Laminated light beam-based light sheet microscopic imaging method and system

Abstract

The invention discloses a light sheet microscopic imaging method and system based on laminated light beams. The microscopic imaging method comprises the following steps: measuring an attenuation coefficient of a sample; calculating a corresponding compensation coefficient through a mathematical model, and calculating parameters required for generating a corresponding phase mask; shaping and filtering light beams emitted by the light source into light beams with preset parameters; loading Fresnel lens phases with different focal lengths by using light beams to carry out phase modulation; the light beam forms a light sheet through a scanning galvanometer, and a sample is excited to generate a fluorescence signal; putting the sample into an imaging system for imaging; carrying out noisy point removal processing on the obtained sample image; the light intensity change in the exciting light propagation process is collected and analyzed through a camera. By adopting the microscopic imaging method, the contrast ratio at the illumination depth is improved by 51.36% compared with that of imaging of an attenuated X-ray plate, and the microscopic imaging method can be suitable for attenuation compensation of common biological samples, namely the attenuation coefficient within 300cm <-1 > is met.

Description

Technical Field

[0001] The present invention relates to the field of biomedical microscopic imaging technology, and in particular to a light sheet microscopic imaging method and system based on stacked light beams. Background Art

[0002] Light Sheet Fluorescence Microscopy (LSFM) is a new technology that takes into account both resolution and imaging depth and is closest to the ideal goal of optimizing the performance of optical microscopes. This technology uses a laser light sheet of a certain thickness to excite the contrast agent inside the sample to emit fluorescence, and then collects the fluorescence signal in a direction orthogonal to the light sheet plane. Thanks to this, compared with traditional microscopic imaging methods, light sheet imaging has excellent characteristics such as high spatial resolution, high signal-to-noise ratio, large field of view, low photobleaching and low phototoxicity, and fast three-dimensional imaging. It has great application potential in many life science research fields such as brain science and embryonic biology.

[0003] During the process of excitation light entering the tissue and emitting fluorescence, the light beam will be reflected, scattered, absorbed, etc., which will reduce the penetration depth of the light signal, affect the "ballistic" characteristics of the photons, and easily attenuate and distort the photon signals at depth, which limits the imaging of light sheet deep in the sample. This directly leads to low spatial resolution and poor signal background for imaging deep in the sample tissue. In the actual biological tissue imaging process, biological samples are mostly highly absorbing and highly scattering media composed of water and fat. During the process of light irradiation and fluorescence excitation, they will be absorbed and scattered, which causes the energy of the light sheet to be severely attenuated during the propagation of the sample, ultimately leading to low spatial resolution, high image background noise, poor contrast, and other problems in imaging deep in the sample. Therefore, the current field of light sheet imaging still faces the problem of poor imaging quality of excitation light at depth. On the other hand, simply relying on increasing the excitation light power to compensate for the attenuation of the sample medium cannot redistribute the light intensity, which will cause additional photobleaching and phototoxicity of shallow tissues.

[0004] Over the past decade, light sheet imaging technology has been continuously combined with other advanced optical imaging concepts. In the field of light sheet imaging, seeing deeper, seeing wider, and seeing more detailed has always been an urgent problem to be solved, and attenuation compensation is the most important means to improve the quality of deep imaging. At present, the attenuation compensation technology proposed in light sheet microscopy mainly uses neutral density filters or spatial light modulators to shape the light field envelope of the Airy beam and modulate the light intensity of the Airy beam in the propagation direction to increase exponentially to offset the decrease in light intensity caused by tissue absorption and scattering. However, these methods are not universal and can only be applied to Airy beams. Moreover, due to the use of shorter wavelengths, the penetration depth is low. Summary of the invention

[0005] The object of the present invention is to provide a light sheet microscopic imaging method and system based on stacked light beams to solve the problems mentioned in the background technology.

