System and method for controlling'optical handle 'to carry out cell excitation regulation and control based on scanning Gaussian beam

Abstract

The invention discloses a system and a method for controlling an'optical handle 'to perform cell excitation regulation based on a scanning Gaussian beam, and belongs to the technical field of optics. The system comprises a lighting source, an objective table, an inverted fluorescence microscope, a dichroscope group, a galvanometer, a spatial light modulator, a 1064nm laser, a fluorescence LED light source and a CMOS camera. The method comprises the following steps: preparing a 3-micron polystyrene microsphere as an optical handle, and culturing K562 cells; a scanning Gaussian beam is controlled by a galvanometer to drive an optical handle to rotate; the method comprises the following steps: optically assembling a target cell and a rotating optical handle in a three-dimensional space, and driving the cell to rotate to apply mechanical stimulation; and the cell calcium ion signal change is monitored in real time by utilizing fluorescence imaging. According to the invention, continuous, stable and controllable rotary stimulation on cells is realized, high-throughput parallel control, high three-dimensional space resolution and real-time dynamic monitoring capability are realized, and a brand new tool is provided for cell mechanics research.

Description

Technical Field

[0001] This invention belongs to the field of optical technology and relates to a method for regulating cell excitation based on a scanning Gaussian beam manipulating an "optical handle". Background Technology

[0002] As the basic unit of life, the precise regulation of cell function is central to life science research and clinical medicine. Traditional cell regulation techniques, such as electrical stimulation, chemical stimulation, or local temperature control, are typically achieved through direct physical or chemical interventions. However, these methods have inherent limitations in terms of biocompatibility, specificity, and spatial resolution: electrical stimulation easily induces membrane potential disturbances and electrolytic damage; chemical stimulation may lead to non-specific binding or metabolic toxicity; and thermal stimulation may disrupt protein stability and cell membrane integrity.

[0003] In recent years, novel optical manipulation technologies, such as optogenetics and optical tweezers, have made non-invasive cell manipulation possible. In particular, a method using Airy beams to project "optical bullets" to mechanically stimulate nerve cells (CN119492715A A method for mechanically modulating nerve cell calcium ion signals using Airy beams to control "optical bullets") has emerged, demonstrating the potential for non-invasive manipulation. However, this approach and other existing technologies still face a series of unresolved technical challenges, specifically: 1. Limitations of stimulation modes: Existing optomechanical stimulation methods (such as "optical bullets") are mostly based on instantaneous, one-time impact-type action modes, unable to provide continuous, stable, and controllable rotational stimulation. Therefore, they are difficult to simulate the complex mechanical microenvironment in vivo (such as blood flow shear force) and cannot meet the needs of studying long-term physiological responses of cells under continuous mechanical action (such as calcium signal oscillations and gene expression changes). 2. Bottlenecks in throughput and efficiency: Existing single-point optical manipulation techniques mainly target individual cells or sites for sequential manipulation, lacking the ability to perform parallel, independent, and synchronous control of multiple cells. This results in low experimental throughput and limited efficiency, making it difficult to meet the needs of cell population statistical studies. 3. Insufficient precision in three-dimensional manipulation: Although optical tweezers can achieve precise positioning in a two-dimensional plane, there are still issues of insufficient precision or complex procedures in achieving precise alignment and stable assembly of cells and micromanipulation tools (such as microspheres) in three-dimensional space. This affects the reliability of mechanical stimulus delivery and the reproducibility of experiments. 4. Trade-offs in tool performance: The size selection of microspheres used for optical manipulation is crucial. If the size is too large, the spatial resolution is low, making it difficult to accurately target subcellular structures; if the size is too small, it is susceptible to Brownian motion and impurities in complex biological media, leading to decreased capture stability and manipulation reliability. Existing technologies lack optimization considerations for this issue. 5. Disconnect between monitoring and stimulation: Many technologies struggle to non-destructively, in real-time, and dynamically monitor key cellular physiological indicators (such as calcium ion signals) while applying mechanical stimulation. This prevents a direct correlation between mechanical stimulation and the immediate excitatory response of cells, limiting the depth of research. 6. Lack of flexibility in stimulation strategies: Existing technologies often employ fixed or limitedly adjustable stimulation pathways and patterns. They cannot flexibly and dynamically adjust the trajectory, direction, and speed of stimulation through software programming, thus limiting the ability to explore complex cellular mechanobiological behaviors.

