expanded. In the field of drug research and development, the nematode model can be used for simulating a human body environment, evaluating drug activity and screening life-prolonging drugs; in the neuroscience research, the research on the relationship between a nervous system and movement and the pathogenesis of neurodegenerative diseases can be assisted; in the aspect of senescence research, senescence indexes of the nematodes are regulated, stimulated and monitored, and a senescence delaying method is explored.

Description

Technical Field

[0001] The present invention relates to the technical field of nematode monitoring, and more specifically, to an intelligent nematode control and monitoring system. Background Art

[0002] Due to its short life cycle, rapid reproduction rate, strong genetic homology, and quantifiable movement behavior, Caenorhabditis elegans has become an important carrier for studying exercise intervention, aging mechanisms, and drug screening.

[0003] However, traditional physical induction experiments face three major technical difficulties: First, the single stimulation method. Existing methods are limited to single stimulation forms such as swimming or mechanical vibration, which makes it difficult to simulate the multi-dimensional interactive effects of complex environments on nematodes; second, the dependence on manual operation. Experimental parameters rely on manual adjustment (such as voltage and temperature), resulting in poor repeatability (error rate as high as 15%-30%) and inability to dynamically respond to real-time data changes; third, data collection is scattered. The collection of motion parameters requires switching between multiple tools (such as ImageJ to measure body length and manual recording of trajectories), lacks automated integration, and it is difficult to generate systematic charts (such as correlation analysis of multiple stimulation conditions). Summary of the Invention

[0004] The present invention provides an intelligent nematode control and monitoring system to solve the problems of the existing intelligent nematode control and monitoring system in terms of single stimulation means, strong dependence on manual operation and scattered data collection.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] An intelligent nematode control and monitoring system includes a physical induction motion system and a nematode motion detection system. The physical induction motion system consists of software one, an intelligent control power supply, and a physical induction motion platform; the nematode motion detection system consists of software two, nematode motion imaging, and a motion observation platform.

[0007] Preferably, the physical induction motion platform includes an electrical induction platform, an ultrasound induction platform, a light stimulation induction platform, a thermal induction platform and a mechanical vibration induction platform.

[0008] Preferably, the software 1 is a hybrid architecture based on LabVIEW and Python / Java. The software 1 can control the physical induced motion system and connect the power supply hardware via the RS-485 bus. The software 1 can perform real-time status monitoring and feedback the power supply status of each platform, including voltage and current values.

[0009] Preferably, the intelligent control power supply is composed of a DC power supply with a continuously adjustable 0-40V and a maximum output current of 2A and an FPGA main control unit, which meets the power requirements of each physical induced motion module and realizes the control and management of various parameters of the power supply.

[0010] Preferably, the electric induction platform includes a circular model, which is made of polymethyl methacrylate. The outer diameter of the circular model is 100 mm and the thickness is 5 mm. Two rectangular nematode placement grooves with a spacing of 10 mm, a length of 20 mm, a width of 10 mm, and a depth of 2 mm are opened in the center of the model. A circular hole with a diameter of 2 mm and a depth that penetrates the thickness of the model is opened on both sides of the nematode placement groove. A platinum rod or a graphite rod is passed through the circular hole.

[0011] Preferably, the ultrasound induction platform is composed of an ultrasound generating module, a transducer focusing system, a microenvironment sample chamber and a behavior analysis unit;

[0012] The ultrasonic generation module can generate an adjustable frequency signal in the range of 20kHz to 3MHz through a high-precision signal generator, and drive the piezoelectric ceramic transducer through a power amplifier to convert the electrical signal into mechanical ultrasonic waves;

[0013] The transducer focusing system includes a concave acoustic lens or a parabolic reflector, which can focus the sound waves into a tiny area of 0.5–1 mm;

[0014] The microenvironment sample chamber is designed using a PDMS or agarose microfluidic chip. A channel is opened inside the microenvironment sample chamber. The channel has a size of 500 μm wide and 100 μm high. The top of the channel is covered with a 50-100 μm thick sound-transmitting film, and deionized water or pre-degassed ultrasound gel is used as a coupling medium.

[0015] The behavioral analysis unit includes an inverted microscope and a high-speed camera. The behavioral monitoring module uses TrackMate or OpenCV algorithms to track the nematode's movement trajectory in real time. The behavioral monitoring module can monitor parameters such as speed, steering angle, and pharyngeal pumping frequency. At the same time, the sample temperature is monitored using a micro-thermocouple and an infrared thermal imager to ensure that the temperature rise throughout the experiment is ≤2°C.

[0016] The nematode fixing area includes a sample fixing frame arranged in the ultrasonic propagation medium tank, and the fixing frame is made of flexible materials such as silica gel.

[0017] Preferably, the light stimulation induction platform includes a light source system, a light path transmission component, and a nematode placement chamber; the light source system includes a high-brightness LED light source that can emit visible light in the wavelength range of 400-700nm; the light intensity of the LED light source is adjustable in the range of 0-500μW / mm 2 ;

[0018] The optical transmission component includes a set of collimating lenses and optical fibers, which are used to collimate the LED light source and transmit light respectively. The end of the optical fiber is connected to a spot diffuser with an adjustable angle.

[0019] The nematode placement chamber is made of transparent quartz glass, has good light transmittance and can tolerate temperature changes, and a light sealing cover is provided on the top of the nematode placement chamber.

[0020] Preferably, the temperature adjustment range of the thermal induction platform is 10-40°C. The thermal induction platform includes a heating element, which is a square thin film heating plate with a side length of 50 mm and a thickness of 0.2 mm. The heating element is connected to the intelligent control power supply through a wire. The thermal induction platform is wrapped with polystyrene foam insulation material; a cooling fan is installed on one side of the thermal induction platform.

