CN117991486A - Optical switch molecule-based light sheet super-resolution microscopic system and method - Google Patents
Optical switch molecule-based light sheet super-resolution microscopic system and method Download PDFInfo
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Abstract
The invention discloses a light sheet super-resolution microscopic system and a method based on optical switch molecules, which firstly emit opening light with set wavelength, and emit the opening light onto a sample to turn the optical switch molecules on the sample into an on state; then, emitting closing light with set wavelength, regulating and controlling the closing light into a hollow annular light sheet, emitting the hollow annular light sheet onto a sample, and converting optical switch molecules in a bright area irradiated by the hollow annular light sheet on the sample into a dark state; finally, reading light is emitted, the reading light is compressed into a common solid light sheet, the common solid light sheet is emitted to a sample, the common solid light sheet and the hollow annular light sheet are coaxially overlapped, and the optical switch molecules in an on state in a dark area in the hollow annular light sheet are excited to emit fluorescence; and collecting fluorescence emitted by the optical switch molecules. The thickness of the practical effective light sheet produced by the invention is far thinner than that of a common solid light sheet, and simultaneously, the signal outside a focal plane can be effectively reduced, so that the axial super-resolution is realized, the imaging signal-to-noise ratio is improved, and an effective tool is provided for super-resolution chromatography of a cell or subcellular level structure.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging, in particular to a light sheet super-resolution microscopic system and method based on optical switch molecules.
Background
Fluorescent microscope is widely used in life science research with the advantages of no damage, no invasion, specific mark and real-time dynamic imaging of living cells. Compared with the point scanning microscopy, the wide-field fluorescence microscope has higher imaging speed, but low axial resolution. To increase the axial resolution, light-sheet fluorescence microscopy imaging techniques (light-sheet fluorescence microscopy, LSFM) have been proposed. LSFM differ from conventional microscopes in the manner of illumination of the excitation light: its illumination (or excitation) light is not coaxial with the imaging light, typically the illumination (or excitation) light path is mutually orthogonal to the optical axis of the imaging light path, i.e. the illumination (or excitation) light is a thin "light sheet" parallel to the imaging plane, only a thin sample on the imaging plane is illuminated, and the samples above and below it are not illuminated by light, their axial resolution is determined by the thickness of the light sheet, and there is no coaxial background light noise as in the case of normal coaxial illumination. The simplest method of producing an optical sheet is to introduce a cylindrical lens in the optical path through which the light passes, the width of which remains unchanged, but is compressed in height into a plane, and then pass through an illumination objective to form an "optical sheet" on the imaging surface. The imaging objective is generally perpendicular to the illumination objective, the imaging surface is coincided with the light sheet, and fluorescent signals excited by the light sheet are obtained, so LSFM has the characteristics of light sheet wide-field excitation and wide-field imaging, the imaging speed is higher, and compared with a common wide-field microscope, the axial resolution is higher, and the background noise is smaller. The width and thickness of the light sheet produced using this method are determined by the NA value of the illumination objective, but the axial resolution of LSFM is constrained by the imaging field of view. For example, using an illumination objective with a smaller NA enables a wider range of uniform illumination, i.e. a larger field of view, but correspondingly a greater thickness of the light sheet, resulting in a reduced axial resolution; the high NA objective lens produces a smaller field of view of the light sheet, but better axial resolution. Currently, for larger volumes of samples (e.g., organoids, thickness >100 um), one can produce optical sheets about 1-5um thick by optical focusing to obtain good optical slice tomographic images. However, due to limitations in optical diffraction limits, one cannot produce optical sheets less than 1um thick, limiting the use of optical sheets in chromatographic imaging of smaller subcellular structures (about 1-10um thick).
