Four-beam lattice light sheet super-resolution microscopic imaging device and method based on spatial spectrum estimation

Abstract

The application provides a four-beam lattice light sheet super-resolution microscopic imaging device and method based on spatial spectrum estimation. The device belongs to the technical field of super-resolution fluorescence microscopy, and comprises the following optical components: a linearly polarized input light beam, a cylindrical lens group, an SLM, λ a 1 / 4 wave plate, a PBS, a mask, a relay lens group, a galvanometer, an excitation objective O1, a fluorescent sample, a mirror, a collection objective O2, and an sCMOS camera. The SLM is combined with a high reflectivity mirror on the side of the excitation objective to generate a fine periodic structure of the lattice light sheet illumination field. According to the threshold value, the signal and noise subspaces are divided. The mapping vector is used to project the ratio of the signal subspace and the noise subspace which are orthogonal to each other to obtain a high-resolution image. According to the noise model of the imaging system, the number of signal eigenvalues is obtained by maximum likelihood estimation to determine the optimal signal-to-noise ratio threshold value of any point in the field of view. Without selecting the threshold value, the influence of subjective factors is avoided, and finally the isotropic resolution is improved.

Description

Technical Field

[0001] This invention relates to the field of super-resolution fluorescence microscopy, and in particular to a four-beam lattice light sheet super-resolution microscopy imaging device and method based on spatial spectrum estimation. Background Technology

[0002] Existing super-resolution light-sheet fluorescence microscopy techniques inevitably require trade-offs between spatial resolution, temporal resolution, and phototoxicity. Lattice light-sheet microscopy, a super-resolution fluorescence microscopy technique, utilizes a quasi-diffraction-free lattice light field with a two- or three-dimensional periodic structure, formed by the coherent superposition of multiple plane waves with wave vectors located on a conical surface. This combines structured light illumination with light-sheet microscopy, achieving 150 nm super-resolution imaging in a single transverse direction using a light-sheet microscopy system with mutually perpendicular illumination and probe objectives. However, axial resolution is not improved, which is detrimental to more detailed three-dimensional long-term observations. Summary of the Invention

[0003] The purpose of this invention is to solve the problem of spatial resolution anisotropy in fluorescence microscopy of lattice sheets, and to propose a four-beam lattice sheet super-resolution microscopy device and method based on spatial spectrum estimation.

[0004] This invention is achieved through the following technical solution: This invention proposes a four-beam lattice light sheet super-resolution microscopic imaging device based on spatial spectrum estimation. The microscopic imaging device includes a linearly polarized input beam 1, a first cylindrical lens group 2, a second cylindrical lens group 3, and an SLM 4, which are sequentially distributed along the optical path direction. λ 5. Waveplate 4, PBS 6. Lens L1 7. Mask 8. First relay lens group 9. z Galvanometer 10, Second relay lens group 11 x Galvanometer 12, third relay lens group 13, excitation objective O114, fluorescent sample 15, mirror 16, collection objective O217, lens 18, filter 19 and sCMOS camera 20; A linearly polarized input beam 1 is sequentially projected onto the surface of an SLM4 after passing through a first cylindrical lens group 2 and a second cylindrical lens group 3. The SLM4 is loaded with... xz The planar lattice pattern modulates the light beam, and the light reflected from the SLM4 passes through... λ After being converted into orthogonally linearly polarized light by waveplate 5, the beam is incident perpendicularly on PBS6, then focused by lens L17, and ineffective diffraction orders are filtered out by mask 8, which also limits the effective numerical aperture. The beam then passes sequentially through the first relay lens group 9. z Galvanometer 10, Second relay lens group 11 xThe galvanometer 12 and the third relay lens group 13 finally enter the rear pupil of the excitation objective lens O114, and together with the reflector 16 placed on the stage, they form a four-beam interference light field, thereby generating a lattice illumination field with a fine periodic structure. The dynamic scanning of the fluorescent sample 15 is achieved by switching the lattice pattern loaded on the SLM4. The dynamic scanning covers the entire target illumination plane of the fluorescent sample 15, and the cumulative intensity distribution of all illumination patterns is uniform. The fluorescence signal generated by the lattice light sheet in the sample plane is collected by the orthogonally placed collecting objective lens O217, and stray light is filtered out by the lens 18 and the filter 19 in sequence. Finally, the sCMOS camera 20 is synchronized with the clock of the SLM4 and acquires a low-resolution image sequence generated by the fluorescent sample 15 in the illuminated plane during the flashing process at each scanning position.

