A fluorescent microscopic imaging system and method based on static optical sheet of super-oscillatory lens

Abstract

This invention relates to a fluorescence microscopy imaging system and method based on a super-oscillating lens static light sheet, comprising an excitation light emitting device, a super-oscillating lens, a lossy light emitting device, and a grating aperture. The observed sample is located between the super-oscillating lens and the grating aperture. The excitation light emitting device emits linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens. The super-oscillating lens modulates the passing linearly polarized light to generate high-energy sidelobes and a sub-diffraction-limited light sheet. The lossy light emitting device emits a parallel lossy beam and reshapes the parallel lossy beam so that it only covers the high-energy sidelobes. This eliminates the influence of the high-energy sidelobes generated by the super-oscillating lens on the light sheet fluorescence microscopy imaging, achieving high signal-to-noise ratio, large field of view, and enhanced resolution static light sheet fluorescence microscopy imaging.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, and in particular to a fluorescence microscopic imaging system and method based on a super-oscillating lens static light sheet. Background Technology

[0002] Light sheet fluorescence microscopy (LSFM) technology (Acta Optica Sinica, 2017, 37(3):71) uses two orthogonal microscope objectives. One objective emits a thin light sheet to excite fluorescent molecules on a thin layer of the sample, while the other objective's focal plane coincides with the excitation light sheet to collect the fluorescence signal. Because the light sheet selectively excites a thin layer of the sample, the fluorescence emitted by this thin layer is entirely in-focus useful signal, effectively reducing the phototoxicity and photobleaching of the illumination light on the sample, suppressing the generation of defocus background, and improving the imaging resolution. Light sheet fluorescence microscopy has become an important fluorescence imaging method in fields such as cell biology, biophysics, and neuroscience. According to the type of light sheet used, light sheet fluorescence microscopy systems can be divided into two categories: one is a fluorescence microscopy system based on a virtual light sheet, and the other is a fluorescence microscopy system based on a static light sheet. Regarding fluorescence microscopy imaging systems based on virtual light sheets, virtual light sheets are light sheets formed by scanning light needles or other light beams. They require precise and expensive mechanical components or spatial light modulators to achieve spatial scanning. The system itself is complex and bulky, and cannot meet the requirements of high-speed real-time acquisition (Opt Lett., 2015, 40(21):5121). Regarding fluorescence microscopy imaging systems based on static light sheets, static light sheets do not require any spatial scanning and can be directly acquired in real time. The system structure is simpler and more conducive to rapid real-time information acquisition and system miniaturization. The static light sheets used in existing fluorescence microscopy imaging systems are mainly Gaussian light sheets. With the numerical aperture unchanged, the thickness of Gaussian light sheets is constrained by the diffraction limit and cannot be thinned further, which limits the further improvement of its resolution. Even if the thickness of the light sheet is reduced by increasing the numerical aperture, the discretization speed of the light sheet will also increase, resulting in a smaller imaging field of view. Therefore, the light sheet fluorescence microscopy imaging system built with traditional Gaussian light sheets has two limitations: on the one hand, it is difficult to further improve the resolution; on the other hand, it is impossible to increase the imaging field of view while improving the resolution. That is, the imaging field of view and resolution are mutually constrained.

[0003] Optical superoscillation refers to the phenomenon where the band-limited function of an optical field oscillates at a frequency faster than its fastest Fourier component (Nano Lett., 2009, 9(3): 1249). A superoscillation lens is a lens that utilizes micro / nano structures to precisely control the optical field behind the lens, achieving specific interference at specific locations to generate superoscillation phenomena (Sci. Appl., 2019, 8: 56). Therefore, superoscillation lenses can achieve focusing beyond the diffraction limit in the far field. With the development of micro / nano optics technology, superoscillation lenses, as an imaging illumination source, have attracted widespread attention due to their advantages such as overcoming the diffraction limit, customizability, and mass production via silicon microprocessors, and have already demonstrated significant technological advantages in the field of super-resolution imaging. In recent years, superoscillation lenses have begun to be used to generate light sheets (Opt. Lett., 2022, 47(13): 3267), the principle of which is as follows: Figure 2 As shown in (a), by optimizing the position and width of the slit on lens 6, the light field behind the lens can be precisely controlled, resulting in a light sheet 7 with a thickness less than the diffraction limit. The static light sheet generated by the super-oscillating lens, compared to a traditional Gaussian light sheet, is as follows: Figure 3 As shown, it has a thinner thickness (less than the diffraction limit) and a smaller dispersion velocity, which improves the resolution of light sheet microscopy and also increases the field of view of the light sheet imaging. However, while the super-oscillating lens generates a light sheet, it is accompanied by high-energy sidelobes 10 that are difficult to eliminate (sidelobes outside the field of view of the super-oscillating lens are called high-energy sidelobes), such as Figure 2 As shown in (b), it generates severe background noise, which reduces the resolution of its imaging and affects the imaging quality, thus hindering its application in the field of fluorescence microscopy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a fluorescence microscopy system and method based on a static light sheet with a super-oscillating lens that can effectively avoid the influence of high-energy side lobes of a super-oscillating lens on fluorescence microscopy imaging of a light sheet, thereby achieving high signal-to-noise ratio, large field of view and enhanced resolution.

