A multi-region adaptive optical multi-photon microscopic imaging system and method based on holographic wavefront scanning

Abstract

The application discloses a multi-region adaptive optical multi-photon microscopic imaging system and method based on holographic wavefront scanning. The system comprises a dispersion compensation module, an acousto-optic deflector, a mirror group, a multi-angle adapter, a first 4f lens group, a phase coding module, a decoding module and a multi-photon imaging device. The dispersion compensation module is arranged in front of the input end of the acousto-optic deflector, the acousto-optic deflector generates adjustable scanning angles through the mirror group and the multi-angle adapter, the multi-angle adapter is conjugated through the first 4f lens group and the phase coding module, and the phase coding module is conjugated through the decoding module and the multi-photon imaging device. When the scanning beam reaches different spatial regions, the application utilizes the holographic wavefront scanning technology to dynamically switch different correction wavefront phase maps, so that customized aberration compensation is applied to multiple spatial regions in a single scanning process, and the problem that different regions in multi-photon imaging have different aberrations and cannot be effectively corrected at the same time is solved.

Description

Technical Field

[0001] This invention relates to the field of adaptive optics, and more specifically to a multi-region adaptive optics multiphoton microscopy imaging system and method based on holographic wavefront scanning. Background Technology

[0002] Multiphoton microscopy (MPM) has become a core tool for high-resolution imaging of live biological samples due to its ability to image deep tissues, excellent optical sectioning properties, and low phototoxicity. However, the inhomogeneity of refractive index within biological tissues introduces complex optical aberrations that vary spatially, leading to excitation light focus distortion, signal intensity attenuation, and resolution reduction, severely limiting its imaging performance in deep tissues.

[0003] Adaptive optics (AO) technology, by measuring and compensating for these optical aberrations in real time, has been successfully applied to multiphoton microscopy to restore its diffraction-limited resolution. Traditional AO systems typically employ a single wavefront corrector (such as a deformable mirror or spatial light modulator) and a single wavefront measurement scheme, applying the wavefront correction obtained from a single point or small region to the entire imaging field of view. This approach has a fundamental limitation: due to the significant spatial heterogeneity of aberrations generated by biological tissues, a wavefront that is effectively corrected in one region may have limited effect or even produce negative effects when applied to another. This effective correction area is limited by the "isohalo zone" of the optical system. Therefore, while traditional AO technology can improve point imaging quality, it is difficult to simultaneously obtain uniform high-resolution imaging across the entire large field of view.

[0004] To overcome the limitations of the isohalo region and expand the effective correction field of view, the academic community has conducted several explorations. Among them, Multi-Pupil Adaptive Optics (MPAO) is a representative scheme. This technique divides the large field of view into multiple sub-regions by placing a pair of polyhedral prism arrays on the conjugate plane of the sample, and spatially separates the pupil images corresponding to each sub-region. This allows a single spatial light modulator to simultaneously load the correction wavefronts corresponding to different sub-regions, achieving parallel aberration correction for a large field of view.

[0005] However, MPAO and other similar technologies rely on complex optical path designs and sophisticated optical components (such as customized polyhedral prism arrays and their complementary arrays) to achieve physical separation of pupil images and subsequent re-merging. This system is not only structurally complex and costly, and difficult to calibrate its optical path, but its field-of-view segmentation strategy (the number, size, and arrangement of sub-regions) is determined by fixed physical components. Once fabricated, it lacks flexibility and is difficult to adaptively adjust according to the aberration distribution characteristics of different samples.

[0006] Therefore, there is an urgent need in this field for a new adaptive optics multiphoton microscopy imaging technology that can achieve large field of view and multi-region parallel aberration correction, while also possessing advantages such as system simplicity, flexible adjustment, and easy integration. Summary of the Invention

[0007] To address the problems and needs existing in the background technology, this invention proposes a multi-region adaptive optics multiphoton microscopy imaging system and method based on holographic wavefront scanning. This invention utilizes the encoding and decoding technology described in the prior art patent application number CN202411348062.1, multiplexing different wavefronts in different frequency domains. During the scanning imaging process, the acousto-optic deflector decodes the corresponding compensation phase in the corresponding scanning region, achieving the purpose of multi-region aberration compensation.

[0008] The technical solution adopted in this invention is as follows: I. Multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning It includes a dispersion compensation module, an acousto-optic deflector, a mirror group, a multi-angle adapter, a first 4f mirror group, a phase encoding module, a decoding module, and a multiphoton imaging device; the dispersion compensation module is spaced in front of the input end of the acousto-optic deflector, the acousto-optic deflector and the multi-angle adapter are spaced apart by mirror groups, the multi-angle adapter is conjugate with the phase encoding module through the first 4f mirror group, and the phase encoding module is conjugate with the multiphoton imaging device through the decoding module.

[0009] The multi-region adaptive optics multiphoton microscopy system is equipped with an external femtosecond laser that emits laser light to the dispersion compensation module. After entering the dispersion compensation module, the laser light is reflected by an acousto-optic deflector and a mirror group to the multi-angle adapter. After exiting the multi-angle adapter, the laser light passes through the first 4f mirror group, the phase encoding module, and the decoding module before entering the multiphoton imaging device.

[0010] The multi-angle adapter includes a primary mirror group and a secondary mirror group arranged at intervals along the optical path, with the secondary mirror group close to the mirror group and the primary mirror group close to the first 4f mirror group.

[0011] The first-stage reflector assembly includes multiple isosceles triangular tilted reflectors and a frame. The included angle formed by the isosceles sides of each isosceles triangular tilted reflector points towards the center. The frame has a hollow hole in the middle. The isosceles triangular tilted reflectors are arranged closely in a regular polygon along the circumference and mounted on the frame. There is a gap in the middle of the regular polygon for laser light to pass through. The mirror surfaces of the multiple isosceles triangular tilted reflectors are at a certain angle to the light path. The multiple isosceles triangular tilted reflectors are mounted using either a reflector patch or a prism coating. The second-stage reflector assembly includes a mounting base, a tilted mounting block, a compact reflector frame, and a trapezoidal reflector. The mounting base is connected to the compact reflector frame via the tilted mounting block. The trapezoidal reflector is connected circumferentially to the side of the compact reflector frame away from the mounting base. The compact reflector frame is configured to adjust the reflection angle of the trapezoidal reflector.

[0012] The laser beam enters the multi-angle adapter. The laser beam is first reflected by the isosceles triangular tilted reflector and then reflected onto the trapezoidal reflector. The trapezoidal reflector adjusts the reflection angle of the laser beam so that the laser beam, after being reflected by the trapezoidal reflector, converges with laser beams from other angles at a single convergence point. This convergence point is located on the side of the primary reflector group away from the secondary reflector group.

[0013] The dispersion compensation module includes two dispersion prisms, which are spaced apart between the acousto-optic biaser and the femtosecond laser. The mirror group includes multiple mirrors, which are spaced apart between the acousto-optic biaser and the multi-angle adapter. The multiple mirrors are used to reflect the laser output from the acousto-optic biaser to the multi-angle adapter. The first 4f mirror group includes a first lens, a first mirror, and a second lens. The first lens is spaced apart with the first mirror and the second lens to form a 4f mirror group. The first lens is close to the multi-angle adapter. The phase encoding module includes a waveplate, a spatial modulator, and a second mirror. The waveplate is spaced apart with the spatial modulator and the second mirror. The waveplate is close to the spatial light modulator.

[0014] The decoding module includes a third lens, an aperture stop, and a fourth lens. The third lens is arranged at intervals with the aperture stop and the fourth lens to form a 4f lens group. The third lens is close to the reflector of the phase encoding module.

