A method for mechanically modulating calcium ion signals in nerve cells using an Airy beam-manipulated "optical bullet"

Abstract

The present invention relates to a method for mechanically regulating calcium ion signals in nerve cells using an Airy beam-manipulated “optical bullet.” The method involves first preparing the “optical bullet” and culturing HT-22 cells. The cellular calcium ion signals are then dynamically regulated, and finally, images of the regulation process are monitored and recorded in real time using a CMOS camera. This method enables non-contact mechanical stimulation of nerve cells. It not only breaks through the spatial resolution limitations of traditional neuromodulation techniques but also provides a non-invasive, highly biocompatible means of regulating neural signals. Furthermore, combined with real-time fluorescence monitoring technology, the present invention enables intuitive assessment of regulatory effects, providing a novel tool for neuroscience research and clinical applications.

Description

Technical Field

[0001] The present 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 neural cell signaling is crucial for a deeper understanding of nervous system function and disease mechanisms. Driven by the rapid advancement of biomedicine, neuroscience, and related interdisciplinary fields, there is a growing demand for technologies capable of precisely regulating neural cell signaling. These technologies hold great promise for advancing basic research, improving disease treatment strategies, and optimizing drug screening processes. However, existing technologies are limited in terms of control accuracy, operational complexity, and biocompatibility, which restrict their application in achieving high-precision, non-invasive, 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. Ultrasound control technology, as a non-invasive means, 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 limitations, but the viral transfection process may also bring toxicity and stability issues. 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 urgently need new methods for regulating neural cell signaling. These methods should have higher regulatory precision, be non-invasive, enable real-time dynamic regulation, and have better biocompatibility and ease of use than existing technologies. These demands are driving the exploration of new non-invasive neuromodulation technologies, hoping to overcome the limitations of existing technologies and provide powerful tools for in-depth research on nervous system function and the development of new treatments. Summary of the Invention

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

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

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

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

[0009] Furthermore, an illumination light source is provided above the fluorescence microscope. 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. Therefore, the illumination light directly passes through dichroic mirror A and dichroic mirror B, and is then reflected by the first reflector and focused by the tubular lens into the CMOS camera. The CMOS camera records the pictures and videos presented in the fluorescence microscope.

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

[0011] S1: Preparation of polystyrene "optical bullets" and culture 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 cellular calcium ion signals, comprising the following steps:

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

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

[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: This invention adopts the method of manipulating 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 beam parameters and 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 adopted by 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 a fluorescence microscopy imaging system, the present invention can monitor the response of nerve cells to the stimulation of the "optical bullet" in real time, providing intuitive and real-time data support for the study of nerve cell signal conduction.

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

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

[0026] Figure 2 This is a graph showing the experimental results of 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 the experimental results of the cell nucleus acting as an "optical bullet" in an embodiment of the present invention based on the Airy-Gauss beam manipulation of the "optical bullet". DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0029] Example 1

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

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

[0032] An illumination light source 1 is disposed above the fluorescence microscope 3. The illumination light source 1 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 1 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 images and videos presented in the fluorescence microscope 3.

[0033] Example 2

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

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

[0036] S11: Dissolve a certain amount of polystyrene microspheres in cell culture medium to obtain a solution with a concentration of 0.01 mg / mL. Place the prepared solution in an ultrasonic oscillator at room temperature and oscillate at a frequency of 20-80 kHz for 2-10 minutes to prepare a dispersed polystyrene "optical bullet" solution.

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

[0038] S12: In DMEM complete medium, use a sterile pipette to repeatedly pipette to form a single-cell suspension. The neurons were then seeded onto a culture dish containing a cell slide that had been coated with polyethanolamine and laminin antibody 1 hour in advance to obtain adherent neurons. Finally, the cells were cultured in a 37°C, 50 mL / L CO2 incubator to obtain HT-22 cells.

[0039] S13: Grow the HT-22 cells from S12 to 70–80% confluency and then wash the cells with serum-free medium. Next, add Fluo-4 AM (a fluorescent intracellular calcium stain) to a working concentration (e.g., 2 μM) and incubate the cells for 30–45 minutes. After staining, wash the cells again with serum-free medium to remove unbound Fluo-4 AM probe. After the wash step, place the cells in saline or a buffer containing 1–2 mM CaCl₂ to maintain intracellular calcium levels.

