Self-adaptive dynamic adjustment automatic microinjection method

Abstract

The invention relates to the technical field of microinjection, and discloses a self-adaptive dynamic adjustment automatic microinjection method, which comprises the step of transferring cells with mark point arrays formed on the surfaces in a culture dish containing marking substances. The temperature, humidity and vibration parameters of the environment where the cells are located are collected in real time through multiple sensors. The cells are irradiated with light sources of different wavelengths, and images are captured. Acquiring position information of cell surface mark points, and establishing and optimizing a cell three-dimensional dynamic model. And determining an injection target, planning a path and generating a control instruction. The flexible injection needle is controlled to move and inject substances, the cell state and environmental parameters are monitored in real time during injection, the upper computer adjusts related parameters according to a self-adaptive control algorithm, and the needle is withdrawn to the initial position after injection. The micro-injection precision and stability can be effectively improved, cell damage is reduced, the experiment success rate is increased, and powerful support is provided for cell research and biotechnology application.

Description

Technical Field

[0001] The present invention relates to the technical field of microinjection, in particular to an automated microinjection method with adaptive dynamic adjustment. Background Art

[0002] Microinjection plays a crucial role in modern biological science research. It enables the precise introduction of cells, genetic material, or other reagents into embryos, cells, or tissues, providing a key tool for cell transfection, signaling pathway exploration, and organelle function studies. Taking zebrafish embryos as an example, due to their relative transparency during the embryonic stage, allowing for the observation of early morphological changes, microinjection of zebrafish embryos is widely used in numerous disciplines, including genetics, virology, toxicology, endocrinology, immunology, and oncology. It has significantly promoted advances in cell biology in areas such as transgenics, assisted reproduction, and drug development.

[0003] Traditional microinjection techniques rely primarily on researchers manually adjusting and fixing the injected cells within the microscope's field of view, then using a microinjection needle to inject the exogenous genetic material into the designated location within the cell. This method has numerous limitations, including being time-consuming and labor-intensive, requiring a high level of technical proficiency and experience from the experimenter, and significant differences in operation between operators, resulting in poor repeatability and stability in experimental results. Furthermore, traditional methods struggle to meet the high-precision injection requirements when faced with complex situations such as tiny and easily deformable cells and the transparent membranes of zebrafish embryos.

[0004] Therefore, it is necessary to design an automated microinjection method with adaptive dynamic adjustment to solve the problem that traditional microinjection technology is difficult to meet the high-precision injection requirements. Summary of the Invention

[0005] In view of this, the present invention proposes an automated microinjection method with adaptive dynamic adjustment, aiming to solve the problem that traditional microinjection technology is difficult to meet the high-precision injection requirements.

[0006] The present invention proposes an automated microinjection method with adaptive dynamic adjustment, comprising:

[0007] The cells are placed in a culture dish containing a labeled substance, the labeled substance is evenly distributed, and an image acquisition device is used for real-time observation, and the cells with an array of labeled points formed on the surface are transferred;

[0008] Temperature, humidity, and vibration parameters of the cell environment are collected using temperature, humidity, and vibration sensors at different locations around the microinjection device.

[0009] Using multiple light sources of different wavelengths to illuminate cells in a predetermined order, corresponding image acquisition devices capture the light to form cell images of different wavelengths;

[0010] Obtain cell surface marker location information and cell image data sets of different wavelengths, establish a three-dimensional dynamic model of the cell, call the three-dimensional dynamic model of the cell to determine the injection target, obtain environmental parameters, and generate a planned injection path and injection parameters;

[0011] The flexible injection needle is controlled to move along the planned injection path. After reaching the target, the substance is injected according to the injection parameters. During the injection process, the cell status and environmental parameters are monitored in real time through sensors and fed back to the host computer. The host computer adaptively controls and adjusts the injection parameters. After the injection is completed, the injection needle is withdrawn from the cell and returned to its initial position.

[0012] Furthermore, the step of placing the cells in a culture dish containing a marker substance comprises:

[0013] Microfluidic technology is used to control the flow of the solution in the culture dish, wherein the concentration of the labeled molecules of the labeled substance is ΔC, and a first preset concentration C1, a second preset concentration C2, a third preset concentration C3 and a fourth preset concentration C4 are preset, and C1<C2<C3<C4;

[0014] Selecting a flow rate level of the microfluidic technology based on the relationship between the labeled molecule concentration ΔC and each preset concentration;

[0015] When C1≤△C<C2, the first preset flow rate gear V1 is selected as the flow rate of the microfluidic technology;

[0016] When C2≤△C<C3, the second preset flow rate gear V2 is selected as the flow rate of the microfluidic technology;

[0017] When C3≤△C<C4, the third preset flow rate gear V3 is selected as the flow rate of the microfluidic technology;

[0018] When C4≤ΔC, the fourth preset flow rate level V4 is selected as the flow rate of the microfluidic technology.

[0019] Furthermore, after selecting the i-th preset flow rate gear Vi as the flow rate of the microfluidic technology, i=1, 2, 3, 4, the cells are observed in real time by the image acquisition device;

[0020] A first preset observation time T1, a second preset observation time T2, a third preset observation time T3 and a fourth preset observation time T4 are preset, and T1 < T2 < T3 < T4;

[0021] When the flow rate of the microfluidic technology is V1, the fourth preset observation time T4 is used as the observation judgment time;

[0022] When the flow rate of the microfluidic technology is V2, the third preset observation time T3 is used as the observation judgment time;

[0023] When the flow rate of the microfluidic technology is V3, the second preset observation time T2 is used as the observation judgment time;

[0024] When the flow rate of the microfluidic technology is V4, the first preset observation time T1 is used as the observation judgment time;

[0025] The cell surface is observed by an image acquisition device within the observation judgment time. If an array of evenly distributed fluorescent marker points appears, the addition of the marker substance is stopped.

[0026] Furthermore, the temperature sensors, humidity sensors and vibration sensors at different locations around the microinjection device are used to measure the ambient temperature and humidity at fixed time intervals and monitor the vibration in real time, including:

[0027] A first time interval Δt1, a second time interval Δt2, a third time interval Δt3 and a fourth time interval Δt4 are preset, and Δt1 < Δt2 < Δt3 < Δt4;

[0028] A first vibration sensitivity level S1, a second vibration sensitivity level S2, a third vibration sensitivity level S3, and a fourth vibration sensitivity level S4 are preset, and S1<S2<S3<S4;

[0029] Selecting the time intervals for temperature and humidity measurements and the sensitivity of vibration monitoring according to the vibration sensitivity level corresponding to the cell type;

[0030] When the vibration sensitivity level corresponding to the cell type is S1, the fourth time interval Δt4 is selected as the time interval for temperature and humidity measurement, and vibration is monitored with the lowest sensitivity;

[0031] When the vibration sensitivity level corresponding to the cell type is S2, the third time interval Δt3 is selected as the time interval for temperature and humidity measurement to monitor vibration with lower sensitivity;

[0032] When the vibration sensitivity level corresponding to the cell type is S3, the second time interval Δt2 is selected as the time interval for temperature and humidity measurement to monitor vibration with higher sensitivity;

[0033] When the vibration sensitivity level corresponding to the cell type is S4, the first time interval Δt1 is selected as the time interval for measuring temperature and humidity, and vibration is monitored with the highest sensitivity.