[0015] The femtosecond laser emits laser light, which passes sequentially through two dispersive prisms, an acousto-optic deflector, a mirror group, and a multi-angle adapter before entering the first lens. After exiting the first lens, the laser light is reflected by the first mirror onto the second lens. After exiting the second lens, the laser light passes through a waveplate and enters a spatial light modulator for phase encoding before exiting. It is then reflected by the second mirror onto the third lens. After exiting the third lens, the laser light passes through a pinhole at the center of the aperture for phase decoding before entering the fourth lens.

[0016] The multiphoton imaging device includes a scanning galvanometer module, a fourth 4f mirror group, a fluorescence collection module, and a wavefront calibration module; the decoding module is arranged alternately via the scanning galvanometer module and the fourth 4f mirror group, and the scanning galvanometer module is conjugate via the entrance pupil of the objective lens in the fourth 4f mirror group and the wavefront calibration module.

[0017] The scanning galvanometer module includes an x-axis galvanometer, a fifth lens, a sixth lens, and a y-axis galvanometer. The fourth lens is spaced apart from the x-axis galvanometer and the fifth lens. The fifth lens and the sixth lens form a 4f array that conjugates the x and y galvanometers. The sixth lens and the y-axis galvanometer are spaced apart on the other side of the fifth lens. The x-axis galvanometer, the fifth lens, the sixth lens, and the y-axis galvanometer are arranged sequentially and spaced apart along the light propagation direction.

[0018] The fourth 4f lens group includes a scanning lens and a tube lens; the sixth lens is spaced apart from the scanning lens via a y-axis galvanometer, and a tube lens is spaced apart on the other side of the scanning lens via a detachable reflector, the scanning lens and the tube lens forming the 4f lens group; the detachable reflector is used to split the laser beam into two optical paths, the two optical paths respectively entering the objective lens and the wavefront calibration module.

[0019] The fluorescence collection module includes a dichroic mirror, an objective lens, a collecting lens, a filter, and a photomultiplier tube. The dichroic mirror is close to the tube lens, and the objective lens and the collecting lens are arranged on opposite sides of the dichroic mirror. The objective lens is located on the side of the dichroic mirror away from the tube lens. On the side of the collecting lens away from the dichroic mirror, a filter and a photomultiplier tube are arranged at intervals in sequence. The dichroic mirror, collecting lens, filter, and photomultiplier tube are arranged at intervals in sequence along the light propagation direction.

[0020] The wavefront calibration module includes a seventh lens, a third reflecting mirror, a detachable reflecting mirror, a microlens array, and a CMOS sensor. The scanning lens is arranged alternately with the detachable reflecting mirror and the seventh lens to form a 4f mirror group. The seventh lens is arranged alternately with the third reflecting mirror and the microlens array. The CMOS sensor is located on the side of the microlens array away from the third reflecting mirror.

[0021] After exiting the fourth lens, the laser beam passes through the x-axis galvanometer, the fifth lens, the sixth lens, the y-axis galvanometer, and the scanning lens before entering the detachable reflector. The laser beam is split into a first beam and a second beam by the detachable reflector. The first beam is reflected by the detachable reflector and then passes through the seventh lens, the third reflector, and the microlens array before entering the CMOS sensor. The second beam passes through the detachable reflector and the tube lens before entering the dichroic mirror. The second beam is excited by fluorescence by the dichroic mirror and then enters the objective lens. The fluorescence is collected by the objective lens and reflected by the dichroic mirror, then passes through the collecting lens and the filter before entering the photomultiplier tube.

[0022] The multi-region adaptive optics multiphoton microscopy imaging system further includes an acquisition control module, which is located in the multiphoton imaging device. The acquisition control module is connected to an acousto-optic deflector, an x-axis galvanometer, a y-axis galvanometer, and a photomultiplier tube. The acquisition control module is used to control the scanning waveform and acquire fluorescence signals.

[0023] II. Multi-region adaptive optics multiphoton microscopy imaging method for multi-region adaptive optics multiphoton microscopy imaging systems S1. The laser enters the dispersion module for dispersion pre-compensation. After exiting the dispersion module, the laser is rapidly deflected by an acousto-optic deflector and then enters the multi-angle adapter through a mirror group. After passing through the multi-angle adapter, spatial light modulator, and aperture, the laser enters the scanning galvanometer module, which scans the laser. S2. The laser beam is split and reflected by the detachable mirror in the fourth 4f mirror group to the calibration module for wavefront calibration; the laser beam is reflected by the scanning galvanometer module and enters the objective lens through the fourth 4f mirror group; the laser-excited fluorescence is collected by the objective lens in the fluorescence collection module. S3. The scanning galvanometer module scans and images the imaging field of view. The acquisition control module obtains the compensation phase corresponding to each of the different sub-regions in the imaging field of view through an algorithm. After performing phase composite encoding on multiple compensation phases, a composite phase map is generated, and the encoded composite phase map is loaded into the spatial light modulator. Finally, according to the different acousto-optic deflector angles corresponding to different regions, the acousto-optic deflector deflects and decodes the corresponding compensation phase when the galvanometer scans and images to different regions.

[0024] In step S1, the laser is converted into an angle that matches the multiplexing grating of the spatial light modulator in the multi-angle adapter. After the laser enters the spatial light modulator, it is modulated to obtain a modulated laser. The modulated laser enters the aperture for wavefront decoding and then exits and enters the scanning galvanometer module to be scanned.

[0025] In step S3, the scanning imaging of the imaging field of view is as follows: During the scanning imaging process, whenever the x-axis galvanometer finishes scanning one row of the imaging field of view, the y-axis galvanometer moves in the column direction to switch to the next row, so that the x-axis galvanometer performs the next row of the imaging field of view. When the x-axis galvanometer or the y-axis galvanometer scans the correction sub-regions corresponding to different sub-regions, the acquisition control module controls the acousto-optic deflector to perform random scanning, so that when the laser passes through different sub-regions of the imaging field of view, the compensation phase corresponding to the sub-region is decoded, and multiple corrected wavefront phase maps corresponding to different sub-regions are obtained.

[0026] The phase multiplexing encoding method is as follows: multiple corrected wavefront phase maps corresponding to different sub-regions are superimposed and summed with grating phases of different spatial frequencies, so that the encoded phases are distributed at equal angular intervals along the circumference on the focal plane of the third lens.

[0027] The sub-region division method of the imaging field of view is one or more of the following: dividing the imaging field of view into equal intervals according to preset rules, adaptively dividing the imaging field of view, and freely dividing the imaging field of view into arbitrary shapes.

[0028] The specific details of controlling the acousto-optic deflector to perform random scanning are as follows: The scanning waveform of the acousto-optic deflector is generated by the sub-region distribution of the imaging field of view and the scanning waveform of the galvanometer, which together generate the scanning control signal and the galvanometer scanning signal. The random scanning control signal and the galvanometer scanning signal are matched at the pixel level to obtain the matched signal. The matched signal is output through the acquisition control module to drive the acousto-optic deflector to perform random scanning.

[0029] In step S2, the compensation phase corresponding to each sub-region in the imaging field of view is obtained as follows: 1) The algorithm is an adaptive optics algorithm that recovers the initial compensated wavefront for the sub-region in the imaging field of view; 2) The algorithm classifies the initial compensation wavefront using a clustering algorithm, classifying it into a specific number of categories, each category corresponding to a sub-region, and generating a compensation wavefront for each category, wherein the compensation wavefront serves as the compensation phase; In step 1), the adaptive algorithm adopts an image feedback-based wavefront sensorless adaptive optics algorithm. The adaptive optics algorithm is one of the following: wavefront sensorless adaptive optics algorithm, wavefront detection method based on direct detection by wavefront sensor, and interferometric detection method based on the principle of interference.