[0021] Preferably, the mechanical vibration induction platform includes an M12-24V eccentric vibration motor, and the vibration frequency range of the eccentric vibration motor is 10-100Hz; the mechanical vibration induction platform is provided with a vibration transmission and amplification device composed of a spring and a lever, the spring elastic coefficient and the lever length ratio are adjustable, and the amplitude adjustment range is 0.01-0.1mm; the shell of the mechanical vibration induction platform is made of medical grade polycarbonate, the outer diameter of the mechanical vibration induction platform is 150mm, the inner diameter is 100mm, and the height is 40mm, which is suitable for a 90mm culture dish, and three holes with a diameter of 10mm and a 120° distribution are opened on the side wall of the mechanical vibration induction platform, and the vibration transmission and amplification device is in contact with the culture dish through the holes.

[0022] Preferably, the second software integrates ImageJ and OpenCV and uses CNN and SVM algorithms; the second software is connected to a microscope and a CCD camera, and the second software displays the image in real time;

[0023] The left side of the software's second operating interface displays the nematode position and number in real time, the middle table displays morphological parameters such as body length and body width accurate to two decimal places, and the right side plots the movement trajectory and displays parameters such as speed;

[0024] The second software uses a machine learning model to analyze images, identify nematode behavior patterns, and display changes on a timeline, with different color icons corresponding to different behaviors.

[0025] Preferably, the nematode motion imaging system comprises a microscope and a CCD camera, and the CCD camera is connected to a computer via a Camera Link interface.

[0026] Preferably, the motion observation platform includes active motion observation and physically induced motion observation, wherein the active motion observation includes recording motion parameters, analyzing the individual motion differences of nematodes under different developmental and nutritional conditions, and classifying motion patterns using machine learning algorithms.

[0027] Preferably, the physical induced motion observation records the nematode motion after application of physical stimulation such as electricity or ultrasound, including changes in the nematode motion direction and speed under electrical stimulation, and changes in the nematode motion trajectory under ultrasonic stimulation.

[0028] The principle and beneficial effects of this technical solution:

[0029] (1) The present invention can realize multi-module physical induction. The physical induction motion platform provided in the present invention integrates electric, ultrasonic, optical, thermal and mechanical vibration platforms; the electric platform uses a polymethyl methacrylate model to adjust the 0-40V electric field; the ultrasonic platform can generate an adjustable frequency signal in the range of 20kHz to 3MHz, and drive the piezoelectric ceramic transducer through a power amplifier to convert the electrical signal into a mechanical ultrasonic wave; the optical platform emits 400-700nm light and accurately adjusts the light intensity; the thermal platform relies on a thin film heater and a thermistor to achieve 10-40℃, ±0.1℃ temperature control; the mechanical vibration platform simulates natural disturbances, adjusts the frequency to 10-100Hz and the amplitude to 0.01-0.1mm; it is used to regulate the nematode environment, monitor its behavior and physiological changes, and assist in the screening of anti-aging drugs.

[0030] (2) The present invention can realize full-process automated control, constructing a closed loop with the RS-485 bus and FPGA main control unit; the software adopts a hybrid architecture of LabVIEW and Python / Java, connects the computer and power hardware via RS-485, regulates the 0-40V, 2A DC power supply, and can set parameters, remotely and in real time; the software interface is intuitive, with safety and status monitoring functions; automation reduces the experimental error to within 5%, and dynamic response ensures stable and efficient experiments.

[0031] (3) Software 2 provided in the present invention integrates software such as ImageJ and OpenCV as well as CNN and SVM algorithms to process microscope images. On the one hand, it can reduce noise, adjust contrast, identify the position of nematodes, and measure morphological and movement parameters. On the other hand, it can filter data according to date and experimental conditions, generate various charts, and realize cross-experimental comparison, providing accurate data for nematode research and helping to discover patterns. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the intelligent nematode movement monitoring and control platform (A) and nematode speed analysis (B);

[0033] Figure 2 This is a schematic diagram of the contraction frequency of the nematode pharyngeal pump;

[0034] Figure 3 This is a schematic diagram of the nematode body length measurement;

[0035] Figure 4 This is a schematic diagram comparing the lipofuscin content in different groups of nematodes;

[0036] Figure 5 This is a table recording experimental data of nematode body length, pharyngeal pump contraction, and lipofuscin quantitative indicators;

[0037] Figure 6 This is a graph showing the change in the number of nematodes surviving over time under different stimulation conditions;

[0038] Figure 7 This is a statistical chart comparing the number of pharyngeal pumps of nematodes under experimental conditions;

[0039] Figure 8 This is the recording diagram of nematodes in the experimental group;

[0040] Figure 9 This is the recording image of nematodes in the control group;

[0041] Figure 10 This is a statistical chart of the nematode body length comparison;

[0042] Figure 11 This is a graph recording the fluorescence intensity of nematodes;

[0043] Figure 12 This is a diagram showing the analysis of the movement behavior of Caenorhabditis elegans under electrical stimulation;

[0044] Figure 13 This is a simplified diagram of the composition of the device; DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0046] Example 1:

[0047] An intelligent nematode movement monitoring and control platform explores the effects of vibration and electrical stimulation on nematode lifespan.

[0048] (1) Experimental purpose

[0049] Using an intelligent nematode movement monitoring and control platform, we studied the effects of vibration stimulation and electrical stimulation on nematode lifespan, compared the survival of nematodes under different stimulation conditions, and combined with indicators such as body length, pharyngeal pump contraction, and lipofuscin quantification to provide comprehensive data support for nematode movement intervention-related research.