[0034] Furthermore, the method of using a plurality of light sources of different wavelengths and corresponding image acquisition devices to sequentially turn on the light sources of different wavelengths in a predetermined order to irradiate the cells includes:

[0035] A first wavelength group λ1, a second wavelength group λ2, a third wavelength group λ3 and a fourth wavelength group λ4 are preset, each wavelength group including light sources of different numbers and wavelength values;

[0036] A first cell type classification K1, a second cell type classification K2, a third cell type classification K3, and a fourth cell type classification K4 are preset;

[0037] According to the cell type classification, the corresponding wavelength group is selected as the irradiation light source group;

[0038] When the cell type is classified as K1, the first wavelength group λ1 is selected as the illumination light source group;

[0039] When the cell type is classified as K2, the second wavelength group λ2 is selected as the illumination light source group;

[0040] When the cell type is classified as K3, the third wavelength group λ3 is selected as the illumination light source group;

[0041] When the cell type is classified as K4, the fourth wavelength group λ4 is selected as the illumination light source group;

[0042] The light sources are turned on in sequence according to the wavelength sequence of the light sources in the selected light source group to irradiate the cells.

[0043] Furthermore, the path planning algorithm plans the injection path from the initial position of the injection needle to the target position, taking into account parameters such as the movement speed, acceleration and injection pressure of the injection needle, including:

[0044] A first injection pressure range P1, a second injection pressure range P2, a third injection pressure range P3 and a fourth injection pressure range P4 are preset, and P1 < P2 < P3 < P4;

[0045] A first movement speed range V1, a second movement speed range V2, a third movement speed range V3 and a fourth movement speed range V4 are preset, and V1 < V2 < V3 < V4;

[0046] A first acceleration range A1, a second acceleration range A2, a third acceleration range A3 and a fourth acceleration range A4 are preset, and A1<A2<A3<A4;

[0047] According to the hardness △H of the cell, the corresponding injection pressure range, movement speed range and acceleration range are selected;

[0048] A first hardness threshold H1, a second hardness threshold H2, a third hardness threshold H3 and a fourth hardness threshold H4 are preset, and H1<H2<H3<H4;

[0049] When H1≤△H<H2, the first injection pressure range P1, the first movement speed range V1 and the first acceleration range A1 are selected;

[0050] When H2≤△H<H3, the second injection pressure range P2, the second movement speed range V2 and the second acceleration range A2 are selected;

[0051] When H3≤△H<H4, the third injection pressure range P3, the third movement speed range V3 and the third acceleration range A3 are selected;

[0052] When H4≤△H, the fourth injection pressure range P4, the fourth movement speed range V4 and the fourth acceleration range A4 are selected;

[0053] Based on the selected parameter range, the path planning algorithm is used to plan the injection path and determine the specific injection needle movement speed, acceleration and injection pressure parameters.

[0054] Furthermore, after planning the injection path using a path planning algorithm and determining specific injection needle movement speed, acceleration, and injection pressure parameters according to the selected parameter range, controlling the flexible injection needle to move along the planned path and controlling the injection needle movement speed and acceleration during the movement process include:

[0055] A first speed error tolerance value ε1, a second speed error tolerance value ε2, a third speed error tolerance value ε3 and a fourth speed error tolerance value ε4 are preset, and ε1<ε2<ε3<ε4;

[0056] A first acceleration error tolerance value δ1, a second acceleration error tolerance value δ2, a third acceleration error tolerance value δ3, and a fourth acceleration error tolerance value δ4 are preset, and δ1<δ2<δ3<δ4;

[0057] According to the complexity of the injection path △L, select the error tolerance value of speed and acceleration;

[0058] A first complexity threshold L1, a second complexity threshold L2, a third complexity threshold L3 and a fourth complexity threshold L4 are preset, and L1 < L2 < L3 < L4;

[0059] When L1≤△L<L2, the first velocity error tolerance value ε1 and the first acceleration error tolerance value δ1 are selected;

[0060] When L2≤△L<L3, the second speed error tolerance value ε2 and the second acceleration error tolerance value δ2 are selected;

[0061] When L3≤△L<L4, the third speed error tolerance value ε3 and the third acceleration error tolerance value δ3 are selected;

[0062] When L4≤△L, the fourth speed error tolerance value ε4 and the fourth acceleration error tolerance value δ4 are selected;

[0063] During the movement of the flexible injection needle, its speed and acceleration are monitored in real time. When the error between the actual speed and the command speed exceeds the selected speed error tolerance value, or the error between the actual acceleration and the command acceleration exceeds the selected acceleration error tolerance value, the movement parameters of the flexible injection needle are adjusted.

[0064] Furthermore, during the injection process, the position and state of the injection needle and the reaction of the cells are monitored in real time by a sensor and fed back to the host computer. The host computer controls the adaptive control device to dynamically adjust the injection speed and injection volume of the syringe and the movement speed and position of the positioning platform, including:

[0065] The cell response includes the cell deformation degree ΔD;

[0066] A standard deformation degree D0 is preset, and the difference between the cell deformation degree ΔD and the standard deformation degree D0, |ΔD-D0|, is obtained;

[0067] A first deformation difference threshold D1, a second deformation difference threshold D2, a third deformation difference threshold D3 and a fourth deformation difference threshold D4 are preset, and D1<D2<D3<D4;

[0068] A first injection speed adjustment coefficient α1, a second injection speed adjustment coefficient α2, a third injection speed adjustment coefficient α3 and a fourth injection speed adjustment coefficient α4 are preset, and α1<α2<α3<α4;

[0069] According to the relationship between the difference |△D-D0| and each preset deformation difference threshold, an injection speed adjustment coefficient is selected to adjust the current injection speed;

[0070] When D1≤|△D-D0|<D2, the first injection speed adjustment coefficient α1 is selected to adjust the current injection speed;

[0071] When D2≤|△D-D0|<D3, the second injection speed adjustment coefficient α2 is selected to adjust the current injection speed;

[0072] When D3≤|△D-D0|<D4, the third injection speed adjustment coefficient α3 is selected to adjust the current injection speed;

[0073] When D4≤|ΔD−D0|, the fourth injection speed adjustment coefficient α4 is selected to adjust the current injection speed.

[0074] Furthermore, after selecting the i-th injection speed adjustment coefficient αi to adjust the current injection speed, i=1, 2, 3, 4, the host computer controls the adaptive control device to dynamically adjust the injection speed and injection volume of the syringe and the moving speed and position of the positioning platform according to the feedback information, further comprising:

[0075] The cell's response also includes the diffusion degree of intracellular substances ΔS;

[0076] A standard diffusion degree S0 is preset, and a difference value |ΔS-S0| between the diffusion degree ΔS of the intracellular substance and the standard diffusion degree S0 is obtained;

[0077] A first diffusion difference threshold S1, a second diffusion difference threshold S2, a third diffusion difference threshold S3 and a fourth diffusion difference threshold S4 are preset, and S1<S2<S3<S4;

[0078] A first injection volume adjustment coefficient β1, a second injection volume adjustment coefficient β2, a third injection volume adjustment coefficient β3 and a fourth injection volume adjustment coefficient β4 are preset, and β1 < β2 < β3 < β4;

[0079] According to the relationship between the difference |△S-S0| and each preset diffusion difference threshold, an injection volume adjustment coefficient is selected to adjust the current injection volume;

[0080] When S1≤|△S-S0|<S2, the first injection volume adjustment coefficient β1 is selected to adjust the current injection volume;

[0081] When S2≤|△S-S0|<S3, the second injection volume adjustment coefficient β2 is selected to adjust the current injection volume;

[0082] When S3≤|ΔS-S0|<S4, the third injection volume adjustment coefficient β3 is selected to adjust the current injection volume;

[0083] When S4≤|ΔS−S0|, the fourth injection volume adjustment coefficient β4 is selected to adjust the current injection volume.

