An Airy two-photon microscopy imaging method and device for global enhanced aberration correction

Abstract

The present invention discloses an Airy two-photon microscopy imaging method and device with global enhanced aberration correction, belonging to the technical field of biological imaging. The imaging method includes the following steps: S1. Expand the laser beam through a laser beam expansion module to ensure that the laser illumination beam meets the beam size requirements for subsequent modulation; S2. Generate an Airy beam and perform wavefront correction through an AO correction module; S3. Split and combine two beams through a beam splitting and combining module; S4. Drive the beam to scan the entire sample through a beam scanning module; S5. Collect the sample signal through a fluorescence collection module. By adopting the above-mentioned Airy two-photon microscopy imaging method and device with global enhanced aberration correction, the present invention simultaneously realizes the generation of an Airy beam and real-time aberration correction through a single spatial light modulator, improves the imaging resolution of the two-photon imaging device, and helps to observe more detailed tissue information in in vivo imaging applications.

Description

Technical Field

[0001] The present invention relates to the field of bioimaging technology, and particularly to a method and device for Airy two-photon microscopy imaging with global enhanced aberration correction. Background Art

[0002] Microscopy imaging technology with large depth and high resolution plays a crucial role in the field of biomedicine, providing scientists with a key tool to reveal complex biological structures and dynamic processes. At the microscopic level, studying the fine structure and function of biological tissues and cells is of great significance for understanding life processes, disease mechanisms, and their treatment strategies. However, the effects of light scattering, absorption, and aberration pose many challenges to traditional microscopy imaging technology in terms of imaging depth, resolution, etc., hindering the effective observation and analysis of deep biological tissues. Therefore, a live imaging technology that can correct aberrations in real time, improve imaging depth, and resolution is particularly important.

[0003] Adaptive Optics (AO) refers to a technology that compensates for optical aberrations caused by atmospheric turbulence or other factors by adjusting the wavefront corrector in the optical system in real time, thereby improving the imaging quality. Its basic principle is to dynamically adjust the wavefront in the optical system so that the wavefront distortion of the incident light after passing through the system can be corrected in real time. AO technology was first developed in the field of astronomy to compensate for the imaging blur problem of astronomical telescopes caused by atmospheric turbulence. These aberrations are caused by the refraction of light passing through different temperature and density air layers in the atmosphere. AO technology can dynamically compensate for these aberrations by monitoring and adjusting optical elements in real time, enabling the telescope to obtain clearer images. With the development of technology, AO technology has gradually been introduced into the field of biomedical imaging to overcome the optical aberrations caused by biological tissues and improve imaging depth and imaging quality.

[0004] By combining the tissue penetration ability and low light damage characteristics of long-wavelength excitation light, researchers introduced AO technology into two-photon excitation fluorescence microscopy (2PEF) to achieve high-resolution imaging within biological tissues. Then, in traditional 2PEF, a femtosecond laser with a small depth of field is usually used as the excitation light source. The conventional beam propagating in free space will gradually broaden due to the diffraction effect, and only the sample information at the focal point can be obtained. Through specific mathematical transformations, a beam whose transverse distribution remains unchanged during propagation can be designed, and such beams are called non-diffracting beams. One of the core characteristics of non-diffracting beams is self-healing, that is, after the beam is scattered by an object with a finite size, it can automatically recover its original cross-sectional shape during propagation. In optical design, this characteristic of non-diffracting beams is often used to optimize the beam transmission efficiency, ensuring that when propagating in a complex medium, the shape and energy distribution of the beam can remain stable, so as to achieve a more precise optical effect in the target area.

[0005] 2PEF is an advanced optical technology that uses infrared excitation light to achieve high-resolution three-dimensional imaging within biological tissues and is widely used in neuroscience and biomedical research. However, 2PEF is often affected by tissue light scattering and absorption during deep tissue imaging, resulting in aberration generation, thus reducing the resolution and quality of the image. To improve the imaging effect, AO technology has been applied to two-photon imaging for aberration correction.

[0006] Combining the tissue penetration ability and low light damage characteristics of long-wavelength excitation light, the combination of 2PEF and AO technology significantly improves the imaging resolution of deep tissues. The Na Ji team restored the diffraction-limited imaging performance by correcting the distorted wavefront of Bessel focusing in the focal plane of the objective lens. They applied the adaptive optical Bessel focusing scanning two-photon fluorescence microscope to volume imaging of zebrafish larvae and mouse brains up to 500 μm deep, significantly improving the sensitivity and resolution of structural and functional measurements. Lina Streich et al. developed an in vivo imaging method based on three-photon excitation, indirect adaptive optics, and active electrocardiogram gating, achieving near-diffraction-limited imaging of mouse hippocampal cortical spines and (sub)cortical dendrites at 1.4 mm. However, in two-photon microscopy imaging, the method of combining AO technology and Bessel beams has a certain optical path complexity. Specifically, the traditional adaptive optics correction optical path requires an even number of lenses to be configured behind the spatial light modulator to achieve the conjugate effect, while the generation of Bessel beams requires an odd number of lenses for Fourier transform of the focal plane, which leads to the problem of optical path mismatch.

