Method for mechanically regulating and controlling nerve cell calcium ion signal by using Airy beam to control optical bullet

Abstract

The invention relates to a method for mechanically regulating and controlling a nerve cell calcium ion signal by using an Airy beam to control an optical bullet, which comprises the following steps: firstly preparing the optical bullet and culturing HT-22 cells, then dynamically regulating and controlling the cell calcium ion signal, and finally monitoring and recording an image in the regulation and control process in real time through a CMOS (Complementary Metal Oxide Semiconductor) camera. According to the method, non-contact mechanical stimulation on nerve cells can be realized. The spatial resolution limitation of the traditional nerve regulation and control technology is broken through, and a non-invasive and high-biocompatibility nerve signal regulation and control means is provided. Besides, in combination with a real-time fluorescence monitoring technology, the regulation and control effect can be intuitively evaluated, and a brand new tool is provided for neuroscience research and clinical application.

Description

Technical Field

[0001] The invention belongs to the field of optical technology and relates to a method for mechanically regulating nerve cell signals by manipulating an "optical bullet" with an Airy beam. Background Art

[0002] Precise regulation of nerve cell signals is crucial for a deeper understanding of the functions of the nervous system and disease mechanisms. Driven by the rapid development of biomedicine, neuroscience, and related interdisciplinary disciplines, there is a growing demand for technologies that can precisely regulate nerve cell signals. These technologies have extremely broad application prospects in advancing basic research, improving disease treatment strategies, and optimizing drug screening processes. However, existing technologies have limitations in terms of regulation accuracy, operational complexity, and biocompatibility, which limit their ability to achieve high-precision, non-invasive, and real-time dynamic regulation.

[0003] Specifically, electrical stimulation and magnetic stimulation techniques affect the activity of nerve cells through electric current or magnetic field. Although these methods are easy to operate, they are somewhat damaging and have low spatial resolution. For example, high current stimulation may cause damage to cells, while magnetic field stimulation is limited by its accuracy in spatial positioning. As a non-invasive means, ultrasound regulation technology can produce stimulation in deep tissues, but it still faces the challenge of improving spatial resolution and accuracy. Optogenetics expresses light-sensitive proteins through gene modification. Although it can achieve precise control of specific nerve cells, this technology requires genetic manipulation, which not only has ethical and regulatory restrictions, but the viral transfection process may also bring toxicity and stability problems. In addition, although mechanical electrode stimulation can provide precise quantitative control, it is highly invasive and may cause physical damage to cells.

[0004] Given these limitations of existing technologies, researchers and clinicians are in urgent need of a new means of regulating nerve cell signals, which should have higher regulation accuracy, no need to invade cells, be able to regulate dynamically in real time, and have better biocompatibility and ease of operation compared to existing technologies. These needs have driven the exploration of new non-invasive neuromodulation technologies in order to overcome the limitations of existing technologies and provide powerful tools for in-depth research on nervous system functions and the development of new treatments. Summary of the invention

[0005] The purpose of the present invention is to provide an Airy beam controlled "optical bullet" to mechanically regulate nerve cell signals, thereby solving the problems of invasiveness, damage and low precision in regulating cell signals in the prior art.

[0006] The technical solution adopted by the present invention is:

[0007] A system for mechanically regulating calcium ion signals of nerve cells by manipulating an "optical bullet" with an Airy beam, the system comprises an object stage, an illumination light source is arranged above the object stage, an inverted fluorescence microscope, a dichroic mirror A, a dichroic mirror B and a first reflector are arranged in sequence directly below the object stage, a plano-convex lens group and a spatial light modulator are arranged in sequence on the left side of the dichroic mirror A, a second reflector is arranged at the lower right of the spatial light modulator, a beam expander, a polarizer and a 1064nm laser are arranged in sequence on the left side of the second reflector; a fluorescent LED laser is arranged to the right of the dichroic mirror B; and a tubular lens and a CMOS camera are arranged in sequence on the right side of the first reflector.

[0008] Furthermore, the lens of the inverted fluorescence microscope is a 60x water lens (NA1.2).

[0009] Furthermore, an illumination light source is arranged above the fluorescence microscope, and the illumination light source passes through the inverted fluorescence microscope, dichroic mirror A and dichroic mirror B. The dichroic mirror only reflects light of a specific wavelength, so the illumination light directly passes through the dichroic mirror A and dichroic mirror B, and then is reflected by the first reflector and focused by the tubular lens to the CMOS camera, and the CMOS camera records the pictures and videos presented in the fluorescence microscope.