[0004] Therefore, there is an urgent need in this field for a novel cell excitation regulation technology that can simultaneously solve the above problems. It should have the characteristics of providing continuous and stable stimulation, high-throughput parallel manipulation, three-dimensional precise positioning, optimized manipulation tools, real-time multimodal monitoring, and high flexibility and programmability. Summary of the Invention

[0005] The purpose of this invention is to provide a method for regulating cell excitation based on a scanning Gaussian beam manipulating an "optical handle," which solves the problems of damage, low flexibility, and low precision in existing cell regulation technologies.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a system for regulating cell excitation based on a scanning Gaussian beam manipulating an "optical handle". The system includes an illumination source, a stage, an inverted fluorescence microscope, a first dichroic mirror, a second dichroic mirror, a first reflecting mirror, a galvanometer, a beam expander, a spatial light modulator, a second reflecting mirror, a second plano-convex lens group, a polarizer, a 1064 nm laser, a fluorescent LED light source, a tubular lens, and a CMOS camera.

[0007] Furthermore, the laser emitted by the 1064 nm laser is collimated sequentially by a polarizer and a second plano-convex lens group, and then reflected by a second mirror to a spatial light modulator for wavefront modulation. The modulated beam is then expanded by a beam expander before entering a galvanometer. This galvanometer is used for high-speed deflection of the laser beam, with a maximum scanning angle of ±20° and a small-signal step response time of 0.2 ms, making it crucial for dynamic control. The deflected beam is then shaped by a first plano-convex lens group and finally reflected by a first dichroic mirror into the objective lens of an inverted fluorescence microscope, forming a dynamically controllable optical potential well on the sample plane.

[0008] Furthermore, the illumination source is used for transmission bright-field imaging. The light path passes sequentially through the microscope, the first dichroic mirror, and the second dichroic mirror, and after being reflected by the first reflecting mirror, it is converged by a tubular lens to the CMOS camera. The 490 nm excitation light emitted from the fluorescent LED source is reflected by the second dichroic mirror and enters the objective lens to excite the fluorescent probe (such as Fluo-4 AM) in the sample. The resulting 520 nm fluorescence signal is finally received and recorded by the CMOS camera.

[0009] Secondly, the present invention provides a method for regulating cell excitation based on the above-described system. The method includes the following specific steps: S1: Preparation of optical handle and cell sample; S2: Establish and execute optical rotation drive; S3: Enables the assembly and rotation control of the optical handle and cell; S4: Perform real-time fluorescence monitoring and image recording.

[0010] Furthermore, step S1 specifically includes: S1.1: Prepare polystyrene microspheres with a size of 3 μm as "optical handles" and disperse them in cell culture medium by ultrasonication (20~80 kHz, 3~7 min) to form a solution with a concentration of 0.01 mg / mL.

[0011] S1.2: Culture K562 cells to 70-80% density, incubate with 2 μm Fluo-4 AM calcium ion fluorescent dye for 30-45 min, wash with serum-free medium after staining, and place in buffer containing 1-2 mM CaCl2 for later use.

[0012] Furthermore, step S2 specifically includes: S2.1: Construct a closed sample well on a glass slide.

[0013] S2.2: Add the cell suspension and optical handle solution to the sample well and seal the slide, then place it on the stage.

[0014] S2.3: Start the 1064 nm laser and execute a circular scanning path under computer control via a galvanometer (16) to make the captured polystyrene microspheres rotate continuously along the set trajectory. By adjusting the deflection angle and switching speed of the beam, the rotation path and speed of the microspheres can be precisely controlled.

[0015] Furthermore, step S3 specifically includes: By moving the optical potential trap, the target cell is moved in three-dimensional space (XY plane and Z axis) until it comes into contact with and coaxially rotates with the rotating "optical handle". Under the synergistic effect of the optical gradient force and the cell surface viscosity, the rotating polystyrene microspheres act as the "handle" to drive the target cell to rotate synchronously, thereby achieving dynamic mechanical stimulation of the cell.

[0016] Furthermore, step S4 specifically includes: Turn off the bright field light source and turn on the 490 nm fluorescence excitation light. Use a CMOS camera (9) to record in real time the changes in intracellular calcium ion signal (520 nm fluorescence) caused by cell rotation stimulation, so as to realize the visualization and monitoring of the regulation process.