[0005] Furthermore, by switching the lattice pattern loaded on SLM4 to change the illumination intensity distribution of the front focal plane of the excitation objective lens O114, the fluorescent sample 15 is subjected to non-uniform excitation over time, and the cumulative intensity distribution of all illumination patterns is uniform.

[0006] Furthermore, the linearly polarized input beam 1 is projected onto the first cylindrical lens group 2 via the first reflecting mirror, the output beam of the first cylindrical lens group 2 enters the second cylindrical lens group 3 via the second reflecting mirror, and the output beam of the second cylindrical lens group 3 enters the surface of the SLM4 via the third reflecting mirror.

[0007] Furthermore, the first cylindrical lens group 2 includes two cylindrical lenses arranged in parallel; the second cylindrical lens group 3 includes two cylindrical lenses arranged in parallel.

[0008] Furthermore, the first relay lens group 9 includes two relay lenses arranged in parallel; the second relay lens group 11 includes two relay lenses arranged in parallel; and the third relay lens group 13 includes two relay lenses arranged perpendicularly.

[0009] The present invention also proposes a microscopic imaging method based on the aforementioned four-beam lattice light sheet super-resolution microscopic imaging device based on spatial spectrum estimation, the method comprising the following steps: S1. The linearly polarized input beam 1 is incident on the reflective surface of SLM4 along the light transmission path. After being modulated by SLM4, it is combined with the reflector 16 placed on the stage to form a lattice illumination with a fine periodic structure on the fluorescent sample 15. S2. The phase modulation of the illumination light is achieved by changing the lattice pattern of SLM4, and the fluorescent sample 15 is excited non-uniformly over time. S3. Use sCMOS camera 20 to acquire a low-resolution image sequence generated by fluorescent sample 15 under time-varying lattice light sheet illumination; S4. Use the spatial spectrum estimation algorithm to reconstruct the low-resolution image sequence to obtain a high-resolution image.

[0010] Further, step S1 specifically includes: Linearly polarized input beam 1 first passes through the first cylindrical lens group 2 to polarize the beam along... x directional stretching, then passing through the second cylindrical lens group, three pairs of beams along... z Directional compression ultimately forms a thin stripe of illumination at the center of the SLM4; the surface of the SLM4 is loaded with a preset... xz A planar lattice pattern, combined with a reflector 16 placed on the stage, causes four laser beams to interfere with each other to generate a lattice light sheet illumination field with a fine periodic structure; through polarization path separation technology, spatial decoupling of incident and reflected light is achieved, and then invalid diffraction levels are filtered out by lens L17 and mask 8; different lattice patterns generated by SLM4 can change the illumination intensity distribution of the front focal plane of the excitation objective lens O114, and perform time-varying non-uniform excitation of the fluorescent sample 15.

[0011] Furthermore, in step S2, the illumination light is modulated by SLM4 and mirror 16 to form a lattice light sheet illumination field with a fine periodic structure. SLM4 is based on the principle of optical phased array and uses an external electric field to control the refractive index of the liquid crystal layer to achieve phase modulation and beam deflection, thereby flexibly generating the required lattice pattern.

[0012] Further, in step S3, The formula for the light intensity distribution of a low-resolution image sequence is: (1) In the formula, Light intensity, Represents the spatial coordinates of the object. Represents image space coordinates, The material function representing the fluorescent sample. Represents the point spread function. This indicates the distribution of illumination intensity on the sample surface.

[0013] Further, step S4 specifically includes: The low-resolution image sequence is described in the form of a mathematical matrix, and the low-resolution image sequence forms a matrix. I : (2) In the formula, K represents the total number of original image frames acquired; calculate its covariance matrix: (3) For matrix REigenvalue decomposition is performed. Based on the minimum description length criterion (MDL) and the imaging system noise model, the number of signal eigenvalues ​​is obtained through maximum likelihood estimation to determine the optimal signal-to-noise ratio threshold σ0 at any point in the field of view. The eigenvectors corresponding to eigenvalues ​​greater than σ0 represent the signal S:{u σi =0}, other feature vectors represent N:{u σi =0};The eigenvalues ​​corresponding to the signal-to-noise subspace and the noise subspace satisfy the following equation: (4) In the formula, M represents the number of fluorescent probes in fluorescent sample 15. λ The eigenvalues ​​obtained from the eigenvalue decomposition are used to perform spectral peak search using the orthogonality of the two subspaces, resulting in a high-resolution image.