[0030] The beneficial effects of this invention are: (1) Breaking through the limitations of the iso-halo region: By using holographic wavefront scanning technology to dynamically switch the wavefront during the pixel dwell time, synchronous parallel compensation for heterogeneous aberrations in multiple sub-regions within a large field of view can be achieved.

[0031] (2) Enhanced system flexibility: Based on the programmable field segmentation and wavefront loading strategy, the number, size and shape of the correction area can be flexibly adjusted without changing the hardware to adapt to different sample characteristics; and the system can be directly integrated with the traditional multiphoton microscopy system, adding multi-region adaptive correction capability without affecting the original imaging function. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the overall structure of the optical system in one embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the working principle and optical path of the multi-angle adapter in this invention; Figure 3 These are three views of the specific installation structure of the multi-angle adapter of the present invention; wherein, a is a side view of the multi-angle adapter, b is a top view of the multi-angle adapter, and c is a front view of the multi-angle adapter. Figure 4 This is a schematic diagram of the multi-region adaptive optics algorithm for aberration recovery in this invention; where a is the image acquired by the pattern method, b is the aberration diagram of sub-region segmentation and pattern method fitting, c is the dimension-reduced aberration and the distribution of Zernike polynomial coefficients of all sub-regions, and d is the clustered aberration and the representative Zernike polynomial coefficients after clustering. Figure 5 This is a schematic diagram of the random scanning waveform generation process of the acousto-optic deflector of the present invention; wherein, a is the AOD driving voltage diagram, and b is the AOD and galvanometer control voltage waveform diagram; Figure 6 This diagram illustrates the comparison between the compensation effects of the multi-region adaptive optics method and the traditional adaptive optics method. In the diagram, a represents the aberration map of the region variation, b represents the multi-region adaptive aberration fitting map, c represents the traditional adaptive optics map, d represents the spread function map of the region variation point, e represents the multi-region adaptive aberration compensation map, f represents the traditional adaptive aberration compensation map, g represents the magnified aberration map of the uncompensated region, h represents the magnified aberration map of the multi-region adaptive region, i represents the magnified aberration map of the traditional adaptive region, j represents the focused spot map of the uncompensated region, k represents the focused spot map of the multi-region adaptive region, and l represents the focused spot map of the traditional adaptive region.

[0033] In the diagram: 1-2, dispersive prism; 3, acousto-optic deflector; 4-8, mirror; 9, multi-angle adapter; 10, first lens; 11, mirror; 12, second lens; 13, waveplate; 14, spatial light modulator; 15, mirror; 16, third lens; 17, aperture; 18, fourth lens; 19, x-axis galvanometer; 20, fifth lens; 21, sixth lens; 22, y-axis galvanometer; 23, scanning lens; 24, detachable mirror; 25, tube lens; 26, dichroic mirror; 27, objective lens; 28, collecting lens; 29, filter; 30, photomultiplier tube; 31, seventh lens; 32, mirror; 33, microlens array; 34, CMOS; MA1, first-stage mirror; MA2, second-stage mirror. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] The embodiments of the present invention and their specific processes are as follows: A multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning includes a femtosecond laser, a dispersion compensation module, an acousto-optic deflector 3, a mirror group, a multi-angle adapter 9, a first 4f mirror group, a phase encoding module, a decoding module, and a multiphoton imaging device arranged sequentially along the optical path. The femtosecond laser is positioned before the dispersion compensation module to emit laser light into the multi-region adaptive optics multiphoton microscopy imaging system. The dispersion compensation module is positioned at intervals before the input end of the acousto-optic deflector 3. The laser light passes through the acousto-optic deflector 3 and the mirror group to reach the multi-angle adapter 9. The multi-angle adapter 9 is conjugate with the first 4f mirror group and the phase encoding module. The phase encoding module is conjugate with the multiphoton imaging device through the decoding module. The multi-region adaptive optics multiphoton microscopy system has an externally mounted femtosecond laser that emits laser light to the dispersion compensation module. After entering the dispersion compensation module, the laser light is reflected by the acousto-optic deflector 3 and the mirror group to the multi-angle adapter 9 to adjust the laser tilt angle. After exiting the multi-angle adapter 9, the laser light passes through the first 4f mirror group, the phase encoding module, and the decoding module before entering the multiphoton imaging device.

[0036] Specifically, after being emitted by a femtosecond laser, the laser undergoes time dispersion pre-compensation through a dispersion compensation module, then enters an acousto-optic deflector 3 for beam deflection before exiting. The laser then enters a reflector group and exits before entering a multi-angle adapter 9 to adjust the beam tilt angle. After that, the laser passes through a first 4f mirror group and enters a phase encoding module. After phase encoding by a spatial light modulator 14 in the phase encoding module, the laser exits. The laser then enters a decoding module, undergoes specific wavefront decoding through a second 4f mirror group and a pinhole in the aperture 17 of the focal plane, and finally enters a multiphoton imaging device.

[0037] The multi-angle adapter includes a primary mirror group MA1 and a secondary mirror group MA2 arranged at intervals along the optical path, with the secondary mirror group MA2 close to the mirror group and the primary mirror group MA1 close to the first 4f mirror group.

[0038] The first-stage reflector group MA1 includes multiple isosceles triangular tilted mirrors and a mirror frame. The angle formed by the isosceles sides of each isosceles triangular tilted mirror points towards the center of the circle. The mirror frame has a hollow hole in the middle. The isosceles triangular tilted mirrors are closely arranged in a regular polygon along the circumference and mounted on the mirror frame. There are gaps in the middle of the regular polygons for laser light to pass through. The mirror surfaces of the multiple isosceles triangular tilted mirrors are at a certain angle to the light path. The multiple isosceles triangular tilted mirrors are mounted by either mirror patches or prism coatings. Specifically, the mirror frame is circular, with a hollow, near-circular hole in the middle for mounting the isosceles triangular tilted mirrors. The hollow, near-circular hole matches the regular polygon formed by the multiple isosceles triangular tilted mirrors. The mirror frame is filled with triangular through holes along the circumference of the hollow hole for light to pass through.

[0039] The secondary reflector assembly MA2 includes a mounting base, an inclined mounting block, a compact reflector frame, and a trapezoidal reflector. The mounting base is connected to the compact reflector frame via the inclined mounting block. The trapezoidal reflector is circumferentially fixed to the side of the compact reflector frame away from the mounting base. The compact reflector frame is configured to adjust the reflection angle of the trapezoidal reflector.

[0040] Among them, such as Figure 2 As shown, h is the distance between the beam reaching the secondary mirror group and the optical axis, d is the lateral distance between the beam reaching the position of the secondary mirror group and the convergence point, and a is the tilt angle of the beam regenerated by the multi-angle adapter.

[0041] The laser beam enters the multi-angle adapter 9. After passing through the isosceles triangular tilted reflector, the laser beam is guided and reflected onto the trapezoidal reflector. The trapezoidal reflector is configured to adjust the reflection angle of the laser beam so that after being reflected by the trapezoidal reflector, the laser beam converges with laser beams from other angles at a single convergence point. In other words, the beam is guided to the convergence point, which is located on the side of the first-stage reflector group MA1 away from the second-stage reflector group MA2.

[0042] The dispersion compensation module includes two dispersion prisms 1 and 2, which are spaced apart between the acousto-optic biaser 3 and the femtosecond laser. The reflector group includes multiple reflectors 4, 5, 6, 7, and 8, which are spaced apart between the acousto-optic biaser 3 and the multi-angle adapter 9. The multiple reflectors 4-8 are used to reflect the laser output from the acousto-optic biaser 3 to the multi-angle adapter 9. Reflectors 6 and 7 form an extension line. Specifically, the laser is reflected multiple times by the multiple reflectors 4-8, and the multiple reflectors 4, 5, 6, 7, and 8 are used to lengthen the laser light path output from the acousto-optic biaser 3. The first 4f lens group includes a first lens 10, a first reflector 11, and a second lens 12. The first lens 10 is arranged at intervals with the first reflector 11 and the second lens 12 to form a 4f lens group. The first lens 10 is close to the multi-angle adapter 9. The phase encoding module includes a waveplate 13, a spatial modulator 14, and a second reflector 15. The waveplate 13 is arranged at intervals with the spatial modulator 14 and the second reflector 15. The waveplate 13 is close to the second lens 12.

