CN119335714A

CN119335714A - A light sheet imaging system and method with large field of view and high temporal and spatial resolution

Info

Publication number: CN119335714A
Application number: CN202411254788.9A
Authority: CN
Inventors: 严伟; 郭勇; 翁哓羽; 向睿捷; 屈军乐
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2024-09-09
Filing date: 2024-09-09
Publication date: 2025-01-21

Abstract

The invention provides a large-view-field high-space-time resolution optical sheet imaging system and a method, which relate to the field of optical microscope imaging, wherein the system comprises a laser for generating Gaussian laser; the device comprises a 1/2 wave plate, a spatial light modulator, a scanning galvanometer, a scanning lens, a tube mirror I, a sample pool, a four-dimensional displacement table, a detection objective, a filter and a camera, wherein the 1/2 wave plate is used for adjusting the polarization direction of laser light, the spatial light modulator is used for regulating an optical field, gaussian laser is modulated into a curved beam through the spatial light modulator, the scanning galvanometer is used for synchronously scanning the modulated excitation light and controlling the beam to form an area array image on a sample, the scanning lens is positioned behind the galvanometer and used for collecting the laser beam which is scanned by the area array so as to focus the laser to a target area of the sample, the tube mirror I and an illumination objective are matched to form a microscope system, and the sample pool is used for placing and fixing the sample. The invention realizes high-flux three-dimensional imaging with large field of view and high space-time resolution by LSFM imaging of the curved light beam with obviously stretched axial focal length.

Description

Large-view-field high-space-time resolution optical sheet imaging system and method

Technical Field

The invention relates to the field of optical microscope imaging, in particular to a large-view-field high-space-time resolution light sheet imaging system and method.

Background

The optical microscope has the advantages of non-contact, no damage and specificity, plays an important role in revealing cell morphology, dynamics, interaction, positioning and the like, and is an indispensable important tool in modern biomedical research all the time as an important means for observing the microscopic world. From the initial two-dimensional low-resolution observation of stained plant cells, evolution has been made to high-resolution, multi-dimensional real-time observation of single-cell resolution of tissue organs or living small animals. However, the presence of the diffraction limit limits of light limits its resolution capability for microscopic objects, and thus biological structures with dimensions below 200nm cannot be clearly distinguished. At the same time, phototoxicity, photobleaching, etc. make long-term in vivo imaging difficult. However, a mild microscopic observation means is needed for visualization and quantitative analysis of biological processes in samples such as organisms, tissues, cells and the like, and meanwhile, rapid high-resolution three-dimensional observation can be performed in a large field of view. The Light-sheet fluorescence microscope (Light-Sheet Fluorescence Microscopy, LSFM) plays an increasingly important role in the field of modern biomedicine by virtue of the characteristics of high space-time resolution, low Light damage, suitability for long-time three-dimensional imaging of the dynamic life process of a living sample, and the like.

The Light Sheet Fluorescence Microscope (LSFM) is a wide field three-dimensional microscopic imaging technique that will illuminate and detect orthogonal layouts. The sample is illuminated by a laminar light field (called a light sheet) emitted by an excitation (illumination) objective, the excited fluorescence is collected by a detection objective perpendicular to the light sheet, and by changing the relative position of the light sheet and the sample, a series of two-dimensional fluorescence images are photographed layer by a camera and finally three-dimensional microscopic imaging is realized. The light sheet only illuminates the in-focus area of the sample, and the out-of-focus area is not excited to emit fluorescent signals, so LSFM has the advantages of tomography, small optical damage and the like, and in addition, the wide-field imaging mode based on area array detection also ensures the imaging speed advantage, so that LSFM is very suitable for long-time three-dimensional dynamic observation. LSFM have superior chromatographic capabilities and lower phototoxicity and photobleaching properties compared to wide field fluorescence microscopy, thus enabling rapid three-dimensional imaging of samples with low damage. Compared with a laser scanning confocal microscope, the wide-field detection mode is far higher than confocal imaging in imaging flux, and phototoxicity and photobleaching performance are far smaller than those of confocal imaging. This extremely efficient illumination, detection approach makes it possible to study rapid vital activity processes with fine structures. The low phototoxicity and photobleaching properties enable long-term observation of living samples, making them an irreplaceable role in three-dimensional high-speed imaging of tissue and organs.

