Two-photon volume imaging device and method based on phase regulation annular Airy beam

Abstract

The invention provides a two-photon volume imaging device and method based on a phase regulation annular Airy beam, and belongs to the technical field of optical microscope imaging and optical control. The device comprises a laser beam expanding module, a light beam deflection module, an annular Airy beam modulation module, a light beam scanning module and an imaging module, the annular Airy beam modulation module is innovatively introduced on the basis of a traditional two-photon microscope, a spatial light modulator SLM is used for adjusting the phase, a Fourier lens is used for generating a frequency spectrum, and the imaging precision is improved. The shape of the excitation light beam at the focus is accurately regulated and controlled; the annular Airy light beam and the novel vortex annular Airy light beam are indirectly generated by using the SLM, a smaller focusing focus and a long-focus deep light needle can be generated, the imaging resolution is directly improved and the axial depth of field is expanded by improving the scanning precision of the two-photon microscope, and meanwhile, the axial change is generated on the basis of the annular Airy light needle, so that the imaging precision of the two-photon microscope is improved. And deep analysis of axial three-dimensional information is realized by fitting the shape of the light beam and the axial depth.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical microscope imaging and optical manipulation technology, in particular to a two-photon volume imaging device and method based on phase control annular Airy beam. BACKGROUND

[0002] In the field of optical microscopic imaging technology, confocal microscopy has become an important tool for three-dimensional imaging of thick tissues due to its ability to provide high spatial resolution and contrast at remarkable scales and depths within scattering tissues; two-photon microscopy (TPM) uses longer wavelength illumination and nonlinear excitation, which is superior to traditional confocal microscopy in terms of resistance to scattering and improvement of spatial resolution. In summary, the combination of two-photon and volume imaging technology can achieve deeper penetration depth while maintaining acceptable lateral resolution, and can also improve the ability of volume microscopy based on annular Airy beam to locate the lateral offset position on the two-dimensional projection plane.

[0003] However, since the excitation of confocal microscopy only occurs at the focal point of the diffraction-limited microscope, volume imaging of thick biological samples is usually achieved by scanning the focal point in three dimensions, and the volume imaging speed is limited by mechanical Z-axis scanning, and the tissue scattering effect restricts the quality of deep imaging; two-photon microscopy (TPM) uses long-wavelength excitation and nonlinear optical effects to significantly improve the penetration depth and scattering suppression capability, but its volume imaging efficiency is still limited by the scanning mechanism. To break through the above technical bottlenecks, annular Airy beam is introduced, which realizes axial large depth-of-field imaging when generating an axially elongated needle-shaped beam in the focusing field, reducing the time cost of layer scanning volume imaging. However, since this method projects all volume information onto a compact two-dimensional plane, it cannot provide sample depth information from different layers, i.e. this platform lacks axial resolution. SUMMARY

[0004] The purpose of the present application is to provide a two-photon volume imaging device and method based on phase control annular Airy beam, by adding an additional vortex phase control on the basis of the annular Airy beam, the generated needle-shaped beam produces axial changes, by fitting this axial change and depth information, combining with the calculation fitting method to reconstruct three-dimensional spatial information, realizing three-dimensional volume imaging depth information analysis.

[0005] In order to achieve the above object, the application provides a two-photon volume imaging device based on phase control annular Airy beam, which comprises a laser beam expansion module, a beam deflection module, an annular Airy beam modulation module, a beam scanning module and an imaging module; the laser beam expansion module comprises a laser, a half-wave plate, a polarization beam splitter, a lens one and a lens two; the beam deflection module comprises a mirror one, a mirror two and a mirror three; the annular Airy beam modulation module comprises a spatial light modulator, a Fourier lens and a diaphragm; the beam scanning module comprises a lens three, a lens four, an X-Y scanning galvanometer, a scanning lens, a sleeve lens and a mirror four; and the imaging module comprises a dichroic mirror, an objective lens, a sample, a filter and a photomultiplier tube.

[0006] Preferably, the mirror one, the mirror two and the mirror three are flip mirrors.