[0005] In a first aspect, the technical solution adopted by the present invention is a fluorescence microscopy imaging system based on a super-oscillating lens static light plate, comprising an excitation light emitting device, a super-oscillating lens located on one side of the excitation light emitting device, a lossy light emitting device, a grating aperture located on one side of the lossy light emitting device and opposite to the super-oscillating lens, a stage for placing the observed sample located between the super-oscillating lens and the grating aperture, and an imaging device located above the stage; the excitation light emitting device emits linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens, the linearly polarized light passing through the super-oscillating lens, and the super-oscillating lens... The optical field of the linearly polarized light is modulated to generate high-energy sidelobes and a sub-diffraction-limited light plate that are emitted towards the direction of the observed sample. The lossy light emitting device emits a parallel lossy beam, which passes through the grating stop. The grating stop reshapes the parallel lossy beam, which is then emitted towards the direction of the observed sample. The shaped parallel lossy beam is a parallel lossy beam that only covers the high-energy sidelobes. The sub-diffraction-limited light plate excites the observed sample with fluorescence, and the excited fluorescence is received by the imaging device and used for imaging.

[0006] The beneficial effects of this invention are as follows: By employing the above-mentioned fluorescence microscopy imaging system based on a super-oscillating lens static light sheet, while the super-oscillating lens emits excitation light toward the observed sample, a loss light emitting device emits loss light. By shaping the loss light, the shaped loss light becomes a parallel loss beam that only covers the high-energy sidelobes. This eliminates the influence of the high-energy sidelobes generated by the super-oscillating lens on the light sheet fluorescence microscopy imaging, thereby achieving static light sheet fluorescence microscopy imaging with high signal-to-noise ratio, large field of view, and enhanced resolution.

[0007] Preferably, the thickness of the sub-diffraction-limited plate is less than the thickness of the diffraction-limited plate and less than the thickness of the Gaussian plate. This structure increases both the resolution and field of view of the plate microscopy.

[0008] Preferably, the excitation light emitting device includes an excitation laser, a first beam expander, an aperture stop, a polarizer, and a reflector, all sequentially located on the same optical axis. The excitation laser emits an excitation light beam to the first beam expander, which shapes the beam into a large-diameter parallel beam. The parallel beam is then projected onto the aperture stop, which adjusts the diameter of the parallel beam. The parallel beam after passing through the aperture stop is projected onto the polarizer, which converts the parallel beam into linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens. The linearly polarized light is reflected by the reflector and then projected perpendicularly onto the super-oscillating lens. With this structure, linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens is emitted by the excitation light emitting device, and the linearly polarized light is finally projected perpendicularly onto the super-oscillating lens. This structure is simple and easy to operate.

[0009] Preferably, the lossy light emitting device includes a lossy laser and a second beam expander sequentially located on the same optical axis. The lossy laser emits a lossy light beam to the second beam expander, which shapes the lossy light beam into a large-diameter parallel beam. The parallel beam is then projected onto a grating stop. The parallel beam passing through the grating stop is shaped into a parallel beam that only covers the high-energy sidelobes. With this structure, the lossy laser is expanded and shaped before being projected onto the grating stop. This structure is simple and easy to operate.