[0010] A method for mechanically regulating calcium ion signals of nerve cells by manipulating an "optical bullet" with an Airy beam comprises the following steps:

[0011] S1: Preparation of polystyrene “optical bullets” and cultivation of HT-22 cells;

[0012] S2: Using polystyrene microspheres as “optical bullets” to dynamically regulate cellular calcium signals;

[0013] S3: The images of the control process are monitored and recorded in real time through the CMOS camera.

[0014] A method for preparing a cell nucleus "optical bullet" and regulating a cell calcium ion signal comprises the following steps:

[0015] S1: Preparation of cell nucleus "optical bullets" to culture HT-22 cells;

[0016] S2: Using the cell nucleus as an “optical bullet” to dynamically regulate cellular calcium signals;

[0017] S3: The images of the control process are monitored and recorded in real time through the CMOS camera.

[0018] The beneficial effects of the present invention are:

[0019] 1. Non-invasive regulation: The present invention adopts the method of controlling the "optical bullet" with Airy-Gaussian beam to achieve non-contact regulation of nerve cell signals, avoiding the damage to cells that may be caused by traditional electrode stimulation methods, and improving the safety and reliability of the experiment.

[0020] 2. High-precision positioning: By utilizing the characteristics of the Airy-Gaussian beam, the present invention can achieve precise control of the "optical bullet" at the subcellular structure level, thereby achieving precise stimulation of specific nerve cells or their substructures, meeting the needs of high-precision spatial positioning.

[0021] 3. Dynamic programmable control: By changing the light beam parameters and the control path, the present invention can dynamically program and control the motion trajectory and action mode of the "optical bullet", thereby realizing real-time and dynamic control of nerve cell signals.

[0022] 4. High biocompatibility: The optical manipulation method used in the present invention does not require the use of any invasive tools or chemical reagents that may be harmful to cells, reducing interference with biological samples. The fully biocompatible cell nucleus can be used as an "optical bullet" to improve the biocompatibility of the method.

[0023] 5. Real-time dynamic monitoring: Combined with the fluorescence microscopy imaging system, the present invention can monitor the response of nerve cells to the stimulation of "optical bullets" in real time, providing intuitive and real-time data support for the study of nerve cell signal transduction.

[0024] 6. Versatility: The present invention can not only be used for basic research on nerve cell signals, but can also be extended to areas such as drug screening, disease model research, and nerve regeneration, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the experimental device based on Airy-Gauss beam manipulation of "optical bullets";

[0026] Figure 2 This is a diagram of experimental results of the polystyrene microspheres used as "optical bullets" to regulate HT-22 cells at different times in an embodiment of the present invention based on Airy-Gaussian beam manipulation of "optical bullets";

[0027] Figure 3 This is a diagram of experimental results of using the cell nucleus as an "optical bullet" in an embodiment of the present invention based on Airy-Gaussian beam manipulation of the "optical bullet". DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] A system for mechanically regulating calcium ion signals of nerve cells by manipulating an Airy beam with an "optical bullet", the system comprising a stage 1, an illumination light source 2 is arranged above the stage 1, an inverted fluorescence microscope 3, a dichroic mirror A4, a dichroic mirror B5 and a first reflector 9 are arranged in sequence directly below the stage 1, a plano-convex lens group 15 and a spatial light modulator 14 are arranged in sequence on the left side of the dichroic mirror A4, a second reflector 10 is arranged at the lower right of the spatial light modulator 14, a beam expander 11, a polarizer 12 and a 1064nm laser 13 are arranged in sequence on the left side of the second reflector 10; a fluorescent LED laser 6 is arranged to the right of the dichroic mirror B5; a tubular lens 7 and a CMOS camera 8 are arranged in sequence on the right side of the first reflector 9.

[0031] The lens of the inverted fluorescence microscope 3 is a 60x water lens (NA1.2).