At the same time, in conventional LSFM, the imaging and illumination objectives are perpendicular to each other, and the imaging objective needs to be kept at a distance from the sample. In order to prevent collision of the two objective lenses, a low power objective lens with a long working distance is required, and the high power objective lens with a high NA value is not suitable. This presents difficulties in detecting cellular or subcellular level structures. The novel LSFM design based on a bevel plane microscope (oblique plane microscopy, OPM) solves the challenges of sample accessibility and detection efficiency, allowing imaging in conventional samples such as high NA slides and multiwell plates. However, due to the diffraction characteristics of light, even a high NA OPM system (e.g., NA 1.3) optical sheet cannot break through the optical diffraction limit.
Disclosure of Invention
The invention aims to provide an optical switch molecule-based light sheet super-resolution microscopic system and method, which can break through the optical diffraction limit, realize an ultrathin light sheet with thinner thickness and realize light sheet microscopic imaging with higher axial resolution.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides an optical switch molecule-based light sheet super-resolution microscopic system, which comprises: opening the light path module, closing the light path module, reading the light path module, and the sample imaging module and the fluorescence acquisition module; the sample imaging module comprises a sample provided with optical switch molecules;
the opening light path module is used for emitting opening light with set wavelength and transmitting the opening light to the sample imaging module to turn the optical switch molecules on the sample into an on state;
the light path closing module is used for emitting closing light with set wavelength, regulating and controlling the light path closing module into a hollow annular light sheet, emitting the hollow annular light sheet to the sample imaging module, and converting light switch molecules in a bright area irradiated by the hollow annular light sheet on a sample into a dark state;
The reading light path module is used for emitting reading light, compressing the reading light into a common solid light sheet, transmitting the common solid light sheet to the sample imaging module, coaxially overlapping the common solid light sheet and the hollow annular light sheet, and exciting an optical switch molecule in an on state in a dark area in the hollow annular light sheet to emit fluorescence;
the sample imaging module emits fluorescence emitted by the optical switch molecules to the fluorescence acquisition module;
the fluorescence acquisition module is used for acquiring fluorescence emitted by the optical switch molecules to form a fluorescence microscopic image.
Further, the opening light path module comprises a second pulse light source, a second excitation filter and a third reflector which are sequentially arranged on the same horizontal axis;
The second pulse light source is used for emitting opening light with set wavelength, the second excitation filter is used for filtering stray light in the opening light, and the third reflector is used for reflecting the opening light to the sample imaging module.
Further, the light path closing module comprises a first pulse light source, a first excitation filter, a first half-reflecting half-lens, a second reflecting mirror, a vortex phase plate, a second half-reflecting half-lens and a cylindrical lens, wherein the first pulse light source, the first excitation filter and the first half-reflecting half-lens are arranged on the same horizontal axis, and the second reflecting mirror, the vortex phase plate, the second half-reflecting half-lens and the cylindrical lens are arranged on the same horizontal axis;
The first pulse light source is used for emitting pulse laser with set wavelength, the first excitation filter is used for filtering stray light in the pulse laser, the first half-reflecting half-mirror is used for splitting the pulse laser into two light beams, and the light beams reflected by the first half-reflecting half-mirror are used as closing light and reflected to the second reflecting mirror; after the light is closed, the light is subjected to phase modulation through the vortex phase plate, modulated into a doughnut-shaped hollow annular light beam, transmitted through the second half-reflecting half-lens, converged in one direction through the cylindrical lens and then emitted to the sample imaging module.
Further, the reading light path module comprises the first pulse light source, a first excitation filter, a first half-reflecting half-lens, a second half-reflecting half-lens, a cylindrical lens and a first reflecting mirror, wherein the first reflecting mirror and the first half-reflecting half-lens are arranged on the same horizontal axis;
the first half-reflecting half-mirror is used for splitting the pulse laser into two beams, wherein the beam transmitted by the first half-reflecting half-mirror is used as reading light and is emitted to the first reflecting mirror; the reading light is reflected to the second half-reflecting half-lens by the first reflecting mirror, reflected by the second half-reflecting half-lens and is coaxial with the closed light, enters the cylindrical lens to be compressed into a common solid light sheet, and is emitted to the sample imaging module.