[0084] Furthermore, after the injection is completed, the injection needle is withdrawn from the cell and returned to the initial position, including:

[0085] A first evacuation speed range E1, a second evacuation speed range E2, a third evacuation speed range E3 and a fourth evacuation speed range E4 are preset, and E1<E2<E3<E4;

[0086] A first cell fragility threshold F1, a second cell fragility threshold F2, a third cell fragility threshold F3 and a fourth cell fragility threshold F4 are preset, and F1 < F2 < F3 < F4;

[0087] According to the fragility of the cell, △F, the corresponding evacuation speed range is selected;

[0088] When F1≤△F<F2, the first evacuation speed range E4 is selected;

[0089] When F2≤△F<F3, the second evacuation speed range E3 is selected;

[0090] When F3≤△F<F4, the third evacuation speed range E2 is selected;

[0091] When F4≤△F, the fourth evacuation speed range E1 is selected;

[0092] The flexible injection needle is controlled to evacuate cells along the planned evacuation path at an appropriate speed within the selected evacuation speed range.

[0093] Compared with the prior art, the present invention has the following beneficial effects:

[0094] 1. By irradiating cells with multiple light sources of different wavelengths in a predetermined order and capturing cell images at different wavelengths, it is possible to obtain cell information from multiple angles, providing richer data for cell analysis and research, contributing to a more comprehensive understanding of cell structure and function, and providing a more detailed basis for decision-making on cell injection.

[0095] 2. During the injection process, sensors monitor cell status and environmental parameters in real time and feed them back to the host computer, enabling timely detection and resolution of problems that arise during the injection process, improving the safety and success rate of cell injection, while also providing rich real-time data for subsequent data analysis and research.

[0096] 3. Control the flexible injection needle to move along the planned path. Combined with real-time monitoring of cell status and environmental parameters during the injection process and adaptive control of the host computer to adjust the injection parameters, the injection operation can be dynamically adjusted in time according to the actual cell response and environmental changes, reducing damage to cells, improving cell survival rate and injection success rate, while ensuring the stability and consistency of the injection process. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0098] Figure 1 This is a flow chart of the adaptive dynamic adjustment automated microinjection method provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0099] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, unless there is a conflict, the embodiments of the present disclosure and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0100] Reference Figure 1 In some embodiments of the present application, an adaptive dynamic adjustment automated microinjection method comprises:

[0101] Step S100: placing cells in a culture dish containing a labeled substance, evenly distributing the labeled substance, observing in real time using an image acquisition device, and transferring cells with an array of labeled points formed on their surface;

[0102] Step S200: using temperature, humidity, and vibration sensors at different locations around the microinjection device to collect temperature, humidity, and vibration parameters of the cell environment;

[0103] Step S300: irradiating cells with a plurality of light sources of different wavelengths in a predetermined order, and corresponding image acquisition devices capturing the light to form cell images of different wavelengths;

[0104] Step S400: obtaining cell surface marker location information and cell image data sets of different wavelengths, establishing a three-dimensional dynamic model of the cell, retrieving the three-dimensional dynamic model of the cell to determine the injection target, obtaining environmental parameters, and generating a planned injection path and injection parameters;

[0105] Step S500: Control the flexible injection needle to move along the planned injection path. After reaching the target, inject the substance according to the injection parameters. During the injection process, the cell status and environmental parameters are monitored in real time through sensors and fed back to the host computer. The host computer adaptively controls and adjusts the injection parameters. After the injection is completed, the injection needle is withdrawn from the cell and returned to the initial position.

[0106] Specifically, the labeling substance contains molecules that can specifically bind to the cell membrane and fluorescent groups. When the cell comes into contact with the labeling substance, the molecule will specifically bind to the cell membrane surface, causing the fluorescent group to attach to the cell. During this process, image acquisition equipment and a high-resolution microscope camera are used to observe the cell state in real time at a frequency of 5-10 frames per second. When a regular and evenly distributed array of fluorescent marker points is observed on the cell surface, the cell is transferred to a new culture dish by a micro-manipulation robotic arm to complete the cell pretreatment. This pretreatment process provides a clear mark for the subsequent accurate identification and positioning of the cells.

[0107] Specifically, temperature, humidity, and vibration sensors are strategically placed around the microinjection equipment. The temperature sensor uses a thermocouple, the humidity sensor uses a capacitive humidity sensor, and the vibration sensor uses a piezoelectric vibration sensor. The temperature and humidity sensors collect data every 30 seconds, while the vibration sensor monitors environmental vibrations in real time. Each sensor transmits the collected temperature and humidity data, as well as parameters such as vibration frequency and amplitude, via wired data transmission to a data storage unit. The data storage unit then organizes and stores these parameter data in timestamp order, providing data support for considering environmental factors during subsequent injections.

[0108] It is understandable that by irradiating cells with multiple light sources of different wavelengths in a predetermined order and capturing cell images at different wavelengths, it is possible to obtain cell information from multiple angles, provide richer data for cell analysis and research, contribute to a more comprehensive understanding of the structure and function of cells, and provide a more detailed basis for cell injection decisions. During the injection process, the cell status and environmental parameters are monitored in real time by sensors and fed back to the host computer, which can promptly detect and deal with problems that arise during the injection process, improve the safety and success rate of cell injection, and also provide rich real-time data for subsequent data analysis and research. Controlling the flexible injection needle to move along the planned path, combined with real-time monitoring of cell status and environmental parameters during the injection process and adaptive control of the host computer to adjust the injection parameters, can dynamically adjust the injection operation in a timely manner according to the actual cell response and environmental changes, reduce damage to cells, improve cell survival rate and injection success rate, and ensure the stability and consistency of the injection process.

[0109] In some embodiments of the present application, when placing the cells in a culture dish containing a labeled substance in step S100, microfluidic technology is used to control the flow of a solution in the culture dish, wherein the labeled molecule concentration of the labeled substance is ΔC, and a first preset concentration C1, a second preset concentration C2, a third preset concentration C3, and a fourth preset concentration C4 are preset, and C1<C2<C3<C4;

[0110] According to the relationship between the concentration of labeled molecules △C and each preset concentration, the flow rate gear of the microfluidic technology is selected;

[0111] When C1≤△C<C2, the first preset flow rate gear V1 is selected as the flow rate of the microfluidic technology;

[0112] When C2≤△C<C3, the second preset flow rate gear V2 is selected as the flow rate of the microfluidic technology;

[0113] When C3≤△C<C4, the third preset flow rate gear V3 is selected as the flow rate of the microfluidic technology;

[0114] When C4≤ΔC, the fourth preset flow rate level V4 is selected as the flow rate of the microfluidic technology.

[0115] Specifically, microfluidics is a technology that can precisely manipulate tiny volumes of fluid. Its core is to use tiny channels (usually micrometer-sized) to guide and control the flow of fluids.

[0116] Specifically, different flow rate gears (V1, V2, V3, V4) are selected according to the relationship between the marker molecule concentration △C and the preset concentration (C1, C2, C3, C4). This is because the marker molecule concentration will affect the physical properties of the solution (such as viscosity, etc.), and different flow rate gears can adapt to the flow requirements of solutions under different concentrations. When the marker molecule concentration is high (C4≤△C), the solution viscosity may be large. At this time, a higher flow rate gear V4 is selected to ensure that the solution can flow smoothly and the marker substance is evenly distributed in the culture dish; when the concentration is low (C1≤△C<C2), the solution viscosity is small, and a lower flow rate gear V1 is selected to avoid the solution flowing too fast and causing impact on the cells.

[0117] As you can understand, microfluidics relies on microchannels to achieve precise control of fluids. By constructing or connecting microchannel structures in a culture dish, the solution flows through the microchannels, and precise manipulation of the fluid is achieved by controlling factors such as the size and shape of the microchannels and the pressure difference of the fluid.