[0050] (2) Experimental materials

[0051] ① Caenorhabditis elegans: Select nematode larvae of the same period with good growth status and consistent developmental stage.

[0052] ② Intelligent nematode movement monitoring and control platform: including mechanical vibration induction platform and electrical induction platform, as well as supporting monitoring system. The mechanical vibration induction platform uses a small eccentric vibration motor of model M12-24V as the vibration source ( Figure 1 ); The electric induction platform is made of polymethyl methacrylate (PMMA) and is made into a circular model with an outer diameter of 100 mm and a thickness of 5 mm.

[0053] ③ Nematode culture medium: standard nematode culture medium (NGM)

[0054] (3) Experimental groups

[0055] ① Control group (Group C): 85 nematodes were placed in a normal culture environment without additional vibration or electrical stimulation.

[0056] ②Vibration stimulation group (L group): A mechanical vibration induction platform was used, with the vibration frequency set to 30 Hz and the vibration amplitude set to 0.05 mm. The stimulation was performed for 60 minutes per day, divided into two sessions, each lasting 30 minutes, for a total of 85 nematodes.

[0057] ③Electrical stimulation group (M group): Using an electrical induction platform, set the voltage to 20V, the current to 1A, the stimulation time to 40 minutes each time, twice a day, for a total of 87 nematodes.

[0058] (4) Experimental process

[0059] ① Nematode culture: Different groups of nematodes were inoculated into culture dishes containing NGM medium and cultured in a constant temperature incubator at 20°C.

[0060] ② Stimulation application: Based on the group settings, the intelligent nematode movement monitoring and control platform is used to apply vibration and electrical stimulation to the nematodes in group L and group M respectively, and this lasts for the entire nematode life cycle. During the vibration stimulation, the vibration motor passes through 0.5mm 2 A 500mm long polyvinyl chloride insulated copper core wire is connected to an intelligent control power supply to generate stable vibration; during electrical stimulation, nematodes are placed in two rectangular grooves with a length of 20mm, a width of 10mm, and a depth of 2mm set in the center of the circular model of the electric induction platform. The grooves are 10mm apart, and a circular hole with a diameter of 3mm and a depth that penetrates the thickness of the model is set on both sides of the corresponding groove. A platinum rod is inserted to form a stable electric field to encourage the nematodes to move.

[0061] (5) Data Recording

[0062] Pharyngeal pump contraction: Samples were collected at a fixed time every Monday and Thursday (2 hours after stimulation). A 1-minute swallowing video (200 fps) was recorded using a Keyence VHX-7000 digital microscope. The number of pharyngeal muscle contractions was counted independently by three researchers and the average value was calculated ( Figure 2 ).

[0063] Length measurement: Fifteen nematodes were randomly selected every three days (sampled from surviving individuals), and dorsal and ventral images were taken using an Olympus CX23 microscope (40×). Body length (from head to tail) and body width (at the widest point) were measured using ImageJ software. The measurement criteria were to exclude individuals with curvature greater than 30° ( Figure 3 ).

[0064] Lipofuscin quantification: Each batch of dead nematodes was immediately transferred to M9 buffer containing 0.1% sodium azide, and photographed using a Nikon Eclipse Ti fluorescence microscope (excitation 470 nm / emission 525 nm). The mean fluorescence intensity of the intestinal region (30-70% of the body length) was analyzed by ImageJ. Figure 4 ).

[0065] Lifespan record: Observe and record the number of surviving nematodes in each group regularly every day until all nematodes die.

[0066] (6) Experimental results

[0067] Depend on Figure 5 It can be seen that:

[0068] ①Morphological data (body length)

[0069] Judging from the image data, the body length of nematodes in the control group (Group C) was relatively stable. There were differences in body length between the vibration stimulation group (Group L) and the electrical stimulation group (Group M) and the control group. The body length of the vibration stimulation group (Group L) was smaller than that of the control group. This may be because the vibration stimulation interfered with the growth and development process of the nematodes and affected their physiological activities such as cell division and elongation. The body length of the electrical stimulation group (Group M) was greater than that of the control group, indicating that electrical stimulation promoted the growth and development of nematodes. Perhaps electrical stimulation affected the signal transduction pathways in the nematodes and promoted the expression of growth-related genes.

[0070] ② Pharyngeal pump contraction data

[0071] The frequency of pharyngeal pump contractions reflects the feeding and digestion ability of nematodes. The control group (Group C) had a relatively stable pharyngeal pump contraction frequency. The pharyngeal pump contraction frequency of the vibration stimulation group (Group L) was lower than that of the control group, which means that vibration stimulation may have affected the nervous system or muscle function of the nematodes, resulting in a decrease in their feeding and digestion ability. The pharyngeal pump contraction frequency of the electrical stimulation group (Group M) was higher than that of the control group, indicating that electrical stimulation also had an effect on pharyngeal pump contraction, which may affect the contraction activity of the pharyngeal muscles by changing the cell membrane potential and interfering with the transmission of nerve signals.

[0072] ③ Lipofuscin quantitative data

[0073] Lipofuscin accumulation is one of the hallmarks of aging. The lipofuscin content in the nematodes of the control group (Group C) slowly increased over time. The lipofuscin content in the vibration stimulation group (Group L) was slightly higher than that in the control group, indicating that vibration stimulation accelerated the aging process of the nematodes. It was the oxidative stress response triggered by vibration that led to the accumulation of free radicals in the cells, thereby promoting the production of lipofuscin. The lipofuscin content in the electrical stimulation group (Group M) was lower than that in the control group and different from that in the vibration stimulation group, indicating that electrical stimulation also affects the aging of nematodes. This may be because electrical stimulation interferes with the metabolic process in the cells, causing an imbalance between the synthesis and decomposition of lipofuscin, thereby increasing the lifespan of the nematodes.