[0014] The beneficial effects of this invention are: 1. Using SLM combined with a high-reflectivity mirror on the opposite side of the excitation objective to generate a lattice light sheet illumination field with a fine periodic structure.

[0015] 2. High-resolution images are obtained by using the projection ratio of the mapping vector onto the mutually orthogonal signal subspace and noise subspace. Based on the noise model of the imaging system, the number of signal feature values ​​is obtained through maximum likelihood estimation to determine the optimal signal-to-noise ratio threshold σ0 at any point in the field of view. This eliminates the need to select a threshold and avoids the influence of subjective factors, ultimately achieving isotropic resolution improvement. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of a four-beam lattice light sheet super-resolution microscopy imaging device based on spatial spectrum estimation. Figure 2 yes Figure 1 The imaging device shown illuminates the lattice pattern of the fluorescent sample through the focal plane of the excitation objective lens. Figure 3 This is a schematic diagram of the contrast results of super-resolution microscopy of a four-beam lattice optical sheet based on spatial spectrum estimation.

[0018] Explanation of markings in the diagram: 1. Linearly polarized input beam; 2. First cylindrical lens group; 3. Second cylindrical lens group; 4. SLM; 5. λ 4. Waveplate; 6. PBS; 7. Lens L1; 8. Mask; 9. First relay lens group; 10.z 11. Galvanometer; 12. Second relay lens group; x 13. Galvanometer; 14. Third relay lens group; 15. Excitation objective O1; 16. Fluorescent sample; 17. Reflector; 18. Collection objective O2; 19. Lens; 20. sCMOS camera. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Combination Figures 1-3 This invention proposes a four-beam lattice light sheet super-resolution microscopy imaging device based on spatial spectrum estimation. Figure 1 This is a schematic diagram of the microscopic imaging device in this embodiment. The microscopic imaging device includes a linearly polarized input beam 1, a first cylindrical lens group 2, a second cylindrical lens group 3, and an SLM 4, which are sequentially distributed along the optical path. λ 5. Waveplate 4, PBS 6. Lens L1 7. Mask 8. First relay lens group 9. z Galvanometer 10, Second relay lens group 11 x Galvanometer 12, third relay lens group 13, excitation objective O114, fluorescent sample 15, mirror 16, collection objective O217, lens 18, filter 19 and sCMOS camera 20; A linearly polarized input beam 1 is sequentially projected onto the surface of an SLM4 after passing through a first cylindrical lens group 2 and a second cylindrical lens group 3. The SLM4 is loaded with... xz The planar lattice pattern modulates the light beam, and the light reflected from the SLM4 passes through... λ After being converted into orthogonally linearly polarized light by waveplate 5, the beam is incident perpendicularly on PBS6, then focused by lens L17, and ineffective diffraction orders are filtered out by mask 8, which also limits the effective numerical aperture. The beam then passes sequentially through the first relay lens group 9. z Galvanometer 10, Second relay lens group 11 xThe galvanometer 12 and the third relay lens group 13 finally enter the rear pupil of the excitation objective lens O114, and together with the reflector 16 placed on the stage, they form a four-beam interference light field, thereby generating a lattice illumination field with a fine periodic structure. The dynamic scanning of the fluorescent sample 15 is achieved by switching the lattice pattern loaded on the SLM4. The dynamic scanning covers the entire target illumination plane of the fluorescent sample 15, and the cumulative intensity distribution of all illumination patterns is uniform. The fluorescence signal generated by the lattice light sheet in the sample plane is collected by the orthogonally placed collecting objective lens O217, and stray light is filtered out by the lens 18 and the filter 19 in sequence. Finally, the sCMOS camera 20 is synchronized with the clock of the SLM4 and acquires a low-resolution image sequence generated by the fluorescent sample 15 in the illuminated plane during the flashing process at each scanning position.

[0021] By switching the lattice pattern loaded on SLM4 to change the illumination intensity distribution on the front focal plane of the excitation objective lens O114, the fluorescent sample 15 is subjected to non-uniform excitation over time, and the cumulative intensity distribution of all illumination patterns is uniform.