[0043] The decoding module includes a third lens 16, an aperture 17, and a fourth lens 18. The third lens 16 is spaced apart by the aperture 17 and the fourth lens 18 to form a 4f lens group. The third lens 16 and the fourth lens 18 form a second 4f lens group. The third lens 16 is close to the reflector 15 of the phase encoding module.

[0044] The femtosecond laser emits a laser beam, which passes sequentially through two dispersive prisms 1 and 2, an acousto-optic deflector 3, a mirror group, and a multi-angle adapter 9 before entering the first lens 10. After exiting the first lens 10, the laser beam is reflected by the first mirror 11 onto the second lens 12. After exiting the second lens 12, the laser beam passes through the waveplate 13 and enters the spatial light modulator 14 for phase encoding before exiting. It is then reflected by the second mirror 15 onto the third lens 16. After exiting the third lens 16, the laser beam undergoes phase decoding through a pinhole in the center of the aperture 17 before entering the fourth lens 18.

[0045] The multiphoton imaging device includes a scanning galvanometer module, a fourth 4f mirror group, a fluorescence collection module, and a wavefront calibration module. The decoding module is conjugate between the scanning galvanometer module and the fourth 4f mirror group. The scanning galvanometer module is conjugate between the fourth 4f mirror group, the objective lens, and the calibration module. That is, the fluorescence collection module and the calibration module are set on two orthogonal sides of the fourth 4f mirror group.

[0046] The scanning galvanometer module includes an x-axis galvanometer 19, a fifth lens 20, a sixth lens 21, and a y-axis galvanometer 22. A fourth lens 18 is spaced apart from the x-axis galvanometer 19 and the fifth lens 20. The fifth lens 20 and the sixth lens 21 form a third 4f lens group. The x-axis galvanometer is conjugate with the y-axis galvanometer via the fourth lens 18 and the fifth lens 19. The sixth lens 21 and the y-axis galvanometer 22 are spaced apart on the other side of the fifth lens 20. The x-axis galvanometer 19, the fifth lens 20, the sixth lens 21, and the y-axis galvanometer 22 are arranged sequentially and spaced apart along the light propagation direction.

[0047] The fourth 4f lens group includes a scanning lens 23 and a tube lens 25; the sixth lens 21 is set at intervals between the y-axis galvanometer 22 and the scanning lens 23, and the scanning lens 23 and the tube lens 24 conjugate the y-axis galvanometer 22 and the objective lens; the detachable mirror 24 is used to split the laser beam into two optical paths, and the two optical paths are respectively injected into the objective lens and the wavefront calibration module.

[0048] The fluorescence collection module includes a dichroic mirror 26, an objective lens 27, a collecting lens 28, a filter 29, and a photomultiplier tube 30. The dichroic mirror 26 is close to the tube lens 25. The objective lens 27 and the collecting lens 28 are arranged on two orthogonal sides of the dichroic mirror 26, with the objective lens 27 arranged on the side of the dichroic mirror 26 away from the tube lens 25. The collecting lens 28 is arranged with the filter 29 and the photomultiplier tube 30 spaced apart in sequence on the side away from the dichroic mirror 26. The dichroic mirror 26, the collecting lens 28, the filter 29, and the photomultiplier tube 30 are arranged with the filter 29 and the photomultiplier tube 30 spaced apart in sequence along the direction of light propagation.

[0049] The wavefront calibration module includes a seventh lens 31, a third reflector 32, a detachable reflector 24, a microlens array 33, and a CMOS 34. The scanning lens 23 is arranged in a 4f configuration with the detachable reflector 24 and the seventh lens 31 at intervals. The y-axis galvanometer 22 and the microlens array 33 are conjugate. The CMOS 34 is located on the side of the microlens array 33 away from the third reflector 32. The CMOS 34 uses a camera containing a CMOS chip.

[0050] After the laser beam exits the fourth lens 18, it passes through the x-axis galvanometer 19, the fifth lens 20, the sixth lens 21, the y-axis galvanometer 22, and the scanning lens 23 before entering the detachable reflector 24. The laser beam is split into a first beam and a second beam by the detachable reflector 24. The first beam is reflected by the detachable reflector 24 and then passes through the seventh lens 31, the third reflector 32, and the microlens array 33 before entering the CMOS 34 for calibration. The second beam passes through the detachable reflector 24, the tube lens 25, and the dichroic mirror 26 before entering the objective lens. The excited fluorescence is collected by the objective lens and then reflected by the dichroic mirror 26 before entering the photomultiplier tube 30 through the collecting lens 28 and the filter 29.

[0051] The beam convergence point of the multi-angle adapter 9, the modulation plane of the spatial light modulator 14, and the planes of the x-axis galvanometer 19, y-axis galvanometer 22, and entrance pupil plane of the objective lens 27 of the multiphoton imaging device are optically conjugate through the 4f lens group.

[0052] The multi-region adaptive optics multiphoton microscopy imaging system also includes an acquisition control module, which is located in the multiphoton imaging device, i.e., CMOS 34. The acquisition control module is connected to the acousto-optic deflector 3, the x-axis galvanometer 19, the y-axis galvanometer 22, and the photomultiplier tube 30. The acquisition control module is used to control the scanning waveform and acquire fluorescence signals.

[0053] A multi-region adaptive optics multiphoton microscopy imaging method for a multi-region adaptive optics multiphoton microscopy imaging system includes the following steps: S1. The laser enters the dispersion module for dispersion pre-compensation. After exiting the dispersion module, the laser is rapidly deflected by the acousto-optic deflector 3 and then enters the multi-angle adapter 9 through the reflector group. After passing through the multi-angle adapter 9, the spatial light modulator 14 and the aperture 17, the laser enters the scanning galvanometer module. The scanning galvanometer module scans the laser. The laser is converted into an angle in the multi-angle adapter 9 that matches the multiplexing grating of the spatial light modulator 14. S2. The laser is reflected by the scanning galvanometer module and enters the fluorescence collection module through the fourth 4f mirror group. The fluorescence in the laser is collected by the objective lens 27 in the fluorescence collection module. The laser is split and reflected by the detachable reflector 24 in the fourth 4f mirror group to the calibration module for wavefront calibration. S3. The scanning galvanometer module scans the imaging field of view and obtains the compensation phase corresponding to each of the different sub-regions in the imaging field of view through an algorithm. After phase reset encoding of multiple compensation phases, a composite phase map is generated and loaded into the spatial light modulator. Finally, according to the different acousto-optic deflector angles corresponding to different regions, the acousto-optic deflector deflects and decodes the corresponding compensation phase when the galvanometer scans and images to different regions.

[0054] Step S3 is an adaptive optics method without a wavefront sensor. The steps are as follows: first, the sample is scanned and imaged using Zernike aberration modes with different coefficients to obtain the image sequence corresponding to the Zernike aberration. Then, the correct coefficients corresponding to different Zernike aberrations are fitted according to the image evaluation index using the mode method, thereby obtaining the compensated wavefront.

[0055] In step S1, the laser is converted into an angle that matches the multiplexing grating of the spatial light modulator 14 in the multi-angle adapter 9. After the laser enters the spatial light modulator 14, it is loaded onto the multiplexing wavefront that is distributed in a circular pattern in the frequency domain and modulated to obtain the modulated laser. The modulated laser enters the aperture 17 for wavefront decoding and then exits and enters the scanning galvanometer module to be scanned.