[0118] In some embodiments of the present application, in step S100, after selecting the i-th preset flow rate gear Vi as the flow rate of the microfluidic technology, i=1, 2, 3, 4, the cells are observed in real time by an image acquisition device;

[0119] A first preset observation time T1, a second preset observation time T2, a third preset observation time T3 and a fourth preset observation time T4 are preset, and T1 < T2 < T3 < T4;

[0120] When the flow rate of the microfluidic technology is V1, the fourth preset observation time T4 is used as the observation judgment time;

[0121] When the flow rate of the microfluidic technology is V2, the third preset observation time T3 is used as the observation judgment time;

[0122] When the flow rate of the microfluidic technology is V3, the second preset observation time T2 is used as the observation judgment time;

[0123] When the flow rate of the microfluidic technology is V4, the first preset observation time T1 is used as the observation judgment time;

[0124] The cell surface is observed by an image acquisition device within the observation judgment time. If an array of evenly distributed fluorescent marker points appears, the addition of the marker substance is stopped.

[0125] Specifically, different microfluidic flow rates will affect the efficiency of the interaction between the labeled substance and the cells and the speed of uniform distribution. When the flow rate is low (such as V1), the process of the labeled substance diffusing and binding to the cells in the culture dish is relatively slow, and it takes a longer time to form a uniform array of fluorescent markers on the cell surface. Therefore, a longer fourth preset observation time T4 is set; and when the flow rate is high (such as V4), the labeled substance can be distributed and bound around the cells faster, and the required observation time is correspondingly shortened, so a shorter first preset observation time T1 is set. The gradient setting of each preset observation time is based on a large amount of previous experimental data and the kinetic analysis of the labeling process at different flow rates.

[0126] Specifically, if an evenly distributed array of fluorescent markers still does not appear after the observation period, the system automatically records the current observation data and processes it according to a preset strategy. It can automatically increase the microfluidic flow rate by one level and restart the observation timer; or it can pause the labeling process, prompting the operator to check whether parameters such as the marker concentration and the operating status of the microfluidic system are normal. After manual intervention and adjustment, the observation process is restarted until a satisfactory array of fluorescent markers is formed on the cell surface.

[0127] It is understandable that real-time image acquisition and analysis can timely capture the formation of fluorescent marker arrays on the cell surface. Once the standard is met, the addition of marker substances will be stopped immediately, reducing unnecessary reagent consumption, while reducing the potential toxic effects of marker substances on cells and ensuring cell activity. The abnormal situation handling mechanism further ensures the smooth progress of the labeling process, ultimately improving the success rate, uniformity and consistency of cell labeling, and laying a solid foundation for subsequent operations such as microinjection based on cell labeling.

[0128] In some embodiments of the present application, in step S200, the ambient temperature and humidity are measured at fixed time intervals by temperature sensors, humidity sensors, and vibration sensors at different locations around the microinjection device, and the vibration condition is monitored in real time, including:

[0129] A first time interval Δt1, a second time interval Δt2, a third time interval Δt3 and a fourth time interval Δt4 are preset, and Δt1 < Δt2 < Δt3 < Δt4;

[0130] A first vibration sensitivity level S1, a second vibration sensitivity level S2, a third vibration sensitivity level S3, and a fourth vibration sensitivity level S4 are preset, and S1<S2<S3<S4;

[0131] According to the vibration sensitivity level corresponding to the cell type, the time interval for temperature and humidity measurement and the sensitivity of vibration monitoring are selected;

[0132] When the vibration sensitivity level corresponding to the cell type is S1, the fourth time interval Δt4 is selected as the time interval for temperature and humidity measurement, and vibration is monitored with the lowest sensitivity;

[0133] When the vibration sensitivity level corresponding to the cell type is S2, the third time interval Δt3 is selected as the time interval for temperature and humidity measurement to monitor vibration with lower sensitivity;

[0134] When the vibration sensitivity level corresponding to the cell type is S3, the second time interval Δt2 is selected as the time interval for temperature and humidity measurement to monitor vibration with higher sensitivity;

[0135] When the vibration sensitivity level corresponding to the cell type is S4, the first time interval Δt1 is selected as the time interval for measuring temperature and humidity, and vibration is monitored with the highest sensitivity.

[0136] Specifically, the temperature sensor uses a high-precision platinum resistance temperature sensor with a measurement accuracy of ±0.1°C, meeting the high temperature monitoring requirements of cell culture. The humidity sensor uses a capacitive polymer humidity sensor with a fast response speed and a wide measurement range. The vibration sensor uses a piezoelectric accelerometer, which can sensitively capture tiny vibrations. These three types of sensors are installed in a triangular pattern around the microinjection device. The temperature and humidity sensors are placed 10-20 cm from the device to ensure that the measurement environment is close to the actual cell environment. The vibration sensor is directly fixed to the device base to accurately monitor the vibration of the device during operation.

[0137] Specifically, when setting the time intervals for temperature and humidity measurements, the different metabolic rates and tolerance to environmental changes of different cell types are taken into consideration. For example, primary cells metabolize slowly and are sensitive to environmental changes, corresponding to the high vibration sensitivity level S4. A short time interval △t1 (such as 1 minute) is selected to capture small changes in ambient temperature and humidity in a timely manner; while the domesticated cell lines have strong tolerance, corresponding to the low vibration sensitivity level S1. A long time interval △t4 (such as 10 minutes) is used to reduce the amount of data processing while ensuring the effectiveness of monitoring. The sensitivity of vibration monitoring is determined based on the mechanical strength and internal structural stability of the cells. For cells with fragile structures and sensitive to vibrations (S4 level) such as nerve cells, high-sensitivity monitoring is set, which can detect 0.01m / s 2 The following small acceleration changes; while plant protoplasts and other relatively tolerant cells (S1 level) can withstand 0.1m / s 2 As the vibration monitoring threshold.

[0138] It is understandable that real-time and graded environmental monitoring can quickly respond to temperature, humidity, and vibration anomalies, and automatically adjust related equipment to create a stable injection environment for cells, effectively avoiding the impact of environmental fluctuations on cell activity, while improving microinjection accuracy, reducing injection errors caused by environmental factors, and ensuring the reliability and repeatability of experimental results.

[0139] In some embodiments of the present application, in step S300, using a plurality of light sources of different wavelengths and a corresponding image acquisition device, the light sources of different wavelengths are sequentially turned on in a predetermined order to irradiate the cells, including:

[0140] A first wavelength group λ1, a second wavelength group λ2, a third wavelength group λ3 and a fourth wavelength group λ4 are preset, each wavelength group including light sources of different numbers and wavelength values;

[0141] A first cell type classification K1, a second cell type classification K2, a third cell type classification K3, and a fourth cell type classification K4 are preset;

[0142] According to the cell type classification, the corresponding wavelength group is selected as the irradiation light source group;

[0143] When the cell type is classified as K1, the first wavelength group λ1 is selected as the illumination light source group;

[0144] When the cell type is classified as K2, the second wavelength group λ2 is selected as the illumination light source group;

[0145] When the cell type is classified as K3, the third wavelength group λ3 is selected as the illumination light source group;

[0146] When the cell type is classified as K4, the fourth wavelength group λ4 is selected as the illumination light source group;

[0147] The light sources are turned on in sequence according to the wavelength sequence of the light sources in the selected light source group to irradiate the cells.

[0148] It is understandable that different cell types have different material compositions and structural characteristics, and they exhibit different absorption, reflection, and transmission characteristics at different wavelengths. By classifying and matching specific wavelength groups, we can maximize the highlighting of characteristic details such as the internal structure and component distribution of cells, and obtain clearer, more comprehensive, and multi-dimensional image information. At the same time, sequential irradiation avoids light interference and energy accumulation damage caused by simultaneous irradiation from multiple light sources, reducing the adverse effects on cell activity.