[0007] Preferably, the spatial light modulator receives the collimated and expanded illumination beam, the spatial light modulator is loaded with a designed phase hologram in advance, the angle is adjusted to be less than 15° between the incident light and the outgoing light, and the spatial light modulator can be replaced by a digital micromirror device (DMD), a customized phase element or a liquid crystal phase wafer; when the spatial light modulator is a reflective type, it can be replaced by a transmissive spatial light modulator and placed in front of the objective lens.

[0008] Preferably, the diaphragm is placed at the focal point of the Fourier lens.

[0009] Preferably, the sample receives the excitation light in the form of an annular Airy beam or a new type of vortex annular Airy beam.

[0010] Preferably, the excitation light is focused on the sample by the objective lens, the sample generates signal light after being excited, the signal light is reflected by the dichroic mirror, filtered by the filter and then enters the photomultiplier tube.

[0011] The application further provides a two-photon volume imaging method based on phase control annular Airy beam, which comprises the following steps: Step S1: a femtosecond laser illumination beam is emitted by a laser, the laser power is adjusted by a half-wave plate and a polarization beam splitter, and the adjusted illumination beam is collimated and expanded by a lens one and a lens two, so that the beam diameter is adjusted to be matched with a spatial light modulator, and the light path is switched by a mirror one, a mirror two and a mirror three; Step S2: a designed phase hologram is loaded in the spatial light modulator, the collimated and expanded beam is sent into the spatial light modulator through the mirror one, and the incident beam is converted into an annular Airy beam or a new type of vortex annular Airy beam by phase modulation of the spatial light modulator; Step S3, adjust the angle of the spatial light modulator so that the incident light and the outgoing light angle is less than 15°, the light beam modulated by the spatial light modulator is converted from spatial domain to frequency domain through the Fourier lens, the stop is placed at the focal point of the Fourier lens, the +1 order diffracted light is filtered out from the two blazed gratings, a new vortex annular Airy beam carrying vortex phase is generated, and then the frequency domain light beam is conjugated to the entrance pupil surface of the objective lens through lens three, lens four, scanning lens and sleeve lens; Step S4, the modulated light beam is collimated and X-Y scanned by the scanning galvanometer through lens three and lens four, the sample is scanned point by point in the X-Y plane by adjusting the reflection angle, the axial range of the sample is covered by using the long focal depth characteristics of the light beam, and the spectrum surface is transmitted to the entrance pupil of the objective lens through the scanning lens, the sleeve lens and the mirror four; Step S5, the excitation light is focused on the sample through the objective lens, the sample is excited to generate signal light, the signal light is collected by the objective lens, reflected by the dichroic mirror, processed by the filter, and finally sent into the photomultiplier tube, so as to complete the collection and recording of the imaging signal; Step S6, axial depth analysis is performed, the light intensity distribution or fluorescence intensity distribution at different scanning positions is recorded, the sidelobe rotation characteristic and radius change are extracted and analyzed, and the three-dimensional structure of the sample is fitted and analyzed through the mapping relationship between the rotation angle and the axial depth.

[0012] Preferably, in step S1, the switching light path includes a Gaussian light path and a structured light path.

[0013] Preferably, in step S2, the phase hologram is calculated by a diffraction grating algorithm, and the specific calculation formula is as follows: ; ; ; wherein, is a normalized bounded positive function of amplitude, is an inverse function of function, is used to obtain the modulus of the complex parameter, is an expression of the annular Airy beam, is an analytical function of the amplitude and phase profile of the desired field, is used to obtain the argument of the complex parameter, is a phase distribution function, and are pixel coordinates, is an imaginary unit, is a modulus operation function, is an analytical function of the target field amplitude and phase distribution, is a period.

[0014] In step S5, the new vortex ring Airy beam is obtained by fitting the beam shape and axial depth, and the expression of the new vortex ring Airy beam in the cylindrical coordinate system is as follows: wherein, is the amplitude index of the ring Airy beam, represents the Airy function, is the main ring radius, is the radial coordinate, is an arbitrary scaling factor, is the vortex function, and the expression is as follows: wherein, is the azimuth angle, which is always in the range of to , represents the number of topological charges, represents the power index of the spiral phase, represents the mode function.