[0056] In step S3, the scanning imaging of the imaging field of view is specifically performed as follows: During the scanning imaging process, whenever the x-axis galvanometer 19 completes scanning one row of the image field of view, the y-axis galvanometer 22 moves in the column direction to switch to the next row, so that the x-axis galvanometer 19 performs scanning in the row direction of the next image field of view. When the x-axis galvanometer 19 or the y-axis galvanometer 22 scans different sub-regions, the acquisition control module controls the acousto-optic deflector to perform random scanning, so that when the laser passes through different sub-regions of the imaging field of view, the compensation phase corresponding to the sub-region, i.e. the scanning area, is decoded, and multiple corrected wavefront phase maps corresponding to different sub-regions are obtained to achieve parallel aberration compensation for multiple sub-regions in the imaging field of view.

[0057] The phase multiplexing coding method is as follows: multiple corrected wavefront phase maps corresponding to different sub-regions are superimposed and summed with grating phases of different spatial frequencies, so that the coded phases are distributed at equal angular intervals along the circumference on the focal plane of the third lens 16.

[0058] The sub-region division method of the imaging field of view can be one or more of the following: dividing the imaging field of view into equal intervals according to preset rules, adaptively dividing it according to the region of interest of the imaging field of view of the sample, or freely dividing the imaging field of view into arbitrary shapes.

[0059] The random scanning of the acousto-optic deflector is controlled as follows: the random scanning waveform of the acousto-optic deflector is generated in the following manner: The random scanning control signal of the acousto-optic deflector 3 is generated based on the sub-region division information of the imaging field of view and the scanning waveform of the galvanometer. After pixel-level timing matching of the galvanometer scanning control signal, it is synchronously output through the acquisition control module to drive the acousto-optic deflector 3 to perform random scanning.

[0060] In step S2, obtaining the compensation phase corresponding to each sub-region in the imaging field of view is specifically as follows: 1) The algorithm is an adaptive optics algorithm that recovers the initial compensated wavefront for any sub-region in the imaging field of view; 2) The algorithm classifies the initial compensation wavefront using a clustering algorithm, categorizing it into a specific number of categories. Each category corresponds to a specific sub-region, and a representative compensation wavefront is generated for each category. The compensation wavefront serves as the compensation phase.

[0061] In step 1), the adaptive algorithm adopts an image feedback-based wavefront sensorless adaptive optics algorithm. The adaptive optics algorithm is one of the following: wavefront sensorless adaptive optics algorithm, wavefront detection method based on direct detection by wavefront sensor, and interferometric detection method based on the principle of interference.

[0062] The system includes a femtosecond laser for providing the excitation source; a dispersion compensation module for pre-compensating for temporal dispersion of the laser pulse; an acousto-optic deflector for high-speed random scanning of the beam; a multi-angle adapter for adjusting the beam tilt angle; a spatial light modulator for multiplexing phase encoding; a 4f mirror group and aperture 17 for wavefront decoding; a conventional multiphoton microscopy imaging system for final imaging; and an acquisition and control module for system control and signal acquisition.

[0063] The convergence point of the multi-angle adapter 9, the spatial light modulator 14, the galvanometer plane of the traditional multiphoton microscope system, and the entrance pupil plane of the objective lens are all conjugated through the 4f lens group.

[0064] The multi-angle adapter 9 consists of a primary reflector group MA1 and a secondary reflector group MA2. Small-angle beams are first reflected by the primary reflector group MA1 and then reach the secondary reflector group MA2. The secondary reflector group MA2 adjusts the reflection angle to reach the convergence point.

[0065] The primary reflector group MA1 consists of multiple isosceles triangular tilted reflectors forming an arbitrary regular polygon reflector group. The mirrors can be mounted using either reflector patches or prism coatings.

[0066] The secondary reflector assembly MA2 consists of a mounting base, a tilting mounting block, a compact reflector frame, and trapezoidal reflector mirrors. Angle adjustment is achieved using the compact reflector frame.

[0067] The specific methods of microscopic imaging are as follows: First, customized aberration compensation phases for each sub-region in the imaging field of view are obtained through an adaptive algorithm. Then, these phases are multiplexed and encoded to generate a composite phase map, which is then loaded into the spatial light modulator 14. During the scanning process, the acousto-optic deflector 3 operates according to a random scanning waveform synchronized with the galvanometer scanning waveform, enabling the corresponding compensation phase to be decoded in real time when the scanning beam is in different regions, thus achieving parallel aberration correction across multiple regions within a single frame scan. The phase multiplexing encoding method involves encoding different phases in the frequency domain and distributing them at equal intervals along a circumference in the frequency domain.

[0068] The adaptive algorithm steps are as follows: recover the compensation wavefront of any sub-region using the algorithm, and then classify the compensation wavefront into several specific compensation wavefronts corresponding to different sub-regions using a clustering algorithm.

[0069] The algorithm is as follows: acquire fluorescence images of Zernike aberration mode under different coefficients, divide the image into multiple sub-regions, and obtain the corresponding compensation phase in each sub-region by fitting the total fluorescence intensity curve mode method.

[0070] The random scanning waveform of the acousto-optic deflector is generated by scanning the waveform through divided sub-regions and galvanometers, and then passing the same trigger source after pixel matching.

[0071] Unlike traditional adaptive optics which uses a single corrected phase, this scheme utilizes holographic wavefront scanning technology during scanning imaging. By switching wavefronts at a rate corresponding to the pixel dwell time, different corrected wavefront phase maps are dynamically switched as the scanning beam reaches different spatial regions. This allows for customized aberration compensation to be applied to multiple spatial regions during a single-frame scan. This method solves the problem of simultaneously and effectively correcting varying aberrations in different regions during multiphoton imaging.

[0072] Example 1: Sensorless mode measurement like Figure 1 As shown, the multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning of the present invention includes a holographic wavefront scanning component and a multiphoton imaging component. The multiphoton imaging component is used to realize the scanning imaging function, while the holographic wavefront scanning component is used to rapidly switch wavefronts during scanning. The holographic wavefront scanning component includes dispersion compensation modules 1 and 2, an acousto-optic scanning module 3, an angle adaptation module 9, phase encoding modules 13 and 14, and decoding modules 16, 17, and 18; the multiphoton imaging component includes scanning galvanometer modules 19 and 22, fluorescence collection modules 28, 29, and 30, and an acquisition control module.

[0073] The input laser beam first undergoes dispersion pre-compensation via dispersion compensation modules 1 and 2, and then rapid beam deflection is achieved through acousto-optic scanning module 3. The deflected beam is guided by mirrors 4, 5, 6, 7, and 8 to angle adaptation module 9 to adjust the scanning angle. Subsequently, the beam passes through the first 4f mirror group 10 and 12, conjugating the beam convergence point generated by the angle adapter with the spatial light modulator 14. The spatial light modulator 14 performs phase encoding on the laser beam, and the encoded beam then undergoes specific wavefront decoding via the second 4f mirror group 16 and 18 and aperture 17, maintaining conjugation with galvanometer scanning modules 19 and 22. Finally, the laser beam passes through a 4f system composed of scanning lens 23 and tube lens 25, conjugating with the entrance pupil plane of objective lens 27. The fluorescence collection module includes a collection lens 28, a filter 29, and a photomultiplier tube 30. The excitation fluorescence collected by objective lens 27 is reflected by dichroic mirror 26 and enters this module for detection. The acquisition and control module is used to synchronously control the acousto-optic deflector, galvanometer scanning, and fluorescence signal acquisition; during the adaptive phase compensation process, such as Figure 4 As shown in a, b, c, and d, the compensation phase corresponding to different sub-regions is first obtained through an adaptive algorithm, and each phase is multiplexed and encoded before being loaded into the spatial light modulator 14. Taking a 3x3 square sub-region with equal intervals as an example, during scanning imaging, the acousto-optic deflector 3 is adjusted according to... Figure 5 The random scanning waveform is generated in the manner shown in 'a', and the corresponding compensation phase is decoded in different scanning areas, thereby realizing the partition aberration correction.