[0074] (7) Lifespan data

[0075] Depend on Figure 6 It can be seen that:

[0076] ① At the beginning of the experiment, the survival rate of all three groups of nematodes was 100%. As time went on, the survival rate of all three groups of nematodes showed a downward trend.

[0077] ②Control group (Group C): The number of surviving fish decreased relatively slowly. On the 13th day, the number of surviving fish was 78, accounting for about 89.7%; on the 18th day, the number of surviving fish was 59, accounting for about 67.8%; on the 23rd day, the number of surviving fish dropped to 0.

[0078] ③ Vibration induction group (L group): The number of surviving fish decreased relatively quickly. On the 13th day, the number of surviving fish was 76, accounting for about 89.4%. However, it decreased rapidly thereafter. On the 18th day, the number of surviving fish was 34, accounting for about 40%. On the 22nd day, the number of surviving fish had dropped to 0.

[0079] ④ Electric induction group (M group): The downward trend of the number of surviving fish was between the control group and the vibration stimulation group. On the 13th day, the number of surviving fish was 78, accounting for about 89.7%. On the 18th day, the number of surviving fish was 43, accounting for about 49.4%. On the 23rd day, the number of surviving fish dropped to 0.

[0080] (8) Analysis and discussion

[0081] Effects of stimulation on lifespan and related indicators

[0082] ① The nematodes in the vibration stimulation group (L group) had the shortest survival time. Not only was their lifespan significantly affected, but their body length, pharyngeal pumping frequency, and lipofuscin content also changed. The 30Hz vibration frequency, 0.05mm amplitude, and 60 minutes of daily stimulation may affect the nematodes' growth and development, feeding and digestion abilities, and accelerate aging, leading to a shortened lifespan, by interfering with physiological processes such as nerve conduction and muscle contraction.

[0083] ② The electrically stimulated group (Group M) had higher survival rates than the control group (Group C), and also showed changes in body length, pharyngeal pump contraction, and lipofuscin content. While the effects of electrical stimulation at 20V, 1A, and 80 minutes per day were less severe than those of vibration stimulation, it still disrupted some physiological processes in the nematodes, potentially affecting intracellular signaling and metabolism, leading to stunted growth and development, accelerated aging, and ultimately, reduced lifespan.

[0084] Example 2:

[0085] Pharyngeal pumps for drug screening using the Caenorhabditis elegans model

[0086] Experimental principle:

[0087] The pharynx of the nematode Caenorhabditis elegans (C. elegans) is a highly specialized neuromuscular organ that rhythmically contracts to facilitate feeding. Pharyngeal pump function shows a significant decline with aging, with a strong negative correlation with age, making it an ideal biomarker for studying motility mechanisms.

[0088] By observing the contraction of the pharynx of the N2 nematode under a dissecting microscope and counting the number of pharyngeal pumps within a certain period of time, the movement of the nematode can be determined.

[0089] Experimental plan: We divided the nematodes into two groups: an experimental group and a control group. The experimental group was treated with three different concentrations of drugs: L, M, and H. After a certain period of time, we observed and counted the contractions of the nematodes' pharynx under a microscope within 10 seconds.

[0090] like Figure 7 As shown, the number of pharyngeal pumping after drug treatment of nematodes was significantly increased compared with the control group, and the number of pharyngeal pumping at concentration M was the largest, indicating the strongest nematode vitality.

[0091] Evaluation conclusion:

[0092] 1. After a comparative experiment with 10 nematodes in each group, the number of pharyngeal pumps in the drug-treated group increased compared with the control group, showing an inverted V-shaped curve, among which the motility of nematodes was the highest under the M concentration.

[0093] 2. This experiment confirmed that the drug has no effect on disrupting nematode feeding.

[0094] Example 3:

[0095] Locomotor Assessment Using the Caenorhabditis elegans Model—Crawling Speed

[0096] Evaluation Principle: Caenorhabditis elegans (C. elegans) is an excellent animal model. It contains 302 neurons, and various motor phenotypes such as crawling speed are used as evaluation indicators of C. elegans' motor ability.

[0097] Furthermore, nematodes are highly sensitive to chemicals in their environment. In this study, we exposed nematodes to chemicals and measured their crawling speed to assess their locomotion. Nematode crawling traces are typically expressed as positive and negative spurs. Quantifying the number of positive and negative spurs in a nematode's crawling trajectory over a specified time period directly assesses crawling speed.

[0098] Experimental plan:

[0099] The nematodes to be tested were divided into two groups, an experimental group and a control group, wherein the experimental group took in the drug to be tested.

[0100] Transfer the nematodes to a new nematode culture medium. After the nematodes adapt to the new environment, the number of positive and negative nematodes in the nematode crawling trajectory can be quantitatively counted within a fixed time.

[0101] like Figure 8 As shown, 1. The crawling speed of nematodes in the experimental group decreased; 2. This experiment shows that the drug has an effect on the movement ability of nematodes.

[0102] Example 4:

[0103] Locomotion Assessment Using the Caenorhabditis elegans Model—Swing Frequency

[0104] Evaluation principle: Caenorhabditis elegans (C. elegans) is very sensitive to external environmental and drug stimuli. Movement behavior is a rapid evaluation indicator reflecting whether the nematode's nervous system is damaged. In the evaluation of movement ability, the two behavioral indicators of the nematode's head swing frequency and body bending frequency are often used to measure the nematode's movement ability.