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

[0041] S21: Use pointed tweezers to remove 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, 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 1064 nm laser 13 emits a Gaussian beam with a wavelength of 1064 nm, which is modulated into a linearly polarized collimated beam by the polarizer 12. The beam is expanded by the beam expander 11 composed of two plano-convex lenses, and then reflected into the spatial light modulator 14 by the second reflector 10. The beam is collimated by the plano-convex lens group 15, and then the collimated beam passes through the dichroic mirror A4 (the mirror has high reflectivity for beams with a wavelength of 1064 nm and high transmittance for beams with wavelengths of 490 nm and 520 nm) and the objective lens of the inverted fluorescence microscope 3 to be focused onto the slide. Polystyrene microspheres are captured at the focal point in the light field due to the action of the light gradient force. The Airy beam phase is loaded by the spatial light modulator in collaboration 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 subjected to a light scattering force greater 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". By setting the deflection angle of the light beam and the incident laser power, the ejection direction and speed of the "optical bullet" can be controlled.

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

[0044] To observe intracellular fluorescence changes, turn off illumination source 1 and turn on fluorescent LED laser 6, setting the excitation wavelength to 490 nm. The excitation beam from this LED laser 6 is reflected by dichroic mirror B5 (which has high reflectivity for 490 nm wavelengths and high transmittance for 520 nm wavelengths), then coupled through dichroic mirror A4 into the objective lens of fluorescence microscope 3, where it excites the calcium ion fluorescent dye in the cells within the sample chamber. The instantaneous momentum transfer generated by the elastic contact of the "optical bullet" with the cells exerts force on the cell membrane surface, opening mechanosensitive channels and modulating changes in the cellular calcium ion signal. The image signal of this process is coupled through an amplification system consisting of the objective lens and tube lens 7 to a CMOS camera 8, which monitors and records the modulation process in real time.

[0045] Figure 2 These are the experimental results of polystyrene microspheres used as "optical bullets" to regulate HT-22 cells at different times in an embodiment of the present invention based on the manipulation of "optical bullets" by Airy-Gaussian beams; wherein, a1 and a2 are the bright-field and dark-field fluorescence images of HT-22 cells before polystyrene microspheres are used as "optical bullets" for stimulation, b1 and b2 are the experimental images and dark-field fluorescence images of the cells after stimulation to regulate calcium ion signals, and c1 and c2 are the 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 cellular calcium ion signals, comprising the following steps:

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

[0049] S31: First, fresh blood is collected from a chicken via cardiac puncture. An anticoagulant is added during the collection process to prevent blood clotting. The collected blood is then mixed with a lysis buffer and stirred thoroughly with a syringe until the red blood cells are completely lysed. Next, the blood is centrifuged at 3000 rpm for 5 minutes to allow the nuclei to settle at the bottom of the centrifuge tube. The isolated nuclei are then washed with an appropriate buffer to remove residual cell debris and lysis buffer components, ultimately yielding pure nuclei.

[0050] The anticoagulant is EDTA or heparin.

[0051] S12: In DMEM complete medium, use a sterile pipette to repeatedly pipette to form a single-cell suspension. The neurons were then seeded onto a culture dish containing a cell slide that had been coated with polyethanolamine and laminin antibody 1 hour in advance to obtain adherent neurons. Finally, the cells were cultured in a 37°C, 50 mL / L CO2 incubator to obtain HT-22 cells.

[0052] S13: Grow the HT-22 cells from S12 to 70–80% confluency and then wash the cells with serum-free medium. Next, add Fluo-4 AM (a fluorescent intracellular calcium stain) to a working concentration (e.g., 2 μM) and incubate the cells for 30–45 minutes. After staining, wash the cells again with serum-free medium to remove unbound Fluo-4 AM probe. After the wash step, place the cells in saline or a buffer containing 1–2 mM CaCl₂ to maintain intracellular calcium levels.

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

[0054] S21: Use pointed tweezers to remove 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, 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 1064 nm laser 13 emits a Gaussian beam with a wavelength of 1064 nm, which is modulated into a linearly polarized collimated beam by the polarizer 12. The beam is expanded by the beam expander 11 composed of two plano-convex lenses, and then reflected into the spatial light modulator 14 by the second reflector 10. The beam is collimated by the plano-convex lens group 15, and then the collimated beam passes through the dichroic mirror A4 (the mirror has high reflectivity for beams with a wavelength of 1064 nm and high transmittance for beams with wavelengths of 490 nm and 520 nm) and the objective lens of the inverted fluorescence microscope 3 to be focused onto the slide. The cell nucleus is captured at the focal point in the light field due to the action of the light gradient force. The spatial light modulator and the computer are used to load the Airy beam phase, 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 with a force greater 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". By setting the deflection angle of the beam and the incident laser power, the ejection direction and speed of the "optical bullet" can be controlled.

