An optical sheet super-resolution microscopy system and method based on optical switch molecules

Abstract

This invention discloses a light-sheet super-resolution microscopy system and method based on light-switching molecules. First, a set wavelength of turn-on light is emitted and projected onto the sample, switching the light-switching molecules on the sample to the "on" state. Then, a set wavelength of turn-off light is emitted and modulated into a hollow ring light sheet, which is also emitted onto the sample, turning the light-switching molecules in the bright region illuminated by the hollow ring light sheet into the "dark" state. Finally, a readout light is emitted and compressed into a regular solid light sheet, which is then projected onto the sample. The regular solid light sheet and the hollow ring light sheet are coaxially overlapped, exciting the light-switching molecules in the "on" state within the dark region of the hollow ring light sheet to emit fluorescence. The fluorescence emitted by the light-switching molecules is then collected. The actual effective light sheet thickness generated by this invention is much thinner than that of a regular solid light sheet. Simultaneously, it effectively reduces signals outside the focal plane, thereby achieving axial super-resolution and improving the imaging signal-to-noise ratio, providing an effective tool for super-resolution tomography of cellular or subcellular structures.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy imaging technology, and in particular to a light-sheet super-resolution microscopy system and method based on light-switching molecules. Background Technology

[0002] Fluorescence microscopy, with its advantages of being non-destructive, non-invasive, allowing for specific labeling, and enabling real-time dynamic imaging of living cells, has been widely used in life science research. Compared to point scanning microscopy, wide-field fluorescence microscopy offers faster imaging speeds, but suffers from lower axial resolution. To improve axial resolution, light-sheet fluorescence microscopy (LSFM) has been proposed. LSFM differs from traditional microscopy in its illumination method: the illumination (or excitation) light and the imaging light are not coaxial. Typically, the optical axes of the illumination (or excitation) light path and the imaging light path are orthogonal, meaning the illumination (or excitation) light is a thin "light sheet" parallel to the imaging plane. Only a thin layer of sample on the imaging plane is illuminated, while the samples above and below are not. Its axial resolution is determined by the thickness of the light sheet, and it avoids the coaxial background noise common in coaxial illumination. The simplest way to generate a light sheet is to introduce a cylindrical lens into the optical path. The light passing through this lens maintains a constant width but is compressed into a plane in height, then passes through the illumination objective to form a "light sheet" on the imaging plane. Typically, the imaging objective is placed perpendicular to the illumination objective, with the imaging plane coinciding with the light plate, capturing the fluorescence signal excited by the light plate. Therefore, LSFM (Laser-Screen Tomography) features wide-field excitation and wide-field imaging via light plates, resulting in faster imaging speeds and higher axial resolution and lower background noise compared to conventional wide-field microscopes. The width and thickness of the light plate generated using this method are determined by the NA (Number of Atmosphere) value of the illumination objective, but the axial resolution of LSFM is mutually constrained by the imaging field of view. For example, using an illumination objective with a smaller NA allows for a wider range of uniform illumination, i.e., a larger field of view, but correspondingly, the light plate thickness is also greater, leading to a decrease in axial resolution; conversely, a high NA objective produces a light plate with a smaller field of view but better axial resolution. Currently, for larger samples (e.g., organoids, thickness > 100 μm), optical focusing can produce light plates approximately 1-5 μm thick, obtaining good optical section tomographic images. However, due to the limitations of the optical diffraction limit, it is impossible to produce light plates thinner than 1 μm, limiting the application of light plates in tomographic imaging of smaller subcellular structures (approximately 1-10 μm thick).

[0003] Meanwhile, in traditional LSFM, the imaging and illumination objectives are perpendicular to each other, and the imaging objective needs to maintain a certain distance from the sample. To prevent the two objectives from colliding, a low-power objective with a long working distance is required, which is not suitable for high-power objectives with high NA values. This poses a challenge for detecting cellular or subcellular level structures. A novel LSFM design based on oblique plane microscopy (OPM) solves the challenges of sample accessibility and detection efficiency, allowing imaging in conventional samples such as high-NA slides and well plates. However, due to the diffraction properties of light, even in high-NA OPM systems (e.g., NA ~ 1.3), the thickness of the slide cannot exceed the optical diffraction limit. Summary of the Invention

