Airy beam attenuation compensation method based on neutral density filter and imaging system

Abstract

The invention discloses an Airy beam attenuation compensation method based on a neutral density filter and an imaging system, and belongs to the technical field of microscopic imaging, the method comprises the following steps: shaping and filtering a light beam emitted by a light source through a shaping device, and then generating an Airy beam; light beams pass through a neutral density optical filter, and attenuation compensation of different degrees is achieved on the light beams at different positions on a light beam propagation transverse plane; enabling the light beam to pass through a scanning galvanometer to form a light sheet, enabling the light beam to enter from one side of the sample, and exciting the sample to generate a fluorescence signal; the sample is imaged, an imaging result is collected through a camera, and the light intensity change in the exciting light propagation process is analyzed; and measuring an attenuation coefficient by adopting a biological spectrophotometer, and determining parameters of the neutral density filter. According to the attenuation compensation method, the advantages of Airy beams are combined, and a new optical element (neutral density filter) is introduced to achieve the purpose of attenuation compensation.

Description

Technical Field

[0001] The invention relates to the technical field of microscopic imaging, and in particular to an attenuation compensation method for Airy beams based on a neutral density filter and an imaging system. Background Art

[0002] As biomedical research gradually deepens into the exploration of complex tissue structures and functions, the demand for high-resolution, high signal-to-noise ratio, and large field of view imaging technology has become increasingly prominent. However, traditional confocal microscopy technology is mainly limited to the observation of two-dimensional, transparent biological thin samples, with long imaging time and unable to achieve rapid three-dimensional imaging. Light sheet fluorescence microscopy (LSFM) uses a cylindrical lens or a scanning galvanometer to generate a thin sheet as illumination light, and the detection light path collects the excited fluorescence in the normal direction, and images the opposite side through wide-field illumination. This illumination method can effectively reduce the exposure time of the sample, reduce phototoxicity and photobleaching, and can achieve continuous observation of living cells and large-scale biological tissue samples. It has great application potential in many life science research fields such as brain science and organoids. The radius trajectory of the traditional Gaussian beam on the interface shows the characteristics of a hyperbola, and the Rayleigh length is relatively short, resulting in a very limited field of view of the Gaussian light sheet fluorescence microscope: beyond the Rayleigh range, the diffraction effect of the Gaussian beam will quickly appear, and its resolution ability will be significantly reduced. At the edge of the field of view, the axial resolution usually exceeds 15μm.

[0003] Light sheet fluorescence microscopy technology uses thin-sheet illumination to reduce phototoxicity and photobleaching while achieving large field of view and fast three-dimensional imaging. However, traditional Gaussian light sheets have a small field of view and energy attenuation due to absorption and scattering during propagation, significantly reducing the imaging quality deep in the sample.

[0004] Diffraction-free light beams, especially Airy beams, have been widely used to improve the imaging field of light sheet fluorescence microscopes. Airy beams have non-diffraction properties, self-acceleration properties, and self-healing properties, which make them perform well in biological imaging. The non-diffraction properties of Airy beams make the diffraction intensity and pulse profile of the beam remain almost unchanged during the propagation process. However, the degradation rate of the beam profile of a Gaussian beam during the propagation process is relatively fast. The present invention concludes that while maintaining the same resolution, the field of view of an Airy light sheet microscope is approximately 5 times that of a Gaussian light sheet microscope. Combined with deconvolution technology, the Airy light sheet microscopy imaging system can image a large field of view. Although Airy beams can significantly improve the imaging field of view in light sheet fluorescence microscopes, the complex optical properties of biological samples and the resulting attenuation of light intensity are still a problem that cannot be ignored.

[0005] At present, the most common method to compensate for Airy beam attenuation is to use a spatial light modulator (SLM) to perform wavefront modulation, which modulates the light intensity of the Airy beam in the propagation direction to increase exponentially by shaping the light field envelope of the Airy beam to offset the decrease in light intensity caused by tissue absorption and scattering. In addition, using SLM to generate a stacked light sheet can also change the light intensity distribution of the beam to compensate for the light intensity attenuation caused by tissue absorption.