Further, the sample imaging module comprises a first dichroic mirror, a second dichroic mirror, a first tube mirror, a first objective lens and a sample which are coaxially arranged in sequence, wherein an optical molecular switch is arranged on the sample;
the first dichroic mirror is used for receiving the opening light, the closing light or the reading light, transmitting the opening light, the closing light or the reading light to the second dichroic mirror, transmitting the opening light, the closing light or the reading light to the first tube mirror through the second dichroic mirror, and focusing the opening light, the closing light or the reading light on a sample through the first objective lens;
The fluorescence is reflected to the fluorescence acquisition module by the second dichroic mirror after passing through the first objective lens and the first tube lens.
Further, the fluorescence collection module comprises a second tube lens, a second object lens, a third tube lens, an emission filter and a camera, wherein the second tube lens receives fluorescence emitted by the optical switch molecule, and after passing through the second tube lens, the third object lens and the third tube lens, stray light is filtered by the emission filter, and then reaches a detection surface of the camera to be received.
Further, the included angle between the axes of the second objective and the third objective is matched with the inclination angle of the open light irradiation sample.
Further, the opening light is a pulse laser with a wavelength of 405 nm, the closing light is a pulse laser with a wavelength of 488: 488 nm, and the reading light has the same wavelength as the closing light.
The invention also provides a light sheet super-resolution microscopy method based on the optical switch molecules, which is applied to the light sheet super-resolution microscopy system based on the optical switch molecules, and comprises the following steps:
emitting opening light with set wavelength, emitting the opening light to the sample, and converting the optical switch molecules on the sample into an on state;
Emitting closing light with set wavelength, regulating the closing light into a hollow annular light sheet, emitting the hollow annular light sheet onto a sample, and converting light switch molecules in a bright area irradiated by the hollow annular light sheet on the sample into a dark state;
Emitting reading light, compressing the reading light into a common solid light sheet, and transmitting the common solid light sheet to a sample, wherein the common solid light sheet and the hollow annular light sheet are coaxially overlapped, and exciting light switch molecules in an on state in a dark area in the hollow annular light sheet to emit fluorescence;
and collecting fluorescence emitted by the optical switch molecules to form a fluorescence microscopic image.
According to the specific embodiment provided by the invention, the invention discloses the following technical effects: optical switch molecules play an important role in the production of ultra-thin optical sheets. The optical switch molecules have two different states under different wavelengths of laser light, called an on (fluorescent) and an off (dark) state. The optical switch molecule is converted into an on state under the excitation of the opening light; and the optical switch molecule is changed into a dark state again under the irradiation of the off light. While fluorescent molecules in the on state emit fluorescent light under excitation of the read light. According to the optical switch molecule-based light sheet super-resolution microscopic system and method provided by the invention, the optical switch molecules are arranged on the sample, the characteristics of the optical switch molecules are fully utilized, the optical switch molecules are turned into an on state by utilizing the on light with the set wavelength, then the off light with the set wavelength is regulated and controlled into the hollow annular light sheet, the optical switch molecules in the bright area irradiated by the hollow annular light sheet are turned into a dark state, and the hollow dark area irradiated by the hollow annular light sheet still maintains the on state. Finally, a beam of reading light is compressed into a common solid light sheet to excite the optical switch molecules in the on state in the dark area to emit fluorescence. At this time, the optical switch molecules in the overlapping area of the light sheet and the hollow annular light sheet are turned into a dark state, no fluorescence is emitted, and the area which emits fluorescence actually is only the hollow dark area of the light sheet, so that the effective thickness of the light sheet which emits fluorescence actually is far thinner than that of the common light sheet, thereby realizing the generation of the light sheet with a thinner thickness, and finally realizing the axial resolution improvement of LSFM.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a light sheet super-resolution microscopic system based on optical switch molecules;
Reference numerals illustrate: 1. a first pulsed light source; 2. a first excitation filter; 3. a first half mirror; 4. a first mirror; 5. a second mirror; 6. a vortex phase plate; 7. a second half mirror; 8. a cylindrical lens; 9. a second pulsed light source; 10. a second excitation filter; 11. a third mirror; 12. a first dichroic mirror; 13. a second dichroic mirror; 14. a first tube mirror; 15. a first objective lens; 16. a sample; 17. a second tube mirror; 18. a second objective lens; 19. a third objective lens; 20. a third tube mirror; 21. an emission filter; 22. and a camera.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The optical switch molecule-based light sheet super-resolution microscopy system and method provided by the invention have the advantages that the thickness of the generated actual effective light sheet is far thinner than that of a common solid light sheet, and meanwhile, the signal outside a focal plane can be effectively reduced, so that the axial super-resolution is realized, the imaging signal to noise ratio is improved, and an effective tool is provided for super-resolution chromatography of a cell or subcellular level structure.