[0015] Therefore, the present application proposes a two-photon volumetric imaging device and method based on phase-controlled ring Airy beams, which has the following beneficial effects: (1) The imaging system proposed in the present application uses a structured light field to excite a sample, and has a faster volumetric imaging speed compared with a traditional confocal scanning imaging system.

[0016] (2) The present application can achieve an axial depth of 130-150 μm without sacrificing its imaging resolution, and can accurately position the sample depth position according to the side lobe.

[0017] (3) The present application uses a structured light field to excite a sample, realizes a two-photon fluorescence imaging system with a faster volumetric imaging speed, can stably obtain three-dimensional volumetric imaging of biological samples without labeling, and can realize depth resolution.

[0018] (4) In addition to using a phase-type spatial light modulator to modulate the amplitude and phase of incident light to generate a new vortex ring Airy beam with special amplitude and phase, the present application can also generate other forms of excitation light sources, such as Bessel light and various structured light, through the spatial light modulator to form an imaging system that is more convenient to switch the excitation light source. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 FIG. 1 is a structural schematic diagram of a two-photon volumetric imaging device based on phase-controlled ring Airy beams; Figure 2 ​​This is a flow chart of a two-photon volume imaging method based on phase-controlled annular Airy beam; Figure 3 is the simulated normalized intensity distribution of the new vortex annular Airy beam in the xz plane and yz plane; Figure 4 Schematic diagram of two-photon fluorescence intensity scanning of a single fluorescent microsphere using a novel vortex annular Airy beam.

[0020] Reference numerals 1. Laser; 2. Half-wave plate; 3. Polarization beam splitter; 4. Lens 1; 5. Lens 2; 6. Mirror 1; 7. Mirror 2; 8. Spatial light modulator; 9. Fourier lens; 10. Aperture; 11. Mirror 3; 12. Lens 3; 13. Lens 4; 14. XY scanning galvanometer; 15. Scanning lens; 16. Tube lens; 17. Mirror 4; 18. Dichroic mirror; 19. Objective lens; 20. Sample; 21. Filter; 22. Photomultiplier tube. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0023] Example 1 like Figure 1 As shown, the present invention provides a two-photon volume imaging device based on a phase-controlled annular Airy beam. The device adopts a compact modular design and has a reasonable overall structure. It can provide a two-photon fluorescence imaging device with a faster volume imaging speed, including a laser beam expansion module, a beam deflection module, an annular Airy beam modulation module, a beam scanning module and an imaging module; the laser beam expansion module includes a laser 1, a half-wave plate 2, a polarization beam splitter 3, a lens 1 4 and a lens 2 5; the beam deflection module includes a reflector 1 6, a reflector 2 7 and a reflector 3 11; the annular Airy beam modulation module includes a spatial light modulator 8, a Fourier lens 9 and an aperture 10; the beam scanning module includes a lens 3 12, a lens 4 13, an XY scanning galvanometer 14, a scanning lens 15, a sleeve lens 16 and a reflector 4 17; the imaging module includes a dichroic mirror 18, an objective lens 19, a sample 20, a filter 21 and a photomultiplier tube 22.

[0024] The laser beam expander module is used for expanding the laser beam to meet the subsequent modulation of the beam size, the laser 1 provides a femtosecond laser illumination beam, which is the light source basis of the whole imaging device; the half wave plate 2 and the polarization beam splitter 3 are used for adjusting the laser power to adapt to the demand of different samples for the laser intensity; the lens one 4 and the lens two 5 collimate and expand the illumination beam, and finally adjust the beam diameter to match the spatial light modulator 8, to ensure the effectiveness of the subsequent phase modulation.