[0006] As an advanced wavefront control tool, SLM has demonstrated extremely high flexibility and dynamic control capabilities in light sheet fluorescence microscopy. It can accurately adjust the phase and amplitude of the light beam to achieve complex wavefront modulation purposes, and plays an important role in the research of beam attenuation compensation. However, SLM has high costs, complex system configurations, and the need for real-time control. This method has poor rigidity, is complex to use, is expensive, and has low light intensity utilization, all of which limit its further application in complex scenarios.

[0007] The Beer-Lambert Law explains the quantitative relationship between the degree of light absorption by a substance and the thickness of the absorbing medium and the concentration of absorbing particles: when a beam of monochromatic light passes through a certain absorbing medium, its light intensity will be gradually weakened by the absorption of the medium. Specifically, the degree of light intensity reduction is not only closely related to the medium's ability to absorb light (usually determined by the chemical composition of the medium), but is also directly affected by the concentration and thickness of the medium. The higher the concentration of the medium, the more light-absorbing substances there are per unit volume, and therefore the stronger the absorption of light; at the same time, the increase in the thickness of the medium also means that the light will experience a longer absorption path during the passage, further exacerbating the attenuation of light intensity. The relationship is:

[0008] A = lg (1 / T) = Kbc;

[0009] Among them, A is absorbance, T is transmittance (transmittance), which is the ratio of the outgoing light intensity (I) to the incident light intensity (I0), K is the molar absorptivity, which is related to the properties of the absorbing substance and the wavelength λ of the incident light, c is the concentration of the absorbing substance, and b is the thickness of the absorbing layer.

[0010] In the actual biological tissue imaging process, most samples are highly absorbing and scattering media composed of water and fat. According to the Lambert-Beer law, the absorption of light by the sample causes the light sheet to decay exponentially during the propagation of the sample, and the scattering of light by the sample also causes energy attenuation. A large number of experiments have verified that the combined effect of absorption and scattering still causes the exponential decay of light intensity. Therefore, this paper introduces the attenuation coefficient based on the Lambert-Beer law. The light still decays exponentially in the sample, which ultimately leads to problems such as low spatial resolution, high image background noise, and poor contrast in deep imaging of the sample. Summary of the invention

[0011] The purpose of the present invention is to provide an attenuation compensation method for Airy beam based on a neutral density filter and an imaging system to solve the problems mentioned in the background technology.

[0012] To achieve the above object, the present invention provides a method for compensating the attenuation of an Airy beam based on a neutral density filter, comprising the following steps:

[0013] Step 101, the light beam emitted by the light source is shaped and filtered by a shaping device, and then an Airy beam is generated;

[0014] Step 102, the light beam passes through a neutral density filter, and different degrees of attenuation compensation are achieved for the light beam at different positions on the transverse plane of light beam propagation;

[0015] Step 103, the light beam is passed through a scanning galvanometer to form a light sheet, so that the light beam is incident from one side of the sample to excite the sample to generate a fluorescence signal;

[0016] Step 104, imaging the sample, collecting the imaging results through a camera, and analyzing the light intensity change during the propagation of the excitation light;

[0017] Step 105: Use a biological spectrophotometer to measure the attenuation coefficient and determine the neutral density filter parameters.

[0018] Preferably, the shaping device includes a laser beam expansion system and a spatial filtering system.

[0019] Preferably, in step 101, the Airy beam is generated by wavefront modulation by passing through a cubic phase mask or using a spatial light modulator to load a cubic phase.

[0020] Preferably, a spatial light modulator is used to generate an Airy beam, and the phase of the spatial light modulator needs to be superimposed and corrected in sequence. wavecorrect , blazed grating phase M blaze , aperture phase M apodization and the cubic phase M desired , and finally the phase M loaded into the liquid crystal panel of the spatial light modulator is obtained SLM The formula is as follows:

[0021] M SLM =M wavecorrect +M blaze +M apodization +M desired ;

[0022] Among them, the correction phase needs to be measured according to the actual situation of the spatial light modulator and the selected laser wavelength, and the aperture phase needs to be determined according to the specific parameters of the subsequent optical path and matched with the entrance pupil beam diameter.