As shown in fig. 1, the optical switch molecule-based optical sheet super-resolution microscopy system provided by the invention comprises: opening the light path module, closing the light path module, reading the light path module, and the sample imaging module and the fluorescence acquisition module; the sample imaging module comprises a sample 16 provided with optical switch molecules;
the opening light path module is used for emitting opening light with set wavelength and transmitting the opening light to the sample imaging module to turn the optical switch molecules on the sample 16 into an on state;
The light path closing module is used for emitting closing light with set wavelength, regulating and controlling the light path closing module into a hollow annular light sheet, emitting the hollow annular light sheet to the sample imaging module, and converting light switch molecules in a bright area irradiated by the hollow annular light sheet on the sample 16 into a dark state;
the reading light path module is used for emitting reading light, compressing the reading light into a common solid light sheet, transmitting the common solid light sheet to the sample imaging module, coaxially overlapping the common solid light sheet and the hollow annular light sheet on the sample 16, and exciting the optical switch molecules in the dark area of the hollow annular light sheet to emit fluorescence;
the sample imaging module emits fluorescence emitted by the optical switch molecules to the fluorescence acquisition module;
the fluorescence acquisition module is used for acquiring fluorescence emitted by the optical switch molecules to form a fluorescence microscopic image.
Specifically, the light path opening module includes a second pulse light source 9, a second excitation filter 10, and a third reflecting mirror 11 sequentially disposed on the same horizontal axis.
The light path closing module comprises a first pulse light source 1, a first excitation filter 2, a first half-reflecting half-lens 3, a second reflecting mirror 5, a vortex phase plate 6, a second half-reflecting half-lens 7 and a cylindrical lens 8, wherein the first pulse light source 1, the first excitation filter 2 and the first half-reflecting half-lens 3 are arranged on the same horizontal axis, and the second reflecting mirror 5, the vortex phase plate 6, the second half-reflecting half-lens 7 and the cylindrical lens 8 are arranged on the same horizontal axis.
The reading light path module comprises the first pulse light source 1, a first excitation filter 2, a first half-reflecting half-lens 3, a second half-reflecting half-lens 7, a cylindrical lens 8 and a first reflecting mirror 4, wherein the first reflecting mirror 4 and the first half-reflecting half-lens 3 are arranged on the same horizontal axis; the sample imaging module comprises a first dichroic mirror 12, a second dichroic mirror 13, a first tube mirror 14, a first objective lens 15 and a sample 16 which are coaxially arranged in sequence, wherein an optical molecular switch is arranged on the sample 16.
The fluorescence acquisition module comprises a second tube mirror 17, a second object mirror 18, a third object mirror 19, a third tube mirror 20, an emission filter 21 and a camera 22.