[0074] The first reflector group uses a prism coating method for lens mounting; the adaptive optics method uses a sensorless mode; and the sub-region is divided into 6×6 equally spaced areas according to the field of view.

[0075] The first 4f system includes a first lens 10 and a second lens 11 arranged coaxially and having coincident focal points; the second 4f system includes a third lens 16 and a fourth lens 18 arranged coaxially and having coincident focal points, with a coaxial aperture 17 between the two lenses. The dispersion compensation module consists of two identical dispersion prisms.

[0076] The implementation process is as follows: 1. The femtosecond laser first undergoes dispersion pre-compensation at points 1 and 2 via a dispersive prism to counteract the positive dispersion generated by the acousto-optic deflector and other optical components, thus maintaining a narrower pulse width for the beam reaching the sample, for example, compressing it from 550 fs to 150 fs. Subsequently, the beam is rapidly deflected by the acousto-optic deflector 3 and incident on the angle adaptation module 9. This module uses the first and second mirror groups 9 to convert the small-angle incident beam into an angle that matches the multiplexing grating of the spatial light modulator 14. The angle adapter structure is as follows: Figure 3 As shown in a, b, and c, the reflection principle is as follows: Figure 2 As shown. The acousto-optic modulator 3 used is the MT110-A1-IR from AAOptoelectronics of France, and the spatial light modulator 14 is a 1920×1200 from Meadowlark Optics of the United States, with a suitable wavelength of 850–1300 nm.

[0077] 2. After angle adaptation, the different mode beams converge behind the module, and then the convergence point is conjugate with the spatial light modulator 14 through the first 4f system 10, 12. The spatial light modulator is loaded with a multiplexed wavefront distributed circumferentially in the frequency domain. After modulation, the beam is decoded by the second 4f mirror group 16, 18 and its focal plane aperture 17. The first-stage reflector group of the angle adapter 9 consists of 9 tilted triangular prism coated reflectors.

[0078] 3. The decoded light beam reaches scanning mirrors 19 and 22. The X and Y axis mirrors are conjugated through a 4f system 20 and 21. After being reflected by mirror 22, the beam passes through a 4f system consisting of scanning lens 23 and tube lens 25 and is incident on objective lens 27. The excited fluorescence is collected by objective lens 27, reflected by dichroic mirror 26, and enters the fluorescence collection module consisting of collection lens 28, filter 29, and photomultiplier tube 30. The system guides the laser to Shakhartmann wavefront detectors 33 and 34 through detachable reflector 24 for wavefront calibration at different angles. The galvanometer used was a CTI 6215h (USA), the scanning lens was a Thorlabs SL-50-2P (USA), the tube lens was a Thorlabs TTL-200MP, the objective lens was an Olympus XLUPLanFI 20×, the dichroic mirror was a Semrock FF665-Di02-35×37 (USA), the filter was a Semrock FF665-Di02-35×37, the photomultiplier tube was a Hamamatsu H7422 (Japan), the microlens array was an Edmund 64-483 (USA), and the camera was a Dahua Technology A3B00MU000 (China).

[0079] 4. When performing multi-region adaptive optics correction, the process is as follows: Figure 4 As shown: First, Zernike aberrations of different coefficients are loaded onto the spatial light modulator 14, and fluorescence images under the corresponding coefficients are acquired. The images are then divided into 36 equally spaced and equally sized sub-regions (6×6). The compensation phase of the corresponding region is obtained by fitting the fluorescence intensity curve pattern within each sub-region. Then, dimensionality reduction and clustering algorithms are used to classify the compensation wavefront into 9 specific types, each corresponding to a different sub-region. After obtaining the aberration region distribution, according to... Figure 5 As shown in a and b, the control waveform of the acousto-optic deflector 3 is generated based on the synchronization relationship between the sub-region pixels and the scanning waveform of the galvanometer. During the scanning imaging process, when the galvanometer 19 scans to different sub-regions, the acousto-optic deflector 3 deflects the beam angle accordingly, decoding the corresponding compensation phase. Figure 6 The figure 1a shows a comparison of the correction effects of traditional single-region adaptive optics and the multi-region method of the present invention. It can be seen that when there are regional differences in aberrations, the method of the present invention has a better correction capability.

[0080] Example 2: Measurement using the direct wavefront sensor method like Figure 1As shown, the multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning of the present invention includes a holographic wavefront scanning component and a multiphoton imaging component. The multiphoton imaging component is used to realize the scanning imaging function, while the holographic wavefront scanning component is used to rapidly switch wavefronts during scanning. The holographic wavefront scanning component includes dispersion compensation modules 1 and 2, an acousto-optic scanning module 3, an angle adaptation module 9, phase encoding modules 13 and 14, and decoding modules 16, 17, and 18; the multiphoton imaging component includes scanning galvanometer modules 19 and 22, fluorescence collection modules 28, 29, and 30, and an acquisition control module.

[0081] The input laser beam first undergoes dispersion pre-compensation via dispersion compensation modules 1 and 2, and then rapid beam deflection is achieved through acousto-optic scanning module 3. The deflected beam is guided by mirrors 4, 5, 6, 7, and 8 to angle adaptation module 9 to adjust the scanning angle. Subsequently, the beam passes through the first 4f mirror group 10 and 12, conjugating the beam convergence point generated by the angle adapter with the spatial light modulator 14. The spatial light modulator 14 performs phase encoding on the laser beam, and the encoded beam then undergoes specific wavefront decoding via the second 4f mirror group 16 and 18 and aperture 17, maintaining conjugation with galvanometer scanning modules 19 and 22. Finally, the laser beam passes through a 4f system composed of scanning lens 23 and tube lens 25, conjugating with the entrance pupil plane of objective lens 27. The fluorescence collection module includes a collection lens 28, a filter 29, and a photomultiplier tube 30. The excitation fluorescence collected by objective lens 27 is reflected by dichroic mirror 26 and enters this module for detection. The acquisition and control module is used to synchronously control the acousto-optic deflector, galvanometer scanning, and fluorescence signal acquisition; during the adaptive phase compensation process, such as Figure 4 As shown, firstly, the compensation phase corresponding to different sub-regions is obtained through an adaptive algorithm, and each phase is multiplexed and encoded before being loaded into the spatial light modulator 14. During scanning imaging, the acousto-optic deflector 3 is activated according to... Figure 5 The method shown generates a random scanning waveform, and decodes the corresponding compensation phase in different scanning areas, thereby realizing partition aberration correction.

[0082] The first reflector group uses a triangular reflector patch method for lens mounting; the adaptive optics method uses the direct wavefront sensor method; and the sub-region is arbitrarily divided according to the region of interest.

[0083] The first 4f system includes a first lens 10 and a second lens 11 arranged coaxially and having coincident focal points; the second 4f system includes a third lens 16 and a fourth lens 18 arranged coaxially and having coincident focal points, with a coaxial aperture 17 between the two lenses. The dispersion compensation module consists of two identical gratings.