[0010] Preferably, the imaging device includes an objective lens, a filter, and a CCD camera arranged sequentially on the same optical axis. The sub-diffraction-limited light sheet is located at the focal length of the objective lens. The sub-diffraction-limited light sheet excites the observed sample with fluorescence. The excited fluorescence is collected by the objective lens, filtered by the filter, and finally captured by the CCD camera. With this structure, the CCD camera ultimately captures the fluorescence emitted by the observed sample, eliminating the influence of high-energy sidelobes generated by the super-oscillating lens on the light sheet fluorescence microscopy imaging, and realizing static light sheet fluorescence microscopy imaging with high signal-to-noise ratio, large field of view, and enhanced resolution.

[0011] Secondly, the technical solution adopted in this invention is a fluorescence microscopy imaging method based on a super-oscillating lens static light sheet, which includes the following steps:

[0012] S1. The excitation laser emits an excitation beam into the first beam expander, and the laser beam is shaped into a large-diameter parallel beam by the first beam expander.

[0013] S2. The parallel beam of light passing through the first beam expander is projected onto the aperture stop;

[0014] S3. The parallel beam of light passing through the aperture stop is projected onto the polarizer, converting the parallel beam into linearly polarized light with the polarization direction parallel to the slit direction of the super-oscillating lens.

[0015] S4. The linearly polarized light is reflected by the mirror and projected perpendicularly onto the super-oscillating lens. The super-oscillating lens modulates the light field of the linearly polarized light, generating high-energy side lobes and a sub-diffraction-limited light sheet behind the super-oscillating lens.

[0016] S5. The depleted laser emits a depleted light beam, which is shaped into a large-diameter parallel beam by the second beam expander. The parallel beam is then projected onto the grating stop, and the parallel beam passing through the grating stop is shaped into a parallel beam that only covers the high-energy side lobes.

[0017] S6. Drive the stage to make the observed sample move step by step, so that the sub-diffraction-limited light plate scans the observed sample. The sub-diffraction-limited light plate excites the observed sample with fluorescence. The excited fluorescence is collected by the objective lens, filtered by the filter, and finally captured by the CCD camera. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the structure of a fluorescence microscopy imaging system based on a super-oscillating lens static light sheet according to the present invention;

[0019] Figure 2 This is a schematic diagram of the super-oscillating lens in this invention;

[0020] Figure 3 This is a comparison diagram of the thickness and dispersion velocity of the light sheet and the Gaussian light sheet generated by the super oscillating lens in this invention;

[0021] Figure 4 This is a schematic diagram illustrating the principle of covering the high-energy side lobes of the super-oscillating lens in this invention.

[0022] Figure 5 for Figure 1 A cross-sectional view along the AA direction;

[0023] Figure 6 This is a schematic diagram illustrating how the computer reassembles each captured frame of image in this invention;

[0024] As shown in the figure: 1. Excitation laser; 2. First beam expander; 3. Aperture stop; 4. Polarizer; 5. Mirror; 6. Superoscillating lens; 7. Sub-diffraction-limited filter; 8. Observation sample; 9. Stage; 10. High-energy sidelobe; 11. Objective lens; 12. Filter; 13. CCD camera; 14. Computer; 15. Lossy laser; 16. Grating stop; 17. Second beam expander. Detailed Implementation

[0025] The invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can implement it based on the description. The scope of protection of the invention is not limited to these specific embodiments.

[0026] Those skilled in the art should understand that, in the disclosure of this invention, the terms "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the above terms should not be construed as limiting this invention.

[0027] Furthermore, the terms "first," "second," and "third" are used only to distinguish descriptions and should not be interpreted as indicating or implying relative importance.

[0028] In the description of the embodiments of this application, it should also be noted that, unless otherwise expressly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0029] This invention relates to a fluorescence microscopy imaging system based on a super-oscillating lens 6 static light plate, such as... Figure 1 As shown, it includes an excitation light emitting device, a super-oscillating lens 6 located on one side of the excitation light emitting device, a loss light emitting device, a grating aperture 16 located on one side of the loss light emitting device and opposite to the super-oscillating lens 6, a stage 9 located between the super-oscillating lens 6 and the grating aperture 16 for placing the observation sample 8, and an imaging device located above the stage 9.