[0004] The purpose of this invention is to provide a super-resolution optical sheet microscopy system and method based on optically switched molecules, which can break through the optical diffraction limit, realize ultra-thin optical sheets with thinner thickness, and achieve optical sheet microscopy imaging with higher axial resolution.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a light-sheet super-resolution microscopy system based on optically switching molecules. The system includes: an on-light optical path module, an off-light optical path module, a readout light optical path module, a sample imaging module, and a fluorescence acquisition module; the sample imaging module includes a sample incorporating optically switching molecules. The activation light path module is used to emit activation light of a set wavelength and transmit it to the sample imaging module to switch the optical switch molecules on the sample to the on state. The shut-off light path module is used to emit shut-off light of a set wavelength and adjust it into a hollow ring light sheet, which is then emitted to the sample imaging module to turn the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample into a dark state. The readout light optical path module is used to emit readout light and compress it into a regular solid light sheet, which is then emitted to the sample imaging module. The regular solid light sheet and the hollow ring light sheet are coaxially overlapped, which excites the light switch molecules in the dark region of the hollow ring light sheet to emit fluorescence. The sample imaging module emits fluorescence from the light-switching molecules into the fluorescence acquisition module; The fluorescence acquisition module is used to collect the fluorescence emitted by the photo-switching molecules and form a fluorescence microscopic image.

[0006] Furthermore, the optical path module for opening the light includes a second pulse light source, a second excitation filter, and a third reflector, which are sequentially arranged on the same horizontal axis; The second pulsed light source is used to emit turn-on light of a set wavelength, the second excitation filter is used to filter out stray light in the turn-on light, and the third reflector is used to reflect the turn-on light to the sample imaging module.

[0007] Furthermore, the optical path shut-off module includes a first pulse light source, a first excitation filter, a first semi-reflective lens, a second reflector, a vortex phase plate, a second semi-reflective lens, and a cylindrical lens. The first pulse light source, the first excitation filter, and the first semi-reflective lens are arranged on the same horizontal axis, and the second reflector, the vortex phase plate, the second semi-reflective lens, and the cylindrical lens are arranged on the same horizontal axis. The first pulsed light source is used to emit pulsed laser light of a set wavelength. The first excitation filter is used to filter out stray light in the pulsed laser light. The first half-reflecting lens is used to split the pulsed laser light into two beams. The beam reflected by the first half-reflecting lens is used as the shut-off light and is reflected to the second mirror. After passing through the second mirror, the shut-off light is phase-modulated by a vortex phase plate and modulated into a hollow ring beam in the shape of a donut. Then it passes through the second half-reflecting lens, and then the beam is focused in one direction by a cylindrical lens before being emitted to the sample imaging module.

[0008] Furthermore, the readout optical path module includes the first pulse light source, the first excitation filter, the first semi-reflective lens, the second semi-reflective lens, and the cylindrical lens, and also includes a first reflector, wherein the first reflector and the first semi-reflective lens are arranged on the same horizontal axis; The first half-reflective lens is used to split the pulsed laser beam into two beams. The beam transmitted by the first half-reflective lens is used as the readout light and is emitted to the first reflector. The readout light is reflected by the first reflector to the second half-reflective lens, and is reflected by the second half-reflective lens and coaxially with the turn-off light. It then enters the cylindrical lens to be compressed into a regular solid light sheet and emitted to the sample imaging module.

[0009] Furthermore, the sample imaging module includes a first dichroic mirror, a second dichroic mirror, a first tube mirror, a first objective lens, and a sample arranged coaxially in sequence, and a photomolecule switch is provided on the sample; The first dichroic mirror is used to receive the on light, off light, or read light, and emits it to the second dichroic mirror. The light is then transmitted through the second dichroic mirror to the first tube mirror and focused onto the sample by the first objective lens. The fluorescence is reflected by the second dichroic mirror after passing through the first objective lens and the first tube lens to the fluorescence acquisition module.

[0010] Furthermore, the fluorescence acquisition module includes a second tube mirror, a second objective lens, a third objective lens, a third tube mirror, an emission filter, and a camera. The second tube mirror receives the fluorescence emitted by the photo-switching molecules, and after passing through the second objective lens, the third objective lens, and the third tube mirror, the stray light is filtered out by the emission filter before reaching the detection surface of the camera for reception.

[0011] Furthermore, the angle between the axes of the second and third objectives matches the tilt angle of the sample illuminated by the on-light.

[0012] Furthermore, the turn-on light is a pulsed laser with a wavelength of 405 nm, the turn-off light is a pulsed laser with a wavelength of 488 nm, and the wavelength of the readout light is the same as that of the turn-off light.