[0006] To achieve the above object, the present invention provides a light sheet microscopy imaging method based on stacked light beams, comprising the following steps:

[0007] Step S1: measuring the attenuation coefficient of the sample;

[0008] Step S2: Calculate the corresponding compensation coefficient through the mathematical model, and calculate and generate the required parameters of the corresponding phase mask;

[0009] Step S3: shaping and filtering the light beam emitted by the light source into a light beam with preset parameters;

[0010] Step S4: using the light beam to load the phase of the Fresnel lens with different focal lengths to perform phase modulation;

[0011] Step S5: The light beam is passed through a scanning galvanometer to form a light sheet, which excites the sample to generate a fluorescent signal, and a fluorescent probe is used to mark the biological sample during the excitation process;

[0012] Step S6: cutting and transparentizing the sample, and placing the sample into an imaging system for imaging;

[0013] Step S7: removing noise from the obtained sample image to obtain a picture of the deep part of the sample;

[0014] Step S8: The light intensity variation during the propagation of the excitation light is collected and analyzed by a camera, and the phase area ratio of the Fresnel lens with different focal lengths loaded on the spatial light modulator is changed to realize attenuation compensation of light sheet microscopy imaging based on stacked beams.

[0015] Preferably, step S4 is specifically as follows: subdividing the circular area modulated by the spatial light modulator into a plurality of dense sector-shaped areas, and loading a periodically changing regional Fresnel lens phase on the sector-shaped areas, adjusting the number of focal points according to the phase change period, and adjusting the stacking distance by adjusting the offset distance of the Fresnel lens phase.

[0016] Preferably, before performing phase modulation on the plane light beam in step S4, the following steps need to be performed:

[0017] Correct the original flatness of the spatial light modulator LCD panel and add the correction phase M wavecorrect ;

[0018] Adding a blazed grating phase M to the spatial light modulator blaze , reducing the unmodulated area of ​​the spatial light modulator;

[0019] Add truncation function phase M apodization, controls the actual numerical aperture at the entrance pupil;

[0020] The final phase pattern loaded on the liquid crystal panel of the spatial light modulator is shown in the formula:

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

[0022] Among them, M desire is the desired Fresnel lens phase.

[0023] Preferably, step S8 is specifically:

[0024] Step S81: Collect imaging results through a camera, analyze the light intensity change of the excitation light sheet during the sample propagation process, and calculate the attenuation coefficient;

[0025] Step S82: generating preset parameters of the phase by calculating the attenuation coefficient and the attenuation compensation model;

[0026] Step S83: changing the phase area ratio of the Fresnel lenses with different focal lengths loaded on the spatial light modulator to form a stacked light beam in the incident direction;

[0027] Step S84: Compensate for the light intensity attenuation problem caused by absorption deep in the sample at different focal points.

[0028] Preferably, step S81 is specifically as follows:

[0029] According to the Lambert-Beer law, the attenuation of light intensity in the absorbing medium follows the law, and the output light intensity is obtained:

[0030]

[0031] Where I0 is the incident light intensity, I det is the output light intensity, μ a is the tissue absorption coefficient, G is the geometric correlation factor, and A is the absorbance;

[0032] In the attenuation simulation process of biological imaging, the influence of medium geometry-related factors is ignored, and the organism is regarded as a uniform medium. The point spread function (PSF) of the attenuation light sheet in the uniform medium is obtained. ill_attn for:

[0033]

[0034] In the formula, C atten is the sample attenuation coefficient, x k is the light sheet propagation distance;

[0035] Calculate the attenuation coefficient of the sample: fit the attenuation coefficient along the x-direction based on the average intensity projection of the xz or xy plane imaging results; estimate the tissue attenuation coefficient by compensating the biological imaging results to a uniform intensity distribution along the propagation direction.

[0036] Preferably, step S82 is specifically as follows:

[0037] Establish the relationship between the attenuation coefficient and the compensation coefficient to obtain the light field distribution of the stacked beam And light intensity I coh The calculation formula is as follows:

[0038]

[0039] I coh =|∑ n E n | 2 ;

[0040] Where r is the radial distance, x is the distance from the center of the beam in the propagation direction, and E n0 represents the electric field at the center of the light beam, k = 2π / λ is the wave number, λ is the wavelength of the illumination beam, n is the refractive index, E n represents the electric field distribution of Gaussian beams with different centers, ω0 represents the beam waist width, Indicates the beam width at different positions from the center of the field of view, For the ancient phase shift, represents phase shift, i represents complex exponential, Indicates the direction of propagation of the light beam from a point x to x n The radius of curvature between

[0041] The point spread function of the simulated light sheet propagation is convolved with the one-dimensional resolution target with a fixed scale in the z direction, and the imaging line widths of different medium light sheets at different locations are selected and the contrast is calculated to compare the imaging performance of the laminated light sheets with and without compensation in the attenuating medium.