[0084] The implementation process is as follows: 1. The femtosecond laser first undergoes dispersion pre-compensation on points 1 and 2 via a dispersion grating to counteract the positive dispersion generated by the acousto-optic deflector and other optical components, thus maintaining a narrower pulse width for the beam reaching the sample, for example, compressing it from 550 fs to 150 fs. Subsequently, the beam is rapidly deflected by the acousto-optic deflector 3 and incident on the angle adaptation module 9. This module converts the small-angle incident beam into an angle that matches the multiplexing grating of the spatial light modulator 14 through the first and second mirror groups 9. The angle adapter structure is as follows: Figure 3 As shown, its reflection principle is as follows: Figure 2 As shown. The acousto-optic modulator 3 used is the MT110-A1-IR from AA Optoelectronics of France, and the spatial light modulator 14 is a 1920×1200 from Meadowlark Optics of the United States, with a suitable wavelength of 850–1300 nm.

[0085] 2. After angle adaptation, the different mode beams converge behind the module, and then the convergence point is conjugate with the spatial light modulator 14 through the first 4f system 10, 12. The spatial light modulator is loaded with a multiplexed wavefront distributed circumferentially in the frequency domain. After modulation, the beam is decoded by the second 4f mirror group 16, 18 and its focal plane aperture 17. The first-stage reflector group of the angle adapter 9 consists of 9 tilted triangular prism coated reflectors.

[0086] 3. The decoded light beam reaches scanning mirrors 19 and 22. The X and Y axis mirrors are conjugated through a 4f system 20 and 21. After being reflected by mirror 22, the beam passes through a 4f system consisting of scanning lens 23 and tube lens 25 and is incident on objective lens 27. The excited fluorescence is collected by objective lens 27, reflected by dichroic mirror 26, and enters the fluorescence collection module consisting of collection lens 28, filter 29, and photomultiplier tube 30. The system guides the laser to Shakhartmann wavefront detectors 33 and 34 through detachable reflector 24 for wavefront calibration at different angles. The galvanometer used was a CTI 6215h (USA), the scanning lens was a Thorlabs SL-50-2P (USA), the tube lens was a Thorlabs TTL-200MP, the objective lens was an Olympus XLUPLanFI 20×, the dichroic mirror was a Semrock FF665-Di02-35×37 (USA), the filter was a Semrock FF665-Di02-35×37, the photomultiplier tube was a Hamamatsu H7422 (Japan), the microlens array was an Edmund 64-483 (USA), and the camera was a Dahua Technology A3B00MU000 (China).

[0087] 4. When performing multi-region adaptive optics correction: The beam is deflected to each sub-region by freely segmenting the region of interest. The compensated phase of each sub-region is directly obtained using a wavefront sensor. Then, dimensionality reduction and clustering algorithms are used to classify the compensated wavefront into user-defined types, each corresponding to a different sub-region. (Based on user-defined region segmentation, ...) Figure 5 As shown, the control waveform of the acousto-optic deflector 3 is generated based on the synchronization relationship between the sub-region pixels and the scanning waveform of the galvanometer. During the scanning imaging process, when the galvanometer 19 scans to different sub-regions, the acousto-optic deflector 3 deflects the beam angle accordingly, decoding the corresponding compensation phase. Figure 6 The figure 1a shows a comparison of the correction effects of traditional single-region adaptive optics and the multi-region method of the present invention. It can be seen that when there are regional differences in aberrations, the method of the present invention has a better correction capability.

[0088] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0089] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0090] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning, characterized in that, It includes a dispersion compensation module, an acousto-optic deflector (3), a mirror group, a multi-angle adapter (9), a first 4f mirror group, a phase encoding module, a decoding module, and a multiphoton imaging device; the dispersion compensation module is spaced in front of the input end of the acousto-optic deflector (3), the acousto-optic deflector (3) and the multi-angle adapter (9) are spaced apart by a mirror group, the multi-angle adapter (9) is conjugate with the phase encoding module through the first 4f mirror group, and the phase encoding module is conjugate with the multiphoton imaging device through the decoding module; The multi-region adaptive optics multiphoton microscopy system is equipped with a femtosecond laser that emits laser light to the dispersion compensation module. After entering the dispersion compensation module, the laser light is reflected by the acousto-optic deflector (2) and the mirror group to the multi-angle adapter (9). After exiting the multi-angle adapter (9), the laser light passes through the first 4f mirror group, the phase encoding module, and the decoding module before entering the multiphoton imaging device.

2. The multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning according to claim 1, characterized in that, The multi-angle adapter includes a first-stage mirror group (MA1) and a second-stage mirror group (MA2) arranged at intervals along the optical path. The second-stage mirror group (MA2) is close to the mirror group, and the first-stage mirror group (MA1) is close to the first 4f mirror group. The first-stage reflector assembly (MA1) includes multiple isosceles triangular tilted mirrors and a mirror assembly frame. The included angle formed by the isosceles sides of each isosceles triangular tilted mirror points towards the center. The mirror assembly frame has a hollow hole in the middle. The isosceles triangular tilted mirrors are arranged closely in a regular polygon along the circumference and mounted on the mirror assembly frame. There is a gap in the middle of the regular polygon for laser to pass through. The mirror surfaces of the multiple isosceles triangular tilted mirrors are at a certain angle to the light path. The multiple isosceles triangular tilted mirrors are mounted by either a mirror patch or a prism coating. The second-stage reflector assembly (MA2) includes a mounting base, a tilted mounting block, a compact reflector frame, and a trapezoidal reflector. The mounting base is connected to the compact reflector frame via the tilted mounting block. The trapezoidal reflector is connected circumferentially to the side of the compact reflector frame away from the mounting base. The compact reflector frame is configured to adjust the reflection angle of the trapezoidal reflector. The laser is injected into the multi-angle adapter (9). The laser is first reflected by the isosceles triangular tilted reflector and then reflected onto the trapezoidal reflector. The trapezoidal reflector adjusts the reflection angle of the laser so that the laser reflected by the trapezoidal reflector converges at a convergence point. The convergence point is on the side of the first-stage reflector group (MA1) away from the second-stage reflector group (MA2).

3. The multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning according to claim 1, characterized in that, The dispersion compensation module includes two dispersion prisms (1, 2), which are spaced apart between the acousto-optic biaser (3) and the femtosecond laser; the reflector group includes multiple reflectors (4, 5, 6, 7, 8), which are spaced apart between the acousto-optic biaser (3) and the multi-angle adapter (9), and the multiple reflectors (4-8) are used to reflect the laser output from the acousto-optic biaser (3) onto the multi-angle adapter (9); the first 4f The mirror assembly includes a first lens (10), a first reflector (11), and a second lens (12). The first lens (10) is spaced apart from the first reflector (11) and the second lens (12), and the first lens (10) is close to the multi-angle adapter (9). The phase encoding module includes a waveplate (13), a spatial modulator (14), and a second reflector (15). The waveplate (13) is spaced apart from the spatial modulator (14) and the second reflector (15), and the waveplate (13) is close to the spatial light modulator (14). The decoding module includes a third lens (16), an aperture (17) and a fourth lens (18). The third lens (16) is arranged at intervals between the aperture (17) and the fourth lens (18). The third lens (16) is close to the reflector (15) of the phase encoding module. The femtosecond laser emits laser light, which passes through two dispersive prisms (1, 2), an acousto-optic deflector (3), a mirror group and a multi-angle adapter (9) in sequence before entering the first lens (10). After exiting the first lens (10), the laser light is reflected by the first mirror (11) onto the second lens (12). After exiting the second lens (12), the laser light passes through a waveplate (13) and enters the spatial light modulator (14) for phase encoding before exiting. It is then reflected by the second mirror (15) onto the third lens (16). After exiting the third lens (16), the laser light passes through a pinhole provided in the center of the aperture (17) for phase decoding before entering the fourth lens (18).

