A light sheet microscopy method and system based on stacked beams

Abstract

The present invention discloses a light sheet microscopy method and system based on stacked light beams, the microscopy method comprising the following steps: determining the attenuation coefficient of the sample; calculating the corresponding compensation coefficient through a mathematical model, and calculating and generating the required parameters of the corresponding phase mask; shaping and filtering the light beam emitted by the light source into a light beam with preset parameters; using the light beam to load the phase of a Fresnel lens with different focal lengths for phase modulation; passing the light beam through a scanning galvanometer to form a light sheet, exciting the sample to generate a fluorescent signal; placing the sample in an imaging system for imaging; performing noise removal on the obtained sample image; and collecting and analyzing the light intensity changes during the propagation of the excitation light through a camera. The present invention adopts the above-mentioned microscopy method, which improves the contrast at the illumination depth by 51.36% compared to light sheet imaging with attenuation, and can be applied to the attenuation compensation of common biological samples, that is, meeting the 300cm ‑1 Attenuation coefficient within.

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 currently balances resolution and imaging depth, most closely approaching the ideal performance optimization of optical microscopy. This technique utilizes a laser light sheet of defined thickness to excite a contrast agent within a sample to emit fluorescence, which is then collected in a direction perpendicular to the light sheet plane. Compared to traditional microscopy methods, light-sheet imaging offers superior characteristics such as high spatial resolution, high signal-to-noise ratio, large field of view, low photobleaching and phototoxicity, and rapid three-dimensional imaging. It holds enormous potential for application in a variety of life science research fields, including brain science and embryonic biology.

[0003] During the process of excitation light entering tissue and emitting fluorescence, the beam is subject to reflection, scattering, and absorption. This reduces the penetration depth of the light signal, affects the "ballistic" properties of photons, and easily attenuates and distorts the photon signal at depth. This limits the ability of light-sheet imaging to image deep within the sample. This directly leads to low spatial resolution and poor signal-to-background imaging. In actual biological tissue imaging, biological samples are mostly composed of highly absorbing and scattering media such as water and fat. During the process of light illumination and fluorescence emission, absorption and scattering occur, causing significant energy attenuation of the light sheet as it propagates through the sample. Ultimately, this leads to low spatial resolution, high background noise, and poor contrast in deep-sample imaging. Therefore, the current field of light-sheet imaging still faces the problem of poor excitation light imaging quality at depth. On the other hand, simply increasing the excitation light power to compensate for the attenuation of the sample medium fails to redistribute the light intensity, resulting in additional photobleaching and phototoxicity in 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. Attenuation compensation is the most important means to improve the quality of deep imaging. The attenuation compensation technologies currently proposed in light sheet microscopy mainly use 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 purpose of the present invention is to provide a light sheet microscopy method and system based on stacked beams to solve the problems mentioned in the background technology.

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

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

[0008] Step S2: Calculate the corresponding compensation coefficients through the mathematical model and calculate the required parameters for generating 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. During the excitation process, a fluorescent probe is used to label the biological sample.

[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 sample image to obtain an image of the deep portion of the sample;

[0014] Step S8: The camera collects and analyzes the light intensity changes during the propagation of the excitation light, and changes the phase area ratio of the Fresnel lens with different focal lengths loaded on the spatial light modulator to achieve attenuation compensation for light sheet microscopy 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 as follows:

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

[0025] Step S82: generating preset phase parameters 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: Compensating 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 as:

[0030]

[0031] Where I0 is the incident light intensity, I det is the outgoing 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] Where 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 width of the beam at different positions from the center of the field of view, For the ancient phase shift, represents phase shift, i represents complex exponential, Indicates that the beam travels from a point x to x in the propagation direction n The radius of curvature between

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

[0042] The intensity variation of the stacked beam in the propagation direction is obtained by superimposing the intensity variation of the Gaussian beams with different focuses. 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 focuses. 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 microscopy 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, wherein the relay device comprises 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 microscopy method and system based on stacked beams, which has the following beneficial effects:

[0045] (1) By enhancing the deep light intensity, attenuation compensation is achieved deep in the sample, 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 converge the excitation light focus 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; by subdividing the circular area modulated by the spatial light modulator into dense fan-shaped areas, the phase change period is determined 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 the 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 superimposed attenuation effect.

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

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

[0050] (6) Using stacked light sheets for imaging can improve the quality of deep imaging without increasing shallow photobleaching, including improving the signal-to-background ratio and spatial resolution at deep depths of the sample, thus enabling further applications 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 This is a flow chart of a light sheet microscopy method based on stacked beams according to an embodiment of the present invention;

[0053] Figure 2 Schematic diagram of the structure 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 illustrating the principle of generating stacked beams in an imaging method according to an embodiment of the present invention;

[0056] Figure 5 A convolution diagram of a point spread function and a one-dimensional resolution target of an 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; 17. Sample chamber; 18. Imaging objective; 19. Tubular 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, not all of the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings herein 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 of 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, it does not need to be further defined or explained in subsequent drawings.