[0149] In some embodiments of the present application, in step S400, the path planning algorithm plans the injection path from the initial position of the injection needle to the target position, taking into account parameters such as the movement speed, acceleration, and injection pressure of the injection needle, including:

[0150] A first injection pressure range P1, a second injection pressure range P2, a third injection pressure range P3 and a fourth injection pressure range P4 are preset, and P1 < P2 < P3 < P4;

[0151] A first movement speed range V1, a second movement speed range V2, a third movement speed range V3 and a fourth movement speed range V4 are preset, and V1 < V2 < V3 < V4;

[0152] A first acceleration range A1, a second acceleration range A2, a third acceleration range A3 and a fourth acceleration range A4 are preset, and A1<A2<A3<A4;

[0153] According to the hardness △H of the cell, the corresponding injection pressure range, movement speed range and acceleration range are selected;

[0154] A first hardness threshold H1, a second hardness threshold H2, a third hardness threshold H3 and a fourth hardness threshold H4 are preset, and H1<H2<H3<H4;

[0155] When H1≤△H<H2, the first injection pressure range P1, the first movement speed range V1 and the first acceleration range A1 are selected;

[0156] When H2≤△H<H3, the second injection pressure range P2, the second movement speed range V2 and the second acceleration range A2 are selected;

[0157] When H3≤△H<H4, the third injection pressure range P3, the third movement speed range V3 and the third acceleration range A3 are selected;

[0158] When H4≤△H, the fourth injection pressure range P4, the fourth movement speed range V4 and the fourth acceleration range A4 are selected;

[0159] Based on the selected parameter range, the path planning algorithm is used to plan the injection path and determine the specific injection needle movement speed, acceleration and injection pressure parameters.

[0160] Specifically, before the injection operation, the cell hardness is measured using an atomic force microscope (AFM). The AFM probe is gently touched to the cell surface, and by applying a small force and measuring the deformation of the cell, the elastic modulus of the cell is calculated according to Hooke's law to quantify the cell hardness △H. To ensure data accuracy, multiple measurements are performed on each cell and the average value is taken. In addition, microfluidic chips combined with optical imaging technology can also be used to infer cell hardness by observing the deformation of cells when they are subjected to fluid forces in microchannels.

[0161] Specifically, the settings of the injection pressure range, movement speed range, and acceleration range are based on a large number of cell experiments and mechanical analysis. For cells with low hardness (H1≤△H<H2), such as nerve cells, their structure is fragile and their ability to withstand external forces is poor. Therefore, a lower first injection pressure range P1 (such as 1-5kPa), a first movement speed range V1 (such as 1-10μm / s), and a first acceleration range A1 (such as 1-5μm / s) are selected. 2 ) to avoid damage to cells due to excessive pressure, excessive speed or sudden acceleration; for plant cells with higher hardness (H4≤△H), since they have thicker cell walls and can withstand greater external forces, a higher fourth injection pressure range P4 (such as 15-25kPa), a fourth movement speed range V4 (such as 30-50μm / s) and a fourth acceleration range A4 (such as 15-25μm / s) are selected. 2 ) to ensure the needle can successfully penetrate the cell wall and complete the injection operation. These parameter ranges have been optimized through repeated experiments to ensure injection efficiency while maximally protecting cell viability.

[0162] Specifically, an improved A* algorithm is used for injection path planning. The algorithm incorporates information about the cell's internal structure, the cell's surrounding environment (such as the location of other cells or obstacles), and a selected parameter range as constraints. Starting from the initial needle position and ending at the target, the algorithm gradually searches for the optimal injection path by calculating a cost function at each node (including the actual cost from the starting point to the current node and the estimated cost from the current node to the end point).

[0163] It is understandable that the injection path and parameter planning method, based on the precise matching of pressure, velocity, and acceleration parameter ranges according to cell hardness, significantly improves the accuracy and safety of microinjection. Cells of different hardness have different structural strengths and tolerances. By classifying and matching parameter ranges, cell damage caused by inappropriate parameters can be avoided. For cells with low hardness, low pressure, low velocity, and low acceleration parameters are used to prevent cells from being punctured or torn; for cells with high hardness, high parameters are used to ensure smooth penetration of the injection needle. Combined with the path planning algorithm, the cell structure and environmental factors are comprehensively considered to plan the optimal path that avoids the vulnerable parts of the cell and obstacles, and the specific parameters are determined on this path, so that the injection needle can reach the target smoothly and accurately, reducing the time and degree to which the cell is affected by external forces, and effectively ensuring cell activity.

[0164] In some embodiments of the present application, in step S500, after planning the injection path using a path planning algorithm according to the selected parameter range and determining specific injection needle movement speed, acceleration, and injection pressure parameters, the flexible injection needle is controlled to move along the planned path, and the movement speed and acceleration of the injection needle are controlled during the movement process, including:

[0165] A first speed error tolerance value ε1, a second speed error tolerance value ε2, a third speed error tolerance value ε3 and a fourth speed error tolerance value ε4 are preset, and ε1<ε2<ε3<ε4;

[0166] A first acceleration error tolerance value δ1, a second acceleration error tolerance value δ2, a third acceleration error tolerance value δ3, and a fourth acceleration error tolerance value δ4 are preset, and δ1<δ2<δ3<δ4;

[0167] According to the complexity of the injection path △L, select the error tolerance value of speed and acceleration;

[0168] A first complexity threshold L1, a second complexity threshold L2, a third complexity threshold L3 and a fourth complexity threshold L4 are preset, and L1 < L2 < L3 < L4;

[0169] When L1≤△L<L2, the first velocity error tolerance value ε1 and the first acceleration error tolerance value δ1 are selected;

[0170] When L2≤△L<L3, the second speed error tolerance value ε2 and the second acceleration error tolerance value δ2 are selected;

[0171] When L3≤△L<L4, the third speed error tolerance value ε3 and the third acceleration error tolerance value δ3 are selected;

[0172] When L4≤△L, the fourth speed error tolerance value ε4 and the fourth acceleration error tolerance value δ4 are selected;

[0173] During the movement of the flexible injection needle, its speed and acceleration are monitored in real time. When the error between the actual speed and the command speed exceeds the selected speed error tolerance value, or the error between the actual acceleration and the command acceleration exceeds the selected acceleration error tolerance value, the movement parameters of the flexible injection needle are adjusted.

[0174] Specifically, a quantization model based on geometric and topological features is adopted to evaluate the complexity △L of the injection path. First, the planned injection path is discretized into a series of spatial nodes, and parameters such as the curvature change rate between nodes, the total turning angle of the path, and the minimum distance from the cell boundary or obstacles are calculated. The local curvature is calculated by fitting the curve equation of adjacent nodes, and the turning angle is obtained through the vector angle formula. After normalizing these parameters, they are weighted and summed to form a quantization index: when △L < L1, the path is approximately straight, without significant turning or obstacles; when L1 ≤ △L < L2, there are a small number of small-angle turns; when L2 ≤ △L < L3, it includes a path with medium curvature or requires avoiding local obstacles; when L3 ≤ △L < L4, the path needs to frequently adjust the direction to cross a complex area; when L4 ≤ △L, the path needs to carefully bypass multiple obstacles or penetrate into the complex structure inside the cell.

[0175] Specifically, the gradient of the error tolerance value is set based on the balance between path complexity and cell safety. For a simple path (L1 ≤ △L < L2), the risk of cell damage is low, and a strict first error tolerance value (ε1, δ1) can be adopted. For example, the velocity error tolerance value ε1 is set to ±0.5 μm / s, and the acceleration error tolerance value δ1 is set to ±0.3 μm / s 2 , ensuring the high-precision operation of the injection needle; while for a complex path (L4 ≤ △L), since it is necessary to frequently adjust the motion posture, in order to avoid mechanical stimulation of the cell caused by control oscillation, the fourth error tolerance value (ε4, δ4) is relaxed to velocity ±3 μm / s and acceleration ±2 μm / s 2 , reducing the risk brought by overcorrection while ensuring the reachability of the path.