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

[0057] To observe intracellular fluorescence changes, turn off illumination source 1 and turn on fluorescent LED laser 6, setting the excitation wavelength to 490 nm. The excitation beam from this LED laser is reflected by dichroic mirror B5 (which has high reflectivity for 490 nm wavelengths and high transmittance for 520 nm wavelengths), then coupled through dichroic mirror A4 into the objective lens of fluorescence microscope 3, where it excites the calcium ion fluorescent dye in the cells within the sample chamber. The instantaneous momentum transfer generated by the elastic contact of the "optical bullet" with the cell exerts force on the cell membrane surface, opening mechanosensitive channels and modulating changes in the cellular calcium ion signal. The image signal of this process is coupled through an amplification system consisting of the objective lens and tube lens 7 to a CMOS camera 8, which monitors and records the modulation process in real time.

[0058] Figure 3 Experimental images showing the cell nucleus acting as an "optical bullet" to regulate cell signaling. Figure a shows a bright-field image of a cell nucleus extracted from chicken blood cells; b shows a dark-field fluorescence image of calcium ions in HT-22 cells before mechanical stimulation; and c shows a dark-field fluorescence image of calcium ion signals in cells stimulated locally using the nucleus.

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

Claims

1. 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 a system for mechanically regulating calcium ion signals of nerve cells by manipulating an "optical bullet" with an Airy beam, the system comprising a stage (2), an illumination light source (1) being arranged above the stage (2), an inverted fluorescence microscope (3), a dichroic mirror A (4), a dichroic mirror B (5) and a first reflector (9) being arranged in sequence directly below the stage (2), a plano-convex lens group (15) and a spatial light modulator (14) being arranged in sequence on the left side of the dichroic mirror A (4), a second reflector (10) being arranged at the lower right of the spatial light modulator (14), and a beam expander (11), a polarizer (12) and a 1064 reflector being arranged in sequence on the left side of the second reflector (10). nm laser (13); a fluorescent LED laser (6) is arranged to the right of the dichroic mirror B (5); a tubular lens (7) and a CMOS camera (8) are arranged in sequence to the right of the first reflector (9); the lens of the inverted fluorescence microscope (3) is a 60x water mirror; an illumination light source (1) is arranged above the fluorescence microscope (3), and the illumination light source (1) 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, so the illumination light source (1) 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); It is characterized by: The steps include: S1: Preparation of polystyrene "optical bullets" and culture of HT-22 cells; S2: Using polystyrene microspheres as "optical bullets" to dynamically regulate cellular calcium signaling; S3: The images of the control process are monitored and recorded in real time through the CMOS camera.

2. A method for preparing a cell nucleus "optical bullet" and regulating cell calcium ion signals, the method is based on a system for mechanically regulating nerve cell calcium ion signals by manipulating an "optical bullet" with an Airy beam, the system comprising a stage (2), an illumination light source (1) being arranged above the stage (2), an inverted fluorescence microscope (3), a dichroic mirror A (4), a dichroic mirror B (5) and a first reflector (9) being arranged in sequence directly below the stage (2), a plano-convex lens group (15) and a spatial light modulator (14) being arranged in sequence on the left side of the dichroic mirror A (4), a second reflector (10) being arranged at the lower right of the spatial light modulator (14), and a beam expander (11), a polarizer (12) and a 1064 optical multimeter being arranged in sequence on the left side of the second reflector (10). nm laser (13); a fluorescent LED laser (6) is arranged to the right of the dichroic mirror B (5); a tubular lens (7) and a CMOS camera (8) are arranged in sequence to the right of the first reflector (9); the lens of the inverted fluorescence microscope (3) is a 60x water mirror; an illumination light source (1) is arranged above the fluorescence microscope (3), and the illumination light source (1) 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, so the illumination light source (1) 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); It is characterized by: 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 signaling; S3: The images of the control process are monitored and recorded in real time through the CMOS camera.

Images