[0023] Preferably, the center of the Airy beam is shifted to the first order of the blazed grating by superimposing the blazed grating phase, and the blazed grating phase is expressed as:

[0024] M blaze =2πdxsinθ / λ;

[0025] Among them, M blaze represents the blazed grating phase, d is the pixel focal length, x is the coordinate of the liquid crystal panel, θ is the deflection angle, and λ is the wavelength.

[0026] Preferably, the blazed grating phase is simplified using the phase slope k as:

[0027] k = dx0 sinθ / λ;

[0028] Wherein, x0 represents the horizontal coordinate corresponding to the first-order diffraction of the blazed grating.

[0029] Preferably, the modulated two-dimensional pupil function superimposed at the spatial light modulator is:

[0030] P Airy (u,v)=R(u,v)exp[2παi·(u 3 +v 3 )];

[0031] Among them, R(u,v) is the pupil aperture function, u and v are the normalized pupil coordinates, and α is the scaling factor.

[0032] Preferably, the spectrum distribution function of the Airy beam finally generated is as follows:

[0033]

[0034] Wherein, a represents the attenuation factor (a>0 and a<<1).

[0035] Preferably, the neutral density filter includes a uniform density filter, a gradient density filter and a complex pattern density filter.

[0036] The present invention also provides an imaging system for attenuation compensation of Airy beams based on neutral density filters, comprising a light source, a first half-wave plate, a polarization beam splitter, an aperture, a second half-wave plate, a first lens, a spatial filtering system, a second lens, a neutral density filter, a spatial light modulator, a third lens, a fourth lens, a scanning galvanometer, a 4F system, an illumination objective lens, a sample chamber, an imaging objective lens, a tube lens, a filter, and a camera, which are arranged in sequence. The light source is a femtosecond laser with a central wavelength of 800nm, the first lens and the second lens constitute a laser beam expansion system, the spatial filtering system includes a pinhole, the galvanometer is placed at the focal position of the fifth lens, the 4F system includes a fifth lens and a sixth lens, and a sample is placed in the sample chamber.

[0037] Therefore, the present invention adopts the above-mentioned attenuation compensation method and imaging system for Airy beam based on neutral density filter, which has the following beneficial effects:

[0038] (1) As a low-cost, simple-structured optical element, the neutral density filter has good rigidity and high beam utilization. The present invention adjusts its parameters to make the transmittance vary exponentially along the length, thereby achieving effective compensation for the attenuation of the Airy beam. The present invention avoids the high cost and complex regulation brought by the spatial light modulator, and provides a stable attenuation compensation effect without increasing the complexity of the system, thereby directing more light energy to the deep part of the sample and significantly improving the imaging depth and image quality.

[0039] (2) The present invention uses an Airy beam instead of a traditional Gaussian beam as an excitation light, which can expand the field of view by 5 times. By placing a neutral density filter whose density changes linearly along the length direction in the optical path, the parameters of the neutral density filter are adjusted according to the Lambert-Beer law so that the transmittance changes exponentially along the direction of the light sheet. The light intensity of the Airy beam, which is originally weakened due to the increase in the propagation distance, can be effectively compensated after passing through the neutral density filter, thereby restoring the uniformity of the light intensity in the propagation direction.

[0040] (3) The present invention can effectively increase the light intensity deep in the sample without increasing the peak illumination intensity, significantly improving the spatial resolution, contrast and signal-to-noise ratio of deep imaging. The method of using neutral density filters for attenuation compensation has low cost, good system rigidity and better compensation effect.

[0041] (4) The signal-to-noise ratio of two-photon attenuation compensation Airy light sheet fluorescence microscopy imaging based on neutral density filters increased by 39%, effectively increasing the imaging depth to 400μm.