[0176] It can be understood that by dynamically matching the velocity and acceleration error tolerance values in combination with the path complexity, the accuracy and stability of microinjection are significantly improved. By setting different error tolerance thresholds according to the path complexity grading, precise control can be achieved on the premise of ensuring operation safety: for a simple path, a strict error tolerance value is adopted to ensure that the injection needle reaches the target point with high precision and reduces unnecessary disturbance to the cell; for a complex path, the tolerance range is relaxed to avoid damage to the cell caused by mechanical oscillation due to frequent correction.

[0177] In some embodiments of the present application, in step S500, during the injection process, the position and state of the injection needle, as well as the reaction of the cell, are monitored in real time by a sensor and fed back to the host computer, and the host computer controls the adaptive control device to dynamically adjust the injection speed, injection volume of the syringe, and the moving speed and position of the positioning platform, including:

[0178] The reaction of the cell includes the degree of cell deformation △D;

[0179] The standard deformation degree D0 is set in advance, and the difference between the cell deformation degree △D and the standard deformation degree D0 |△D-D0| is obtained;

[0180] A first deformation difference threshold D1, a second deformation difference threshold D2, a third deformation difference threshold D3 and a fourth deformation difference threshold D4 are preset, and D1<D2<D3<D4;

[0181] A first injection speed adjustment coefficient α1, a second injection speed adjustment coefficient α2, a third injection speed adjustment coefficient α3 and a fourth injection speed adjustment coefficient α4 are preset, and α1<α2<α3<α4;

[0182] According to the relationship between the difference |△D-D0| and each preset deformation difference threshold, the injection speed adjustment coefficient is selected to adjust the current injection speed;

[0183] When D1≤|△D-D0|<D2, the first injection speed adjustment coefficient α1 is selected to adjust the current injection speed;

[0184] When D2≤|△D-D0|<D3, the second injection speed adjustment coefficient α2 is selected to adjust the current injection speed;

[0185] When D3≤|△D-D0|<D4, the third injection speed adjustment coefficient α3 is selected to adjust the current injection speed;

[0186] When D4≤|ΔD−D0|, the fourth injection speed adjustment coefficient α4 is selected to adjust the current injection speed.

[0187] Specifically, the D0 value is determined based on cell type specificity and injection safety. A "deformation-survival" relationship curve for different cells was established through preliminary experiments: a gradient injection pressure test was performed on each cell, the degree of cell deformation and 24-hour survival rate under different pressures were recorded, and the maximum deformation value corresponding to a survival rate ≥ 90% was selected as D0. The D0 of neural stem cells is set to a volume change rate of 5%, and the plant protoplast can be relaxed to 15% due to cell wall protection. It is understandable that with the degree of cell deformation as the core feedback indicator, a graded response strategy based on preset thresholds and adjustment coefficients can initiate refined speed regulation when the cell undergoes slight deformation, thereby avoiding excessive deformation or rupture of the cell due to improper injection speed.

[0188] In some embodiments of the present application, in step S500, after selecting the i-th injection speed adjustment coefficient αi to adjust the current injection speed, i=1, 2, 3, 4, the host computer controls the adaptive control device to dynamically adjust the injection speed and injection volume of the syringe and the movement speed and position of the positioning platform according to the feedback information, further comprising:

[0189] The cell's response also includes the degree of diffusion of substances within the cell, ΔS;

[0190] The standard diffusion degree S0 is set in advance, and the difference between the diffusion degree △S of the intracellular substance and the standard diffusion degree S0 |△S-S0| is obtained;

[0191] A first diffusion difference threshold S1, a second diffusion difference threshold S2, a third diffusion difference threshold S3 and a fourth diffusion difference threshold S4 are preset, and S1<S2<S3<S4;

[0192] A first injection volume adjustment coefficient β1, a second injection volume adjustment coefficient β2, a third injection volume adjustment coefficient β3 and a fourth injection volume adjustment coefficient β4 are preset, and β1 < β2 < β3 < β4;

[0193] According to the relationship between the difference |△S-S0| and each preset diffusion difference threshold, the injection volume adjustment coefficient is selected to adjust the current injection volume;

[0194] When S1≤|△S-S0|<S2, the first injection volume adjustment coefficient β1 is selected to adjust the current injection volume;

[0195] When S2≤|△S-S0|<S3, the second injection volume adjustment coefficient β2 is selected to adjust the current injection volume;

[0196] When S3≤|△S-S0|<S4, the third injection volume adjustment coefficient β3 is selected to adjust the current injection volume;

[0197] When S4≤|ΔS−S0|, the fourth injection volume adjustment coefficient β4 is selected to adjust the current injection volume.

[0198] Specifically, the setting of the S0 value is based on the characteristics of the cell type and the experimental objectives. For different cells, an "injection volume-diffusion degree" relationship curve is established through preliminary experiments: the cells are tested with a gradient injection volume, and the diffusion range and uniformity of the substance in the cells under each injection volume are recorded. The diffusion degree that can ensure the effective distribution of the substance without causing cytotoxicity is selected as S0. For metabolically active cancer cells, S0 is set to a uniform diffusion covering 70% of the volume of the cell; for nerve cells, because they are sensitive to changes in substance concentration, S0 is adjusted to 50% volume diffusion. In addition, S0 is also dynamically calibrated in combination with material properties (such as molecular size and hydrophilicity) to ensure parameter adaptability.

[0199] It is understandable that with the degree of intracellular substance diffusion as the key feedback indicator, the graded response mechanism based on preset thresholds and adjustment coefficients can timely fine-tune the injection volume when there is a slight deviation in the diffusion of substances, so as to avoid imbalance in the concentration of substances in the cells due to abnormal diffusion of substances; as the diffusion deviation increases, the adjustment force is gradually strengthened to prevent excessive or insufficient diffusion of substances from causing adverse effects on cells.

[0200] In some embodiments of the present application, in step S500, when the injection is completed and the injection needle is withdrawn from the cell and returned to the initial position, the following steps are included:

[0201] A first evacuation speed range E1, a second evacuation speed range E2, a third evacuation speed range E3 and a fourth evacuation speed range E4 are preset, and E1<E2<E3<E4;

[0202] A first cell fragility threshold F1, a second cell fragility threshold F2, a third cell fragility threshold F3 and a fourth cell fragility threshold F4 are preset, and F1 < F2 < F3 < F4;

[0203] According to the fragility of the cell, △F, the corresponding evacuation speed range is selected;

[0204] When F1≤△F<F2, the first evacuation speed range E4 is selected;

[0205] When F2≤△F<F3, the second evacuation speed range E3 is selected;

[0206] When F3≤△F<F4, the third evacuation speed range E2 is selected;

[0207] When F4≤△F, the fourth evacuation speed range E1 is selected;

[0208] The flexible injection needle is controlled to evacuate cells along the planned evacuation path at an appropriate speed within the selected evacuation speed range.

[0209] Specifically, multimodal detection technology is used to comprehensively evaluate the degree of cell fragility △F. First, an atomic force microscope (AFM) is used to perform nanoscale mechanical scanning of the cell surface to obtain mechanical parameters such as cell elastic modulus and adhesion force; at the same time, a confocal microscope is used to observe the integrity of the cytoskeleton structure and quantify the degree of breakage of microfilaments and microtubules; combined with cell membrane potential detection technology, the activity and permeability of cell membrane ion channels are analyzed. After normalizing the above parameters, the principal component analysis (PCA) is used for dimensionality reduction, and the support vector machine (SVM) model is used for training to output a △F value in the range of 0-100. Because the cell membrane of newborn stem cells is soft and the skeleton is not fully developed, the △F value is often higher than 80.