[0062] Example

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

[0064] Step S1: Measure 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 beam with preset parameters. The monochromatic laser light emitted by the light source is passed through shaping devices such as a laser beam expansion system and a spatial filtering system to produce a beam with preset parameters. 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 the 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 on them. 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] The superimposed Fresnel lens phases are effectively equivalent to lens optical elements. Introducing Fresnel lens phases of varying focal lengths is equivalent to converging a normally propagating light beam through a convex lens, focusing it before its original focal point, or diverging it through a concave lens, focusing it after its original focal point. The formula for correlating the Fresnel lens phase with its focal length derives that the preset focal length parameter f and the offset Δx are inversely proportional. Experimental calculations can be used to calculate the offset Δx corresponding to parameter f, providing a basis for the focal distribution of the stacked beams.

[0071] like Figure 3-4 As shown, before phase modulating the plane 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 of the spatial light modulator (the area between the lattices), the blazed grating phase M is added to the spatial light modulator.blaze , 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 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 zero-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 passed through a scanning galvanometer to form a light sheet, which is used to excite the sample to produce a fluorescent signal. Specifically, the light sheet is formed by scanning a stack of light beams, which are incident on the sample from one side, exciting the sample to produce fluorescence. During the excitation process, fluorescent probes are used to label the biological sample to obtain the tissue sample to be imaged. Different contrast agents can affect the final imaging results, so choosing the right fluorescent probe is particularly important.

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

[0076] Step S7: performing noise removal and other processing operations on the obtained sample image to obtain a picture of the deep portion of the sample.

[0077] Step S8: The camera collects and analyzes the light intensity changes during the propagation of the excitation light, and changes the phase area ratio of the Fresnel lens with different focal lengths loaded on the spatial light modulator to achieve attenuation compensation for light sheet microscopy based on stacked beams.

[0078] Step S8 is specifically as follows:

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

[0080] Step S82: generating preset phase parameters 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: Compensating 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 allows for more accurate offsets near the beam waist. Generating a multi-focus stack requires a phase diagram that includes the phases of several Fresnel lenses with different focal points. This phase diagram is divided into periods based on angle, and the phase of each offset focus is assigned to each period. To minimize errors, the offsets used in the experiment must be remeasured in the optical system.

[0084] Furthermore, when generating multi-focus stacked beams, a large distance between the focal points can cause a "hollow" axial intensity profile between the focal points of the stacked beam, compromising the axial performance of the beam. Therefore, excessive stacking distances are undesirable. On the other hand, simply increasing the excitation light power to boost the intensity deep within the medium can cause additional photodamage in the superficial tissue layers, failing to redistribute the light intensity and resulting in significant photodamage 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 a law, and the output light intensity is obtained:

[0086]

[0087] Where I0 is the incident light intensity, I det is the outgoing 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 homogeneous medium. Experimental measurements show that the combined effect of absorption and scattering also results in exponential decay. In this case, the point spread function (PSF) of the attenuated light sheet in the homogeneous medium is obtained. ill_attn for:

[0089]

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

[0091] Calculate the attenuation coefficient of the sample: The biospectrophotometer can also be used to calculate the absorption coefficient of the medium. For imaging targets that are focused and the fluorescence imaging is relatively large in the three-dimensional space, the attenuation coefficient is fitted based on the intensity attenuation along the x-direction based on the average intensity projection of the xz or xy plane imaging results. For biological tissue attenuation coefficient measurement, due to the particularity of the image signal, the distribution of fluorophores in the tissue is related to the structure of the biological sample. 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. Therefore, 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 width of the beam at different positions from the center of the field of view, For the ancient phase shift, represents phase shift, i represents complex exponential, Indicates that the beam travels from a point x to x in the propagation direction 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 linewidths of different dielectric light sheets at x = 0 μm (beam waist), x = 50 μm, and x = 120 μm were selected and contrast was calculated to compare the imaging performance of the laminated light sheet with and without compensation in an attenuating medium. This method can improve the contrast at x = 120 μm by 51.36%. Because attenuation suppresses the off-focus intensity of the laminated light sheet, the contrast at x = 50 μm in the attenuating medium is slightly improved, indicating that laminated light sheets have great potential for application in weakly attenuating 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 beam in the propagation direction, and the relationship between the compensation coefficient and the attenuation coefficient is calculated, as shown in the following example: Figure 6 The attenuation coefficient C of the brain and other tissues is shown. attn At 50cm -1 to 200cm -1 The simulation calculated the compensation coefficients for different focal lengths from 0 to 300 cm. -1 Changes in attenuation coefficient, select a compensation coefficient of 100cm -1 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 complete. 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 microscopy imaging system based on a stacked 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. The relay device includes a third lens 11 and a fourth lens 12.