[0042] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a flow chart of a method for compensating the attenuation of an Airy beam based on a neutral density filter according to an embodiment of the present invention;

[0044] Figure 2 Schematic diagram of the structure of an imaging system for attenuation compensation of Airy beam based on a neutral density filter according to an embodiment of the present invention;

[0045] Figure 3 This is a comparison diagram of the effects of different attenuation compensation methods according to an embodiment of the present invention;

[0046] Figure 4 is a schematic diagram of a light beam passing through a spatial modulator according to an embodiment of the present invention;

[0047] Figure 5 A phase mask diagram for finally achieving attenuation compensation by the attenuation compensation method of an embodiment of the present invention;

[0048] Reference numerals

[0049] 1. Light source; 2. First half-wave plate; 3. Polarization beam splitter; 4. Aperture; 5. Second half-wave plate; 6. First lens; 7. Pinhole; 8. Second lens; 9. Neutral density filter; 10. Spatial light modulator; 11. Third lens; 12. Fourth lens; 13. Scanning galvanometer; 14. Fifth lens; 15. Sixth lens; 16. Illumination objective; 17. Sample chamber; 18. Imaging objective; 19. Tube lens; 20. Filter; 21. Camera. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0051] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0052] Example

[0053] like Figure 1As shown, the present invention provides a method for compensating the attenuation of an Airy beam based on a neutral density filter, comprising the following steps:

[0054] Step 101: The light beam emitted by the light source is shaped and filtered by a shaping device, and then an Airy beam is generated. The light source adopts a femtosecond laser with a central wavelength of 800nm, the shaping device includes a laser beam expansion system and a spatial filtering system, the laser beam expansion system includes a first lens 6 and a second lens 8, and the spatial filtering system includes a pinhole 7. The Airy beam can be generated by a cubic phase mask or by using a spatial light modulator to load a cubic phase for wavefront modulation. In order to better compare the attenuation compensation performance of NDF and spatial light modulator in the experiment, a spatial light modulator is placed in the optical path, and the spatial light modulator is used to generate the Airy beam. In the application process, a cubic phase mask can be used instead.

[0055] In this embodiment, a spatial light modulator is used to apply a cubic phase mask to the light beam to generate an Airy beam, such as Figure 5 To generate an Airy beam, the phase of the spatial light modulator needs to be superimposed and corrected in turn. wavecorrect , blazed grating phase M blaze , aperture phase M apodization and the cubic phase M desired , and finally the phase M loaded into the liquid crystal panel of the spatial light modulator is obtained SLM , the final phase pattern loaded on the liquid crystal panel of the spatial light modulator is shown in the formula:

[0056] M SLM =M wavecorrect +M blaze +M apodization +M desired ;

[0057] These phases are generated using Matlab software. The correction phase needs to be determined based on the actual conditions of the spatial light modulator and the selected laser wavelength. The aperture phase needs to be determined based on the specific parameters of the subsequent optical path and should match the entrance pupil beam diameter.

[0058] The spatial light modulator is not an ideal optical element. During its operation, the diffraction efficiency of the spatial light modulator is not high due to the pixel gap and the laser wavelength not completely matching the set working wavelength. The unmodulated light beam is converged and focused at the focal position by the lens to form a beam waist similar to Gaussian light, which is superimposed on the modulated light beam and cannot be separated, affecting the quality of the modulated light beam. Figure 4 shown.

[0059] In order to solve this problem, the present invention superimposes the blazed grating phase to shift the center of the Airy beam to the first order of the blazed grating, and blocks the zero-order diffraction in the optical path to obtain a higher quality Airy beam. The blazed grating phase can be expressed as:

[0060] M blaze =2πdxsinθ / λ;

[0061] Among them, M blaze represents the blazed grating phase, d is the pixel focal length, x is the coordinate of the liquid crystal panel, and θ is the deflection angle. The present invention uses the phase slope k to simplify the blazed grating phase:

[0062] k = dx0 sinθ / λ;

[0063] Wherein, x0 represents the horizontal coordinate corresponding to the first-order diffraction of the blazed grating.