[0210] Specifically, the gradient design of the evacuation speed range follows the principle of "fragility is inversely proportional to speed". For extremely fragile cells (F4≤△F), such as synapses and early embryonic cells, the lowest E1 speed range (0.1-0.5μm / s) is used to avoid tearing the cell membrane due to the fluid shear force generated by rapid evacuation; for cells with stronger tolerance (F1≤△F<F2), such as plant callus cells, the E4 speed range (5-10μm / s) can be used to improve operational efficiency. A safety redundancy value is set for each speed range. For example, within the E1 range, the actual execution speed does not exceed 0.4μm / s to prevent sudden vibrations from causing impact on the cells.

[0211] It is understandable that differentiating the evacuation speed for cells of different fragility levels can effectively avoid secondary damage to cells caused by improper evacuation speed. Using low-speed evacuation for highly fragile cells can minimize the mechanical pull and fluid shear force between the injection needle and the cells, preventing cell membrane rupture or cell structure damage. Using relatively high-speed evacuation for cells with strong tolerance can improve operational efficiency while ensuring cell safety.

[0212] Those skilled in the art will appreciate that the embodiments of the present application may be provided as methods, systems, or computer program products. Therefore, the present application may take the form of a complete hardware embodiment, a complete software embodiment, or a combination of software and hardware embodiments. Furthermore, the present application may take the form of a computer program product implemented on one or more computer-usable storage media (including but not limited to magnetic disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0213] The present application is described with reference to the flowcharts and / or block diagrams of the methods, devices (systems) and computer program products according to the embodiments of the present application. It should be understood that each process and / or box in the flowchart and / or block diagram, as well as the combination of the processes and / or boxes in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 a process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0214] These computer program instructions may also be stored in a computer readable memory that can direct a computer or other programmable data processing device to work in a specific manner, so that the instructions stored in the computer readable memory produce an article of manufacture comprising an instruction device, which implements the process Figure 1a process or multiple processes and / or boxes Figure 1 The function specified in one or more boxes.

[0215] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operational steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing the instructions executed on the computer or other programmable device for implementing the process. Figure 1 a process or multiple processes and / or boxes Figure 1 A step that specifies a function in one or more boxes.

[0216] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. An automated microinjection method with adaptive dynamic adjustment, characterized in that: include: The cells are placed in a culture dish containing a labeled substance, the labeled substance is evenly distributed, and an image acquisition device is used for real-time observation, and the cells with an array of labeled points formed on the surface are transferred; Temperature, humidity, and vibration parameters of the cell environment are collected using temperature, humidity, and vibration sensors at different locations around the microinjection device. Using multiple light sources of different wavelengths to illuminate cells in a predetermined order, corresponding image acquisition devices capture the light to form cell images of different wavelengths; Obtain cell surface marker location information and cell image data sets of different wavelengths, establish a three-dimensional dynamic model of the cell, call the three-dimensional dynamic model of the cell to determine the injection target, obtain environmental parameters, and generate a planned injection path and injection parameters; The flexible injection needle is controlled to move along the planned injection path. After reaching the target, the substance is injected according to the injection parameters. During the injection process, the cell status and environmental parameters are monitored in real time through sensors and fed back to the host computer. The host computer adaptively controls and adjusts the injection parameters. After the injection is completed, the injection needle is withdrawn from the cell and returned to its initial position.

2. The automated microinjection method according to claim 1, wherein Placing the cells in a culture dish containing a marker substance comprises: Microfluidic technology is used to control the flow of the solution in the culture dish, wherein the concentration of the labeled molecules of the labeled substance is ΔC, and a first preset concentration C1, a second preset concentration C2, a third preset concentration C3 and a fourth preset concentration C4 are preset, and C1<C2<C3<C4; Selecting a flow rate level of the microfluidic technology based on the relationship between the labeled molecule concentration ΔC and each preset concentration; When C1≤△C<C2, the first preset flow rate gear V1 is selected as the flow rate of the microfluidic technology; When C2≤△C<C3, the second preset flow rate gear V2 is selected as the flow rate of the microfluidic technology; When C3≤△C<C4, the third preset flow rate gear V3 is selected as the flow rate of the microfluidic technology; When C4≤ΔC, the fourth preset flow rate level V4 is selected as the flow rate of the microfluidic technology.

3. The automated microinjection method according to claim 2, wherein: After selecting the i-th preset flow rate gear Vi as the flow rate of the microfluidic technology, i=1, 2, 3, 4, the cells are observed in real time by the image acquisition device; A first preset observation time T1, a second preset observation time T2, a third preset observation time T3 and a fourth preset observation time T4 are preset, and T1 < T2 < T3 < T4; When the flow rate of the microfluidic technology is V1, the fourth preset observation time T4 is used as the observation judgment time; When the flow rate of the microfluidic technology is V2, the third preset observation time T3 is used as the observation judgment time; When the flow rate of the microfluidic technology is V3, the second preset observation time T2 is used as the observation judgment time; When the flow rate of the microfluidic technology is V4, the first preset observation time T1 is used as the observation judgment time; The cell surface is observed by an image acquisition device within the observation judgment time. If an array of evenly distributed fluorescent marker points appears, the addition of the marker substance is stopped.

4. The automated microinjection method according to claim 3, wherein: The temperature sensors, humidity sensors and vibration sensors at different locations around the microinjection device are used to measure the ambient temperature and humidity at fixed time intervals and monitor the vibration in real time, including: A first time interval Δt1, a second time interval Δt2, a third time interval Δt3 and a fourth time interval Δt4 are preset, and Δt1 < Δt2 < Δt3 < Δt4; A first vibration sensitivity level S1, a second vibration sensitivity level S2, a third vibration sensitivity level S3, and a fourth vibration sensitivity level S4 are preset, and S1<S2<S3<S4; Selecting the time intervals for temperature and humidity measurements and the sensitivity of vibration monitoring according to the vibration sensitivity level corresponding to the cell type; When the vibration sensitivity level corresponding to the cell type is S1, the fourth time interval Δt4 is selected as the time interval for temperature and humidity measurement, and vibration is monitored with the lowest sensitivity; When the vibration sensitivity level corresponding to the cell type is S2, the third time interval Δt3 is selected as the time interval for temperature and humidity measurement to monitor vibration with lower sensitivity; When the vibration sensitivity level corresponding to the cell type is S3, the second time interval Δt2 is selected as the time interval for temperature and humidity measurement to monitor vibration with higher sensitivity; When the vibration sensitivity level corresponding to the cell type is S4, the first time interval Δt1 is selected as the time interval for measuring temperature and humidity, and vibration is monitored with the highest sensitivity.

5. The automated microinjection method according to claim 4, wherein: The method comprises: utilizing a plurality of light sources of different wavelengths and a corresponding image acquisition device to sequentially turn on the light sources of different wavelengths in a predetermined order to irradiate the cells, including: A first wavelength group λ1, a second wavelength group λ2, a third wavelength group λ3 and a fourth wavelength group λ4 are preset, each wavelength group including light sources of different numbers and wavelength values; A first cell type classification K1, a second cell type classification K2, a third cell type classification K3, and a fourth cell type classification K4 are preset; According to the cell type classification, the corresponding wavelength group is selected as the irradiation light source group; When the cell type is classified as K1, the first wavelength group λ1 is selected as the illumination light source group; When the cell type is classified as K2, the second wavelength group λ2 is selected as the illumination light source group; When the cell type is classified as K3, the third wavelength group λ3 is selected as the illumination light source group; When the cell type is classified as K4, the fourth wavelength group λ4 is selected as the illumination light source group; The light sources are turned on in sequence according to the wavelength sequence of the light sources in the selected light source group to irradiate the cells.