[0030] like Figure 1 As shown, the excitation light emitting device includes an excitation laser 1, a first beam expander 2, an aperture stop 3, a polarizer 4, and a reflector 5, which are sequentially located on the same optical axis. Figure 1 In the process, the excitation laser 1 is a helium-neon laser with a wavelength of 488 nm, the first beam expander 2 is a beam expander with a magnification of 5 times, the observation sample 8 consists of several fluorescent microspheres, each with a diameter of 1 μm, the excitation wavelength is 488 nm, the emission wavelength is 518 nm, the numerical aperture of the super-oscillating lens 6 is 0.138, the glass thickness d1 = 300 μm, the chromium film thickness of the functional layer d2 = 100 nm, and the focal length F = 1428 μm;

[0031] like Figure 1 As shown, the lossy light emitting device includes a lossy laser 15 and a second beam expander 17 located sequentially on the same optical axis; Figure 1 In the process, the depleted laser 15 is a 592-nanometer fiber laser, and the second beam expander 17 is a beam expander with a magnification of 5.

[0032] Figure 1In the process, the excitation laser 1 emits an excitation beam to the first beam expander 2, which shapes the excitation beam into a large-diameter parallel beam. The parallel beam is then projected onto the aperture stop 3, which is used to adjust the diameter of the parallel beam. The parallel beam passing through the aperture stop 3 is projected onto the polarizer 4, which is used to convert the parallel beam into linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens 6. The linearly polarized light is reflected by the mirror 5 and then projected perpendicularly onto the super-oscillating lens 6. The super-oscillating lens 6 modulates the light field of the linearly polarized light, thereby generating a high-energy sidelobe 10 emitted towards the direction of the observed sample 8 and a sub-diffraction-limited light plate 7. The sub-diffraction-limited light plate 7 has a thickness less than the diffraction limit (W = ... The light sheet microscopy imaging field of D=32 μm and the field of view of the super-oscillating lens 6=80 μm are as follows: the depleted light laser 15 emits a depleted light beam to the second beam expander 17, which shapes the depleted light beam into a large-diameter parallel beam. The parallel beam is then projected onto the grating stop 16. The parallel beam passing through the grating stop 16 is shaped into a parallel beam that only covers the high-energy sidelobe 10. Once the high-energy sidelobe 10 is covered by the depleted light, it will not excite the fluorescence of the observed sample 8. In the end, only the sub-diffraction-limited light sheet 7 is retained to excite the fluorescence of the observed sample 8. In this way, the influence of the high-energy sidelobe 10 of the super-oscillating lens 6 on the light sheet fluorescence microscopy imaging can be eliminated, and a large field of view and enhanced resolution light sheet fluorescence microscopy imaging that is not affected by the high-energy sidelobe 10 can be achieved.

[0033] like Figure 1 As shown, the imaging device includes an objective lens 11, a filter 12, and a CCD camera 13, all sequentially located on the same optical axis. The sub-diffraction-limited filter 7 is located at the focal length of the objective lens 11. The sub-diffraction-limited filter 7 excites the observed sample 8 with fluorescence. The excited fluorescence is collected by the objective lens 11, filtered by the filter 12, and finally captured by the CCD camera 13. For each step the observed sample 8 moves, the CCD camera 13 captures the cross-sectional image excited by the filter. Figure 6 As shown, each captured frame is eventually reconstructed by computer 14 to reconstruct a three-dimensional image of the sample.

[0034] In existing technologies, the super-oscillating lens 6 generates unavoidable high-energy side lobes 10 while producing light sheets. The background noise caused by these high-energy side lobes 10 severely impairs the imaging quality and reduces the imaging resolution, hindering the application of the super-oscillating lens 6 in the field of light sheet microscopy. Figure 4As shown, this invention utilizes stimulated emission depletion (STED) technology (Acta Physica Sinica, 2020, 69(10):77) to eliminate the influence of the high-energy sidelobe 10 of the superoscillating lens 6 on the imaging of the optical sheet. STED technology requires two wavelengths of light: one is the excitation light, used to excite the fluorescence of the observed sample 8; the other is the depletion light, which, even when irradiated by the excitation light, will not excite fluorescence in the sample portion irradiated by the depletion light. Therefore, the incident depletion light is shaped using the grating aperture 16, and the shaped depletion light only irradiates the portion of the high-energy sidelobe 10 generated by the optical sheet. Thus, during the imaging of the fluorescent sample, the high-energy sidelobe 10 will not excite the fluorescence of the observed sample 8. The fluorescence emitted by the observed sample 8 is collected by the back-end CCD camera 13 through the filter 12, effectively eliminating the high-energy sidelobe 10 in the imaging result.