[0007] In terms of algorithms, with the development of computing technology, a variety of optimization algorithms have been applied to the wavefront correction of Zernike polynomial patterns. The application of these algorithms has significantly improved the efficiency and accuracy of adaptive optical systems during the wavefront correction process. The Hill Climbing (HC) method is a gradient-based local search algorithm that gradually approaches the optimal solution by iteratively increasing the value of the objective function. Although this method may get trapped in local optima. The Genetic Algorithm (GA) explores approximate optimal solutions in a complex search space by simulating natural selection and genetic mechanisms, adopting a global search strategy and being able to handle multimodal and nonlinear problems. The Simulated Annealing (SA) algorithm is a probabilistic global optimization algorithm that jumps out of local optima and searches for the global optimal solution by simulating the physical annealing process and accepting worse solutions with a certain probability. The Stochastic Parallel Gradient Descent (SPGD) algorithm takes advantage of parallel computing to accelerate the convergence of the optimization process through stochastic gradient estimation, especially suitable for large-scale and high-dimensional optimization problems. Traditional adaptive algorithms usually use the maximum intensity as the feedback mechanism. However, this method results in an error convergence curve showing a relatively high error level without significant decline and is prone to the problem of local overcorrection.

[0008] For non-diffracting beams, the research by Dholakia et al. first demonstrated the ability to use Bessel beams for two-photon excitation in 2008. The Na Ji team used Bessel beams to achieve high-throughput imaging in vivo with more than 1000 excitatory input synapses per volume mapping and more than 500 dendritic spines per neuron. In addition, the research by Dufour et al. further improved the depth of field in two-photon fluorescence microscopy by using an axicon lens, successfully obtaining a high-resolution projection image with a depth of field of 1 mm while maintaining a lateral resolution of 2 μm. Xiao-Jie Tan et al. verified the imaging ability of Airy beams in a scattering environment by utilizing their non-diffracting characteristics.

[0009] Generally speaking, the development of related technologies has provided breakthrough solutions for deeper biomedical imaging, helping to overcome the limitations of the resolution and penetration depth of traditional imaging technologies.

[0010] The self-accelerating property enables Airy beams to effectively reduce the influence of optical scattering and tissue absorption on the imaging effect during the imaging of biological tissues. By adjusting the initial conditions of the Airy beam, a focal point can be formed at the target position, reducing the influence of scattering and refraction on imaging and improving the imaging resolution and signal-to-noise ratio. Licheng Yu et al. used a camera to measure the far-field intensity distribution and combined a ramp algorithm and an open-loop control method to achieve indirect adaptive correction, experimentally verifying the optical properties such as non-diffraction, self-acceleration, and self-healing of the generated Airy beam.

[0011] Although Airy beams have attracted much attention in the past decade or so, due to their complex generation method and the need for a high-precision system, their research in adaptive systems is still in a relatively early stage. Currently, most adaptive imaging systems based on Airy beams use direct wavefront detection methods, which are costly and not easy to implement. In addition, in these studies, the aberration caused by biological tissues still affects the imaging depth and resolution. In view of this, in actual biological imaging, researchers are still seeking a simple optical path construction method that can significantly improve the imaging resolution and imaging speed on the basis of ensuring real-time aberration correction. Summary of the Invention

[0012] The purpose of the present invention is to provide a global enhanced aberration correction Airy two-photon microscopy imaging method and device to solve the problems mentioned in the background technology.

[0013] To achieve the above object, the present invention provides a global enhanced aberration correction Airy two-photon microscopy imaging method, including the following steps:

[0014] S1. Expand the laser beam through a laser beam expander module to ensure that the laser illumination beam meets the beam size required for subsequent modulation;

[0015] S2. Generate an Airy beam and perform wavefront correction through an AO correction module;

[0016] S3. Split and combine two beams of light through a beam splitting and combining module;

[0017] S4. Drive the beam to scan the entire sample through a beam scanning module;

[0018] S5. Collect the sample signal through a fluorescence collection module.

[0019] Preferably, the S1 specifically includes:

[0020] A femtosecond laser illumination beam is provided by a laser; the laser illumination beam is collimated and expanded by a first lens and a second lens; the laser illumination beam is divided into two switchable illumination beams by a first half-wave plate, and at the same time, the power of the illumination beam is dynamically adjusted by the combined action of the first half-wave plate and a first polarization beam splitter.