[0032] An illumination light source 2 is arranged above the fluorescence microscope 3. The illumination light source 2 passes through the inverted fluorescence microscope 3, the dichroic mirror A4 and the dichroic mirror B5. The dichroic mirror only reflects light of a specific wavelength (a laser with a wavelength of 1064 nm is used in the present invention). Therefore, the illumination light source 2 directly passes through the dichroic mirror A4 and the dichroic mirror B5, and then is reflected by the first reflector 9 and focused by the tubular lens 7 to the CMOS camera 8. The CMOS camera 8 records the pictures and videos presented in the fluorescence microscope 3.

[0033] Example 2

[0034] A method for mechanically regulating calcium ion signals of nerve cells by manipulating an "optical bullet" with an Airy beam comprises the following steps:

[0035] S1: Preparation of polystyrene "optical bullets" and cultivation of HT-22 cells

[0036] S11: dissolving a certain amount of polystyrene microspheres in a cell culture medium to obtain a solution with a concentration of 0.01 mg / mL, placing the prepared solution in an ultrasonic oscillator at room temperature, and oscillating at a frequency of 20 to 80 KHz for 2 to 10 minutes to prepare a dispersed polystyrene "optical bullet" solution;

[0037] The "optical bullet" used in the present invention is a polystyrene microsphere of 1 micron in size. Microspheres larger than this size will result in a decrease in the spatial resolution of the regulatory signal, while microspheres smaller than this size will reduce the light force that affects the mechanical force of collision with cells.

[0038] S12: In DMEM complete medium, use a sterile pipette to repeatedly blow and beat to form a single cell suspension, and inoculate the neural cells into a cell culture dish. The culture dish is placed with a cell slide and coated with polyethanolamine and laminin antibody 1 hour in advance to obtain attached neural cells. Finally, the cells are cultured in a 37°C, 50mL / L CO2 incubator to obtain HT-22 cells.

[0039] S13: Cultivate the HT-22 cells in S12 to a density of 70-80%, and then wash the cells with serum-free medium. Next, prepare Fluo-4 AM (intracellular calcium ion fluorescent stain) to a working concentration (e.g., 2 μM) and incubate with the cells for 30-45 minutes. After staining, wash the cells again with serum-free medium to remove unbound Fluo-4 AM probes. After the washing step is completed, place the cells in physiological saline or a specific buffer containing 1-2 mM CaCl2 to maintain the stability of intracellular calcium ions.

[0040] S2: Using polystyrene microspheres as “optical bullets” to dynamically regulate cellular calcium signals

[0041] S21: Use pointed tweezers to take out the cell slide from the culture dish, place it upside down on a glass slide, use a pipette to take out the "optical bullet" solution prepared in step S11, and drop it between the cell slide and the glass slide, place the prepared sample upside down on the stage 2, and turn on the top lighting source 1 to clearly see the sample morphology.

[0042] S22: Use holographic optical tweezers to set the spatial position and laser power of the capture point. The 1064nm laser 13 emits a Gaussian beam with a wavelength of 1064nm, which is modulated into a linearly polarized collimated beam by a polarizer 12. The beam is expanded by a beam expander 11 composed of two plano-convex lenses, and then reflected into a spatial light modulator 14 by a second reflector 10. The beam is collimated by a plano-convex lens group 15, and then the collimated beam passes through a dichroic mirror A4 (the mirror has high reflectivity for beams with a wavelength of 1064nm and high transmittance for beams with wavelengths of 490nm and 520nm) and the objective lens of an inverted fluorescence microscope 3 and is focused onto a glass slide. Polystyrene microspheres are captured at the focus in the light field due to the light gradient force. The Airy beam phase is loaded through the spatial light modulator and the computer, and the phase of the input Gaussian beam is modulated into an Airy beam. The main characteristic of the Airy beam is that the central light intensity (main lobe) propagates along a parabola. At this time, the particles are scattering light more than the gradient force, so the captured polystyrene microspheres are ejected along the trajectory to the cell surface. The polystyrene microspheres ejected by the light beam are usually called "optical bullets". The ejection direction and speed of the "optical bullet" can be controlled by setting the deflection angle of the light beam and the incident laser power.