[0105] Head oscillations in nematodes are driven by coordinated neuromuscular activity. When the nematode is freely moving, it oscillates its head slightly, then randomly chooses a direction to move forward. Each head oscillation is counted as one head movement, from midline to side and back to midline. The number of head oscillations within a fixed timeframe is often used to assess nematode locomotion.

[0106] Experimental plan:

[0107] The nematodes were divided into two groups: an experimental group and a control group. Both groups were N2 nematodes, and the experimental group was set to gradually increase the concentration of L, M, and H.

[0108] After a period of drug treatment, the number of times the nematode's head swung within 30 seconds was observed and recorded.

[0109] like Figure 9 As shown, 1. After a comparative experiment with 10 nematodes in each group, the head swing frequency of the nematodes treated with the drug was significantly higher than that of the nematodes in the control group; 2. This experiment shows that the drug has the effect of enhancing the movement ability of nematodes.

[0110] Example 5:

[0111] Movement Assessment Using the Caenorhabditis elegans Model—Body Length Evaluation

[0112] Experimental principle:

[0113] A key task in evolutionary biology and ecology is to explain why organisms grow to their characteristic sizes. Similarly, a complete description of an organism's development must include an explanation of how its growth and body size are regulated. During development, various genetic pathways regulating cell growth, proliferation, and apoptosis are activated appropriately to define body size and proportions, ultimately determining individual body size.

[0114] Caenorhabditis elegans (C. elegans) is an excellent model organism. It undergoes four molts (L1-L2-L3-L4-adult) and develops into an adult worm with a complete body structure. In experimental studies, drug treatments and environmental exposures may affect individual growth and development. Body length assessment can, to a certain extent, reflect changes in growth and development following drug treatment or environmental exposure.

[0115] Usually, drug-treated N2 nematodes are placed under a microscope and photographed, and then the body length of the nematodes in the image is measured using ImageJ software to assess whether the drug affects the growth and development of the nematodes.

[0116] Experimental plan:

[0117] The nematodes were divided into two groups: an experimental group and a control group. The experimental group was treated with increasing concentrations of L, M, and H. After a period of treatment, the adult nematodes were photographed under a microscope, and the body length of the nematodes in the images was measured using ImageJ software.

[0118] like Figure 10 As shown, the body length of nematodes in the drug-treated group was longer than that in the experimental group.

[0119] Evaluation conclusion:

[0120] 1. After a comparative experiment with 10 nematodes in each group, the body length of the nematodes in the drug-treated group was longer than that in the experimental group.

[0121] 2. This experiment verified that the drug treatment has an effect on the growth and development of nematodes.

[0122] Example 6:

[0123] Anti-aging evaluation using the Caenorhabditis elegans model - lipofuscin

[0124] Principle: Lipofuscin is a pigment used to assess lipid aging. This pigment is primarily composed of senescent proteins, cross-linked by lipid peroxidation products and different lipid groups. Lipofuscin accumulates during normal aging, earning it the nickname "aging pigment," and is a prominent marker of cellular aging.

[0125] The lipofuscin fluorescence (blue) of Caenorhabditis elegans (C. elegans) was observed under a fluorescence microscope. The fluorescence intensity was used to determine the lipofuscin deposition and thus the aging of the C. elegans.

[0126] Experimental plan:

[0127] We divided the tested N2 nematodes into two groups: a control group and an experimental group. The experimental group was treated with increasing concentrations of L, M, and H. After a period of treatment, the changes in the overall fluorescence intensity of the nematodes were observed.

[0128] like Figure 11 As shown, after a comparative experiment with 10 nematodes in each group, the fluorescence of the experimental group was stronger than that of the control group, which was statistically significant.

[0129] The present invention provides an intelligent nematode control and monitoring system, including a physical induced motion system and a nematode motion detection system. The physical induced motion system consists of software one, an intelligent control power supply, and a physical induced motion platform; the nematode motion detection system consists of software two, nematode motion imaging, and a motion observation platform.

[0130] The physically induced motion system consists of software (control functions), a control power supply, and a physically induced motion platform. Developed based on a hybrid LabVIEW and Python / Java architecture, the software connects the computer system to the power supply hardware via an RS-485 bus. This connection enables convenient power supply control, providing strong support for each physically induced motion platform, enabling a range of functions such as parameter setting, remote control, and real-time control. The software also collects real-time power supply operating status data, allowing operators to quickly monitor system performance and provide comprehensive technical support for the smooth conduct of experiments.

[0131] Physically induced motion platforms include electrical induction platforms, ultrasound induction platforms, light stimulation induction platforms, thermal induction platforms, and mechanical vibration induction platforms.

[0132] Software 1, based on a hybrid architecture of LabVIEW and Python / Java, controls the physical-induced motion system. Connecting to the power supply hardware via an RS-485 bus, this connection allows for convenient power control, providing robust support for various physical-induced motion platforms and enabling a range of functions, including parameter setting, remote control, and real-time control. The software also collects real-time power supply operating status data, enabling operators to monitor system performance and provide comprehensive technical support for the smooth conduct of experiments. Software 1 also provides real-time status monitoring, providing feedback on the power supply status of each platform, including voltage and current values.

[0133] Software 1 enables three functions, including automated precision control. On the electrical stimulation platform, users use a slider to adjust the voltage from 0-40V and a digital box to set the maximum current of 2A. The software automatically transmits instructions to the power supply to establish a stable electric field. On the ultrasound induction platform, the software automatically controls the transducer power supply to adjust the frequency from 20-100kHz and regulates the power supply to the focusing component, improving experimental preparation efficiency.

[0134] Multi-platform Collaboration: Researchers can pre-plan parameters for each platform in the experimental protocol module, such as parameters for different time periods for electrical stimulation and thermal induction platforms. Once the protocol is saved, the software automatically adjusts the power supply of each platform according to the preset sequence and time points when the protocol is called, simulating complex experimental environments and avoiding tedious manual operations and errors.