[0064] The modulated two-dimensional pupil function is a function applied to a spatial light modulator to control the amplitude and phase distribution of a light beam. In this embodiment, the modulated two-dimensional pupil function superimposed at the spatial light modulator is:

[0065] P Airy (u,v)=R(u,v)exp[2παi·(u 3 +v 3 )];

[0066] Among them, R(u,v) is the pupil aperture function, u and v are normalized pupil coordinates, and α is the scale factor. Let α = 7, and choose to use Airy light sheet fluorescence microscope. The resolution is 3.2μm and the field of view is 450μm. The field of view is 5 times that of Gaussian light sheet microscope with the same resolution.

[0067] The spectrum distribution function of the Airy beam finally generated is as follows:

[0068]

[0069] Among them, a represents the attenuation factor (a>0 and a<<1), which can truncate the light beam to ensure the limited energy. In this formula, the spectrum of the Airy beam is the product of the Gaussian distribution and the cubic distribution. Therefore, the Gaussian beam can be modulated by a cubic phase mask, and then the Airy beam can be obtained through the Fourier transform effect of the first lens 6 and the second lens 8.

[0070] Step 102: The light beam passes through a neutral density filter (NDF) to achieve different degrees of attenuation compensation for the light beam at different positions on the transverse plane of the light beam propagation. Neutral density filters can be divided into many types, including uniform density filters, gradient density filters, and complex pattern density filters. In this embodiment, a gradient density filter is selected, and its transmittance gradually changes along the length direction of the filter. According to the measured attenuation coefficient, the transmittance is adjusted to change exponentially along the direction of the light sheet.

[0071] Step 103: The light beam is passed through a scanning galvanometer to form a light sheet. The light beam is incident from one side of the sample to excite the sample to generate a fluorescence signal.

[0072] Step 104: Image the sample, collect the imaging results through the camera, and analyze the light intensity changes during the propagation of the excitation light. In the sample chamber, the light sheet plane is imaged onto the sCMOS camera, and the filter ensures that only the desired fluorescence signal is captured.

[0073] Step 105: using a biological spectrophotometer to measure the attenuation coefficient, determine the parameters of the neutral density filter, and implement attenuation compensation for light sheet microscopy imaging based on the neutral density filter.

[0074] After determining the parameters of the neutral density filter, the light intensity distribution of the light beam along the propagation direction can be adjusted through the neutral density filter so that the light intensity at its far end, that is, deep in the sample, increases to counteract the exponential decrease in light intensity caused by attenuation. This is achieved by shaping the light field envelope of the Airy beam.

[0075] The density of the neutral density filter used in this embodiment varies linearly along its length, and the transmittance varies exponentially along its length. By adjusting the parameters of the neutral density filter, the intensity of the light beam along the propagation direction of the light sheet is restored to be almost uniform. In other words, without increasing the illumination intensity, the light intensity distribution is changed so that the light intensity deep in the sample is increased, thereby minimizing light damage and improving the spatial resolution and contrast of imaging deep in the sample. The characteristics of the neutral density filter are highly consistent with the light intensity compensation mode required for the Airy beam along the propagation path, making it an ideal choice for achieving Airy beam attenuation compensation. By integrating the gradient density filter into the Airy light sheet microscope system, its light intensity distribution can be effectively adjusted dynamically during the propagation of the beam to achieve on-demand compensation of light intensity. Compared with the spatial modulator solution, the neutral density filter not only greatly reduces the complexity and cost of the system, but also simplifies the operation process, and the compensation effect is better than that of the spatial modulator.

[0076] Attenuation compensation can be achieved by a spatial modulator, or by adding a neutral density filter in front of the spatial modulator. The present invention uses a two-photon attenuation compensation Airy beam fluorescence microscopy system to compare the attenuation compensation of a neutral density filter and a spatial modulator. To facilitate the comparison, the optical path is not changed during the process. A spatial modulator is used to generate an Airy beam, and the attenuation coefficient is measured in a high concentration of fluorescein dye (0.88mM, Thermo Fisher Scientific, FITC). The beam intensity within a depth range of 400μm is measured and normalized. The experimental results are shown in Figure 2. Figure 3 As shown in the figure, the normalized intensity of the beam center along the propagation direction with and without attenuation compensation by the spatial modulator and neutral density filter is shown. It can be seen that the intensity compensation of the neutral density filter is better than that of the spatial modulator. Both methods can achieve the effect of intensity compensation, and the imaging depth reaches 400μm. At this depth, the signal-to-noise ratio of the neutral density filter and spatial modulator attenuation compensation is increased by 39% and 27%, respectively.