[0035] use Figure 1 The fluorescence microscopy imaging system based on a super-oscillating lens 6 static light plate described herein, using, as... Figure 2 The super-oscillating lens 6, as shown, generates a light sheet with a thickness smaller than the diffraction limit and a slow dispersion velocity, enabling large-field-of-view and resolution-enhanced fluorescence microscopy imaging unaffected by the high-energy sidelobes 10. Simultaneously, while the super-oscillating lens 6 emits excitation light toward the observed sample 8, a lossy light emitting device emits lossy light. By shaping the lossy light to create a parallel lossy beam that only covers the high-energy sidelobes 10, the influence of the high-energy sidelobes 10 generated by the super-oscillating lens 6 on the light sheet fluorescence microscopy imaging can be eliminated, achieving high signal-to-noise ratio, large field of view, and enhanced resolution static light sheet fluorescence microscopy imaging.

[0036] This invention also relates to a fluorescence microscopy imaging method based on a super-oscillating lens 6 static light plate, the method comprising the following steps:

[0037] S1. The helium-neon laser emits an excitation beam with a wavelength of 488 nanometers to the first beam expander 2. The laser beam is shaped into a large-diameter parallel beam by the first beam expander 2.

[0038] S2. The parallel beam of light passing through the first beam expander 2 is projected onto the aperture stop 3, and the diameter of the beam can be adjusted by the aperture stop 3.

[0039] S3. The parallel beam passing through the aperture stop 3 is projected onto the polarizer 4, converting the parallel beam into linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens 6.

[0040] S4. The linearly polarized light, after being reflected by the reflector 5, is projected perpendicularly onto the super-oscillating lens 6. The working principle of the super-oscillating lens 6 is as follows: Figure 2As shown, the super-oscillating lens 6 modulates the optical field of linearly polarized light, generating a high-energy sidelobe 10 and a sub-diffraction-limited plate 7 behind the super-oscillating lens 6.

[0041] S5. The depleted laser 15 emits a depleted light beam with a wavelength of 592 nanometers. The depleted light beam is shaped into a large-diameter parallel beam by the second beam expander 17. The parallel beam is then projected onto the grating stop 16, where it is shaped to cover only the high-energy sidelobes 10. Figure 4 As shown, the aperture of the grating is M1 = 150 micrometers and M2 = 70 micrometers; the parallel beam is irradiated only through the aperture M1 with an aperture of 150 micrometers, thus achieving the purpose of covering only the high-energy sidelobe 10.

[0042] S6. Drive the stage 9 using a piezoelectric nano-displacement stage (minimum step 10 nm) to make the observed sample 8 move according to... Figure 5 The scan direction shown produces a 10-nanometer step movement;

[0043] S7 and the light plate 7 are located at the focal length of the objective lens 11. The objective lens 11 has a magnification of 20× and a focal length of 8.5 mm. The cross-sectional image of the observed sample 8 excited by the light plate is acquired by the objective lens 11. The acquired information is filtered by the filter 12 (center wavelength 520 nm, bandwidth 10 nm). The filter 12 only allows the fluorescence emitted by the observed sample 8 to pass through, and finally it is captured by the CCD camera 13.

[0044] S8. For each step the observed sample 8 moves, the CCD camera 13 captures the cross-sectional image excited by the light sheet, such as... Figure 6 As shown, each captured frame is eventually reconstructed by computer 14 to reconstruct a three-dimensional image of the sample.