[0042] The intensity variation of Gaussian beams with different focal points in the propagation direction is superimposed to obtain the intensity variation of the stacked beam in the propagation direction, and the relationship between the compensation coefficient and the attenuation coefficient is calculated; different compensation coefficients are selected to calculate the attenuation coefficient C that can be fully compensated under different numbers of focal points. atten The value of is obtained by establishing a model between the compensation coefficient and the attenuation coefficient.

[0043] The present invention also provides a light sheet microscopic imaging system based on a stacked light beam, comprising a light source, a first half-wave plate, a polarization beam splitter, a beam terminator, an aperture, a second half-wave plate, a first lens, a pinhole, a second lens, a spatial light modulator, a relay device, a scanning galvanometer, a fifth lens, a sixth lens, an illumination objective lens, a sample chamber, an imaging objective lens, a tube lens, a filter and a camera arranged in sequence, the relay device comprising a third lens and a fourth lens arranged in sequence, and the scanning galvanometer is placed at the focal position of the fifth lens.

[0044] Therefore, the present invention adopts the above-mentioned light sheet microscopic imaging method and system based on stacked light beams, which has the following beneficial effects:

[0045] (1) Attenuation compensation is achieved deep in the sample by enhancing the deep light intensity, and a parameter matching model for attenuation compensation is proposed.

[0046] (2) It can meet the different imaging requirements of different biological tissue samples and biological samples with different attenuation. It can focus the excitation light at different positions in the propagation direction, which is different from simply relying on increasing the excitation light power to compensate for the absorption of the medium, and will not cause additional light damage to the shallow layer of the tissue. It can also subdivide the circular area modulated by the spatial light modulator into dense fan-shaped areas, and determine the period of phase change according to the number of focal points required.

[0047] (3) Based on the preliminary sample imaging results, the axial attenuation ratio of the image light intensity in different areas is found, and the Lambert-Beer law attenuation model is established. In this process, the interference between light beams cannot be ignored. Therefore, the light intensity is calculated based on the phase, and the relationship between the attenuation coefficient and the compensation coefficient is established. Then, the size of the fan-shaped area corresponding to the phase of the Fresnel lens with different focal lengths within a modulation cycle on the liquid crystal panel of the spatial light modulator is adjusted to produce intensity compensation, so as to achieve the purpose of redistributing the light intensity evenly along the propagation direction after the superposition attenuation effect.

[0048] (4) The light intensity can be redistributed along the propagation direction, solving the problem that ordinary light sheets cannot compensate for the attenuation of light intensity energy when propagating deep into highly absorbing media.

[0049] (5) This method is based on the Lambert-Beer attenuation model, and a mathematical model of compensation coefficient and attenuation coefficient is established to prove that it can compensate for 0-300cm -1 The attenuation of the laser beam can meet the imaging requirements of biological samples.

[0050] (6) The use of stacked light sheets for imaging can improve the quality of deep-layer imaging while ensuring that shallow-layer photobleaching does not increase, including improving the signal-to-background ratio and spatial resolution at deep depths of the sample, thus achieving further application in the field of light sheet microscopy.

[0051] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A flowchart of a light sheet microscopy imaging method based on stacked beams according to an embodiment of the present invention;

[0053] Figure 2 is a schematic structural diagram of an imaging system according to an embodiment of the present invention;

[0054] Figure 3 A phase generation principle diagram of an imaging method according to an embodiment of the present invention;

[0055] Figure 4 A schematic diagram of the stacked beam generation principle of the imaging method according to an embodiment of the present invention;

[0056] Figure 5 A point spread function and a one-dimensional resolution target convolution map of the imaging method according to an embodiment of the present invention;

[0057] Figure 6 is a simulation relationship diagram of the attenuation coefficient and the compensation coefficient of the imaging method according to an embodiment of the present invention;

[0058] Reference numerals

[0059] 1. Light source; 2. First half-wave plate; 3. Polarization beam splitter; 4. Light screen; 5. Aperture 6. Second half-wave plate; 7. First lens; 8. Pinhole; 9. Second lens; 10. Spatial light modulator; 11. Third lens; 12. Fourth lens; 13. Scanning galvanometer; 14. Fifth lens; 15. Sixth lens; 16. Illumination objective lens; 17. Sample chamber; 18. Imaging objective lens; 19. Tube lens; 20. Filter; 21. Camera. DETAILED DESCRIPTION

[0060] 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.

[0061] 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.