[0017] Compared with the prior art, the present invention has the following significant advantages: 1. It achieves stable, controllable, and long-duration rotational stimulation, overcoming the limitations of the instantaneous impact mode. To address the limitations of existing technologies (such as "optical bullets") that only provide instantaneous, one-time stimulation, this invention utilizes a galvanometer to execute a circular scanning path, driving a "optical handle" to rotate continuously and stably. This galvanometer features a maximum scanning angle of ±20° and a rapid response time of 0.2 ms, ensuring precise control and high-speed switching of the rotation trajectory. Experiments have demonstrated that this system can drive cells to rotate continuously for over one hour, providing an unprecedented stable platform for studying long-term physiological responses of cells under sustained shear or torsional forces (such as calcium signal oscillations and changes in gene expression).

[0018] 2. It possesses powerful capabilities for high throughput and parallel manipulation, significantly improving experimental efficiency. To address the issue of low throughput in single-point operations, this invention leverages the high-speed beam deflection capability of the galvanometer, combined with computer control, to rapidly switch the spatial position of the optical potential well using time-division multiplexing technology. This means that multiple "optical handles" can be quickly and sequentially captured and driven within the same field of view, enabling independent and synchronous rotational control of multiple target cells. This high-throughput parallel manipulation capability significantly improves experimental efficiency and is suitable for cell population statistical studies.

[0019] 3. Provides high-precision three-dimensional spatial positioning and assembly capabilities, ensuring reliable stimulus delivery. To overcome the limitations of three-dimensional manipulation precision, in step S3, this invention not only moves the optical potential well in the XY plane to bring the cell closer to the handle (approximately 5 μm away), but also achieves precise alignment and contact between the cell and the "optical handle" in the Z-axis direction by finely adjusting the fine focusing knob of the microscope objective (approximately 2 μm). This precise positioning and assembly in three-dimensional space ensures that mechanical stimulation can be efficiently and reliably delivered to the target cell, which is key to achieving highly reproducible experiments.

[0020] 4. Through optimized optical handle design and non-destructive real-time monitoring, stability, resolution, and dynamic observation are balanced. In step S1.1, this invention explicitly selects polystyrene microspheres with a size of 3 μm as the "optical handle". This specific size ensures the optimal balance between the stability of light capture in complex media and the high spatial resolution of cell-mediated localization. The system integrates both bright-field imaging and fluorescence imaging modes. By excitation with a 490 nm fluorescent LED light source (6) and acquisition of a 520 nm fluorescence signal, combined with a CMOS camera (9), it is possible to monitor changes in intracellular calcium ion concentration in real time and dynamically without interrupting rotational stimulation, thus realizing a direct correlation between stimulation and cellular excitation response (such as calcium scintillation).

[0021] 5. It exhibits high flexibility and programmability, supporting dynamic adjustment of complex stimulus patterns. To address the lack of flexibility in stimulation strategies, the entire manipulation process is controlled by a computer program. Through the software interface, the operator can flexibly set the scanning path of the galvanometer (e.g., circular, linear), the rotation direction (clockwise / counterclockwise), and the switching speed. This software-defined control method allows the mechanical stimulation patterns applied to cells (e.g., steady-state rotation, oscillation, acceleration) to be programmed and adjusted in real-time according to experimental needs, providing unparalleled experimental flexibility.

[0022] 6. It ensures low damage and high biocompatibility throughout the process, maintaining the natural physiological state of cells. As a fundamental and crucial advantage, this invention ensures the harmlessness of the modulation process. The system's capture and drive optical path employs a 1064 nm near-infrared laser, a wavelength within the "bio-optical window" with low water absorption, resulting in minimal phototoxicity and thermal damage to cells. Simultaneously, the entire stimulation process is purely optical and non-contact, avoiding the physical punctures of traditional electrodes or the potential toxicity of chemical reagents, thus maximally maintaining the natural physiological state of cells and ensuring the reliability of experimental data. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of an experimental setup that uses a scanning Gaussian beam to manipulate an "optical handle". Figure 2 This is an example of the invention based on a scanning Gaussian beam manipulating an "optical handle," showing the rotation of polystyrene microspheres as an "optical handle," optical assembly with cells, and experimental results of regulating K562 cells at different times. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 A system for regulating cell excitation based on a scanning Gaussian beam-controlled "optical handle" includes an illumination source 1, a stage 2, an inverted fluorescence microscope 3, a first dichroic mirror 4, a second dichroic mirror 5, a first reflecting mirror 7, a second reflecting mirror 13, a first plano-convex lens group 17, a galvanometer 16, a beam expander 15, a spatial light modulator 14, a second plano-convex lens group 12, a polarizer 11, a 1064 nm laser 10, a fluorescent LED light source 6, a tubular lens 8, and a CMOS camera 9.