[0013] This invention also provides a super-resolution microscopy method for light-switching molecules, applied to the aforementioned super-resolution microscopy system for light-switching molecules, comprising the following steps: It emits a set wavelength of turn-on light and sends it onto the sample, turning the optical switch molecules on the sample into the on state; It emits a set wavelength of shut-off light, which is then modulated into a hollow ring light sheet and emitted onto the sample, turning the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample into a dark state. The reading light is emitted and compressed into a regular solid light sheet, which is then emitted onto the sample. The regular solid light sheet and the hollow ring light sheet are coaxially overlapped, exciting the light-switching molecules in the dark region of the hollow ring light sheet to emit fluorescence. The fluorescence emitted by the photo-switching molecules is collected to form a fluorescence microscopic image.

[0014] According to specific embodiments provided by the present invention, the following technical effects are disclosed: Optically switching molecules play a crucial role in generating ultrathin optical sheets. Optically switching molecules exhibit two different states under laser irradiation of different wavelengths, referred to as on (fluorescent) and off (dark) states. When excited by on-light, the optical switching molecules transition to the on state; and when irradiated by off-light, they transition back to the dark state. Fluorescent molecules in the on state emit fluorescence when excited by readout light. The optical sheet super-resolution microscopy system and method based on optical switching molecules provided by the present invention sets optical switching molecules on the sample, fully utilizing their characteristics. First, on-light of a set wavelength is used to switch the optical switching molecules to the on state. Then, the off-light of a set wavelength is modulated into a hollow ring-shaped optical sheet, switching the optical switching molecules in the bright region irradiated by the hollow ring-shaped optical sheet to the dark state, while the hollow dark region irradiated by the hollow ring-shaped optical sheet remains in the on state. Finally, a beam of readout light is compressed into a regular solid optical sheet to excite the optical switching molecules in the on state in the dark region to emit fluorescence. Since the light-switching molecules in the overlapping area of ​​the light sheet and the aforementioned hollow annular light sheet are switched to a dark state and do not emit fluorescence, the actual area emitting fluorescence is only the hollow dark area of ​​the light sheet. Therefore, the effective thickness of the light sheet that actually emits fluorescence is much thinner than that of a normal light sheet, thus achieving the generation of a thinner light sheet and ultimately improving the axial resolution of LSFM. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the structure of the optical sheet super-resolution microscopy system based on optically switched molecules provided by the present invention; Explanation of reference numerals in the attached figures: 1. First pulsed light source; 2. First excitation filter; 3. First semi-reflective lens; 4. First reflecting mirror; 5. Second reflecting mirror; 6. Vortex phase plate; 7. Second semi-reflective lens; 8. Cylindrical lens; 9. Second pulsed light source; 10. Second excitation filter; 11. Third reflecting mirror; 12. First dichroic mirror; 13. Second dichroic mirror; 14. First tube mirror; 15. First objective lens; 16. Sample; 17. Second tube mirror; 18. Second objective lens; 19. Third objective lens; 20. Third tube mirror; 21. Emission filter; 22. Camera. Detailed Implementation

[0017] 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.

[0018] The optical sheet super-resolution microscopy system and method based on optically switched molecules provided by this invention produces an actual effective optical sheet thickness that is much thinner than that of ordinary solid optical sheets. At the same time, it can effectively reduce the signal outside the focal plane, thereby achieving axial super-resolution and improving the imaging signal-to-noise ratio, providing an effective tool for super-resolution tomography of cellular or subcellular structures.

[0019] like Figure 1 As shown, the optical sheet super-resolution microscopy system based on optically switching molecules provided by the present invention includes: an on-light optical path module, an off-light optical path module, a readout optical path module, a sample imaging module, and a fluorescence acquisition module; the sample imaging module includes a sample 16 in which optically switching molecules are disposed. The activation light path module is used to emit activation light of a set wavelength and transmit it to the sample imaging module to switch the optical switch molecules on sample 16 to the on state. The shut-off light path module is used to emit shut-off light of a set wavelength and adjust it into a hollow ring light sheet, which is then emitted to the sample imaging module to turn the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample 16 into a dark state. The readout light optical path module is used to emit readout light and compress it into a regular solid light sheet, which is then emitted to the sample imaging module. On sample 16, the regular solid light sheet and the hollow ring light sheet are coaxially overlapped, which excites the light switch molecules in the dark region of the hollow ring light sheet to emit fluorescence. The sample imaging module emits fluorescence from the light-switching molecules into the fluorescence acquisition module; The fluorescence acquisition module is used to collect the fluorescence emitted by the photo-switching molecules and form a fluorescence microscopic image.