[0062] Example

[0063] like Figure 1 As shown, the present invention provides a light sheet microscopic imaging method based on a stacked light beam, comprising the following steps:

[0064] Step S1: Determine the attenuation coefficient of the sample.

[0065] Step S2: Calculate the corresponding compensation coefficients through a mathematical model, and calculate and generate the required parameters of the corresponding phase mask.

[0066] Step S3: shaping and filtering the light beam emitted by the light source into a light beam with preset parameters. The monochromatic laser emitted by the light source generates a light beam with preset parameters through shaping devices such as a laser beam expansion system and a spatial filtering system. The laser beam expansion system includes a first lens and a second lens, and the spatial filtering system includes a pinhole.

[0067] In practical applications, a light beam with preset parameters can be generated before entering the spatial light modulator, or it can be modulated on the spatial light modulator. In this case, the spatial light modulator can simultaneously complete the tasks of generating a preset light beam and loading the Fresnel lens phase in different regions through phase modulation. The light beam phase is a fan-shaped regional Fresnel lens phase in the form of a polar coordinate period, which is scanned by a galvanometer to form a light sheet.

[0068] Step S4: using the light beam to load the phase of the Fresnel lens with different focal lengths to perform phase modulation.

[0069] Specifically, the circular area modulated by the spatial light modulator is subdivided into multiple dense fan-shaped areas, and a periodically changing regional Fresnel lens phase is loaded thereon, the number of focal points is adjusted according to the phase change period, and the stacking distance is adjusted by adjusting the offset distance of the Fresnel lens phase.

[0070] Among them, the superimposed Fresnel lens phase is actually equivalent to a lens optical element. Introducing Fresnel lens phases with different focal lengths is equivalent to making the normally propagating light beam converge through a convex lens so that the light beam is focused before the original focus, or diverge through a concave lens so that the light beam is focused after the original focus. According to the formula of the Fresnel lens phase and its focal length, it is deduced that the preset focal length parameter f and the offset Δx are in an inverse proportional function relationship. After experiments, the offset Δx corresponding to the parameter f can be calculated, providing a basis for the focus distribution of the stacked light beam.

[0071] like Figure 3-4 As shown in the figure, before phase modulating the planar light beam, the original flatness of the liquid crystal panel of the spatial light modulator must be corrected first, and the correction phase M wavecorrect In addition, in order to reduce the unmodulated area (inter-lattice area) of the spatial light modulator, the blazed grating phase M is added to the spatial light modulator.blaze , which is used to separate the ideal first-order diffraction pattern; in order to facilitate the control of the actual numerical aperture at the entrance pupil, it is also necessary to add the toe-truncation function phase M apodization ; The final phase pattern loaded on the liquid crystal panel of the spatial light modulator is shown in the formula,

[0072] M SLM =M wavecorrect ·M blaze ·M apodization ·M desired (1)

[0073] Among them, M desire It is expected that the number of phase blazed gratings of the Fresnel lens can change the distance between the zeroth order diffraction and the first order diffraction and the distribution of the diffraction energy. After testing, the number of blazed gratings is set to 80.

[0074] Step S5: The light beam is formed into a light sheet through a scanning galvanometer, which is used to excite the sample to generate a fluorescent signal. Specifically, the stacked light beam is scanned to form a light sheet, which is incident from one side of the sample to excite the sample to generate fluorescence. During the excitation process, a fluorescent probe is used to mark the biological sample to obtain a tissue sample to be imaged. Different contrast agents will affect the final imaging results, so it is particularly important to choose a suitable fluorescent probe.

[0075] Step S6: After cutting and transparentizing the sample, the sample is placed in an imaging system for imaging.

[0076] Step S7: The obtained sample image is subjected to processing operations such as noise removal to obtain a picture of the deep portion of the sample.

[0077] Step S8: The light intensity variation during the propagation of the excitation light is collected and analyzed by a camera, and the phase area ratio of the Fresnel lens with different focal lengths loaded on the spatial light modulator is changed to realize attenuation compensation of light sheet microscopy imaging based on stacked beams.

[0078] Step S8 is specifically as follows:

[0079] Step S81: Collect imaging results through a camera, analyze the light intensity change of the excitation light sheet during the sample propagation process, and calculate the attenuation coefficient;

[0080] Step S82: generating preset parameters of the phase by calculating the attenuation coefficient and the attenuation compensation model;

[0081] Step S83: changing the phase area ratio of the Fresnel lenses with different focal lengths loaded on the spatial light modulator to form a stacked light beam in the incident direction;

[0082] Step S84: Compensate for the light intensity attenuation problem caused by absorption deep in the sample at different focal points.