[0026] The inverted fluorescence microscope 3, the first dichroic mirror 4, the second dichroic mirror 5, and the first reflecting mirror 7 are arranged sequentially below the stage 2. Along the optical path to the left of the first dichroic mirror 4, a first plano-convex lens group 17, a galvanometer 16, a beam expander 15, and a spatial light modulator 14 are arranged sequentially. To the upper right of the spatial light modulator 14, a second reflecting mirror 13 is arranged, and to its left, a second plano-convex lens group 12, a polarizer 11, and a 1064 nm laser 10 are arranged sequentially. A fluorescent LED light source 6 is arranged to the right of the second dichroic mirror 5. To the right of the first reflecting mirror 7, a tubular lens 8 and a CMOS camera 9 are arranged sequentially.

[0027] The inverted fluorescence microscope 3 is equipped with a 60x water mirror and a numerical aperture (NA) of 1.0, making it suitable for high-resolution cell imaging and optical capture.

[0028] The illumination source 1 is placed above the stage 2, and its emitted light passes sequentially through the inverted fluorescence microscope 3, the first dichroic mirror 4, and the second dichroic mirror 5. Due to the wavelength-selective reflection characteristics of the dichroic mirrors, the illumination light directly passes through the first dichroic mirror 4 and the second dichroic mirror 5, and after being reflected by the first reflecting mirror 7, it is converged by the tubular lens 8 to the CMOS camera 9 for real-time acquisition and recording of bright-field images of the sample.

[0029] The laser emitted by the 1064 nm laser 10 is polarized by the polarizer 11, collimated by the second plano-convex lens group 12, and then reflected by the second mirror 13 into the spatial light modulator 14 for wavefront modulation. The modulated beam is expanded by the beam expander 15, deflected by the galvanometer 16, and further shaped by the plano-convex lens group A17. Finally, it is reflected by the first dichroic mirror 4 into the objective lens of the inverted fluorescence microscope 3, forming a dynamically controllable optical potential well on the sample plane for capturing and driving the "optical handle".

[0030] The excitation light emitted from the fluorescent LED light source 6 has a wavelength of 490 nm. After being reflected by the second dichroic mirror 5, it passes through the first dichroic mirror 4 and enters the objective lens, exciting the fluorescent probe loaded in the sample. The sample emits a fluorescent signal with a wavelength of 520 nm. This signal passes sequentially through the objective lens, the first dichroic mirror 4, the second dichroic mirror 5, and the first reflecting mirror 7, and is finally imaged onto the CMOS camera 9 by the tubular lens 8, realizing real-time monitoring and recording of the fluorescence signal during cell excitation.

[0031] Example 2 A method for regulating cell excitation based on a scanning Gaussian beam manipulating an "optical handpiece" includes the following steps: S1: Preparation of optical handle and cell samples First, an "optical handle" for regulating target cells was prepared and K562 cells were cultured. The "optical handle" was made of polystyrene microspheres, and the specific steps are as follows: S1.1: A certain amount of polystyrene microspheres were mixed in cell culture medium to obtain a solution with a concentration of 0.01 mg / mL. The prepared solution was placed in an ultrasonic shaker and shaken at a frequency of 20~80 kHz for 3~7 min to prepare a dispersed polystyrene "optical handle" solution. The solution was then heated in a water bath and kept at a temperature of about 37℃. The "optical handle" used in this invention is a polystyrene microsphere with a size of 3 μm. Microspheres larger than this size will reduce the spatial resolution of cell regulation and positioning; microspheres smaller than this size will be more affected by viscosity changes and impurity adsorption in complex media (such as cell culture medium), reducing the stability of photomechanical capture and thus affecting the stability and flexibility of cell manipulation.

[0032] S1.2: In RPMI-1640 complete medium, a single-cell suspension was formed by repeated pipetting with a sterile pipette. Tumor cells (human chronic myeloid leukemia cells) were seeded into cell culture dishes and cultured in an incubator at 37°C and 5% carbon dioxide concentration to obtain K562 cells.