Claims

1. A fluorescence microscopy imaging system based on a super-oscillating lens (6) static light sheet, characterized in that: The system includes an excitation light emitting device, a super-oscillating lens (6) located on one side of the excitation light emitting device, a lossy light emitting device, a grating stop (16) located on one side of the lossy light emitting device and opposite to the super-oscillating lens (6), a stage (9) located between the super-oscillating lens (6) and the grating stop (16) for placing the observed sample (8), and an imaging device located above the stage (9). The excitation light emitting device emits linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens (6). The linearly polarized light passes through the super-oscillating lens (6), and the super-oscillating lens (6) modulates the light field of the passing linearly polarized light. The system generates a high-energy sidelobe (10) and a sub-diffraction-limited light plate (7) that are emitted toward the observation sample (8). The lossy light emitting device emits a parallel lossy beam, which passes through the grating aperture (16). The grating aperture (16) shapes the parallel lossy beam, which is then emitted toward the observation sample (8). The shaped parallel lossy beam is a parallel lossy beam that only covers the high-energy sidelobe (10). The sub-diffraction-limited light plate (7) excites the observation sample (8) with fluorescence. The excited fluorescence is received by the imaging device and used for imaging.

2. The fluorescence microscopy imaging system based on a super-oscillating lens (6) static light sheet according to claim 1, characterized in that: The thickness of the sub-diffraction-limited plate (7) is less than the thickness of the diffraction-limited plate and less than the thickness of the Gaussian plate.

3. The fluorescence microscopy imaging system based on a super-oscillating lens (6) static light sheet according to claim 2, characterized in that: The excitation light emitting device includes an excitation laser (1), a first beam expander (2), an aperture stop (3), a polarizer (4), and a reflector (5) located sequentially on the same optical axis. The excitation laser (1) emits an excitation light beam to the first beam expander (2), which shapes the excitation light beam into a large-diameter parallel beam. The parallel beam is then projected onto the aperture stop (3), which is used to adjust the diameter of the parallel beam. The parallel beam passing through the aperture stop (3) is projected onto the polarizer (4), which is used to convert the parallel beam into linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens (6). The linearly polarized light is reflected by the reflector (5) and then projected perpendicularly onto the super-oscillating lens (6).

4. A fluorescence microscopy imaging system based on a super-oscillating lens (6) static light sheet according to claim 3, characterized in that: The lossy light emitting device includes a lossy laser (15) and a second beam expander (17) located sequentially on the same optical axis. The lossy laser (15) emits a lossy light beam to the second beam expander (17), which shapes the lossy light beam into a large-diameter parallel beam. The parallel beam is then projected onto a grating stop (16). The parallel beam passing through the grating stop (16) is shaped into a parallel beam that only covers the high-energy sidelobes (10).

5. A fluorescence microscopy imaging system based on a super-oscillating lens (6) static light sheet according to claim 4, characterized in that: The imaging device includes an objective lens (11), a filter (12), and a CCD camera (13) located sequentially on the same optical axis. The sub-diffraction-limited filter (7) is located at the focal length of the objective lens (11). The sub-diffraction-limited filter (7) excites the observed sample (8) with fluorescence. The excited fluorescence is collected by the objective lens (11), filtered by the filter (12), and finally captured by the CCD camera (13).

6. A fluorescence microscopy imaging method based on a static light sheet with a super-oscillating lens (6), characterized in that: The method includes the following steps: S1. The excitation laser (1) emits an excitation beam to the first beam expander (2), and the laser beam is shaped into a large-diameter parallel beam by the first beam expander (2). S2, The parallel beam of light passing through the first beam expander (2) is projected onto the aperture stop (3); S3. The parallel beam passing through the aperture stop (3) is projected onto the polarizer (4), converting the parallel beam into linearly polarized light with a polarization direction parallel to the slit direction of the super-oscillating lens (6). S4. The linearly polarized light is reflected by the mirror (5) and projected vertically onto the super-oscillating lens (6). The super-oscillating lens (6) modulates the light field of the linearly polarized light, generating a high-energy sidelobe (10) and a sub-diffraction-limited light plate (7) behind the super-oscillating lens (6). S5. The depleted laser (15) emits a depleted light beam. The depleted light beam is shaped into a large-diameter parallel beam by the second beam expander (17). The parallel beam is then projected onto the grating stop (16). The parallel beam passing through the grating stop (16) is shaped into a parallel beam that only covers the high-energy sidelobe (10). S6. Drive the stage (9) to make the observation sample (8) move in a stepping manner, so that the sub-diffraction-limited light plate (7) scans the observation sample (8), the sub-diffraction-limited light plate (7) excites the fluorescence of the observation sample (8), the excited fluorescence is collected by the objective lens (11), then filtered by the filter (12), and finally captured by the CCD camera (13).

Images