[0101] Laser light is emitted from light source 1. The combination of first half-wave plate 2 and polarization beam splitter 3 modifies the overall intensity of the subsequent optical path. The light exiting polarization beam splitter 3 is linearly polarized in a fixed direction. Beam terminator 4 terminates the beam's propagation in an orthogonal direction. Aperture 5 adjusts the beam's diameter. Second half-wave plate 6 adjusts the linearly polarized light to a 45° angle relative to the original polarization direction, aligning it with the polarization direction of the optical axis of the liquid crystal panel in the modulation area of spatial light modulator 10. A beam expansion system consisting of first lens 7 and second lens 9 is spatially filtered by a pinhole 8 positioned at its focal point. The beam is shaped by first lens 7, pinhole 8, and second lens 9 before entering the liquid crystal panel of spatial light modulator 10. A blazed grating and a cubic phase mask are applied to the liquid crystal panel to generate an Airy beam. Third and fourth lenses 11 and 12 form a relay element, relaying the beam to scanning mirror 13. Simultaneously, these lenses reduce the beam to accommodate the size of scanning mirror 13. Scanning galvanometer 13 is placed at the focal point of fifth lens 14. Fifth and sixth lenses 14 and 15 form a 4F system. The light beam passes through these two lenses and is then focused at the input plane of illumination objective 16. The scanning beam is focused by illumination objective 16, forming a scanning light sheet at its focal point, illuminating the sample that has been processed and labeled with a fluorescent probe. The fluorescence signal generated by the excitation passes through imaging objective 18 and tube lens 19, forming a microscopic imaging optical path. A filter 20 filters out all wavelengths of light except for fluorescence, and is then collected by 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 solutions of the present invention rather than to limit the same. 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 solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A light sheet microscopy method based on stacked beams, characterized by: The following steps are involved: Step S1: measuring the attenuation coefficient of the sample; Step S2: Calculate the corresponding compensation coefficients through the mathematical model and calculate the required parameters for generating 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. During the excitation process, a fluorescent probe is used to label the biological sample. Step S6: cutting and transparentizing the sample, and placing the sample into an imaging system for imaging; Step S7: removing noise from the sample image to obtain an image of the deep portion of the sample; Step S8: collecting and analyzing the light intensity changes during the propagation of the excitation light through the camera, and changing the phase area ratio of the Fresnel lens with different focal lengths loaded on the spatial light modulator; Step S8 is specifically as follows: Step S81: Collect imaging results through a camera, analyze the change in light intensity of the excitation light sheet during the propagation process of the sample, and calculate the attenuation coefficient; Step S82: generating preset phase parameters 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: Compensating for the light intensity attenuation problem caused by absorption deep in the sample at different focal points; 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 He Guangqiang The calculation formula is as follows: ； ； Where, is the radial distance, is the distance from the beam center in the propagation direction, represents the electric field at the center of the beam, is the wave number, is the wavelength of the illumination beam, is the refractive index, represents the electric field distribution of Gaussian beams with different centers, represents the optical waist width, Indicates the width of the beam at different positions from the center of the field of view, For the ancient phase shift, represents the phase shift, represents a complex exponential, Indicates a point in the propagation direction of the light beam arrive The radius of curvature between The point spread function of the simulated light sheet propagation is convolved with a one-dimensional resolution target with a fixed scale in the z-direction. The imaging linewidths of the light sheets of different media at different locations are selected and the contrast is calculated to compare the imaging performance of the stacked light sheets with and without compensation in attenuating media. The intensity variation of the stacked beam in the propagation direction is obtained by superimposing the intensity variation of the Gaussian beams with different focuses. The relationship between the compensation coefficient and the attenuation coefficient is calculated. Different compensation coefficients are selected to calculate the attenuation coefficient that can be fully compensated under different numbers of focuses. C atten The value of is obtained by establishing a model between the compensation coefficient and the attenuation coefficient.

2. The light sheet microscopy method based on stacked beams according to claim 1, characterized in that: Step S4 specifically comprises: 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 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 correction phase ; Adding Blazed Grating Phase to Spatial Light Modulators , reducing the unmodulated area of the spatial light modulator; Adding truncated function phase , 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: ； in, is the desired Fresnel lens phase.

4. The light sheet microscopy method based on stacked beams according to claim 1, 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, is the incident light intensity, is the outgoing light intensity, is the tissue absorption coefficient, is the geometric correlation factor, is 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 of the attenuation light sheet in the uniform medium is obtained. for: ； Where, is the light sheet propagation distance; Calculate the attenuation coefficient of the sample: Based on or The average intensity projection of the surface imaging result is along x Directional intensity attenuation is fitted to the attenuation coefficient; the tissue attenuation coefficient is estimated by compensating the biological imaging results to a uniform intensity distribution along the propagation direction.

5. A light sheet microscopy system based on stacked beams, based on the light sheet microscopy method based on stacked beams according to any one of claims 1 to 4, 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, wherein 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.

Images