The working principle of the optical switch molecule-based light sheet super-resolution microscopic system is as follows:
the second pulse light source 9 emits laser light as opening light, and stray light is filtered out through the second excitation filter 10. The opening light is reflected by the third reflecting mirror 11, is transmitted by the first dichroic mirror 12 and the second dichroic mirror 13 to the first tube mirror 14, is focused on the back focal plane of the first objective lens 15, and is irradiated onto the sample 16 in a wide field through the first objective lens 15, so that the optical switch molecule is turned into an on state.
Subsequently, the first pulse light source 1 emits laser light, and stray light is filtered out after passing through the first excitation filter 2. Then, the first half mirror 3 splits the beam into two beams, wherein one beam is the beam reflected by the first half mirror 3 as the off light, and the other beam is the beam transmitted by the first half mirror 3 as the reading light. After passing through the second mirror 5, the off-light is phase-modulated via the vortex phase plate 6, so that its beam is modulated into a doughnut-shaped hollow ring-shaped beam, which is transmitted through the second half-mirror 7. Then, after the light beams are converged in one direction through the cylindrical lens 8, the light beams are reflected by the first dichroic mirror 12, the reflected light and the opening light are coaxially and commonly routed, the reflected light is transmitted to the first tube mirror 14 through the second dichroic mirror 13 and then focused on the sample 16 through the first objective lens 15, the hollow annular light sheet-shaped closing light is formed on the sample 16, and the light switch molecules which are originally excited to the opening state by the opening light in the bright area irradiated by the hollow annular light sheet are converted to the dark state. The reading light is reflected to the second half-reflecting half-lens 7 by the first reflecting mirror 4, and is reflected by the second half-reflecting half-lens 7 to be coaxial with the closed light, and enters the cylindrical lens 8 to be compressed into a common solid light sheet, and is reflected to the second dichroic mirror 13 by the first dichroic mirror 12, and then enters the first tube mirror 14, and finally is focused to the sample 16 by the first objective lens 15, so that a diffraction-limited common solid light sheet, namely a reading light sheet, is generated in the sample, and the common solid light sheet and the hollow annular light sheet are coaxially overlapped, the pulse of the common solid light sheet arrives at the sample 16 later than the pulse of the hollow annular light sheet, and the optimal time delay is that the pulse of the hollow annular light sheet has completed converting molecules in the corresponding irradiation light area from an on state to a dark state. At this time, the light-reading sheet causes only the optical switch molecules in the hollow dark region to be excited to generate fluorescence.
The fluorescence is reflected by the second dichroic mirror 13 after passing through the first objective 15 and the first tube mirror 14, and then passes through the second tube mirror 17, the second object mirror 18, the third object mirror 19 and the third tube mirror 20, and the stray light is filtered by the emission filter 21 and then reaches the detection surface of the camera 22 to be received. Wherein the included angle between the axis of the second objective lens 18 and the axis of the third objective lens 19 matches the inclination angle of the open light irradiation sample 16.
Illustratively, the on light is a pulsed laser having a wavelength of 405 nm, the off light is a pulsed laser having a wavelength of 488: 488 nm, and the read light has the same wavelength as the off light.
The invention also provides a light sheet super-resolution microscopy method based on the optical switch molecules, which is applied to the light sheet super-resolution microscopy system based on the optical switch molecules, and comprises the following steps:
emitting opening light with set wavelength, emitting the opening light to the sample, and converting the optical switch molecules on the sample into an on state;
Emitting closing light with set wavelength, regulating the closing light into a hollow annular light sheet, emitting the hollow annular light sheet onto a sample, and converting light switch molecules in a bright area irradiated by the hollow annular light sheet on the sample into a dark state;
Emitting reading light, compressing the reading light into a common solid light sheet, and transmitting the common solid light sheet to a sample, wherein the common solid light sheet and the hollow annular light sheet are coaxially overlapped, and exciting light switch molecules in an on state in a dark area in the hollow annular light sheet to emit fluorescence;
and collecting fluorescence emitted by the optical switch molecules to form a fluorescence microscopic image.