[0021] Preferably, the S2 specifically includes:

[0022] A second half-wave plate is used to adjust the polarization state of the illumination beam to ensure the maximum modulation efficiency of the spatial light modulator; a first reflector is used to adjust the optical path layout; a third lens and a fourth lens are used to collimate and expand the illumination beam so that its diameter matches the liquid crystal working surface of the spatial light modulator; a second reflector is used to adjust the incident angle of the illumination beam to achieve a small-angle incidence of the illumination beam on the spatial light modulator; a pre-generated Airy beam phase diagram and a wavefront correction phase diagram are loaded through the spatial light modulator to generate an Airy beam and achieve wavefront correction; a third reflector is used to adjust the optical path layout; a fifth lens, a diaphragm, and a sixth lens are used to select the multi-order beams generated by the illumination beam passing through the spatial light modulator, and the first-order light is selected as the illumination beam for the subsequent optical path.

[0023] Preferably, the wavefront correction includes:

[0024] S21. An average intensity feedback mechanism is adopted to optimize the wavefront correction process. The average intensity signal during the correction process is collected by a photomultiplier tube as a feedback parameter to ensure the uniformity of the correction and avoid the reduction of the field of view caused by local over-optimization.

[0025] S22. Utilize the high resolution, programmability, and dynamic modulation characteristics of the spatial modulator to identify and compensate for complex wavefront distortions.

[0026] During the compensation process, the modulation parameters of the phase element are continuously adjusted by adjusting the phase pattern on the spatial modulator to quickly respond to the optical distortion problems brought by different tissue depths and complex structures and gradually focus the beam.

[0027] S23. The effect of wavefront correction is judged by monitoring the two-photon signal intensity at the focus. If the average signal intensity tends to be stable and maintains at the highest level in several consecutive iterations, it is judged that the current modulation is close to or reaches the best effect; the key indicators for judging the best modulation effect also include the quality of the focus and the contrast of the imaging.

[0028] S24. To improve the operability of the correction, the MALTAB algorithm is embedded in LabVIEW to analyze the signal intensity and image features in real time. After running the preset number of iterations, the MATLAB program is stopped and called to generate the best correction phase.

[0029] S25. By analyzing the superposition of the modulated optical field and the reference optical field, the electric field and light intensity at the focal point are obtained;

[0030] S26. Perform a Fourier transform on the light intensity signal to obtain the spectra of each modulation frequency component, and extract the phase information from them, thereby determining the correction phase value of the modulation phase element to achieve accurate wavefront correction.

[0031] Preferably, the electric field and light intensity at the focal point obtained by the analysis in S25 include:

[0032] Assume that the modulated optical field is , and the reference optical field is , then the electric field at the focal point is expressed as:

[0033] ;

[0034] Among them, and are the amplitudes of the modulated optical field and the reference optical field respectively, and are the modulation frequency and phase of the modulated optical field , and are the modulation frequency and phase of the reference optical field, represents the imaginary unit, t represents the variation of the optical field with time;

[0035] Since the actual measured value of the electric field is a real number, the electric field at the focal point is expressed as:

[0036] ;

[0037] Since the light intensity at the focal point is expressed as squared, expand and simplify the light intensity expression as follows:

[0038] ;

[0039] Apply the following cosine squared term:

[0040] ;

[0041] Obtain:

[0042] ;

[0043] Among them, depicts the phase change and time evolution of the optical field.

[0044] Preferably, in S26, according to the Nyquist sampling theorem, to accurately reconstruct the signal, the sampling frequency is at least twice the highest modulation frequency in the signal, while ensuring that the lowest modulation frequency can effectively distinguish different phase elements to prevent signal overlap.

[0045] Preferably, S3 specifically includes:

[0046] Adjust the optical path layout using the fourth mirror; through the switchable aperture and the second polarization beam splitter, combined with the first half-wave plate and the first polarization beam splitter, dynamically adjust the beam splitting and beam combining of the two beams.

[0047] Preferably, S4 specifically includes:

[0048] The X-Y scanning galvanometer adjusts the reflection angle by adjusting the voltage value to achieve point-by-point scanning in the X-Y plane; transfer the phase plane to the entrance pupil of the objective lens through the scanning lens and the sleeve lens.

[0049] Preferably, S5 specifically includes:

[0050] The illumination beam is focused on the sample through the dichroic mirror and the objective lens to generate the signal light; after the sample is excited, the signal light is reflected by the dichroic mirror to achieve the separation of the excitation light and the signal light; after the signal light is reflected, it passes through the filter and the seventh lens respectively, and then is collected by the photomultiplier tube; the photomultiplier tube converts the optical signal into an electrical signal and transmits it to the computer for signal processing and image reconstruction.