[0077] The present invention also provides an imaging system for attenuation compensation of Airy beams based on neutral density filters, comprising a light source 1, a first half-wave plate 2, a polarization beam splitter 3, an aperture 4, a second half-wave plate 5, a first lens 6, a spatial filtering system, a second lens 8, a neutral density filter 9, a spatial light modulator 10, a third lens 11, a fourth lens 12, a scanning galvanometer 13, a 4F system, an illumination objective lens 16, a sample chamber 17, an imaging objective lens 18, a tube lens 19, a filter 20, and a camera 21, which are arranged in sequence. The light source 1 is a femtosecond laser with a central wavelength of 800 nm, the first lens 6 and the second lens 8 constitute a laser beam expansion system, the spatial filtering system includes a pinhole 7, the galvanometer 13 is placed at the focal position of the fifth lens 14, the 4F system includes the fifth lens 14 and the sixth lens 15, and a sample is placed in the sample chamber 17.

[0078] Reference Figure 2, the light source 1 emits a laser, and the first half-wave plate 2 and the polarization beam splitter 3 can be combined to change the overall light intensity of the subsequent optical path, and the light emitted by the polarization beam splitter 3 is a fixed-direction linear polarized light. The aperture 4 adjusts the diameter of the light beam at this time, and the second half-wave plate 5 adjusts the linear polarized light in a fixed direction to be 45° with the original polarization direction, and the same polarization direction as the liquid crystal panel optical axis of the modulation area of ​​the spatial light modulator (required for the operation of the spatial light modulator). Through the beam expansion system composed of the first lens 6 and the second lens 8, a pinhole 7 is placed at its focal position for spatial filtering. The light beam passes through the first lens 6, the pinhole 7 and the second lens 8 for beam shaping, and after attenuation compensation is achieved through the neutral density filter 9, it is incident on the liquid crystal panel of the spatial light modulator 10. The liquid crystal panel of the spatial light modulator 10 is loaded with a blazed grating and a three-dimensional phase mask for generating an Airy beam. The third lens 11 and the fourth lens 12 form a relay device to relay the light beam to the scanning galvanometer 13, and at the same time, the third lens 11 and the fourth lens 12 shrink the light beam to adapt to the size of the scanning galvanometer 13. The light beam passes through two lenses (i.e., the fifth lens 14 and the sixth lens 15) to obtain the required light beam at the input plane of the illumination objective lens 16. The scanning light beam is focused by the illumination objective lens 16, and a scanning light sheet is formed at the focal position of the illumination objective lens 16 to illuminate the sample that has been processed and marked with the fluorescent probe. The fluorescence signal generated by the excitation passes through the imaging objective lens 18 and the tube lens 19, and the illumination light is filtered out by the filter 20, and then collected at the camera 21.

[0079] This imaging system can achieve the needs of high-precision imaging at different depths of biological samples at low cost and without increasing system complexity.

[0080] Therefore, the present invention adopts the above-mentioned attenuation compensation method for Airy beam based on neutral density filter and imaging system, and changes the light intensity distribution without increasing the illumination intensity, so that the light intensity in the deep of the sample is increased, thereby minimizing light damage, improving the spatial resolution and contrast of imaging in the deep of the sample, and solving the problems of poor imaging effect at deep depth, small field of view and limited imaging depth of traditional light sheet microscopes.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for compensating the attenuation of an Airy beam based on a neutral density filter, characterized in that: Step 101, the light beam emitted by the light source is shaped and filtered by a shaping device, and then an Airy beam is generated; Step 102, the light beam passes through a neutral density filter, and different degrees of attenuation compensation are achieved for the light beam at different positions on the transverse plane of light beam propagation; Step 103, the light beam is passed through a scanning galvanometer to form a light sheet, so that the light beam is incident from one side of the sample to excite the sample to generate a fluorescence signal; Step 104, imaging the sample, collecting the imaging results through a camera, and analyzing the light intensity change during the propagation of the excitation light; Step 105: Use a biological spectrophotometer to measure the attenuation coefficient and determine the neutral density filter parameters.