[0135] Real-time status monitoring: The system status monitoring page provides real-time feedback on the power status of each platform, including voltage and current values.

[0136] The intelligent control power supply consists of a DC power supply with a continuously adjustable 0-40V and a maximum output current of 2A and an FPGA main control unit. It meets the power requirements of each physical induced motion module and realizes the control and management of various power supply parameters.

[0137] like Figure 12 As shown, the circular model of the electro-inductive platform is made of polymethyl methacrylate (PMMA), with an outer diameter of 100 mm and a thickness of 5 mm. It has excellent insulation and high transparency for easy observation. Two rectangular grooves, 20 mm long, 10 mm wide, and 2 mm deep, are located in the center of the model, separated by 10 mm. These grooves serve as nematode placement areas. These grooves are smooth to minimize physical damage to the nematodes and accommodate a suitable amount of culture medium. A circular hole, 3 mm in diameter and extending through the thickness of the model, is located on either side of the corresponding nematode placement area. These holes connect directly to the nematode placement area and allow the insertion of a platinum or graphite rod, which can precisely reach the nematode's active area. This design creates a specific electric field around the nematodes, thereby influencing their locomotion.

[0138] The core design of the ultrasound intervention nematode movement device is based on controllable sound field generation and precise biological response monitoring. It mainly consists of an ultrasound generation module, a transducer focusing system, a microenvironment sample chamber, and a behavior analysis unit.

[0139] The ultrasonic generator module uses a high-precision signal generator (such as the Tektronix AFG1022) to generate an adjustable frequency signal in the range of 20 kHz to 3 MHz. The power is increased to 0.1–5 W by a power amplifier (such as the Amplifier Research 25A250A) to drive the piezoelectric ceramic transducer (PZT-4 / PZT-8), converting the electrical signal into mechanical ultrasonic waves.

[0140] The transducer focusing system includes a concave acoustic lens or parabolic reflector, which focuses the sound waves into a tiny area of 0.5–1 mm, ensuring that the energy is concentrated on the active site of the nematode (such as the head or tail);

[0141] The microenvironmental sample chamber is designed using a PDMS or agarose microfluidic chip. The channel size precisely matches the nematode body size (500 μm wide × 100 μm high). The top is covered with a 50–100 μm thick acoustically transparent film (such as Parafilm). Deionized water or pre-degassed ultrasound gel is used as the coupling medium to minimize acoustic energy attenuation.

[0142] The behavioral analysis unit integrates an inverted microscope (20× long working distance objective) and a high-speed camera (≥200fps), combined with TrackMate or OpenCV algorithms to track the nematode's movement trajectory in real time, quantifying parameters such as speed, turning angle, and pharyngeal pumping frequency. At the same time, the sample temperature is monitored using micro-thermocouples and infrared thermal imagers (such as FLIR A35) to ensure that the temperature rise throughout the experiment is ≤2°C to avoid thermal damage interference.

[0143] The application of the device requires parameter optimization and strict calibration: low-frequency ultrasound (40–100 kHz, 0.3–0.8 W / cm 2 , pulse duty cycle 10–20%) activates mechanosensitive neurons (such as TRP-4 channels) in nematodes through the non-thermal effect of mechanical force, inducing movement acceleration or turning behavior;

[0144] High-frequency focused ultrasound (1–3 MHz, 1–2 W / cm 2 , continuous exposure ≤ 30 seconds) relies on thermal effects and acoustic radiation forces to precisely inhibit specific parts (such as head muscles). Fluorescent microspheres are required to verify the focus of the sound field (accuracy ±50μm) and establish a baseline motion model.

[0145] In actual experiments, nematodes were pre-acclimated for 5 minutes after being loaded into the microfluidic channel. Behavioral data were recorded simultaneously during ultrasound stimulation. The control and experimental groups were repeated ≥3 times to eliminate individual differences. This design combines non-contact manipulation, high spatiotemporal resolution, and biocompatibility, and can be expanded to neural circuit analysis or aging intervention studies. However, strict control of sound intensity and exposure time is required to prevent mechanical damage to cell membranes.

[0146] The light stimulation induction platform includes a light source system, optical transmission components, and a nematode placement chamber. The light source system uses a high-brightness LED light source that can emit visible light in the 400-700nm wavelength range, meeting the needs of various light stimulation experiments. By changing the power input current and applying PWM (pulse width modulation) technology, the light intensity can be precisely adjusted within a range of 0-500μW / mm 2 The optical transmission component is equipped with a set of collimating lenses and optical fibers. The light emitted by the LED light source is first collimated and then transmitted to the experimental area through the optical fiber. The end of the optical fiber is connected to an adjustable light spot diffuser, which evenly illuminates the nematode sample and ensures that the nematodes receive uniform light intensity. The nematode placement chamber is made of transparent quartz glass, which has good light transmittance and can withstand certain temperature changes. It has a dedicated entrance and exit for replacing the experimental solution containing the nematodes. A light-tight cover is set on the top of the chamber to effectively prevent external light from interfering with the experiment.

[0147] The heating element of the thermal induction platform utilizes a square thin-film heater with a side length of 50mm and a thickness of only 0.2mm. It heats quickly and fits snugly within the nematode placement area. Connected via wires to an intelligently controlled power supply, it efficiently converts the power supply's electrical energy into thermal energy, enabling rapid and precise heat transfer. A high-precision thermistor, tightly fitted within the nematode placement area, serves as a temperature sensor. This temperature sensor monitors the sample temperature in real time and converts the temperature signal into an electrical signal that is fed back to the power supply's FPGA control unit for precise closed-loop temperature control, achieving a temperature measurement accuracy of ±0.1°C. To minimize heat loss, the entire thermal induction plate is wrapped in polystyrene foam insulation. A small axial cooling fan is installed on one side of the platform. When the temperature exceeds the set upper limit, the FPGA control unit activates the fan, accelerating heat dissipation and ensuring a stable temperature range of 10°C-40°C, providing a stable and controllable temperature environment for nematode experiments.