[0051] The present invention also provides an Airy two-photon microscopy imaging device with global enhanced aberration correction, including a laser beam expansion module, an AO correction module, a beam splitting and combining module, a beam scanning module, and a fluorescence collection module arranged in sequence;

[0052] The laser beam expansion module is used to expand the laser beam to meet the beam size required for subsequent modulation, including a laser, a first lens, a second lens, a first half-wave plate, and a first polarization beam splitter arranged in sequence;

[0053] The AO correction module is used to generate Airy beams and aberration correction, including a second half-wave plate, a first mirror, a third lens, a fourth lens, a second mirror, a spatial light modulator, a third mirror, a fifth lens, an aperture, and a sixth lens arranged in sequence;

[0054] The beam splitting and combining module is used for beam splitting and beam combining of two beams, including a fourth mirror, a switchable aperture, and a second polarization beam splitter, and the fourth mirror and the switchable aperture are respectively arranged on the adjacent sides of the second polarization beam splitter;

[0055] The beam scanning module is used to drive the beam to scan the entire sample, including an X-Y scanning galvanometer, a scanning lens, and a sleeve lens arranged in sequence. The X-Y scanning galvanometer is arranged on one side of the second polarization beam splitter and is opposite to the switchable aperture.

[0056] The fluorescence collection module is used to collect the sample signal, including a dichroic mirror, an objective lens, a sample, a filter, a seventh lens, a photomultiplier tube, and a computer. The dichroic mirror, the objective lens, and the sample are arranged in sequence, and the filter, the seventh lens, the photomultiplier tube, and the computer are arranged on the other side of the dichroic mirror.

[0057] Therefore, the present invention adopts the above-mentioned Airy two-photon microscopy imaging method and device with global enhanced aberration correction, and has the following beneficial effects:

[0058] (1) On the basis of 2PEF, the present invention introduces AO technology and non-diffracting beams, and develops a global enhanced iterative two-photon adaptive compensation technology. The generation of Airy beams and real-time wavefront aberration correction are realized through a single SLM, effectively enhancing the imaging penetration and imaging speed of the 2PEF technology for living thick biological samples, solving the device conjugation problem between the non-diffracting optical path and the adaptive optical system, and providing a new strategy for high-resolution imaging of deep tissues.

[0059] (2) The present invention uses a spatial light modulator to generate Airy beams to excite the sample, realizing a two-photon fluorescence imaging system with greater imaging depth and faster volume imaging speed, which helps to observe more deep tissue information in in vivo imaging applications.

[0060] (3) Utilizing the non-diffracting and self-accelerating characteristics of Airy beams, the present invention realizes the generation of Airy beams and real-time aberration correction during the imaging process through a single SLM, improving the integration of the imaging device.

[0061] (4) The present invention uses the average intensity as a feedback mechanism, which can provide a more uniform correction effect with a low overall error. In particular, after the average intensity feedback correction, the frequency distribution of the wavefront is more uniform, and the noise is more effectively suppressed.

[0062] The technical solutions of the present invention will be further described in detail below with reference to the drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a schematic structural diagram of an Airy two-photon microscopy imaging device with global enhanced aberration correction according to an embodiment of the present invention;

[0064] Figure 2 It is an Airy phase diagram of a spatial modulator according to an embodiment of the present invention;

[0065] Figure 3 The calibration phase diagram of the spatial modulator according to the embodiment of the present invention;

[0066] Reference numerals

[0067] 1. Laser; 2. First lens; 3. Second lens; 4. First half-wave plate; 5. First polarization beam splitter; 6. Second half-wave plate; 7. First mirror; 8. Third lens; 9. Fourth lens; 10. Second mirror; 11. Spatial light modulator; 12. Third mirror; 13. Fifth lens; 14. Aperture; 15. Sixth lens; 16. Fourth mirror; 17. Switchable aperture; 18. Second polarization beam splitter; 19. X-Y scanning galvanometer; 20. Scanning lens; 21. Sleeve lens; 22. Dichroic mirror; 23. Objective lens; 24. Sample; 25. Filter; 26. Seventh lens; 27. Photomultiplier tube; 28. Computer. Detailed implementation manners

[0068] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. The components of the embodiments of the present invention usually described and illustrated in the accompanying drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0069] It should be noted that: similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0070] Embodiment

[0071] The present invention provides a global enhanced aberration correction Airy two-photon microscopy imaging method, including the following steps:

[0072] S1. Expand the laser beam through a laser beam expansion module to ensure that the laser illumination beam meets the beam size required for subsequent modulation. The specific steps include:

[0073] A femtosecond laser illumination beam is provided by a laser 1; the laser illumination beam is collimated and expanded by a first lens 2 and a second lens 3; the laser illumination beam is divided into two switchable illumination beams by a first half-wave plate 4, and at the same time, the power of the illumination beam is dynamically adjusted by the combined action of the first half-wave plate 4 and a first polarization beam splitter 5.