[0025] In the beam deflection module, the mirror one 6, the mirror two 7 and the mirror three 11 are three flip mirrors, which are used for switching between the Gaussian light path and the structured light path, and the mirror one 6 and the mirror two 7 are also used for changing the propagation direction of the beam to guide the collimated and expanded beam to accurately enter the spatial light modulator 8. The annular Airy beam modulation module is used for generating an annular Airy beam and modulating a focal point. The spatial light modulator 8 is loaded with a designed phase hologram in advance, which is used for modulating the phase of the incident beam to convert the beam into a required annular Airy beam or a new type of vortex annular Airy beam. The angle of the spatial light modulator 8 is adjusted so that the angle of the incident light and the angle of the outgoing light are less than 15°. The Fourier lens 9 converts the beam modulated by the spatial light modulator 8 from the spatial domain to the frequency domain, to realize the spectrum conversion of the light field. The diaphragm 10 is placed at the focal point of the Fourier lens 9, filters out the +1 order diffracted light from the two blazed gratings, to ensure the purity of the beam. Then the frequency domain beam is conjugated to the entrance pupil surface of the objective lens through the lens three 12, the lens four 13, the scanning lens 15 and the sleeve lens 16.

[0026] The beam scanning module is used for driving the beam to scan the whole sample. The lens three 12 and the lens four 13 send the modulated beam into the X-Y scanning galvanometer 14 after collimating and shrinking the beam, to ensure that the beam enters the scanning galvanometer in a suitable size and state. The X-Y scanning galvanometer 14 realizes point-by-point scanning in the X-Y plane by adjusting the reflection angle, to drive the beam to scan the whole sample. The scanning lens 15, the sleeve lens 16 and the mirror four 17 transfer the phase surface to the entrance pupil of the objective lens 19, to ensure that the beam can be accurately focused on the sample.

[0027] The imaging module is used for collecting the sample signal. The dichroic mirror 18 reflects the signal light generated after the sample is activated, so that the signal light enters the subsequent filtering and detection link. The objective lens 19 focuses the annular Airy beam on the sample 20, to excite the sample 20 to generate signal light and collect the signal light. The optical filter 21 filters the signal light to remove stray light interference and improve the purity of the signal light. The photomultiplier tube 22 collects the filtered signal light and converts the optical signal into an electrical signal, to complete the recording of the imaging signal.

[0028] Embodiment two As Figure 2As shown, the present invention also provides a two-photon volume imaging method based on phase-controlled annular Airy beam, comprising the following steps: Step S1: A femtosecond laser illumination beam is emitted by a laser 1, a half-wave plate 2 and a polarization beam splitter 3 adjust the laser power, a lens 1 4 and a lens 2 5 collimate and expand the adjusted illumination beam, and the beam diameter is adjusted to match the spatial light modulator 8. The optical path is switched through a reflector 1 6, a reflector 2 7 and a reflector 3 11, wherein the switched optical path includes: a Gaussian optical path and a structured optical path; Step S2: Load the designed phase hologram into the spatial light modulator 8, and send the collimated and expanded light beam into the spatial light modulator 8 through the reflector 1 6. The spatial light modulator 8 converts the incident light beam into a ring-Airy beam or a novel vortex ring-Airy beam through phase modulation; Step S3: Adjust the angle of the spatial light modulator 8 so that the angle between the incident light and the outgoing light is less than 15°. The light beam modulated by the spatial light modulator 8 is converted from the spatial domain to the frequency domain through the Fourier lens 9. An aperture 10 is placed at the focal position of the Fourier lens 9 to filter out the +1-order diffraction light from the two blazed gratings to generate a new vortex ring Airy beam carrying a vortex phase. The frequency domain beam is then conjugated to the entrance pupil plane of the objective lens 19 through lens three 12, lens four 13, scanning lens 15, and sleeve lens 16. The phase hologram of the spatial light modulator 8 is calculated using the diffraction grating algorithm. The specific calculation formula is as follows: ; ; ; in, is a normalized bounded positive function of the amplitude, for The inverse function of a function, Used to get the modulus of a complex parameter, is the expression of annular Airy beam, are analytical functions of the amplitude and phase profiles of the desired field, Used to obtain the argument of a complex parameter, is the phase distribution function, and is the pixel coordinate, is the imaginary unit, is the modular operation function, is the analytical function of the target field amplitude and phase distribution, For the cycle; Step S4, the modulated light beam is collimated by lens three 12 and lens four 13, and then is input into the X-Y scanning galvanometer 14, the sample is scanned point by point in the X-Y plane by adjusting the reflection angle, the long focal depth characteristic of the light beam is used to cover the axial range of the sample, the spectrum plane is transmitted to the entrance pupil of the objective lens through the scanning lens 15, the sleeve lens 16 and the reflecting mirror four 17; Step S5, the excitation light is focused on the sample 20 by the objective lens 19, the sample is excited to generate signal light, the signal light is collected by the objective lens 19, is reflected by the dichroic mirror 18, is processed by the filter 21, and finally the processed signal light is input into the photomultiplier tube 22, so that the collection and recording of the imaging signal are completed; Wherein, the new vortex annular Airy beam is obtained by fitting the beam shape and the axial depth, in the cylindrical coordinate system, the expression of the new vortex annular Airy beam is: ; Wherein, is the amplitude index of the annular Airy beam, represents the Airy function, is the main ring radius, is the radial coordinate, is an arbitrary scaling factor, is the vortex function, and the expression is: ; Wherein, is the azimuth angle, and the value range is always between 0 and , represents the number of topological charges, represents the power index of the spiral phase, represents the mode function.