[0148] The mechanical vibration induction platform uses a small eccentric vibration motor of model M12-24V as the vibration source, through a 0.5mm 2A 500mm long PVC insulated copper core wire is connected to an intelligent control power supply, which generates mechanical vibration when operating at a working voltage of 12-24V. The vibration frequency can be adjusted between 10-100Hz by changing the output voltage frequency of the power supply. The vibration transmission and amplification device composed of a spring and a lever can accurately transmit and amplify the vibration to the nematode culture dish by adjusting the spring elastic coefficient and the lever length ratio, and the vibration amplitude can be adjusted within a range of 0.01-0.1mm. Connecting the bottom of the nematode culture dish to the vibration transmission device allows the vibration to act evenly on the agar plate containing the nematodes, causing the C. elegans to move. The outer shell is made of medical-grade polycarbonate (PC) with an outer diameter of 150mm, an inner diameter of 100mm, and a height of 40mm to adapt to common specifications of culture dishes such as 90mm. There are three holes with a diameter of 10mm and a 120° distribution for contact between the vibrator and the culture dish. M3 stainless steel countersunk screws and epoxy resin glue with a shear strength of ≥15MPa are used to fix the vibrator and the outer shell components to ensure stable operation.

[0149] The nematode motion detection system primarily consists of software (real-time detection), nematode motion imaging (microscope + CCD), and a motion observation platform. The software integrates image processing software such as ImageJ and OpenCV. It can apply advanced machine learning algorithms such as convolutional neural networks (CNN) and support vector machines (SVM) to process images captured by the microscope imaging system, such as noise reduction and contrast enhancement. It can also identify nematode positions, measure morphological parameters, analyze motion trajectories to obtain relevant motion parameters, assist in nematode classification, and generate detailed data reports.

[0150] Software 2 integrates ImageJ and OpenCV, and uses CNN and SVM algorithms. It has a well-organized interface and is easy to use. Its real-time monitoring function provides accurate data for nematode research.

[0151] Intelligent Image Acquisition and Preprocessing: The software connects to a microscope and CCD camera, displaying the image in real time at the top of the page. Below, on the left, you'll find noise reduction, contrast adjustment, and annotation functions. On the right, you can set the acquisition rate (1-10 frames per second) and resolution. Click "Apply" to make adjustments. You can also adjust contrast using a slider and highlight key areas, ensuring high-quality images for research.

[0152] Precise Parameter Measurement and Dynamic Analysis: On the parameter measurement page, the software uses advanced algorithms to display the nematode's position and number in real time on the left. A table in the center shows morphological parameters such as body length and width accurate to two decimal places. Click "Refresh" to update. The right side plots the movement trajectory, displaying parameters such as speed. A calibration button is included for easy understanding of the nematode's movement and physiological status.

[0153] In-depth behavioral monitoring and classification statistics: The software uses machine learning models to analyze images, identify nematode behavioral patterns, and display changes over time. Different color icons correspond to different behaviors, and hovering displays reveal details. Statistical charts are generated on the data report page. A bar chart at the top displays classification results and counts, while a bar chart below lists feature parameter ranges and percentages. Reports can be printed, saved, and compared, assisting in nematode research.

[0154] The C. elegans motion imaging system consists of a microscope and a CCD camera, connected to a computer via a CameraLink interface. The microscope clearly displays the nematode's morphology and motion, while the CCD camera captures and transmits images to the computer.

[0155] The motion observation platform includes active motion observation and physically induced motion observation. Active motion observation refers to observing the movement of nematodes without external physical induction. A microscope and a high-frame-rate CCD camera are used to collect data at ≥10 frames per second, record motion parameters, analyze motion differences under different developmental and nutritional conditions, and use machine learning algorithms to classify motion patterns and explore physiological mechanisms.

[0156] Physically induced locomotion observation involves observing nematode movement in response to physical stimulation, such as electrical stimulation or ultrasound. Changes in the nematode's locomotion during stimulation are recorded, such as changes in direction and speed under electrical stimulation and trajectory changes under ultrasound stimulation. The locomotion responses under different physical stimulation conditions are compared, and the effects of these stimulations on locomotion behavior are analyzed. Combined with changes in physiological indicators, the intrinsic connection between physically induced locomotion and the nematode's physiological state is explored.

[0157] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. An intelligent nematode control and monitoring system, characterized by: It includes a physical induced motion system and a nematode motion detection system. The physical induced motion system consists of software one, an intelligent control power supply and a physical induced motion platform; the nematode motion detection system consists of software two, nematode motion imaging and a motion observation platform.

2. The intelligent nematode control and monitoring system according to claim 1, characterized in that: The physical induction motion platform includes an electrical induction platform, an ultrasound induction platform, a light stimulation induction platform, a thermal induction platform and a mechanical vibration induction platform.

3. The intelligent nematode control and monitoring system according to claim 2, characterized in that: The software is based on a hybrid architecture of LabVIEW and Python / Java. The software can control the physical induced motion system and connect to the power supply hardware via an RS-485 bus. The software can perform real-time status monitoring and feedback the power supply status of each platform, including voltage and current values.