[0033] S1.3: Culture K562 cells from S1.2 to a density of 70-80%, and wash the cells with serum-free medium. Then, resuspend the cells in Fluo-4 AM (intracellular calcium fluorescent staining agent) at a working concentration of 2 μm, add it to the cell culture medium, and incubate with the cells for 30-45 min. After staining, wash the cells again with serum-free medium to completely remove unbound Fluo-4 AM probes. After the washing step, place the cells in physiological saline or a specific buffer containing 1-2 mM CaCl2 to maintain intracellular calcium ion stability.

[0034] S2: Establish and execute optical rotation drive Driving the rotation of polystyrene microspheres in the cellular microenvironment specifically includes: S2.1: Adhere two rows of double-sided adhesive layers to the central area of ​​a clean glass slide. The double-sided adhesive layers are approximately 500 μm thick, 3 mm wide, and 20 mm long, with a spacing of approximately 20 mm between each layer, forming a 20 mm × 20 mm sample groove.

[0035] S2.2: Using a pipette, aspirate approximately 50 μL of cell culture medium containing suspended cells from the cell culture dish and drop it into the sample well described in S2.1. Using a pipette, take approximately 10 μL of the "optical handle" solution prepared in step S1.1 and drop it into the center of the sample well. Then, using pointed tweezers, use a coverslip to cover the sample well, ensuring a sealed connection between the double-sided adhesive layer, the lower slide, and the upper coverslip, thus forming a closed cell sample well. Invert the prepared sample onto the stage 2, and turn on the top illumination source 1 to clearly see the sample's appearance.

[0036] S2.3: Using holographic optical tweezers, the spatial position and laser power of the capture optical potential trap are set. A 1064 nm laser beam emitted by the 10 laser 10 is modulated into a linearly polarized beam by the polarizer 11. After being collimated by the second plano-convex lens group, the beam is reflected by the second reflecting mirror 13 into the spatial light modulator 14. The modulated beam is expanded by the beam expander 15 composed of two plano-convex lenses, and then deflected by the galvanometer 16. After passing through the first plano-convex lens group 17, it is focused onto the slide by the first dichroic mirror 4 (which has high reflectivity for 1064 nm wavelength beams and high transmittance for 490 nm and 520 nm wavelength beams) and the objective lens of the inverted fluorescence microscope 3. The polystyrene microspheres are captured at the focal point by the light gradient force in the light field. The laser beam is deflected by the galvanometer 16 under computer control. When a circular scanning path is set, the laser beam will scan along this path, thereby causing the captured polystyrene microspheres to rotate along the set trajectory. By setting the deflection angle and deflection switching speed of the light beam, the path and speed of the polystyrene microspheres can be controlled.

[0037] S3: Enables the assembly and rotation control of the optical handle and cell. Polystyrene microspheres are used as "optical handles" to assemble with cells, driving cell rotation and dynamically regulating cells. Specifically, this includes: The target cells are captured at the focal point by the optical gradient force in the light field. The laser beam is deflected under computer control via galvanometer 16, thereby changing the spatial position of the optical potential trap. The captured cells are moved to the perimeter of the rotating polystyrene microsphere in S2.3, approximately 5 μm apart in the xy plane. The fine focusing knob of the microscope objective is adjusted by approximately 2 μm, further moving the target cells to be coaxial with and in contact with the polystyrene microsphere on the Z-axis, thus completing the optical assembly of the "optical handle" with the K562 cells. Therefore, under the synergistic effect of the optical gradient force and the cell surface viscosity, the rotating polystyrene microsphere can act as an "optical handle" to drive the rotation of the target cells.

[0038] S4: Perform real-time fluorescence monitoring and image recording. The modulation process is monitored and recorded in real time using a CMOS camera 9, specifically including: To observe changes in cell fluorescence in real time, illumination source 1 is turned off, while fluorescence LED source 6 is turned on and its excitation wavelength is set to 490 nm. This excitation beam is reflected by the second dichroic mirror 5 (which has high reflectivity for 490 nm and high transmittance for 520 nm), then transmitted through the first dichroic mirror 4, and coupled into the objective lens of the fluorescence microscope 3, ultimately exciting the calcium ion fluorescent dye loaded in the cells within the sample chamber. As the "optical handle" drives the target cell to rotate rapidly, the resulting momentum is transferred to the cell membrane surface during elastic contact between cells, applying localized mechanical action, thereby opening mechanosensitive channels and regulating changes in calcium ion signals during cell excitation. The image signal of this process is coupled into the CMOS camera 9 via a magnification system composed of the objective lens and tubular lens 8. The CMOS camera 9 monitors and records the image of the regulation process in real time.