[0029] Step S6, axial depth analysis is carried out, the light intensity distribution or the fluorescence intensity distribution of different scanning positions is recorded, the sidelobe rotation characteristic and the radius change are extracted and analyzed, and the three-dimensional structure of the sample is fitted and analyzed through the mapping relationship between the rotation angle and the axial depth.

[0030] The application will be further described through specific implementation cases.

[0031] The experimental device applied in the specific implementation cases of the application is a depth resolution volume two-photon fluorescence microscope based on the new vortex annular Airy beam, and the microscope adopts a common path configuration of a 4f system.

[0032] Step S1, the linearly polarized Gaussian beam output by the 920 nanometer femtosecond laser is divided into two beams by a polarization beam splitter prism PBS, and a lens 4 and a lens 5 are used to collimate and expand the adjusted illumination beam, and the beam diameter is adjusted to match the spatial light modulator; among the two beams, one is a Gaussian optical path, which is used for alignment and calibration of the microscope system; the other is a structured light path, which enters the spatial light modulator SLM (UPOLabs, HDSLM-1080P) to generate a new vortex annular Airy beam; Step S2, load the designed phase hologram in the spatial light modulator, and send the collimated and expanded beam into the spatial light modulator through a mirror 1, and the spatial light modulator converts the incident beam into an annular Airy beam or a new vortex annular Airy beam through phase modulation; Step S3, adjust the angle of the spatial light modulator so that the angle of the incident light and the exit light is less than 15°, and the light beam modulated by the spatial light modulator is converted from spatial domain to frequency domain through a Fourier lens, and an aperture is placed at the focal point of the Fourier lens to filter out the +1 order diffracted light from the two blazed gratings to generate a new vortex annular Airy beam carrying a vortex phase, and then the frequency domain beam is conjugated to the objective entrance pupil plane through a lens 12, a lens 13, a scanning lens and a sleeve lens; Wherein, the spatial light modulator SLM is located on the input plane of the 4f system composed of the lens 12 and the lens 13, and after modulation by the 4f system, the light beam enters the two-photon microscope, which is a Nikon Eclipse Ti microscope here; the phase mask loaded on the spatial light modulator SLM for amplitude modulation is calculated by a diffraction grating algorithm, and the specific calculation formula is as follows: ; ; ; Wherein, is a normalized bounded positive function of amplitude, is the inverse function of the function, is used to obtain the modulus of the complex parameter, is the expression of the annular Airy beam, is the analytical function of the amplitude and phase profile of the desired field, is used to obtain the argument of the complex parameter, is a phase distribution function, and are pixel coordinates, is an imaginary unit, is a modulus operation function, is the analytical function of the target field amplitude and phase distribution, is a period; Step S4, the modulated new vortex annular Airy beam is subjected to beam collimation and X-Y scanning galvanometer by lens three 12 and lens four 13, two-photon of a single fluorescent microsphere is scanned by adjusting the reflection angle, the long focal depth characteristics of the light beam are used to cover the axial range of the sample, and the spectrum plane is transmitted to the entrance pupil of the objective lens through the scanning lens, the sleeve lens and the mirror four; Step S5, the excitation light is focused on the sample by the objective lens, the signal light is generated by exciting the sample, the signal light is collected by the objective lens, is reflected after the dichroic mirror, is processed through the optical filter, and finally the processed signal light is sent into the photomultiplier, so that the collection and recording of the imaging signal are completed; wherein the new vortex annular Airy beam is obtained by fitting the beam shape and the axial depth, and in the cylindrical coordinate system, the expression of the new vortex annular Airy beam is: ; Wherein, is the amplitude index of the annular Airy beam, represents the Airy function, is the main ring radius, is the radial coordinate, is an arbitrary scaling factor, is the vortex function, and the expression is: ; Wherein, is the azimuth angle, and the value range is always between 0 and , represents the number of topological charge, represents the power index of the spiral phase, represents the mode function.