[0020] Specifically, the light-activating optical path module includes a second pulse light source 9, a second excitation filter 10, and a third reflector 11, which are sequentially arranged on the same horizontal axis.

[0021] The optical path shut-off module includes a first pulse light source 1, a first excitation filter 2, a first semi-reflective lens 3, a second reflector 5, a vortex phase plate 6, a second semi-reflective lens 7, and a cylindrical lens 8. The first pulse light source 1, the first excitation filter 2, and the first semi-reflective lens 3 are arranged on the same horizontal axis, and the second reflector 5, the vortex phase plate 6, the second semi-reflective lens 7, and the cylindrical lens 8 are arranged on the same horizontal axis.

[0022] The reading light path module includes a first pulse light source 1, a first excitation filter 2, a first semi-reflective lens 3, a second semi-reflective lens 7, and a cylindrical lens 8, and also includes a first reflecting mirror 4. The first reflecting mirror 4 and the first semi-reflective lens 3 are arranged on the same horizontal axis. The sample imaging module includes a first dichroic mirror 12, a second dichroic mirror 13, a first tube mirror 14, a first objective lens 15, and a sample 16 arranged coaxially in sequence. A photomolecular switch is provided on the sample 16.

[0023] The fluorescence acquisition module includes a second tube lens 17, a second objective lens 18, a third objective lens 19, a third tube lens 20, an emission filter 21, and a camera 22.

[0024] The working principle of the optical sheet super-resolution microscopy system based on optically switched molecules is as follows: The second pulsed light source 9 emits laser light as the turn-on light, which is filtered out by the second excitation filter 10 to remove stray light. Then the turn-on light is reflected by the third mirror 11, and then transmitted by the first dichroic mirror 12 and the second dichroic mirror 13 to the first tube mirror 14 and focused onto the back focal plane of the first objective lens 15. The first objective lens 15 then illuminates the sample 16 in a wide field, turning the optical switch molecules into the open state.

[0025] Subsequently, a laser beam is emitted from the first pulsed light source 1, and stray light is filtered out after passing through the first excitation filter 2. Then, the beam is split into two beams by the first semi-reflective lens 3. One beam, reflected by the first semi-reflective lens 3, serves as the shut-off light, and the other beam, transmitted through the first semi-reflective lens 3, serves as the readout light. The shut-off light passes through the second reflecting mirror 5 and is then phase-modulated by the vortex phase plate 6, thus modulating the beam into a hollow ring-shaped beam in the shape of a donut. This beam then passes through the second semi-reflective lens 7. After being focused in one direction by the cylindrical lens 8, the beam is reflected by the first dichroic mirror 12. The reflected light is coaxial and shares the same path as the turn-on light, and is then transmitted through the second dichroic mirror 13 to the first tube mirror 14, where it is focused onto the sample 16 by the first objective lens 15. This forms a hollow ring-shaped shut-off light on the sample 16, converting the light-switching molecules in the bright area illuminated by the hollow ring-shaped light plate, which were originally excited to the open state by the turn-on light, to the dark state. The readout light is reflected by the first mirror 4 to the second semi-reflective lens 7, and then reflected by the second semi-reflective lens 7 along the same path as the turn-off light. It enters the cylindrical lens 8 to be compressed into a solid light plate, then reflected by the first dichroic mirror 12 to the second dichroic mirror 13, and then incident on the first tube mirror 14. Finally, it is focused onto the sample 16 by the first objective lens 15, creating a diffraction-limited solid light plate in the sample, i.e., the readout light plate. The solid light plate and the hollow annular light plate are coaxially overlapped. The pulse from the solid light plate arrives at the sample 16 later than the pulse from the hollow annular light plate. The optimal time delay is when the pulse from the hollow annular light plate has already completed the transition of molecules in the corresponding illuminated bright region from an open state to a dark state. At this point, the readout light plate only excites the light-switching molecules in the hollow dark region to produce fluorescence.

[0026] After passing through the first objective lens 15 and the first tube mirror 14, the fluorescence is reflected by the second dichroic mirror 13, and then passes through the second tube mirror 17, the second objective lens 18, the third objective lens 19, and the third tube mirror 20. After stray light is filtered out by the emission filter 21, the fluorescence reaches the detection surface of the camera 22 and is received. The angle between the axes of the second objective lens 18 and the third objective lens 19 is matched with the tilt angle of the sample 16 illuminated by the on-light.