[0039] Figure 2 This is an example of the present invention, which uses a scanning Gaussian beam to manipulate an "optical handle," and the experimental results of rotating polystyrene microspheres as an "optical handle," optically assembling with cells, and regulating K562 cells at different times. Among them, a is a time-stacked experimental diagram of polystyrene microsphere rotation, with the rotation direction being counterclockwise; b is a bright-field diagram of K562 cells before regulation; c is an experimental diagram of polystyrene as an "optical handle" optically assembling with target K562 cells; and d1-d3 are experimental diagrams of the "optical handle" driving cell rotation.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A system for cell excitation modulation based on manipulation of "optical handles" using a scanning Gaussian beam, characterized in that, The system comprises a light source (1), a stage (2), an inverted fluorescence microscope (3), a first dichroic mirror (4), a second dichroic mirror (5), a first mirror (7), a galvanometer (16), a beam expander (15), a spatial light modulator (14), a second mirror (13), a second plano-convex lens group (12), a polarizer (11), a 1064 nm laser (10), a fluorescent LED light source (6), a tubular lens (8), and a CMOS camera (9); the stage (2) is sequentially provided below with the inverted fluorescence microscope (3), the first dichroic mirror (4), the second dichroic mirror (5), and the first mirror (7); the left side of the first dichroic mirror (4) is sequentially provided along the light path with a first plano-convex lens group (17), the galvanometer (16), the beam expander (15), and the spatial light modulator (14); the upper right side of the spatial light modulator (14) is provided with the second mirror (13), and the left side of the second mirror (13) is sequentially provided with the second plano-convex lens group (12), the polarizer (11), and the 1064 nm laser (10); the right side of the second dichroic mirror (5) is provided with the fluorescent LED light source (6); and the right side of the first mirror (7) is sequentially provided with the tubular lens (8) and the CMOS camera (9).

2. The system of claim 1, wherein, The objective lens of the inverted fluorescence microscope (3) is a 60x water lens.

3. The system of claim 1, wherein, The maximum scanning angle of the galvanometer (16) is ±20°, and the small signal step response time is 0.2 ms.

4. A method for cell excitation modulation based on manipulation of "optical handle" by a scanning Gaussian beam, characterized in that, The system of any one of claims 1-3 comprises the following steps: S1: preparing optical handles and cell samples: preparing polystyrene microspheres as "optical handles", and culturing K562 cells labeled with calcium ion fluorescent dye; S2: establishing and executing optical rotary drive: controlling the scanning Gaussian light beam by the galvanometer (16) to drive the captured polystyrene microspheres to continuously rotate along the set track; S3: realizing the assembly and rotary regulation of the optical handle and the cell: moving the optical potential well, aligning and contacting the target cell with the rotating "optical handle" in three-dimensional space, and making the "optical handle" drive the cell to rotate synchronously; S4: executing real-time fluorescence monitoring and image recording: exciting the cell under rotary stimulation by the fluorescent LED light source (6) and recording the calcium ion fluorescence signal change of the cell in real time by the CMOS camera (9).

5. The method of claim 4, wherein, In step S1, the size of the polystyrene microspheres is 3 μm.

6. The method of claim 4, wherein, In step S2, the galvanometer (16) is controlled by a computer to execute a ring-shaped scanning path, and the rotation path and speed of the polystyrene microspheres are controlled by adjusting the deflection angle and switching speed of the light beam.

7. The method of claim 4, wherein, In step S3, the aligning and contacting of the target cell with the rotating "optical handle" in three-dimensional space specifically comprises: moving the cell to within about 5 μm from the microsphere in the X-Y plane, and adjusting the focus of the microscope objective lens in the Z-axis direction by about 2 μm, so that the cell and the microsphere are coaxially contacted.

8. The method of claim 4, wherein, In step S1, the calcium ion fluorescent dye is Fluo-4AM, and the concentration used is 2 μm, and the incubation time with the cell is 30-45 min.

9. The method of claim 4, wherein, In step S4, the excitation wavelength of the fluorescent LED light source (6) is 490 nm and the fluorescent signal wavelength received by the CMOS camera (9) is 520 nm.

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