[0022] The linearly polarized input beam 1 is projected onto the first cylindrical lens group 2 via the first reflecting mirror. The output beam of the first cylindrical lens group 2 enters the second cylindrical lens group 3 via the second reflecting mirror. The output beam of the second cylindrical lens group 3 enters the surface of the SLM4 via the third reflecting mirror.

[0023] The first cylindrical lens group 2 includes two cylindrical lenses arranged in parallel; the second cylindrical lens group 3 includes two cylindrical lenses arranged in parallel.

[0024] The first relay lens group 9 includes two relay lenses arranged in parallel; the second relay lens group 11 includes two relay lenses arranged in parallel; and the third relay lens group 13 includes two relay lenses arranged perpendicularly.

[0025] The present invention also proposes a microscopic imaging method based on the aforementioned four-beam lattice light sheet super-resolution microscopic imaging device based on spatial spectrum estimation, the method comprising the following steps: S1. The linearly polarized input beam 1 is incident on the reflective surface of SLM4 along the light transmission path. After being modulated by SLM4, it is combined with the reflector 16 placed on the stage to form a lattice illumination with a fine periodic structure on the fluorescent sample 15. S2. The phase modulation of the illumination light is achieved by changing the lattice pattern of SLM4, and the fluorescent sample 15 is excited non-uniformly over time. S3. Use sCMOS camera 20 to acquire a low-resolution image sequence generated by fluorescent sample 15 under time-varying lattice light sheet illumination; S4. Use the spatial spectrum estimation algorithm to reconstruct the low-resolution image sequence to obtain a high-resolution image.

[0026] Step S1 specifically includes: Linearly polarized input beam 1 first passes through the first cylindrical lens group 2 to polarize the beam along... x directional stretching, then passing through the second cylindrical lens group, three pairs of beams along... z Directional compression ultimately forms a thin stripe of illumination at the center of the SLM4; the surface of the SLM4 is loaded with a preset... xz A planar lattice pattern, combined with a reflector 16 placed on the stage, causes four laser beams to interfere with each other to generate a lattice light sheet illumination field with a fine periodic structure; through polarization path separation technology, spatial decoupling of incident and reflected light is achieved, and then invalid diffraction levels are filtered out by lens L17 and mask 8; different lattice patterns generated by SLM4 can change the illumination intensity distribution of the front focal plane of the excitation objective lens O114, and perform time-varying non-uniform excitation of the fluorescent sample 15.

[0027] In step S2, the illumination light is modulated by SLM4 and mirror 16 to form a lattice light sheet illumination field with a fine periodic structure. SLM4 is based on the principle of optical phased array and uses an external electric field to control the refractive index of the liquid crystal layer to achieve phase modulation and beam deflection, thereby flexibly generating the required lattice pattern.

[0028] In step S3, the fluorescence emitted by the fluorescent sample 15 is collected by the collecting objective lens O217, which is orthogonally placed and has a confocal plane with the excitation objective lens O114. Stray light is filtered out by the lens 18 and filter 19. The camera's photosensitive plane coincides with the front focal plane of the lens 18, thus acquiring the image of the fluorescent sample 15. By switching the lattice pattern loaded on the SLM4, radial dynamic scanning of the lattice light sheet within the sample plane is achieved. This dynamic scanning covers the entire target illumination plane of the fluorescent sample 15, and the cumulative intensity distribution of all illumination patterns is uniform. Finally, the sCMOS camera 20 is synchronized with the clock of the SLM4, acquiring a low-resolution image sequence generated by the fluorescent sample 15 within the illuminated plane during the flashing process at each scanning position.

[0029] The formula for the light intensity distribution of a low-resolution image sequence is: (1) In the formula, Light intensity, Represents the spatial coordinates of the object. Represents image space coordinates, The material function representing the fluorescent sample. Represents the point spread function. This indicates the distribution of illumination intensity on the sample surface.