[0083] In the actual light sheet microscopy imaging process, the interpolation function of the lens phase focal length and offset measured experimentally is used as the Fresnel lens function f fresnel (Δx), which makes the offset near the beam waist more accurate. To produce a multi-focus stacked beam, the phase diagram needs to include the phase of the Fresnel lens with several different focal points, divide the phase diagram into different periods according to the angle, and assign the phase of each offset focus to each period. In order to reduce the error, the offset in the experiment needs to be re-measured in the optical system.

[0084] In addition, when generating multi-focus stacked beams, when the distance between the focuses is large, the axial intensity profile between the focuses of the stacked beam will appear "hollow", and the axial performance of the beam will be affected. Therefore, it is not advisable to have too large a stacking distance. On the other hand, simply increasing the excitation light power to increase the light intensity deep in the medium will cause additional light damage to the shallow tissue, fail to redistribute the light intensity, and have very serious light damage and phototoxicity.

[0085] Step S81 is specifically as follows: According to the Lambert-Beer law, the attenuation of light intensity in the absorbing medium follows the law, and the output light intensity is obtained:

[0086]

[0087] Where I0 is the incident light intensity, I det is the output light intensity, μ a is the tissue absorption coefficient, G is the geometric correlation factor, and A is the absorbance.

[0088] In the attenuation simulation process of biological imaging, the influence of medium geometry-related factors is ignored, and the organism is regarded as a uniform medium. Experimental measurements show that the combined effect of absorption and scattering is also exponential attenuation. At this time, the point spread function (PSF) of the attenuated light sheet in the uniform medium is obtained. ill_attn for:

[0089]

[0090] In the formula, C atten is the sample attenuation coefficient, x k is the light sheet propagation distance.

[0091] Calculate the attenuation coefficient of the sample: The biological spectrophotometer can also be used to calculate the absorption coefficient of the medium; for imaging targets, the fluorescence with a relatively large three-dimensional imaging space is used as the focus, and the attenuation coefficient is fitted according to the intensity attenuation along the x direction based on the average intensity projection of the imaging results on the xz or xy plane; for the attenuation coefficient of biological tissue, due to the particularity of the image signal, the distribution of fluorophores in the tissue is related to the structure of the biological sample, and the tissue attenuation coefficient is estimated by compensating the biological imaging results to a uniform intensity distribution along the propagation direction.

[0092] Step S82 is specifically as follows:

[0093] In the process of light intensity superposition of stacked beams, the interference between beams cannot be ignored. Interference between beams has a negative effect on beam stacking. In this scheme, the stacking distance is changed according to the numerical aperture NA, which is at the μm level, while the coherence length of the laser is mostly above the cm level, so interference cannot be avoided, and the calculation of light intensity needs to consider the phase. The relationship between the attenuation coefficient and the compensation coefficient is established, and the light field distribution of the stacked beam is And light intensity I coh The calculation formula is as follows:

[0094]

[0095] Where r is the radial distance, x is the distance from the center of the beam in the propagation direction, and E n0 represents the electric field at the center of the light beam, k = 2π / λ is the wave number, λ is the wavelength of the illumination beam, n is the refractive index, E n represents the electric field distribution of Gaussian beams with different centers, ω0 represents the beam waist width, Indicates the beam width at different positions from the center of the field of view, For the ancient phase shift, represents phase shift, i represents complex exponential, Indicates the direction of propagation of the light beam from a point x to x n The radius of curvature between .

[0096] The imaging simulation result of the one-dimensional resolution target can be obtained by convolving the point spread function of the simulated light sheet propagation with the one-dimensional resolution target with a fixed scale in the z direction, such as Figure 5 As shown in Table 1. The imaging line widths of different medium light sheets at x = 0 μm (beam waist), x = 50 μm and x = 120 μm are selected and the contrast is calculated to compare the imaging performance of the laminated light sheet with and without compensation in the attenuation medium. This method can improve the contrast at x = 120 μm by 51.36%. Because attenuation suppresses the defocused light intensity of the laminated light sheet, the contrast at x = 50 μm in the attenuation medium is slightly improved, which means that the laminated light sheet has great application potential in weak attenuation media and nonlinear imaging.