[0033] Step S6, axial depth analysis is carried out; Step S61, the simulation normalized intensity distribution of the new vortex annular Airy beam in the x-z plane and the y-z plane is recorded, and the result is as shown in Figure 3 , the side lobe rotation characteristics are extracted, and it is found that the side lobe of the light beam rotates in the propagation process; Step S62, the two-photon fluorescence intensity of the new vortex annular Airy beam scanning a single fluorescent microsphere is recorded, and the result is as shown in Figure 4 , the side lobe radius change is extracted, and it is found that the side lobe at different depths has different angles and radii; Step S63, the three-dimensional structure of the sample is fitted and analyzed through the mapping relationship between the rotation angle and the axial depth.

[0034] It should be noted that the contents not described in detail in the application are all prior art and are well known to those skilled in the art.

[0035] Therefore, the application provides a two-photon volume imaging device and method based on phase control annular Airy beam, which realizes high-resolution and large-depth-of-field imaging of biological tissues or thick biological samples such as blood vessels, has small light damage to biological tissues, and can be used for long-time in-vivo imaging research; compared with a traditional Gaussian beam, the annular Airy beam can realize large-depth-of-field imaging of more than 100 microns, can be used for fast three-dimensional volume imaging of brain blood vessels and three-dimensional volume imaging of deep brain nerve cells in the field of neuroscience, and a new type of vortex annular Airy beam can also realize depth analysis of three-dimensional volume imaging.

[0036] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the present application can still be modified or replaced by equivalents, 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 application.

Claims

1. A two-photon volume imaging device based on a phase-controlled annular Airy beam, characterized by: It includes a laser beam expansion module, a beam deflection module, an annular Airy beam modulation module, a beam scanning module and an imaging module; the laser beam expansion module includes a laser, a half-wave plate, a polarization beam splitter, lens one and lens two; the beam deflection module includes reflector one, reflector two and reflector three; the annular Airy beam modulation module includes a spatial light modulator, a Fourier lens and an aperture; the beam scanning module includes lens three, lens four, an XY scanning galvanometer, a scanning lens, a sleeve lens and reflector four; the imaging module includes a dichroic mirror, an objective lens, a sample, a filter and a photomultiplier tube.

2. The two-photon volume imaging device based on phase-controlled annular Airy beam according to claim 1, characterized in that: The first reflector, the second reflector and the third reflector are flip reflectors.

3. The two-photon volume imaging device based on phase-controlled annular Airy beam according to claim 1, characterized in that: The spatial light modulator receives a collimated and expanded illumination beam. The spatial light modulator is pre-loaded with a designed phase hologram, and the angle is adjusted so that the angle between the incident light and the outgoing light is less than 15°. The spatial light modulator is replaced with a digital micromirror device (DMD), a customized phase element, or a liquid crystal phase glass. When the spatial light modulator is reflective, it is replaced with a transmissive spatial light modulator and placed in front of the objective lens.