[0074] S2. An Airy beam is generated and wavefront correction is performed by an AO correction module. The specific steps include:

[0075] A second half-wave plate 6 is used to adjust the polarization state of the illumination beam to ensure the maximum modulation efficiency of the spatial light modulator 11; a first mirror 7 is used to adjust the optical path layout; a third lens 8 and a fourth lens 9 are used to collimate and expand the illumination beam so that its diameter matches the liquid crystal working surface of the spatial light modulator 11; a second mirror 10 is used to adjust the incident angle of the illumination beam to achieve "small-angle incidence" of the illumination beam onto the spatial light modulator 11, improving the modulation efficiency; a pre-generated Airy beam phase diagram and a wavefront correction phase diagram are loaded through the spatial light modulator 11 to generate an Airy beam and achieve wavefront correction; a third mirror 12 is used to adjust the optical path layout; a fifth lens 13, a diaphragm 14, and a sixth lens 15 are used to select the multi-order beams generated by the illumination beam passing through the spatial light modulator 11, and the first-order light is selected as the illumination beam for the subsequent optical path.

[0076] The wavefront correction includes:

[0077] S21. An average intensity feedback mechanism is adopted to optimize the wavefront correction process. The average intensity signal during the correction process is collected by a photomultiplier tube as a feedback parameter to ensure the uniformity of the correction and avoid the reduction of the field of view caused by local over-optimization.

[0078] S22. Utilizing the high resolution, programmability, and dynamic modulation characteristics of the spatial light modulator (SLM), complex wavefront distortions are effectively identified and compensated, thereby significantly improving the imaging quality and overall performance of the imaging device. During the compensation process, the modulation parameters of the phase element are continuously adjusted by adjusting the phase pattern on the spatial light modulator (SLM) to quickly respond to the optical distortion problems brought about by different tissue depths and complex structures and gradually focus the beam.

[0079] S23. Determine the effect of wavefront correction by monitoring the two - photon signal intensity at the focus. If the average signal intensity tends to be stable and maintains at the highest level in several consecutive iterations, it is determined that the current modulation is close to or has reached the optimal effect. In addition to the signal intensity, the quality of the focus and the contrast of the image are also key indicators for judging the optimal modulation effect. When the wavefront aberration is effectively compensated, the laser beam can be better focused on the target area of the sample. At this time, the diameter of the focus will decrease and the focusing effect will be significantly improved. As the wavefront correction progresses, the contrast of the image will also gradually increase. When the modulation reaches the optimal effect, the details in the image will be clearer, the contrast will be higher, and the background noise will be reduced.

[0080] S24. To improve the operability of the correction, embed the MALTAB algorithm in LabVIEW to analyze the signal intensity and image features in real - time. After running the preset number of iterations, stop and call the MATLAB program to generate the optimal correction phase.

[0081] S25. By modulating the superposition of the optical field and the reference optical field, analyze and obtain the electric field and light intensity at the focus.

[0082] Considering the interaction between the modulation light and the reference light, the electric field at the focus can be expressed as the superposition of the two optical fields. Assume that the modulation optical field is , and the reference optical field is , then the electric field at the focus can be expressed as:

[0083] ;

[0084] Among them, and are the amplitudes of the modulation optical field and the reference optical field respectively, and are the modulation frequency and phase of the modulation optical field , and are the modulation frequency and phase of the reference optical field, represents the imaginary unit, t represents the variation of the optical field with time.

[0085] In physical applications, this method focuses on the actual measured value of the electric field, which is a real number. Therefore, the electric field at the focus can be expressed as:

[0086] ;

[0087] Since the light intensity at the focus can be expressed as the square of , expand and simplify the light intensity expression as follows:

[0088] ;

[0089] Apply the following cosine squared term:

[0090] ;

[0091] Obtain:

[0092] ;

[0093] In an optical field with a frequency of , the coherent superposition of the modulated optical field and the reference optical field generates an interference term, where depicts the phase change and time evolution of the optical field.

[0094] S26. Perform a Fourier transform on the optical intensity signal to obtain the spectra of each modulation frequency component, and extract the phase information therefrom, so as to determine the correction phase value of the modulation phase element and achieve accurate wavefront correction. In the actual wavefront correction process, in order to be able to distinguish the correction phases of different phase elements, the modulation frequency needs to be high enough to provide higher resolution and more accurate phase correction. In addition, the determination of the lowest modulation frequency is also closely related to the sampling frequency and spatial resolution, mainly involving the sampling and reconstruction of the signal in the spatial and temporal domains.

[0095] According to the Nyquist sampling theorem, in order to accurately reconstruct the signal, the sampling frequency should be at least twice the highest modulation frequency in the signal. Therefore, it is necessary to ensure that the lowest modulation frequency can effectively distinguish different phase elements and prevent signal overlap. If the modulation frequency is too low, the imaging device may not be able to distinguish adjacent phase changes, which will lead to signal overlap and thus unable to accurately correct the wavefront.