[0030] In step S4, the original image contains both signal and noise, and the signal subspace and noise subspace are orthogonal. Spectral peak search is performed using the orthogonality of these two subspaces to obtain a high-resolution image. Step S4 specifically includes: The low-resolution image sequence is described in the form of a mathematical matrix, and the low-resolution image sequence forms a matrix. I : (2) In the formula, K represents the total number of original image frames acquired; calculate its covariance matrix: (3) For matrix R Eigenvalue decomposition is performed. Based on the Minimum Description Length (MDL) criterion in information theory, and according to the imaging system noise model, the number of signal eigenvalues ​​is obtained through maximum likelihood estimation to determine the optimal signal-to-noise ratio (SNR) threshold σ0 at any point within the field of view. Eigenvalues ​​greater than σ0 correspond to eigenvectors representing the signal S:{u σi =0}, other feature vectors represent N:{u σi =0};The eigenvalues ​​corresponding to the signal-to-noise subspace and the noise subspace satisfy the following equation: (4) In the formula, M represents the number of fluorescent probes in fluorescent sample 15. λ The eigenvalues ​​obtained from the eigenvalue decomposition are used to perform spectral peak search using the orthogonality of the two subspaces, resulting in a high-resolution image.

[0031] Figure 2 Showing Figure 1 The imaging device shown displays the lattice light sheet distribution of the fluorescent sample within the focal plane of the excitation objective. xz The cross-section visually reveals the fine optical field modulation characteristics achieved by the device on the excitation side. Figure 3 The contrast distribution results of four-beam lattice light sheet super-resolution microscopy obtained by combining a spatial spectrum estimation algorithm are presented. It can be seen that when the distance from the center exceeds 150 nm, the contrast rapidly increases to 1, indicating that this method can achieve a resolution better than 150 nm in all directions of the lateral plane, thus breaking through the constraints of the traditional diffraction limit. Unlike conventional lattice light sheet techniques that can only improve lateral resolution, the method described in this invention significantly improves axial resolution by introducing axial modulation of a high-frequency lattice light field at the excitation end, laying the foundation for achieving more refined three-dimensional super-resolution long-term imaging.

[0032] The above provides a detailed description of the four-beam lattice light sheet super-resolution microscopic imaging device and method based on spatial spectrum estimation proposed in this invention. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this invention. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A four-beam lattice light sheet super-resolution microscopic imaging device based on spatial spectrum estimation, characterized in that, The microscopic imaging device includes a linearly polarized input beam (1), a first cylindrical lens group (2), a second cylindrical lens group (3), and an SLM (4) arranged sequentially along the optical path. λ / 4 waveplate (5), PBS (6), lens L1 (7), mask (8), first relay lens group (9) z Galvanometer (10), second relay lens group (11) x Galvanometer (12), third relay lens group (13), excitation objective O1 (14), fluorescent sample (15), mirror (16), collection objective O2 (17), lens (18), filter (19) and sCMOS camera (20). A linearly polarized input beam (1) passes sequentially through a first cylindrical lens group (2) and a second cylindrical lens group (3) and is projected onto the surface of an SLM (4), which is loaded with... xz The planar lattice pattern modulates the light beam, and the light reflected from the SLM(4) passes through... λ After being converted into orthogonally linearly polarized light by the 4-wave plate (5), the light is incident perpendicularly on the PBS (6), then focused by the lens L1 (7), and the mask (8) filters out invalid diffraction orders and limits the effective numerical aperture; subsequently, the beam passes through the first relay lens group (9) in sequence. z Galvanometer (10), second relay lens group (11) x The galvanometer (12) and the third relay lens group (13) finally enter the pupil of the excitation objective lens O1 (14), and together with the reflector (16) placed on the stage, they form a four-beam interference light field, thereby generating a lattice illumination field with a fine periodic structure. The dynamic scanning of the fluorescent sample (15) is achieved by switching the lattice pattern loaded on the SLM (4). The dynamic scanning covers the target illumination plane of the entire fluorescent sample (15), and the cumulative intensity of all illumination patterns is uniformly distributed. The fluorescence signal generated by the lattice light sheet in the sample plane is collected by the orthogonally placed collecting objective lens O2 (17), and stray light is filtered out by the lens (18) and the filter (19) in sequence. Finally, the sCMOS camera (20) is synchronized with the clock of the SLM (4) to acquire the low-resolution image sequence generated by the fluorescent sample (15) in the illuminated plane during the flashing process at each scanning position.

2. The microscopic imaging device according to claim 1, characterized in that, By switching the lattice pattern loaded on the SLM (4) to change the illumination intensity distribution of the front focal plane of the excitation objective lens O1 (14), the fluorescent sample (15) is subjected to non-uniform excitation over time, and the cumulative intensity distribution of all illumination patterns is uniform.