[0043] S3: Real-time monitoring and recording of images of the control process through CMOS camera

[0044] In order to observe the intracellular fluorescence changes, the illumination light source 1 is turned off, the fluorescent LED laser 6 is turned on, and the excitation wavelength is set to 490nm. The excitation light beam of the fluorescent LED laser 6 is reflected by the dichroic mirror B5 (the mirror has high reflectivity for light beams with a wavelength of 490nm, and high transmittance for light beams with a wavelength of 520nm), and is coupled into the objective lens of the fluorescence microscope 3 through the dichroic mirror A4, thereby exciting the calcium ion fluorescent dye in the cells in the sample chamber. Since the "optical bullet" instantly generates momentum transfer when in elastic contact with the cell, a force is applied to the cell membrane surface to open the mechanical sensitive channel, thereby regulating the change of the cell calcium ion signal. The image signal of this process is coupled into the CMOS camera 8 through the amplification system composed of the objective lens and the tube lens 7, and the CMOS camera 8 monitors and records the image of the regulation process in real time.

[0045] Figure 2 These are graphs of the experimental results of using polystyrene microspheres as "optical bullets" to regulate HT-22 cells at different times in an embodiment of the present invention based on the manipulation of an "optical bullet" by an Airy-Gaussian beam; wherein a1 and a2 are bright-field and dark-field fluorescence images of HT-22 cells before stimulation by polystyrene microspheres as "optical bullets", b1 and b2 are experimental images and dark-field fluorescence images of the stimulated cells regulating calcium ion signals, and c1 and c2 are experimental images and dark-field fluorescence images of the cells after stimulation.

[0046] Example 3

[0047] A method for preparing a cell nucleus "optical bullet" and regulating a cell calcium ion signal comprises the following steps:

[0048] S1: Preparation of nuclear "optical bullets" for culturing HT-22 cells

[0049] S31: First, fresh blood is collected from the chicken by cardiac puncture, and an anticoagulant is added during the collection process to prevent blood coagulation. Subsequently, the collected blood is mixed with a lysis buffer and stirred thoroughly with a syringe until the red blood cells are completely lysed. Next, centrifugation is performed at 3000 rpm for 5 minutes to allow the cell nuclei to settle at the bottom of the centrifuge tube. The separated cell nuclei are washed with an appropriate buffer to remove residual cell debris and lysis buffer components, and finally a pure cell nucleus is obtained.

[0050] The anticoagulant is EDTA or heparin.

[0051] S12: In DMEM complete medium, use a sterile pipette to repeatedly blow and beat to form a single cell suspension, and inoculate the neural cells into a cell culture dish. The culture dish is placed with a cell slide and coated with polyethanolamine and laminin antibody 1 hour in advance to obtain attached neural cells. Finally, the cells are cultured in a 37°C, 50mL / L CO2 incubator to obtain HT-22 cells.

[0052] S13: Cultivate the HT-22 cells in S12 to a density of 70-80%, and then wash the cells with serum-free medium. Next, prepare Fluo-4 AM (intracellular calcium ion fluorescent stain) to a working concentration (e.g., 2 μM) and incubate with the cells for 30-45 minutes. After staining, wash the cells again with serum-free medium to remove unbound Fluo-4 AM probes. After the washing step is completed, place the cells in physiological saline or a specific buffer containing 1-2 mM CaCl2 to maintain the stability of intracellular calcium ions.

[0053] Step S2: Using the cell nucleus as an “optical bullet” to dynamically regulate cellular calcium signals

[0054] S21: Use pointed tweezers to take out the cell slide from the culture dish, place it upside down on a glass slide, use a pipette to take out the "optical bullet" solution prepared in step S11, and drop it between the cell slide and the glass slide, place the prepared sample upside down on the stage 2, and turn on the top lighting source 1 to clearly see the sample morphology.

[0055] S22: Use holographic optical tweezers to set the spatial position and laser power of the capture point. The 1064nm laser 13 emits a Gaussian beam with a wavelength of 1064nm, which is modulated into a linearly polarized collimated beam by a polarizer 12. The beam is expanded by a beam expander 11 composed of two plano-convex lenses, and then reflected into a spatial light modulator 14 by a second reflector 10. The beam is collimated by a plano-convex lens group 15, and then the collimated beam passes through a dichroic mirror A4 (the mirror has high reflectivity for beams with a wavelength of 1064nm and high transmittance for beams with wavelengths of 490nm and 520nm) and the objective lens of an inverted fluorescence microscope 3 and is focused onto a glass slide. The cell nucleus is captured at the focus in the light field due to the light gradient force. The phase of the Airy beam is loaded by the spatial light modulator in coordination with the computer, and the phase of the input Gaussian beam is modulated into an Airy beam. The main characteristic of the Airy beam is that the central light intensity (main lobe) propagates along a parabola. At this time, the particles are scattering light more than the gradient force, so the captured cell nucleus is ejected along the trajectory to the cell surface. The cell nucleus ejected by the light beam is usually called an "optical bullet". The ejection direction and speed of the "optical bullet" can be controlled by setting the deflection angle of the light beam and the incident laser power.