[0096] S3. Split and combine the two beams of light through the beam splitting and combining module. The specific steps include:

[0097] Use the fourth reflector 16 to adjust the optical path layout; through the switchable aperture 17 and the second polarization beam splitter 18, combined with the first half-wave plate 4 and the first polarization beam splitter 5, dynamically adjust the splitting and combining of the two beams of light, and can respectively achieve two-photon imaging based on Gaussian beams, adaptive two-photon imaging based on Gaussian beams, two-photon imaging based on Airy beams, and adaptive two-photon imaging based on Airy beams.

[0098] S4. Drive the beam to scan the entire sample through the beam scanning module. The specific steps include:

[0099] The X-Y scanning galvanometer 19 adjusts the reflection angle by regulating the voltage value to achieve point-by-point scanning in the X-Y plane; the phase plane is transmitted to the entrance pupil of the objective lens 23 through the scanning lens 20 and the sleeve lens 21. The spatial light modulator 11 is conjugate to the first galvanometer of the X-Y scanning galvanometer 19.

[0100] S5. Collect the sample signal through the fluorescence collection module. The specific steps include:

[0101] The illumination beam is focused onto the sample 24 by the dichroic mirror 22 and the objective lens 23 to generate the signal light; after the sample 24 is excited, the signal light is reflected by the dichroic mirror 22 to achieve the separation of the excitation light and the signal light; after reflection, the signal light passes through the filter 25 and the seventh lens 26 respectively, and then is collected by the photomultiplier tube 27; the photomultiplier tube 27 converts the optical signal into an electrical signal and transmits it to the computer 28 for signal processing and image reconstruction.

[0102] As Figures 1 - 3 shown, the present invention also provides an Airy two-photon microscopy imaging device with global enhanced aberration correction, including a laser beam expanding module, an AO correction module, a beam splitting and combining module, a beam scanning module, and a fluorescence collection module arranged in sequence.

[0103] The laser beam expanding module is used to expand the laser beam to meet the beam size required for subsequent modulation, including a laser 1, a first lens 2, a second lens 3, a first half-wave plate 4, and a first polarization beam splitter 5 arranged in sequence;

[0104] The AO correction module is used to generate an Airy beam and wavefront correction, including a second half-wave plate 6, a first mirror 7, a third lens 8, a fourth lens 9, a second mirror 10, a spatial light modulator 11, a third mirror 12, a fifth lens 13, a diaphragm 14, and a sixth lens 15 arranged in sequence;

[0105] The beam splitting and combining module is used for beam splitting and combining of two beams, including a fourth mirror 16, a switchable diaphragm 17, and a second polarization beam splitter 18. The fourth mirror 16 and the switchable diaphragm 17 are respectively arranged on the adjacent sides of the second polarization beam splitter 18;

[0106] The beam scanning module is used to drive the beam to scan the entire sample, including an X-Y scanning galvanometer 19, a scanning lens 20, and a sleeve lens 21 arranged in sequence. The X-Y scanning galvanometer 19 is arranged on one side of the second polarization beam splitter 18 and is opposite to the switchable diaphragm 17;

[0107] The fluorescence collection module is used to collect the sample signal, including a dichroic mirror 22, an objective lens 23, a sample 24, a filter 25, a seventh lens 26, a photomultiplier tube 27 and a computer 28. The dichroic mirror 22, the objective lens 23 and the sample 24 are arranged in sequence, and the filter 25, the seventh lens 26, the photomultiplier tube 27 and the computer 28 are arranged on the other side of the dichroic mirror 22.

[0108] The functions of the modules of the imaging device are as follows:

[0109] Laser beam expansion module: The laser 1 provides a femtosecond laser illumination beam; the first lens 2 and the second lens 3 collimate and expand the laser illumination beam; the first half-wave plate 4 divides the laser illumination beam into two switchable illumination beams; in addition, the first half-wave plate 4 and the first polarization beam splitter 5 jointly act to dynamically adjust the power of the illumination beam.

[0110] AO correction module: The second half-wave plate 6 is used to adjust the polarization state of the illumination beam to ensure the maximum modulation efficiency of the spatial light modulator 11; the first mirror 7 adjusts the optical path layout; the third lens 8 and the fourth lens 9 collimate and expand the illumination beam so that its diameter matches the liquid crystal working surface of the spatial light modulator 11; the second mirror 10 adjusts the incident angle of the illumination beam to achieve "small-angle incidence" of the illumination beam onto the spatial light modulator 11, improving the modulation efficiency; the spatial light modulator 11 loads a pre-generated Airy beam phase diagram and an aberration correction phase diagram to generate an Airy beam and achieve aberration correction; the third mirror 12 adjusts the optical path layout; the fifth lens 13, the aperture 14 and the sixth lens 15 select the multi-order beams generated by the illumination beam passing through the spatial light modulator 11, and select the first-order light as the illumination beam for the subsequent optical path.