[0097] Table 1 Line width and contrast of different dielectric light sheets at different positions

[0098]

[0099] The intensity changes of Gaussian beams with different focuses in the propagation direction are superimposed to obtain the intensity changes of the stacked beams in the propagation direction, and the relationship between the compensation coefficient and the attenuation coefficient is calculated, such as Figure 6 The attenuation coefficient C of the brain and other tissues is shown in attn At 50cm -1 Up to 200cm -1 The simulation calculated the compensation coefficients for different focal numbers from 0 to 300 cm -1 Change of attenuation coefficient, select compensation coefficient as 100cm -1 Up to 200cm -1 The attenuation coefficient C can be obtained from the data between atten The value of is generally smaller than the corresponding compensation coefficient when the compensation is completely compensated. Select different compensation coefficients as 10cm -1 , 100cm -1 , 150cm -1 , 200cm -1 , 250cm -1 The simulation calculated the attenuation coefficient C that can be fully compensated under different numbers of focal points. atten By establishing a model between the compensation coefficient and the attenuation coefficient, it is proved that the stacked beam based on attenuation compensation can meet the attenuation compensation requirements in biological samples.

[0100] like Figure 2 As shown, the present invention also provides a light sheet microscopic imaging system based on a stacked light beam, comprising a light source 1, a first half-wave plate 2, a polarization beam splitter 3, a beam terminator 4, an aperture 5, a second half-wave plate 6, a first lens 7, a pinhole 8, a second lens 9, a spatial light modulator 10, a relay device, a scanning galvanometer 13, a fifth lens 14, a sixth lens 15, 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, and the relay device comprises a third lens 11 and a fourth lens 12.

[0101] The laser is emitted by the light source 1, 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. At the same time, the light emitted by the polarization beam splitter 3 is a fixed-direction linear polarized light. The beam terminator 4 terminates the propagation of the light beam in the orthogonal direction. The aperture 5 adjusts the diameter of the light beam at this time, and the second half-wave plate 6 adjusts the linear polarized light in a fixed direction to be 45° with the original polarization direction, which is the same as the polarization direction of the optical axis of the liquid crystal panel in the modulation area of ​​the spatial light modulator 10. The beam expansion system composed of the first lens 7 and the second lens 9 is placed at the focal position of the pinhole 8 for spatial filtering. The light beam is shaped by the first lens 7, the pinhole 8 and the second lens 9 to be 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. 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 scanning galvanometer 13 is placed at the focal position of the fifth lens 14. The fifth lens 14 and the sixth lens 15 form a 4F system. The light beam passes through two lenses 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 forms a microscopic imaging optical path through the imaging objective lens 18 and the tube lens 19, and the filter 20 filters out light of other wavelengths except fluorescence, and then is collected at the camera 21.

[0102] Therefore, the present invention adopts the above-mentioned light sheet microscopy method and system based on stacked beams, which can change the intensity distribution along the propagation direction and realize attenuation compensation deep in the sample. This method can be applied not only to Gaussian beams, but also to common non-diffraction beams, such as Bessel beams, Airy beams, etc.

[0103] 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 light sheet microscopy imaging method based on stacked beams, characterized in that: The following steps are involved: Step S1: measuring the attenuation coefficient of the sample; Step S2: Calculate the corresponding compensation coefficient through the mathematical model, and calculate and generate the required parameters of the corresponding phase mask; Step S3: shaping and filtering the light beam emitted by the light source into a light beam with preset parameters; Step S4: using the light beam to load the phase of the Fresnel lens with different focal lengths to perform phase modulation; Step S5: The light beam is passed through a scanning galvanometer to form a light sheet, which excites the sample to generate a fluorescent signal, and a fluorescent probe is used to mark the biological sample during the excitation process; Step S6: cutting and transparentizing the sample, and placing the sample into an imaging system for imaging; Step S7: removing noise from the obtained sample image to obtain a picture of the deep part of the sample; Step S8: collecting and analyzing the light intensity changes during the propagation of the excitation light through a camera, and changing the phase area ratio of the Fresnel lens with different focal lengths loaded on the spatial light modulator.

2. The light sheet microscopy imaging method based on stacked beams according to claim 1, characterized in that: Step S4 is specifically as follows: subdividing the circular area modulated by the spatial light modulator into a plurality of dense sector-shaped areas, and loading a periodically changing regional Fresnel lens phase on the sector-shaped areas, adjusting the number of focal points according to the phase change period, and adjusting the stacking distance by adjusting the offset distance of the Fresnel lens phase.