2. The attenuation compensation method for Airy beam based on neutral density filter according to claim 1, characterized in that: The shaping device comprises a laser beam expansion system and a spatial filtering system.

3. The attenuation compensation method for Airy beam based on neutral density filter according to claim 1, characterized in that: In step 101, the Airy beam is generated by wavefront modulation by passing through a cubic phase mask or using a spatial light modulator to load a cubic phase.

4. The attenuation compensation method for Airy beam based on neutral density filter according to claim 3, characterized in that: Using a spatial light modulator to generate an Airy beam, it is necessary to sequentially superimpose the phase correction phase M of the spatial light modulator. wavecorrect , blazed grating phase M blaze , aperture phase M apodization and the cubic phase M desired , and finally the phase M loaded into the liquid crystal panel of the spatial light modulator is obtained SLM The formula is as follows: M SLM =M wavecorrect +M blaze +M apodization +M desired ; Among them, the correction phase needs to be measured according to the actual situation of the spatial light modulator and the selected laser wavelength, and the aperture phase needs to be determined according to the specific parameters of the subsequent optical path and matched with the entrance pupil beam diameter.

5. The attenuation compensation method for Airy beam based on neutral density filter according to claim 4, characterized in that: By superimposing the blazed grating phase, the center of the Airy beam is shifted to the first order of the blazed grating. The blazed grating phase is expressed as: M blaze =2πdxsinθ / λ; Among them, M blaze represents the blazed grating phase, d is the pixel focal length, x is the coordinate of the liquid crystal panel, θ is the deflection angle, and λ is the wavelength.

6. The attenuation compensation method for Airy beam based on neutral density filter according to claim 5, characterized in that: The blazed grating phase is simplified using the phase slope k as: k = dx0 sinθ / λ; Wherein, x0 represents the horizontal coordinate corresponding to the first-order diffraction of the blazed grating.

7. The attenuation compensation method for Airy beam based on neutral density filter according to claim 6, characterized in that: The modulated two-dimensional pupil function superimposed at the spatial light modulator is: P Airy (u,v)=R(u,v)exp[2παi·(u 3 +v 3 )]; Among them, R(u,v) is the pupil aperture function, u and v are the normalized pupil coordinates, and α is the scaling factor.

8. The method for compensating the attenuation of an Airy beam based on a neutral density filter according to claim 7, characterized in that: The spectrum distribution function of the Airy beam finally generated is as follows: Wherein, a represents the attenuation factor (a>0 and a<<1).

9. The method for compensating the attenuation of an Airy beam based on a neutral density filter according to claim 1, characterized in that: The neutral density filters include uniform density filters, gradient density filters and complex pattern density filters.

10. An imaging system for attenuation compensation of Airy beams based on neutral density filters, applied to the attenuation compensation method for Airy beams based on neutral density filters as claimed in any one of claims 1 to 9, characterized in that: It includes a light source, a first half-wave plate, a polarization beam splitter, an aperture, a second half-wave plate, a first lens, a spatial filtering system, a second lens, a neutral density filter, a spatial light modulator, a third lens, a fourth lens, a scanning galvanometer, a 4F system, an illumination objective lens, a sample chamber, an imaging objective lens, a tube lens, a filter, and a camera, which are arranged in sequence; The light source is a femtosecond laser with a central wavelength of 800 nm, the first lens and the second lens constitute a laser beam expansion system, the spatial filtering system includes a pinhole, the galvanometer is placed at the focal position of the fifth lens, the 4F system includes a fifth lens and a sixth lens, and a sample is placed in the sample chamber.

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