4. The two-photon volume imaging device based on phase-controlled annular Airy beam according to claim 1, characterized in that: The aperture is placed at the focal position of the Fourier lens.

5. The two-photon volume imaging device based on phase-controlled annular Airy beam according to claim 1, characterized in that: The excitation light received by the sample is an annular Airy beam or a novel vortex annular Airy beam.

6. The two-photon volume imaging device based on phase-controlled annular Airy beam according to claim 5, characterized in that: The excitation light is focused onto the sample through the objective lens. After the sample is excited, signal light is generated. The signal light is reflected by the dichroic mirror, filtered by the filter, and then enters the photomultiplier tube.

7. A two-photon volume imaging method based on phase-controlled annular Airy beam, characterized in that: The following steps are involved: Step S1: A femtosecond laser illumination beam is emitted from a laser, a half-wave plate and a polarization beam splitter adjust the laser power, lenses 1 and 2 collimate and expand the adjusted illumination beam, and the beam diameter is adjusted to match the spatial light modulator, and the optical path is switched through reflectors 1, 2, and 3; Step S2: Load the designed phase hologram into the spatial light modulator, and send the collimated and expanded light beam into the spatial light modulator through the first reflector. The spatial light modulator converts the incident light beam into a ring-Airy beam or a novel vortex ring-Airy beam through phase modulation; Step S3: Adjust the angle of the spatial light modulator so that the angle between the incident light and the outgoing light is less than 15°. The light beam modulated by the spatial light modulator is converted from the spatial domain to the frequency domain through a Fourier lens. An aperture is placed at the focal position of the Fourier lens to filter out the +1-order diffraction light from the two blazed gratings to generate a new vortex ring Airy beam carrying a vortex phase. The frequency domain beam is then conjugated to the entrance pupil plane of the objective lens through lenses 3, 4, the scanning lens, and the sleeve lens. Step S4: Use lenses 3 and 4 to narrow the modulated light beam and collimate it into the XY scanning galvanometer. Scan the sample point by point in the XY plane by adjusting the reflection angle. Use the long focal depth of the light beam to cover the axial range of the sample. Pass the spectrum surface through the scanning lens, sleeve lens and reflector 4 to the entrance pupil of the objective lens. Step S5: The excitation light is focused onto the sample through the objective lens, exciting the sample to generate signal light, which is collected by the objective lens, reflected by the dichroic mirror, processed by the filter, and finally sent to the photomultiplier tube to complete the collection and recording of the imaging signal; Step S6: perform axial depth analysis, record the light intensity distribution or fluorescence intensity distribution at different scanning positions, extract and analyze the sidelobe rotation characteristics and radius changes, and fit and analyze the three-dimensional structure of the sample through the mapping relationship between the rotation angle and the axial depth.

8. The two-photon volume imaging method based on phase-controlled annular Airy beam according to claim 7, characterized in that: In step S1, the switching light path includes a Gaussian light path and a structured light path.

9. The two-photon volume imaging method based on phase-controlled annular Airy beam according to claim 7, characterized in that: In step S2, the phase hologram is calculated using a diffraction grating algorithm. The specific calculation formula is as follows: ； ； ； in, is a normalized bounded positive function of the amplitude, for The inverse function of a function, Used to get the modulus of a complex parameter, is the expression of annular Airy beam, are analytical functions of the amplitude and phase profiles of the desired field, Used to obtain the argument of a complex parameter, is the phase distribution function, and is the pixel coordinate, is the imaginary unit, is the modular operation function, is the analytical function of the target field amplitude and phase distribution, For the cycle.

10. The two-photon volume imaging method based on phase-controlled annular Airy beam according to claim 7, characterized in that: In step S5, the novel vortex annular Airy beam is obtained by fitting the beam shape and axial depth. In the cylindrical coordinate system, the expression of the novel vortex annular Airy beam is: ； in, is the amplitude index of the annular Airy beam, is the Airy function, is the main ring radius, is the radial coordinate, is an arbitrary scaling factor, is the vortex function, and its expression is: ； in, is the azimuth, represents the amount of topological charge, represents the power index of the spiral phase, Represents a modular function.

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