LSFM produce two forms of light sheet, static light sheet and dynamic scanning light sheet. The static light sheet directly generates the Gaussian light sheet by the cylindrical mirror, the system is simple, the imaging speed is high, but the problems of contradiction between the visual field and the resolution, uneven energy of the static light sheet and the like exist. Dynamic scanning light sheet (DIGITALLY SCANNED LIGHT-sheet Microscopy, DSLM) is a method of generating light sheet fields by using a scanning galvanometer. Compared with the cylindrical mirror focusing generation of the light sheet, the light sheet obtained by scanning has more uniform intensity distribution and higher energy utilization rate. In addition, the scanning beam adopts various types of non-diffraction beams, the thickness of the optical sheet can reach the submicron level under the same field of view, and higher axial resolution is provided. At present, a Gaussian light sheet field is used for static LSFM, an observation field of view and axial resolution are mutually restricted, a large field of view is observed at the expense of axial resolution, the transverse resolution is limited by diffraction limit, a Bessel light sheet field is mostly used for dynamic LSFM, a digital light sheet is formed through scanning of a galvanometer, and the field of view and the resolution are improved, but the requirement of high flux imaging is difficult to meet.

Disclosure of Invention

Accordingly, the present invention is directed to a large field-of-view, high spatial-temporal resolution optical imaging system and method. The imaging view field is greatly improved on the premise of not losing the axial resolution of the system, and then the transverse resolution of the whole LSFM system is improved on the premise of not changing the numerical aperture of the microscope objective lens, so that super-resolution imaging is realized. The invention realizes high-flux three-dimensional imaging with large field of view and high space-time resolution by LSFM imaging of the curved light beam with obviously stretched axial focal length. Firstly, based on a multiple energy oscillation mechanism, a 'snake-shaped' light field with arbitrarily modulated focal length is generated, and the imaging field of view is greatly improved on the premise of not sacrificing the axial resolution of the system. And then, the influence of side lobes of an excitation light beam on image quality and the penetrability of the near infrared illumination enhanced light sheet are effectively reduced by adopting two-photon absorption. Finally, by combining a random optical reconstruction super-resolution imaging microscope (Stochastic Optical Reconstruction Microscopy, STORM) technology, the transverse resolution of the two-photon LSFM system is improved, and finally, large-view-field high-space-time resolution three-dimensional imaging is realized, so that a technical means is provided for long-time high-space-time resolution three-dimensional imaging of large tissues, organs or animals, and the application of the optical sheet imaging technology in biomedical imaging is further promoted.

In view of the above object, the present invention provides, in a first aspect, a large field of view, high spatial-temporal resolution optical sheet imaging system comprising:

the laser is used for generating Gaussian laser and providing high-energy laser beams so as to meet the requirement of high excitation power of two-photon excitation. The laser can be a femtosecond pulse laser for generating femtosecond pulse laser, and the wavelength of the laser can be tuned (680-1300 nm) to meet different experimental requirements.

The 1/2 wave plate is positioned on the light path of Gaussian laser generated by the laser and used for adjusting the polarization direction of the laser so as to ensure that the laser is incident on a Spatial Light Modulator (SLM) in a correct polarization state.

A Spatial Light Modulator (SLM) for light field modulation, wherein a gaussian laser is modulated into a curved beam by the spatial light modulator, wherein the gaussian light field is converted into a curved "serpentine" light field, which is converted into a curved "serpentine" light field having an arbitrary focal length by loading a specific hologram, thereby significantly improving the imaging field of view without degrading the axial resolution. The space light modulator modulates the Gaussian light field into a bending light beam by loading a hologram, the modulated bending light beam is conjugated to the entrance pupil of the excitation objective lens through a 4f system after passing through a one-dimensional scanning galvanometer, and a snake-shaped light sheet field is generated on the focal plane of the detection objective lens by rapid scanning of the galvanometer.

And the scanning galvanometer is used for synchronously scanning the modulated excitation light and controlling the process of forming the area array imaging on the sample by the light beam. The fast scan of the scanning galvanometer can generate the required "serpentine" field of light across the probe objective focal plane.

And the scanning lens is positioned behind the galvanometer and used for collecting the laser beams scanned by the area array to ensure that the excitation light can be accurately focused on a target area of the sample.

And the tube lens I is matched with the illumination objective lens to form a microscope system.

And the illumination objective lens is used for focusing laser to a focal plane and is matched with the scanning galvanometer to form a digital light sheet so as to realize accurate light sheet illumination.

And the sample pool is used for maintaining the water environment, placing and fixing the sample and ensuring the stability and imaging effect of the sample in the imaging process.

The four-dimensional displacement table is used for moving the sample and realizing three-dimensional imaging by matching with the scanning galvanometer, so that the imaging system can scan and capture the sample in multiple angles and all directions.

And the detection objective lens is used for collecting fluorescent signals emitted by the sample and ensuring high-efficiency fluorescent collection and imaging quality.

The optical filter is used for removing stray light and selectively filtering light rays with specific wavelengths so as to improve the signal-to-noise ratio and the quality of images.

And the tube lens II is positioned between the detection objective lens and the camera and is used for receiving and focusing light rays and carrying out object image amplification with different multiplying powers by matching with the detection objective lens.

The camera (sCMOS) is used for collecting and recording fluorescent image data, has high sensitivity and high-speed imaging capability, and is suitable for three-dimensional imaging with high space-time resolution.