[0056] S3: Real-time monitoring and recording of images of the control process through CMOS camera

[0057] In order to observe the intracellular fluorescence changes, the illumination light source 1 is turned off, the fluorescent LED laser 6 is turned on, and the excitation wavelength is set to 490nm. The fluorescent LED laser excitation beam is reflected by the dichroic mirror B5 (the mirror has high reflectivity for beams with a wavelength of 490nm, and high transmittance for beams with a wavelength of 520nm), and is coupled into the objective lens of the fluorescence microscope 3 through the dichroic mirror A4, thereby exciting the calcium ion fluorescent dye in the cells in the sample chamber. Since the "optical bullet" instantly generates momentum transfer when in elastic contact with the cell, a force is applied to the cell membrane surface to open the mechanical sensitive channel, thereby regulating the change of the cell calcium ion signal. The image signal of this process is coupled into the CMOS camera 8 through the amplification system composed of the objective lens and the tube lens 7, and the CMOS camera 8 monitors and records the image of the regulation process in real time.

[0058] Figure 3 Experimental images of cell nuclei as "optical bullets" to regulate cell signals. A is a bright field experimental image of cell nuclei extracted from chicken blood cells; B is a dark field fluorescence image of calcium ions in HT-22 cells before mechanical stimulation; C is a dark field fluorescence image of calcium ion signals in cells stimulated locally using cell nuclei.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A system for mechanically regulating calcium ion signals of nerve cells by manipulating an "optical bullet" with an Airy beam, characterized in that: The system comprises an object stage (1), an illumination light source (2) is arranged above the object stage (1), an inverted fluorescence microscope (3), a dichroic mirror A (4), a dichroic mirror B (5) and a first reflector (9) are arranged in sequence directly below the object stage (1), a plano-convex lens group (15) and a spatial light modulator (14) are arranged in sequence on the left side of the dichroic mirror A (4), a second reflector (10) is arranged at the lower right of the spatial light modulator (14), a beam expander (11), a polarizer (12) and a 1064 nm laser (13) are arranged in sequence on the left side of the second reflector (10); a fluorescent LED laser (6) is arranged to the right of the dichroic mirror B (5); and a tubular lens (7) and a CMOS camera (8) are arranged in sequence on the right side of the first reflector (9).

2. The system according to claim 1, characterized in that The lens of the inverted fluorescence microscope (3) is a 60x water lens.

3. The system according to claim 2, characterized in that An illumination light source (2) is arranged above the fluorescence microscope (3). The illumination light source (2) passes through the inverted fluorescence microscope (3), the dichroic mirror A (4) and the dichroic mirror B (5). The dichroic mirror only reflects light of a specific wavelength. Therefore, the illumination light source (2) directly passes through the dichroic mirror A (4) and the dichroic mirror B (5), and then is reflected by the first reflector (9) and focused by the tubular lens (7) to the CMOS camera (8). The CMOS camera (8) records the pictures and videos presented in the fluorescence microscope (3).

4. A method for mechanically regulating calcium ion signals of nerve cells by manipulating an "optical bullet" with an Airy beam, the method being based on the system according to any one of claims 1 to 3, characterized in that: The steps include: S1: Preparation of polystyrene "optical bullets" and cultivation of HT-22 cells; S2: Using polystyrene microspheres as "optical bullets" to dynamically regulate cellular calcium signals; S3: The images of the control process are monitored and recorded in real time through the CMOS camera.

5. A method for preparing a cell nucleus "optical bullet" and regulating cell calcium ion signals, the method being based on the system according to any one of claims 1 to 3, characterized in that: The steps include: S1: Preparation of cell nucleus "optical bullet" culture HT-22 cells; S2: Using the cell nucleus as an "optical bullet" to dynamically regulate cellular calcium signals; S3: The images of the control process are monitored and recorded in real time through the CMOS camera.

Images