Specifically, first, pulse on light with a wavelength of 405 nm is emitted to turn the optical switch molecules into an on state, then pulse off light with a wavelength of 488 nm is regulated and controlled into a hollow annular light sheet by using a vortex phase sheet and a cylindrical lens, the optical switch molecules in a bright area irradiated by the hollow annular light sheet are turned into a dark state, and a hollow dark area irradiated by the hollow annular light sheet still remains in the on state. Finally, compressing a beam of pulse reading light with the wavelength of 488 nm into a common solid light sheet by using a cylindrical lens so as to excite the optical switch molecules in the on state in a dark area to emit fluorescence; and collecting fluorescence emitted by the optical switch molecules.
At this time, the optical switch molecules in the overlapping area of the light sheet and the hollow annular light sheet are turned into a dark state, no fluorescence is emitted, and the area which emits fluorescence actually is only the hollow dark area of the light sheet, so that the effective thickness of the light sheet which emits fluorescence actually is far thinner than that of the common light sheet, thereby realizing the generation of the light sheet with a thinner thickness, and finally realizing the axial resolution improvement of LSFM.
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to assist in understanding the methods of the present invention and the core ideas thereof; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.
Claims (9)
1. An optical switch molecule-based optical sheet super-resolution microscopy system, comprising: opening the light path module, closing the light path module, reading the light path module, and the sample imaging module and the fluorescence acquisition module; the sample imaging module comprises a sample provided with optical switch molecules;
the opening light path module is used for emitting opening light with set wavelength and transmitting the opening light to the sample imaging module to turn the optical switch molecules on the sample into an on state;
the light path closing module is used for emitting closing light with set wavelength, regulating and controlling the light path closing module into a hollow annular light sheet, emitting the hollow annular light sheet to the sample imaging module, and converting light switch molecules in a bright area irradiated by the hollow annular light sheet on a sample into a dark state;
The reading light path module is used for emitting reading light, compressing the reading light into a common solid light sheet, transmitting the common solid light sheet to the sample imaging module, coaxially overlapping the common solid light sheet and the hollow annular light sheet, and exciting an optical switch molecule in an on state in a dark area in the hollow annular light sheet to emit fluorescence;
the sample imaging module emits fluorescence emitted by the optical switch molecules to the fluorescence acquisition module;
the fluorescence acquisition module is used for acquiring fluorescence emitted by the optical switch molecules to form a fluorescence microscopic image.
2. The optical switch molecule based optical sheet super-resolution microscopy system according to claim 1, wherein the on-optical path module comprises a second pulse light source (9), a second excitation filter (10) and a third mirror (11) sequentially arranged on the same horizontal axis;
The second pulse light source (9) is used for emitting opening light with set wavelength, the second excitation filter (10) is used for filtering stray light in the opening light, and the third reflector (11) is used for reflecting the opening light to the sample imaging module.
3. The optical switch molecule based optical sheet super-resolution microscopy system according to claim 1, wherein the light path closing module comprises a first pulse light source (1), a first excitation filter (2), a first half-reflecting half-lens (3), a second reflecting mirror (5), a vortex phase plate (6), a second half-reflecting half-lens (7) and a cylindrical lens (8), wherein the first pulse light source (1), the first excitation filter (2) and the first half-reflecting half-lens (3) are arranged on the same horizontal axis, and the second reflecting mirror (5), the vortex phase plate (6), the second half-reflecting half-lens (7) and the cylindrical lens (8) are arranged on the same horizontal axis;
The first pulse light source (1) is used for emitting pulse laser with set wavelength, the first excitation filter (2) is used for filtering stray light in the pulse laser, the first half-reflecting half-mirror (3) is used for splitting the pulse laser into two light beams, and the light beam reflected by the first half-reflecting half-mirror (3) is used as closing light and is reflected to the second reflecting mirror (5); after the light is closed through the second reflecting mirror (5), the light is subjected to phase modulation through the vortex phase plate (6), modulated into a doughnut-shaped hollow annular light beam, transmitted through the second half-reflecting half-lens (7), converged in one direction through the cylindrical lens (8), and emitted to the sample imaging module.