6. The automated microinjection method according to claim 5, characterized in that The path planning algorithm plans the injection path from the initial position of the injection needle to the target position, taking into account parameters such as the movement speed, acceleration and injection pressure of the injection needle, including: A first injection pressure range P1, a second injection pressure range P2, a third injection pressure range P3 and a fourth injection pressure range P4 are preset, and P1 < P2 < P3 < P4; A first movement speed range V1, a second movement speed range V2, a third movement speed range V3 and a fourth movement speed range V4 are preset, and V1 < V2 < V3 < V4; A first acceleration range A1, a second acceleration range A2, a third acceleration range A3 and a fourth acceleration range A4 are preset, and A1<A2<A3<A4; According to the hardness △H of the cell, the corresponding injection pressure range, movement speed range and acceleration range are selected; A first hardness threshold H1, a second hardness threshold H2, a third hardness threshold H3 and a fourth hardness threshold H4 are preset, and H1<H2<H3<H4; When H1≤△H<H2, the first injection pressure range P1, the first movement speed range V1 and the first acceleration range A1 are selected; When H2≤△H<H3, the second injection pressure range P2, the second movement speed range V2 and the second acceleration range A2 are selected; When H3≤△H<H4, the third injection pressure range P3, the third movement speed range V3 and the third acceleration range A3 are selected; When H4≤△H, the fourth injection pressure range P4, the fourth movement speed range V4 and the fourth acceleration range A4 are selected; Based on the selected parameter range, the path planning algorithm is used to plan the injection path and determine the specific injection needle movement speed, acceleration and injection pressure parameters.

7. The automated microinjection method according to claim 6, wherein: After planning the injection path using a path planning algorithm according to the selected parameter range and determining specific injection needle movement speed, acceleration, and injection pressure parameters, the flexible injection needle is controlled to move along the planned path, and the movement speed and acceleration of the injection needle during the movement process are controlled, including: A first speed error tolerance value ε1, a second speed error tolerance value ε2, a third speed error tolerance value ε3 and a fourth speed error tolerance value ε4 are preset, and ε1<ε2<ε3<ε4; A first acceleration error tolerance value δ1, a second acceleration error tolerance value δ2, a third acceleration error tolerance value δ3, and a fourth acceleration error tolerance value δ4 are preset, and δ1<δ2<δ3<δ4; According to the complexity of the injection path △L, select the error tolerance value of speed and acceleration; A first complexity threshold L1, a second complexity threshold L2, a third complexity threshold L3 and a fourth complexity threshold L4 are preset, and L1 < L2 < L3 < L4; When L1≤△L<L2, the first velocity error tolerance value ε1 and the first acceleration error tolerance value δ1 are selected; When L2≤△L<L3, the second speed error tolerance value ε2 and the second acceleration error tolerance value δ2 are selected; When L3≤△L<L4, the third speed error tolerance value ε3 and the third acceleration error tolerance value δ3 are selected; When L4≤△L, select the fourth speed error tolerance value ε4 and the fourth acceleration error tolerance value δ4; During the movement of the flexible injection needle, its speed and acceleration are monitored in real time. When the error between the actual speed and the command speed exceeds the selected speed error tolerance value, or the error between the actual acceleration and the command acceleration exceeds the selected acceleration error tolerance value, the movement parameters of the flexible injection needle are adjusted.

8. The automated microinjection method according to claim 7, wherein: During the injection process, the position and state of the injection needle and the reaction of the cells are monitored in real time by sensors and fed back to the host computer. The host computer controls the adaptive control device to dynamically adjust the injection speed and injection volume of the syringe and the movement speed and position of the positioning platform, including: The cell response includes the cell deformation degree ΔD; A standard deformation degree D0 is preset, and the difference between the cell deformation degree ΔD and the standard deformation degree D0, |ΔD-D0|, is obtained; A first deformation difference threshold D1, a second deformation difference threshold D2, a third deformation difference threshold D3 and a fourth deformation difference threshold D4 are preset, and D1<D2<D3<D4; A first injection speed adjustment coefficient α1, a second injection speed adjustment coefficient α2, a third injection speed adjustment coefficient α3 and a fourth injection speed adjustment coefficient α4 are preset, and α1<α2<α3<α4; According to the relationship between the difference |△D-D0| and each preset deformation difference threshold, an injection speed adjustment coefficient is selected to adjust the current injection speed; When D1≤|△D-D0|<D2, the first injection speed adjustment coefficient α1 is selected to adjust the current injection speed; When D2≤|△D-D0|<D3, the second injection speed adjustment coefficient α2 is selected to adjust the current injection speed; When D3≤|△D-D0|<D4, the third injection speed adjustment coefficient α3 is selected to adjust the current injection speed; When D4≤|ΔD−D0|, the fourth injection speed adjustment coefficient α4 is selected to adjust the current injection speed.

9. The automated microinjection method according to claim 8, wherein: After selecting the i-th injection speed adjustment coefficient αi to adjust the current injection speed, i=1, 2, 3, 4, the host computer controls the adaptive control device to dynamically adjust the injection speed and injection volume of the syringe and the moving speed and position of the positioning platform according to the feedback information, and also includes: The cell's response also includes the diffusion degree of intracellular substances ΔS; A standard diffusion degree S0 is preset, and a difference value |ΔS-S0| between the diffusion degree ΔS of the intracellular substance and the standard diffusion degree S0 is obtained; A first diffusion difference threshold S1, a second diffusion difference threshold S2, a third diffusion difference threshold S3 and a fourth diffusion difference threshold S4 are preset, and S1<S2<S3<S4; A first injection volume adjustment coefficient β1, a second injection volume adjustment coefficient β2, a third injection volume adjustment coefficient β3 and a fourth injection volume adjustment coefficient β4 are preset, and β1 < β2 < β3 < β4; According to the relationship between the difference |△S-S0| and each preset diffusion difference threshold, an injection volume adjustment coefficient is selected to adjust the current injection volume; When S1≤|△S-S0|<S2, the first injection volume adjustment coefficient β1 is selected to adjust the current injection volume; When S2≤|△S-S0|<S3, the second injection volume adjustment coefficient β2 is selected to adjust the current injection volume; When S3≤|ΔS-S0|<S4, the third injection volume adjustment coefficient β3 is selected to adjust the current injection volume; When S4≤|ΔS−S0|, the fourth injection volume adjustment coefficient β4 is selected to adjust the current injection volume.

10. The automated microinjection method according to claim 1, wherein After the injection is completed, the injection needle is withdrawn from the cell and returned to the initial position, including: A first evacuation speed range E1, a second evacuation speed range E2, a third evacuation speed range E3 and a fourth evacuation speed range E4 are preset, and E1<E2<E3<E4; A first cell fragility threshold F1, a second cell fragility threshold F2, a third cell fragility threshold F3 and a fourth cell fragility threshold F4 are preset, and F1 < F2 < F3 < F4; According to the fragility of the cell, △F, the corresponding evacuation speed range is selected; When F1≤△F<F2, the first evacuation speed range E4 is selected; When F2≤△F<F3, the second evacuation speed range E3 is selected; When F3≤△F<F4, the third evacuation speed range E2 is selected; When F4≤△F, the fourth evacuation speed range E1 is selected; The flexible injection needle is controlled to evacuate cells along the planned evacuation path at an appropriate speed within the selected evacuation speed range.

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