[0111] Beam splitting and combining module: The fourth mirror 16 adjusts the optical path layout; the switchable aperture 17 and the second polarization beam splitter 18 cooperate with the first half-wave plate 4 and the first polarization beam splitter 5 to jointly act to dynamically adjust the splitting and combining of the two illumination beams, and can respectively achieve two-photon imaging based on Gaussian beams, adaptive two-photon imaging based on Gaussian beams, two-photon imaging based on Airy beams and adaptive two-photon imaging based on Airy beams.

[0112] Beam scanning module: The spatial light modulator 11 is conjugated with the first galvanometer of the X-Y scanning galvanometer 19; the X-Y scanning galvanometer 19 adjusts the reflection angle by adjusting the voltage value to achieve point-by-point scanning in the X-Y plane; the scanning lens 20 and the sleeve lens 21 transfer the phase plane to the entrance pupil of the objective lens 23.

[0113] Fluorescence collection module: The illumination beam is focused onto the sample 24 through the dichroic mirror 22 and the objective lens 23 to generate the signal light; after the sample 24 is excited, the signal light is reflected by the dichroic mirror 22 to separate the excitation light and the signal light; after reflection, the signal light passes through the filter 25 and the seventh lens 26 respectively, and is collected by the photomultiplier tube 27; the photomultiplier tube 27 converts the optical signal into an electrical signal and transmits it to the computer 28 for signal processing and image reconstruction.

[0114] The present invention provides a deep tissue optical imaging method and device based on Airy beams. Verification with agar-fixed fluorescent microsphere samples shows that the device can effectively maintain the light intensity and focusing ability in highly scattering media, and can achieve a 6.23-fold signal enhancement for imaging the same fluorescent bead before and after correction; when testing the imaging depth at a step size of 1 μm, a depth increase of 30 μm is obtained after correction compared to before correction. Further, verification in the in vivo imaging experiment of zebrafish blood vessels shows that the present invention increases the imaging depth from 320 μm before correction to 360 μm, achieving a 20% depth increase.

[0115] Therefore, the present invention adopts the above-mentioned Airy two-photon microscopy imaging method and device with global enhanced aberration correction, combines 2PEF, AO technology and non-diffracting beams, realizes the generation of non-diffracting beams and real-time aberration correction during the imaging process through a single spatial light modulator, increases the imaging depth by 20%, and significantly enhances the fluorescence intensity to 6.23 times that before correction, which is of great significance for the field of in vivo biological imaging.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for Airy two-photon microscopy with global enhanced aberration correction, characterized in that: The following steps are involved: S1. Expand the laser beam through the laser beam expansion module to ensure that the laser illumination beam meets the beam size of subsequent modulation; S2, generating Airy beam and performing wavefront correction through AO correction module; S3, splitting and combining the two light beams through a light splitting and combining module; S4, driving the light beam to scan the entire sample through the light beam scanning module; S5, collecting sample signals through a fluorescence collection module; The S1 specifically includes: A femtosecond laser illumination beam is provided by a laser; the laser illumination beam is collimated and expanded by a first lens and a second lens; the laser illumination beam is divided into two switchable illumination beams by a first half-wave plate, and the illumination beam power is dynamically adjusted by the first half-wave plate and a first polarization beam splitter; The S2 specifically includes: The second half-wave plate is used to adjust the polarization state of the illumination beam to ensure the maximum modulation efficiency of the spatial light modulator; the first reflector is used to adjust the optical path layout; the third lens and the fourth lens are used to collimate and expand the illumination beam so that its diameter matches the liquid crystal working surface of the spatial light modulator; the second reflector is used to adjust the incident angle of the illumination beam to achieve a small angle of incidence of the illumination beam to the spatial light modulator; the pre-generated Airy beam phase diagram and the wavefront correction phase diagram are loaded through the spatial light modulator to generate the Airy beam and achieve wavefront correction; the third reflector is used to adjust the optical path layout; the fifth lens, the aperture and the sixth lens are used to select the multi-level sub-beams generated by the illumination beam through the spatial light modulator, and the first-level light is selected as the illumination beam of the subsequent optical path; The S3 specifically includes: The fourth reflector is used to adjust the optical path layout; the switchable aperture and the second polarization beam splitter are combined with the first half-wave plate and the first polarization beam splitter to dynamically adjust the splitting and combining of the two light beams; The S4 specifically includes: The XY scanning galvanometer adjusts the reflection angle by adjusting the voltage value to achieve point-by-point scanning on the XY plane; the phase plane is transmitted to the entrance pupil of the objective lens through the scanning lens and the sleeve lens; The S5 specifically includes: The illumination light beam is focused onto the sample through a dichroic mirror and an objective lens to generate signal light; after the sample is excited, the signal light is reflected by the dichroic mirror to separate the excitation light and the signal light; after reflection, the signal light passes through the filter and the seventh lens respectively, and then is collected by the photomultiplier tube; the photomultiplier tube converts the optical signal into an electrical signal and transmits it to the computer for signal processing and image reconstruction.