[0027] For example, the turn-on light is a pulsed laser with a wavelength of 405 nm, the turn-off light is a pulsed laser with a wavelength of 488 nm, and the wavelength of the readout light is the same as that of the turn-off light.

[0028] This invention also provides a super-resolution microscopy method for light-switching molecules, applied to the aforementioned super-resolution microscopy system for light-switching molecules, comprising the following steps: It emits a set wavelength of turn-on light and sends it onto the sample, turning the optical switch molecules on the sample into the on state; It emits a set wavelength of shut-off light, which is then modulated into a hollow ring light sheet and emitted onto the sample, turning the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample into a dark state. The reading light is emitted and compressed into a regular solid light sheet, which is then emitted onto the sample. The regular solid light sheet and the hollow ring light sheet are coaxially overlapped, exciting the light-switching molecules in the dark region of the hollow ring light sheet to emit fluorescence. The fluorescence emitted by the photo-switching molecules is collected to form a fluorescence microscopic image.

[0029] Specifically, firstly, a 405 nm pulsed turn-on light is emitted to switch the optical switch molecules to the on state. Then, a vortex phase plate and a cylindrical lens are used to modulate a 488 nm pulsed turn-off light into a hollow ring-shaped optical sheet. This turns the optical switch molecules in the bright region illuminated by the hollow ring-shaped optical sheet into the dark state, while the hollow dark region illuminated by the hollow ring-shaped optical sheet remains in the on state. Finally, a 488 nm pulsed readout light is compressed into a solid optical sheet using a cylindrical lens to excite the optical switch molecules in the on state in the dark region to emit fluorescence; the fluorescence emitted by the optical switch molecules is then collected.

[0030] Since the light-switching molecules in the overlapping area of ​​the light sheet and the aforementioned hollow annular light sheet are switched to a dark state and do not emit fluorescence, the actual area emitting fluorescence is only the hollow dark area of ​​the light sheet. Therefore, the effective thickness of the light sheet that actually emits fluorescence is much thinner than that of a normal light sheet, thus achieving the generation of a thinner light sheet and ultimately improving the axial resolution of LSFM.

[0031] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A light-sheet super-resolution microscopy system based on optically switched molecules, characterized in that, include: The system includes an optical path module for activation, an optical path module for deactivation, an optical path module for reading, a sample imaging module, and a fluorescence acquisition module; the sample imaging module includes a sample equipped with optically switched molecules. The activation light path module is used to emit activation light of a set wavelength and transmit it to the sample imaging module to switch the optical switch molecules on the sample to the on state. The shut-off light path module is used to emit shut-off light of a set wavelength and adjust it into a hollow ring light sheet, which is then emitted to the sample imaging module to turn the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample into a dark state; the shut-off light path module includes a first pulse light source (1), a first excitation filter (2), a first semi-reflective lens (3), a second reflector (5), a vortex phase plate (6), a second semi-reflective lens (7), and a cylindrical lens (8). The first pulse light source (1), the first excitation filter (2), and the first semi-reflective lens (3) are arranged on the same horizontal axis, and the second reflector (5), the vortex phase plate (6), the second semi-reflective lens (7), and the cylindrical lens (8) are arranged on the same horizontal axis; the first pulse light source (1) is used to emit shut-off light of a set wavelength and adjust it into a hollow ring light sheet, which is then emitted to the sample imaging module to turn the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample into a dark state; the shut-off light path module includes a first pulse light source (1), a first excitation filter (2), a first semi-reflective lens (3), a second reflector (5), a vortex phase plate (6), a second semi-reflective lens (7), and a cylindrical lens (8) are arranged on the same horizontal axis; the first pulse light source (1) is used to emit shut-off light of a set wavelength and adjust it into a hollow ring light sheet, which is then emitted to the sample imaging module to turn the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample into a dark state ... The pulsed laser emits a set wavelength. The first excitation filter (2) is used to filter out stray light in the pulsed laser. The first semi-reflective lens (3) is used to split the pulsed laser into two beams. The beam reflected by the first semi-reflective lens (3) is used as the shut-off light and is reflected to the second mirror (5). After passing through the second mirror (5), the shut-off light is phase-modulated by the vortex phase plate (6) and modulated into a hollow ring beam in the shape of a donut. It then passes through the second semi-reflective lens (7) and is then focused in one direction by the cylindrical lens (8) and emitted to the sample imaging module. A hollow ring light is formed on the sample (16) to switch the light switch molecules in the bright area illuminated by the hollow ring light plate from the open state to the dark state. The readout light optical path module is used to emit readout light and compress it into a solid light sheet, which is then emitted to the sample imaging module. The solid light sheet and the hollow ring light sheet are coaxially overlapped, exciting the light-switching molecules in the dark region of the hollow ring light sheet to emit fluorescence. The readout light optical path module includes a first pulse light source (1), a first excitation filter (2), a first semi-reflective lens (3), a second semi-reflective lens (7), a cylindrical lens (8), and a first reflector (4). The first reflector (4) and the first semi-reflective lens (3) are arranged on the same horizontal axis. The first half-reflective lens (3) is used to split the pulsed laser into two beams. The beam transmitted by the first half-reflective lens (3) is used as the readout light and is emitted to the first reflector (4). The readout light is reflected by the first reflector (4) to the second half-reflective lens (7), and is reflected by the second half-reflective lens (7) and coaxially with the shut-off light. It enters the cylindrical lens (8) to be compressed into a solid light sheet and emitted to the sample imaging module. The solid light sheet pulse arrives at the sample (16) later than the hollow ring light sheet pulse. The solid light sheet only excites the light-switching molecules in the hollow dark area to produce fluorescence. The sample imaging module emits fluorescence from the light-switching molecules into the fluorescence acquisition module; The fluorescence acquisition module is used to collect the fluorescence emitted by the photo-switching molecules and form a fluorescence microscopic image.