4. The multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning according to claim 3, characterized in that, The multiphoton imaging device includes a scanning galvanometer module, a fourth 4f mirror group, a fluorescence collection module, and a wavefront calibration module; the decoding module is arranged alternately via the scanning galvanometer module and the fourth 4f mirror group, and the scanning galvanometer module is conjugate via the entrance pupil of the objective lens in the fourth 4f mirror group and the wavefront calibration module. The scanning galvanometer module includes an x-axis galvanometer (19), a fifth lens (20), a sixth lens (21), and a y-axis galvanometer (22); the fourth lens (18) is spaced between the x-axis galvanometer (19) and the fifth lens (20), and the sixth lens (21) and the y-axis galvanometer (22) are spaced between each other on the other side of the fifth lens (20). The x-axis galvanometer (19), the fifth lens (20), the sixth lens (21), and the y-axis galvanometer (22) are arranged sequentially and spaced between each other along the light propagation direction. The fourth 4f mirror group includes a scanning lens (23) and a tube lens (25); the sixth lens (21) is spaced between the y-axis galvanometer (22) and the scanning lens (23), and a tube lens (25) is spaced between the other side of the scanning lens (23); the detachable reflector (24) is used to split the laser beam into two optical paths, and the two optical paths are respectively injected into the fluorescence collection module and the wavefront calibration module; The fluorescence collection module includes a dichroic mirror (26), an objective lens (27), a collecting lens (28), a filter (29), and a photomultiplier tube (30). The dichroic mirror (26) is close to the tube lens (25). The objective lens (27) and the collecting lens (28) are arranged on two orthogonal sides of the dichroic mirror (26). The objective lens (27) is arranged on the side of the dichroic mirror (26) away from the tube lens (25). The collecting lens (28) is arranged with a filter (29) and a photomultiplier tube (30) at intervals on the side away from the dichroic mirror (26). The dichroic mirror (26), the collecting lens (28), the filter (29), and the photomultiplier tube (30) are arranged at intervals along the light propagation direction. The wavefront calibration module includes a seventh lens (31), a third mirror (32), a detachable mirror (24), a microlens array (33), and a CMOS (34); the scanning lens (23) is spaced apart from the detachable mirror (24) and the seventh lens (31), the seventh lens (31) is spaced apart from the third mirror (32) and the microlens array (33), and the CMOS (34) is disposed on the side of the microlens array (33) away from the third mirror (32). After the laser beam exits the fourth lens (18), it passes through the x-axis galvanometer (19), the fifth lens (20), the sixth lens (21), the y-axis galvanometer (22), and the scanning lens (23) and enters the detachable reflector (24). The laser beam is split into a first beam and a second beam by the detachable reflector (24). The first beam is reflected by the detachable reflector (24) and then passes through the seventh lens (31), the third reflector (32), and the microlens array (33) before entering the CMOS (34). The second beam passes through the detachable reflector (24) and the tube lens (25) and enters the dichroic mirror (26). The second beam excites fluorescence through the dichroic mirror (26) and then enters the objective lens. The fluorescence is collected by the objective lens and then reflected by the dichroic mirror (26) and enters the photomultiplier tube (30) through the collecting lens (28) and the filter (29).

5. A multi-region adaptive optics multiphoton microscopy imaging system based on holographic wavefront scanning according to claim 1, characterized in that, The multi-region adaptive optics multiphoton microscopy imaging system further includes an acquisition control module, which is located in the multiphoton imaging device. The acquisition control module is electrically connected to the acousto-optic deflector (3), the x-axis galvanometer (19), the y-axis galvanometer (22), and the photomultiplier tube (30). The acquisition control module is used to control the scanning waveform and acquire fluorescence signals.

6. A multi-region adaptive optics multiphoton microscopy imaging method based on the multi-region adaptive optics multiphoton microscopy imaging system according to any one of claims 1-5, characterized in that, S1. The laser is injected into the dispersion module for dispersion pre-compensation. After the laser is injected into the dispersion module, it is rapidly deflected by the acousto-optic deflector (3) and then injected into the multi-angle adapter (9) through the reflector group. After the laser passes through the multi-angle adapter (9), the spatial light modulator (14) and the aperture (17), it is injected into the scanning galvanometer module. The scanning galvanometer module scans the laser. S2, the laser beam is split and reflected by the detachable mirror (24) in the fourth 4f mirror group to the calibration module for wavefront calibration; the laser beam is reflected by the scanning galvanometer module and enters the objective lens through the fourth 4f mirror group, and the laser-excited fluorescence is collected by the objective lens (27) in the fluorescence collection module; S3. The scanning galvanometer module scans and images the imaging field of view. The acquisition control module obtains the compensation phase corresponding to each of the different sub-regions in the imaging field of view through an algorithm. After performing phase composite encoding on multiple compensation phases, a composite phase map is generated, and the encoded composite phase map is loaded into the spatial light modulator. Finally, according to the different acousto-optic deflector angles corresponding to different regions, the acousto-optic deflector deflects and decodes the corresponding compensation phase when the galvanometer scans and images to different regions.

7. The multi-region adaptive optics multiphoton microscopy imaging method according to claim 6, characterized in that, In step S1, the laser is converted in the multi-angle adapter (9) to an angle that matches the multiplexing grating of the spatial light modulator (14). After the laser enters the spatial light modulator (14), it is modulated to obtain the modulated laser. The modulated laser enters the aperture (17) for wavefront decoding and then exits and enters the scanning galvanometer module to be scanned.

8. A multi-region adaptive optics multiphoton microscopy imaging method according to claim 6, characterized in that, In step S3, the scanning imaging of the imaging field is as follows: during the scanning imaging process, whenever the x-axis galvanometer (19) finishes scanning one row of the imaging field, the y-axis galvanometer (22) moves in the column direction to switch to the next row, so that the x-axis galvanometer (19) performs the next row of the imaging field in the row direction. When the x-axis galvanometer (19) or the y-axis galvanometer (22) scans the correction sub-regions corresponding to different sub-regions, the acquisition control module controls the acousto-optic deflector to perform random scanning, so that when the laser passes through different sub-regions of the imaging field, the compensation phase corresponding to the sub-region is decoded, and multiple correction wavefront phase maps corresponding to different sub-regions are obtained. The phase multiplexing coding method is as follows: multiple corrected wavefront phase maps corresponding to different sub-regions are superimposed and summed with grating phases of different spatial frequencies, so that the coded phases are distributed at equal angular intervals along the circumference on the focal plane of the third lens (16). The sub-region division method of the imaging field of view is one or more of the following: dividing the imaging field of view into equal intervals according to preset rules, adaptively dividing the imaging field of view, and freely dividing the imaging field of view into arbitrary shapes.

9. A multi-region adaptive optics multiphoton microscopy imaging method according to claim 8, characterized in that, The specific details of controlling the acousto-optic deflector to perform random scanning are as follows: The scanning waveform of the acousto-optic deflector (3) is generated by the sub-region distribution of the imaging field of view and the scanning waveform of the galvanometer, which together generate the scanning control signal and the galvanometer scanning signal. The scanning control signal and the galvanometer scanning signal are matched at the pixel level to obtain the matched signal. The matched signal is output through the acquisition control module to drive the acousto-optic deflector (3) to perform random scanning.

10. A multi-region adaptive optics multiphoton microscopy imaging method according to claim 6, characterized in that, In step S2, the compensation phase corresponding to each sub-region in the imaging field of view is obtained as follows: 1) The algorithm is an adaptive optics algorithm that recovers the initial compensated wavefront for the sub-region in the imaging field of view; 2) The algorithm classifies the initial compensation wavefront using a clustering algorithm, classifying it into a specific number of categories, each category corresponding to a sub-region, and generating a compensation wavefront for each category, wherein the compensation wavefront serves as the compensation phase; In step 1), the adaptive optics algorithm is one of the following: an image feedback-based wavefront sensorless adaptive optics algorithm, a wavefront detection method based on direct detection by a wavefront sensor, or an interferometric detection method based on the principle of interference.

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