3. The microscopic imaging device according to claim 1, characterized in that, The linearly polarized input beam (1) is projected onto the first cylindrical lens group (2) via the first reflecting mirror. The output beam of the first cylindrical lens group (2) enters the second cylindrical lens group (3) via the second reflecting mirror. The output beam of the second cylindrical lens group (3) enters the surface of the SLM (4) via the third reflecting mirror.

4. The microscopic imaging device according to claim 1, characterized in that, The first cylindrical lens group (2) includes two cylindrical lenses arranged in parallel; the second cylindrical lens group (3) includes two cylindrical lenses arranged in parallel.

5. The microscopic imaging device according to claim 1, characterized in that, The first relay lens group (9) includes two relay lenses arranged in parallel; the second relay lens group (11) includes two relay lenses arranged in parallel; and the third relay lens group (13) includes two relay lenses arranged perpendicularly.

6. A microscopic imaging method based on the four-beam lattice light sheet super-resolution microscopic imaging device based on spatial spectrum estimation as described in claim 1, characterized in that, The method includes the following steps: S1. The linearly polarized input beam (1) is incident on the reflective surface of the SLM (4) along the light transmission path. After being modulated by the SLM (4), it is combined with the reflector (16) placed on the stage to form a lattice illumination with a fine periodic structure on the fluorescent sample (15). S2. Change the lattice pattern of SLM (4) to achieve phase modulation of the illumination light and perform time-varying non-uniform excitation on the fluorescent sample (15). S3. Use an sCMOS camera (20) to acquire a low-resolution image sequence generated by the fluorescent sample (15) under time-varying lattice light sheet illumination; S4. Use the spatial spectrum estimation algorithm to reconstruct the low-resolution image sequence to obtain a high-resolution image.

7. The microscopic imaging method according to claim 6, characterized in that, Step S1 specifically includes: The linearly polarized input beam (1) first passes through the first cylindrical lens group (2) to polarize the beam along... x The beam is stretched in the direction and then passes through the second cylindrical lens group (3) to guide the beam along the direction. z Directional compression ultimately forms a thin stripe of illumination at the center of the SLM (4); the surface of the SLM (4) is loaded with a preset... xz The planar lattice pattern, combined with the reflector (16) placed on the stage, causes the four laser beams to interfere with each other to generate a lattice light sheet illumination field with a fine periodic structure; through polarization path separation technology, the incident light and the reflected light are spatially decoupled, and then the invalid diffraction level is filtered out by lens L1 (7) and mask (8); the SLM (4) generates different lattice patterns that can change the illumination intensity distribution of the front focal plane of the excitation objective lens O1 (14) and perform non-uniform excitation of the fluorescent sample (15) over time.

8. The microscopic imaging method according to claim 6, characterized in that, In step S2, the illumination light is modulated by the SLM (4) and the reflector (16) to form a lattice light sheet illumination field with a fine periodic structure. The SLM (4) is based on the principle of optical phased array and uses an external electric field to control the refractive index of the liquid crystal layer to achieve phase modulation and beam deflection, thereby flexibly generating the required lattice pattern.

9. The microscopic imaging method according to claim 6, characterized in that, In step S3, The formula for the light intensity distribution of a low-resolution image sequence is: （1） In the formula, Light intensity, Represents the spatial coordinates of the object. Represents image space coordinates, The material function representing the fluorescent sample. Represents the point spread function. This indicates the distribution of illumination intensity on the sample surface.

10. The microscopic imaging method according to claim 6, characterized in that, Step S4 specifically includes: The low-resolution image sequence is described in the form of a mathematical matrix, and the low-resolution image sequence forms a matrix. I : （2） In the formula, K represents the total number of original image frames acquired; calculate its covariance matrix: （3） For matrix R Eigenvalue decomposition is performed. Based on the minimum description length criterion (MDL) and the imaging system noise model, the number of signal eigenvalues ​​is obtained through maximum likelihood estimation to determine the optimal signal-to-noise ratio threshold σ0 at any point in the field of view. The eigenvectors corresponding to eigenvalues ​​greater than σ0 represent the signal S:{u σi =0}, other feature vectors represent N:{u σi =0};The eigenvalues ​​corresponding to the signal-to-noise subspace and the noise subspace satisfy the following equation: （4） In the formula, M represents the number of fluorescent probes in the fluorescent sample (15). λ The eigenvalues ​​obtained from the eigenvalue decomposition are used to perform spectral peak search using the orthogonality of the two subspaces, resulting in a high-resolution image.

Images