2. The optical sheet super-resolution microscopy system based on optically switched molecules according to claim 1, characterized in that, The light-opening optical path module includes a second pulse light source (9), a second excitation filter (10), and a third reflector (11) arranged sequentially on the same horizontal axis. The second pulse light source (9) is used to emit turn-on light of a set wavelength, the second excitation filter (10) is used to filter out stray light in the turn-on light, and the third reflector (11) is used to reflect the turn-on light to the sample imaging module.

3. The optical sheet super-resolution microscopy system based on optically switched molecules according to claim 1, characterized in that, The sample imaging module includes a first dichroic mirror (12), a second dichroic mirror (13), a first tube mirror (14), a first objective lens (15), and a sample (16) arranged coaxially in sequence. The sample (16) is provided with light-switching molecules. The first dichroic mirror (12) is used to receive the turn-on light, turn-off light or read light, and emits it to the second dichroic mirror (13). The light is transmitted from the second dichroic mirror (13) to the first tube mirror (14) and then focused onto the sample (16) by the first objective lens (15). After passing through the first objective lens (15) and the first tube lens (14), the fluorescence is reflected by the second dichroic mirror (13) to the fluorescence acquisition module.

4. The optical sheet super-resolution microscopy system based on optically switched molecules according to claim 1, characterized in that, The fluorescence acquisition module includes a second tube mirror (17), a second objective lens (18), a third objective lens (19), a third tube mirror (20), an emission filter (21), and a camera (22). The second tube mirror (17) receives the fluorescence emitted by the photoswitch molecules, and after passing through the second objective lens (18), the third objective lens (19), and the third tube mirror (20), the stray light is filtered out by the emission filter (21) and then reaches the detection surface of the camera (22) for reception.

5. The optical sheet super-resolution microscopy system based on optically switched molecules according to claim 4, characterized in that, The angle between the axes of the second objective (18) and the third objective (19) matches the tilt angle of the sample (16) illuminated by the light.

6. The optical sheet super-resolution microscopy system based on optically switched molecules according to claim 1, characterized in that, The turn-on light is a pulsed laser with a wavelength of 405 nm, the turn-off light is a pulsed laser with a wavelength of 488 nm, and the wavelength of the readout light is the same as that of the turn-off light.

7. A super-resolution optical sheet microscopy method based on optically switched molecules, characterized in that, The optical sheet super-resolution microscopy system based on optically switched molecules, as described in any one of claims 1-6, comprises the following steps: It emits a set wavelength of turn-on light and sends it onto the sample, turning the optical switch molecules on the sample into the on state; It emits a set wavelength of shut-off light, which is then modulated into a hollow ring light sheet and emitted onto the sample, turning the light-switching molecules in the bright area illuminated by the hollow ring light sheet on the sample into a dark state. The reading light is emitted and compressed into a regular solid light sheet, which is then emitted onto the sample. The regular solid light sheet and the hollow ring light sheet are coaxially overlapped, exciting the light-switching molecules in the dark region of the hollow ring light sheet to emit fluorescence. The fluorescence emitted by the photo-switching molecules is collected to form a fluorescence microscopic image.

Images