4. The intelligent nematode control and monitoring system according to claim 3, characterized in that: The intelligent control power supply is composed of a DC power supply with a continuously adjustable 0-40V and a maximum output current of 2A and an FPGA main control unit, which meets the power requirements of each physical induced motion module and realizes the control and management of various parameters of the power supply.

5. The intelligent nematode control and monitoring system according to claim 4, characterized in that: The electric induction platform includes a circular model made of polymethyl methacrylate, with an outer diameter of 100 mm and a thickness of 5 mm. Two rectangular nematode placement grooves with a spacing of 10 mm, a length of 20 mm, a width of 10 mm, and a depth of 2 mm are opened in the center of the model. A circular hole with a diameter of 2 mm and a depth that penetrates the thickness of the model is opened on both sides of the nematode placement groove. A platinum rod or a graphite rod is inserted into the circular hole.

6. The intelligent nematode control and monitoring system according to claim 5, characterized in that: The ultrasound induction platform consists of an ultrasound generating module, a transducer focusing system, a microenvironment sample chamber and a behavior analysis unit; The ultrasonic generation module can generate an adjustable frequency signal in the range of 20kHz to 3MHz through a high-precision signal generator, and drive the piezoelectric ceramic transducer through a power amplifier to convert the electrical signal into mechanical ultrasonic waves; The transducer focusing system includes a concave acoustic lens or a parabolic reflector, which can focus the sound waves into a tiny area of 0.5–1 mm; The microenvironment sample chamber is designed using a PDMS or agarose microfluidic chip. A channel is opened inside the microenvironment sample chamber. The channel has a size of 500 μm wide and 100 μm high. The top of the channel is covered with a 50-100 μm thick sound-transmitting film, and deionized water or pre-degassed ultrasound gel is used as a coupling medium. The behavioral analysis unit includes an inverted microscope and a high-speed camera. The behavioral monitoring module uses TrackMate or OpenCV algorithms to track the nematode's movement trajectory in real time. The behavioral monitoring module can monitor parameters such as speed, steering angle, and pharyngeal pumping frequency. At the same time, the sample temperature is monitored using a micro-thermocouple and an infrared thermal imager to ensure that the temperature rise throughout the experiment is ≤2°C.

7. The intelligent nematode control and monitoring system according to claim 6, characterized in that: The light stimulation induction platform includes a light source system, an optical transmission component, and a nematode placement chamber; the light source system includes a high-brightness LED light source that can emit visible light in the wavelength range of 400-700nm; the light intensity of the LED light source is adjustable in the range of 0-500μW / mm 2 ; The optical transmission component includes a set of collimating lenses and optical fibers, which are used to collimate the LED light source and transmit light respectively. The end of the optical fiber is connected to a spot diffuser with an adjustable angle. The nematode placement chamber is made of transparent quartz glass, has good light transmittance and can tolerate temperature changes, and a light sealing cover is provided on the top of the nematode placement chamber.

8. The intelligent nematode control and monitoring system according to claim 7, characterized in that: The temperature adjustment range of the thermal induction platform is 10-40°C. The thermal induction platform includes a heating element, which is a square thin film heating plate with a side length of 50mm and a thickness of 0.2mm. The heating element is connected to the intelligent control power supply through a wire. The thermal induction platform is wrapped with polystyrene foam insulation material; a cooling fan is installed on one side of the thermal induction platform.

9. The intelligent nematode control and monitoring system according to claim 8, characterized in that: The mechanical vibration induction platform includes an M12-24V eccentric vibration motor, and the vibration frequency range of the eccentric vibration motor is 10-100Hz; the mechanical vibration induction platform is provided with a vibration transmission and amplification device composed of a spring and a lever, the spring elastic coefficient and the lever length ratio are adjustable, and the amplitude adjustment range is 0.01-0.1mm; the shell of the mechanical vibration induction platform is made of medical-grade polycarbonate, the outer diameter of the mechanical vibration induction platform is 150mm, the inner diameter is 100mm, and the height is 40mm, which is suitable for a 90mm culture dish. Three holes with a diameter of 10mm and a 120° distribution are opened on the side wall of the mechanical vibration induction platform, and the vibration transmission and amplification device is in contact with the culture dish through the holes.

10. The intelligent nematode control and monitoring system according to claim 9, characterized in that: The second software integrates ImageJ and OpenCV and uses CNN and SVM algorithms; the second software connects to a microscope and a CCD camera, and the second software displays images in real time; The left side of the software's second operating interface displays the nematode position and number in real time, the middle table displays morphological parameters such as body length and body width accurate to two decimal places, and the right side plots the movement trajectory and displays parameters such as speed; The second software uses a machine learning model to analyze images, identify nematode behavior patterns, and display changes on a timeline, with different color icons corresponding to different behaviors.

11. The intelligent nematode control and monitoring system according to claim 10, characterized in that: The nematode motion imaging system comprises a microscope and a CCD camera, and the CCD camera is connected to a computer via a Camera Link interface.

12. The intelligent nematode control and monitoring system according to claim 11, characterized in that: The motion observation platform includes active motion observation and physically induced motion observation. The active motion observation includes recording motion parameters, analyzing the individual motion differences of nematodes under different developmental and nutritional conditions, and classifying motion patterns using machine learning algorithms. At the same time, the motion observation platform can observe the beating of the nematode pharyngeal pump, evaluate the nematode lifespan, and measure the nematode body length.

13. The intelligent nematode control and monitoring system according to claim 12, characterized in that: The physical induced motion observation records the movement of nematodes after application of physical stimulation such as electricity and ultrasound, including changes in the movement direction and speed of nematodes under electrical stimulation and changes in the movement trajectory of nematodes under ultrasonic stimulation, so as to compare with nematodes under normal living conditions.

Images