3. The light sheet microscopy imaging method based on stacked beams according to claim 1, characterized in that: Before performing phase modulation on the plane light beam in step S4, the following steps need to be performed: Correct the original flatness of the spatial light modulator LCD panel and add the correction phase M wavecorrect ; Adding a blazed grating phase M to the spatial light modulator blaze , reducing the unmodulated area of ​​the spatial light modulator; Add truncation function phase M apodization , controls the actual numerical aperture at the entrance pupil; The final phase pattern loaded on the liquid crystal panel of the spatial light modulator is shown in the formula: M SLM =M wavecorrect ·M blaze ·M apodization ·M desired ; Among them, M desire is the desired Fresnel lens phase.

4. The light sheet microscopy imaging method based on stacked beams according to claim 1, characterized in that: Step S8 is specifically as follows: Step S81: Collect imaging results through a camera, analyze the light intensity change of the excitation light sheet during the sample propagation process, and calculate the attenuation coefficient; Step S82: generating preset parameters of the phase by calculating the attenuation coefficient and the attenuation compensation model; Step S83: changing the phase area ratio of the Fresnel lenses with different focal lengths loaded on the spatial light modulator to form a stacked light beam in the incident direction; Step S84: Compensate for the light intensity attenuation problem caused by absorption deep in the sample at different focal points.

5. The light sheet microscopy imaging method based on stacked beams according to claim 4, characterized in that: Step S81 is specifically as follows: According to the Lambert-Beer law, the attenuation of light intensity in the absorbing medium follows the law, and the output light intensity is obtained: Where I0 is the incident light intensity, I det is the output light intensity, μ a is the tissue absorption coefficient, G is the geometric correlation factor, and A is the absorbance; In the attenuation simulation process of biological imaging, the influence of medium geometry-related factors is ignored, and the organism is regarded as a uniform medium. The point spread function (PSF) of the attenuation light sheet in the uniform medium is obtained. ill_attn for: In the formula, C atten is the sample attenuation coefficient, x k is the light sheet propagation distance; Calculate the attenuation coefficient of the sample: fit the attenuation coefficient along the x-direction based on the average intensity projection of the xz or xy plane imaging results; estimate the tissue attenuation coefficient by compensating the biological imaging results to a uniform intensity distribution along the propagation direction.

6. The light sheet microscopy imaging method based on stacked beams according to claim 5, characterized in that: Step S82 is specifically as follows: Establish the relationship between the attenuation coefficient and the compensation coefficient to obtain the light field distribution of the stacked beam And light intensity I coh The calculation formula is as follows: I coh =|∑ n E n | 2 ; Where r is the radial distance, x is the distance from the center of the beam in the propagation direction, and E n0 represents the electric field at the center of the light beam, k = 2π / λ is the wave number, λ is the wavelength of the illumination beam, n is the refractive index, E n represents the electric field distribution of Gaussian beams with different centers, ω0 represents the beam waist width, Indicates the beam width at different positions from the center of the field of view, For the ancient phase shift, represents phase shift, i represents complex exponential, Indicates the direction of propagation of the light beam from a point x to x n The radius of curvature between The point spread function of the simulated light sheet propagation is convolved with the one-dimensional resolution target with a fixed scale in the z direction, and the imaging line widths of different medium light sheets at different locations are selected and the contrast is calculated to compare the imaging performance of the laminated light sheets with and without compensation in the attenuating medium. The intensity changes of Gaussian beams with different focuses in the propagation direction are superimposed to obtain the intensity changes of the stacked beams in the propagation direction, and the relationship between the compensation coefficient and the attenuation coefficient is calculated; Select different compensation coefficients and calculate the attenuation coefficient C that can be fully compensated under different numbers of focal points. atten The value of is obtained by establishing a model between the compensation coefficient and the attenuation coefficient.

7. A light sheet microscopy imaging system based on stacked beams, applied to a light sheet microscopy imaging method based on stacked beams as claimed in any one of claims 1 to 6, characterized in that: The invention comprises a light source, a first half-wave plate, a polarization beam splitter, a beam terminator, an aperture, a second half-wave plate, a first lens, a pinhole, a second lens, a spatial light modulator, a relay device, a scanning galvanometer, a fifth lens, a sixth lens, 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 relay device comprises a third lens and a fourth lens which are arranged in sequence, and the scanning galvanometer is placed at the focal position of the fifth lens.

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