4. The optical switch molecule based optical sheet super-resolution microscopy system according to claim 3, wherein the read light path module comprises the first pulse light source (1), a first excitation filter (2), a first half-reflecting half-lens (3), a second half-reflecting half-lens (7), a cylindrical lens (8), and further comprises a first reflecting mirror (4), wherein the first reflecting mirror (4) and the first half-reflecting mirror (3) are arranged on the same horizontal axis;
The first half-reflecting half-mirror (3) is used for splitting the pulse laser into two beams, wherein the beam transmitted by the first half-reflecting half-mirror (3) is used as reading light and is emitted to the first reflecting mirror (4); the reading light is reflected to the second half-reflecting half-lens (7) by the first reflecting mirror (4), and is reflected by the second half-reflecting half-lens (7) to be coaxial and shared with the closed light, enters the cylindrical lens (8) to be compressed into a common solid light sheet, and is emitted to the sample imaging module.
5. The optical switch molecule based optical sheet super-resolution microscopy system according to claim 1, wherein the sample imaging module comprises a first dichroic mirror (12), a second dichroic mirror (13), a first tube mirror (14), a first objective lens (15) and a sample (16) coaxially arranged in sequence, wherein an optical molecule switch is arranged on the sample (16);
The first dichroic mirror (12) is used for receiving the opening light, the closing light or the reading light, emitting the opening light, the closing light or the reading light to the second dichroic mirror (13), transmitting the opening light, the closing light or the reading light to the first tube mirror (14) through the second dichroic mirror (13), and focusing the opening light, the closing light or the reading light on the sample (16) through the first objective lens (15);
The fluorescence is reflected to the fluorescence acquisition module by the second dichroic mirror (13) after passing through the first objective lens (15) and the first tube lens (14).
6. The optical switch molecule-based optical sheet super-resolution microscopy system according to claim 1, wherein the fluorescence acquisition module comprises a second objective lens (17), a second objective lens (18), a third objective lens (19), a third objective lens (20), an emission filter (21) and a camera (22), wherein the second objective lens (17) receives fluorescence emitted by the optical switch molecule, and after passing through the second objective lens (18), the third objective lens (19) and the third objective lens (20), the emission filter (21) filters stray light and then reaches a detection surface of the camera (22) to be received.
7. The optical switch molecule based optical sheet super resolution microscopy system according to claim 6, wherein the included angle between the axes of the second objective lens (18) and the third objective lens (19) is matched to the tilt angle of the open light illuminated sample (16).
8. The optical switch molecule based optical sheet super resolution microscopy system according to claim 1, wherein the on light is a pulsed laser with a wavelength of 405 nm, the off light is a pulsed laser with a wavelength of 488 nm, and the read light has the same wavelength as the off light.
9. The optical switch molecule-based optical sheet super-resolution microscopy method is characterized by being applied to the optical switch molecule-based optical sheet super-resolution microscopy system as claimed in any one of claims 1-8, and comprises the following steps:
emitting opening light with set wavelength, emitting the opening light to the sample, and converting the optical switch molecules on the sample into an on state;
Emitting closing light with set wavelength, regulating the closing light into a hollow annular light sheet, emitting the hollow annular light sheet onto a sample, and converting light switch molecules in a bright area irradiated by the hollow annular light sheet on the sample into a dark state;
Emitting reading light, compressing the reading light into a common solid light sheet, and transmitting the common solid light sheet to a sample, wherein the common solid light sheet and the hollow annular light sheet are coaxially overlapped, and exciting light switch molecules in an on state in a dark area in the hollow annular light sheet to emit fluorescence;
and collecting fluorescence emitted by the optical switch molecules to form a fluorescence microscopic image.
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