2. The method for Airy two-photon microscopy with global enhanced aberration correction according to claim 1, characterized in that: The wavefront correction includes: S21. Use the average intensity feedback mechanism to optimize the wavefront correction process. The average intensity signal collected during the correction process is used as a feedback parameter through a photomultiplier tube to ensure the uniformity of the correction and avoid the reduction of the field of view caused by local over-optimization. S22, using the high resolution, programmability and dynamic modulation characteristics of the spatial light modulator to identify and compensate for complex wavefront distortion; during the compensation process, the modulation parameters of the phase element are continuously adjusted by adjusting the phase pattern on the spatial light modulator, quickly responding to the optical distortion problems caused by different tissue depths and complex structures and gradually focusing the light beam; S23. The effect of wavefront correction is judged by monitoring the intensity of the two-photon signal at the focus. If the average intensity of the signal tends to be stable and maintains at the highest level in several consecutive iterations, it is judged that the current modulation has approached or reached the best effect. The key indicators for judging the best modulation effect also include the quality of the focus and the contrast of the imaging. S24. In order to improve the operability of correction, the MATLAB algorithm is embedded in LabVIEW to analyze the signal strength and image characteristics in real time. After running the preset number of iterations, the MATLAB program is stopped and called to generate the best correction phase; S25, analyzing and obtaining the electric field and light intensity at the focus by superposition of the modulated light field and the reference light field; S26, performing Fourier transform on the light intensity signal to obtain the frequency spectrum of each modulation frequency component, and extracting phase information therefrom, thereby determining the correction phase value of the modulation phase element to achieve accurate wavefront correction.

3. The method for Airy two-photon microscopy with global enhanced aberration correction according to claim 2, characterized in that: The electric field and light intensity at the focus obtained by analysis in S25 include: Assume that the modulated light field is , the reference light field is , then the electric field at the focus It is expressed as: ； in, and are the amplitudes of the modulated light field and the reference light field, and is a modulated light field The modulation frequency and phase, and are the modulation frequency and phase of the reference light field, represents the imaginary unit, Represents the change of light field over time; Since the actual measurement of the electric field is a real number, the electric field at the focus It is expressed as: ； Since the light intensity at the focal point Expressed as The square of , expand and simplify the light intensity expression as follows: ； Apply the following cosine squared term: ； get: ； in, The phase change and time evolution of the light field are depicted.

4. The method for Airy two-photon microscopy with global enhanced aberration correction according to claim 3, characterized in that: In S26, according to the Nyquist sampling theorem, in order to accurately reconstruct the signal, the sampling frequency is at least twice the highest modulation frequency in the signal, while ensuring that the lowest modulation frequency can effectively distinguish different phase elements to prevent signal overlap.

5. A globally enhanced aberration-corrected Airy two-photon microscopy imaging device, applied to a globally enhanced aberration-corrected Airy two-photon microscopy imaging method as claimed in any one of claims 1 to 4, characterized in that: It includes a laser beam expansion module, an AO correction module, a beam splitting and combining module, a beam scanning module and a fluorescence collection module which are arranged in sequence; The laser beam expansion module is used to expand the laser beam to meet the beam size of subsequent modulation, and includes a laser, a first lens, a second lens, a first half-wave plate and a first polarization beam splitter arranged in sequence; The AO correction module is used for generating Airy beam and aberration correction, and includes a second half-wave plate, a first reflector, a third lens, a fourth lens, a second reflector, a spatial light modulator, a third reflector, a fifth lens, an aperture and a sixth lens which are arranged in sequence; The light splitting and beam combining module is used for splitting and combining two light beams, and comprises a fourth reflector, a switchable aperture and a second polarization beam splitter, wherein the fourth reflector and the switchable aperture are respectively arranged on two adjacent sides of the second polarization beam splitter; The beam scanning module is used to drive the beam to scan the entire sample, including an XY scanning galvanometer, a scanning lens and a sleeve lens arranged in sequence, wherein the XY scanning galvanometer is arranged on one side of the second polarization beam splitter and opposite to the switchable aperture; the spatial light modulator is conjugated with the first galvanometer of the XY scanning galvanometer; The fluorescence collection module is used to collect sample signals, and includes a dichroic mirror, an objective lens, a sample, a filter, a seventh lens, a photomultiplier tube and a computer. The dichroic mirror, the objective lens and the sample are arranged in sequence, and the filter, the seventh lens, the photomultiplier tube and the computer are arranged on the other side of the dichroic mirror.

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