Through the cooperative work of the components, the optical sheet imaging system can greatly improve the imaging view field and realize three-dimensional imaging with high space-time resolution on the premise of not sacrificing the axial resolution of the system, and is particularly suitable for long-time and high space-time resolution imaging of large tissues, organs or animals.

In a second aspect, the present invention also provides a large field of view, high spatial-temporal resolution optical sheet imaging method, comprising the steps of:

(1) The light field generation is that a laser beam of Gaussian laser is generated by a laser, the polarization direction of the laser is regulated by a 1/2 wave plate, and a bending light beam, namely a snake-shaped light field, is generated after the modulation by a spatial light modulator.

(2) And (3) light field modulation, namely loading a hologram by using a spatial light modulator, and converting Gaussian laser beams into a 'snake-shaped' light field so as to realize remarkable expansion of an imaging view field, wherein the 'snake-shaped' light field can greatly improve the imaging view field without sacrificing axial resolution.

(3) And (3) sample excitation, namely focusing the modulated snake-shaped optical field on a focal plane through a scanning galvanometer, a tube mirror I and an illumination objective lens, realizing efficient light sheet excitation, and reducing the influence of an excitation beam sidelobe on image quality through a two-photon absorption technology.

(4) Collecting fluorescent signals emitted by a sample under excitation of a serpentine-shaped light sheet field through a detection objective lens, recording the fluorescent signals through a camera, and generating light sheet two-dimensional imaging data;

(5) And three-dimensional fluorescence acquisition, namely moving the relative positions of the sample and the detection surface through a four-dimensional displacement table on the basis of two-dimensional fluorescence acquisition to generate light sheet image data of different positions, and reconstructing three-dimensional data.

The method can also process the acquired image data, and accurately position fluorescent molecules by using the STORM technology so as to improve the transverse resolution of the system and break through the optical diffraction limit. More specifically, an autofluorescence scintillation probe is adopted, a galvanometer is utilized to rapidly perform one-dimensional scanning to realize wide-field illumination of a large-field light sheet in an open-loop time, and meanwhile, the exposure time of a camera is synchronized, as shown on the right side of fig. 2d, so that compatibility of a light sheet system and STORM technology is further solved, and finally, super-resolution imaging is realized after a single-molecule positioning algorithm is utilized. Thereby significantly improving the lateral resolution of the light sheet system. .

Compared with the prior art, the large-view-field and high-space-time resolution optical sheet imaging system and method provided by the invention have the following beneficial effects:

1. Large field imaging the present invention effectively extends the field of view (field 500 x 500 μm ²) of the imaging system by introducing a "serpentine" light sheet field design. The design can cover a larger imaging area without changing the numerical aperture of the microscope objective, thereby realizing three-dimensional imaging of large tissues, organs or whole animals. This feature is particularly suitable for biomedical research requiring high throughput data acquisition, such as neural network imaging and developmental biology research.

2. The method combines STORM technology, and the transverse resolution (30 nm) of the system is obviously improved. By virtue of the super-resolution imaging capability of STORM, the present system is able to resolve structural details within cells at the nanometer level. Meanwhile, due to the use of the femtosecond pulse laser and the efficient fluorescence acquisition strategy, the system also maintains a higher level in terms of time resolution, and is suitable for researching a rapid dynamic process.

3. The invention improves the imaging depth and reduces the phototoxicity and the photo-bleaching effect by the two-photon excitation technology, thereby ensuring the stability of long-time imaging. This is particularly important for continuous observation and long-term experiments of living samples.

4. The invention uses a Spatial Light Modulator (SLM) to regulate and control the 'S-shaped' light sheet field, so that the focal length of the light sheet field can be adjusted according to experimental requirements. This flexibility allows the user to optimize imaging parameters according to different sample types and experimental requirements, thereby improving imaging efficiency and data quality.

5. Light piece super-resolution the invention combines STORM technique, the system breaks through diffraction limit of traditional optical microscope, and super-resolution imaging is realized. This allows researchers to observe microstructures that are not distinguishable by conventional optical microscopy techniques, especially for clear observation of intracellular substructures and molecular levels.

In summary, the large-field-of-view, high-spatial-temporal resolution optical sheet imaging system and method of the present invention reduce system complexity and cost. Compared with the traditional multi-beam or multi-lens scheme, the system can realize diversified imaging requirements by using single optical path configuration and SLM regulation, thereby reducing equipment investment and maintenance cost. The invention realizes super-resolution imaging under a large view field, greatly improves the three-dimensional imaging speed, and provides a powerful tool for three-dimensional imaging with large flux.

These and other aspects of the application will be more readily apparent from the following description of the embodiments. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application as claimed.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are necessary for the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention and that other embodiments may be obtained according to these drawings without inventive effort for a person skilled in the art.

In the figure:

FIG. 1 is a schematic diagram of a large field of view, high spatial-temporal resolution optical imaging system according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of light field modulation and light sheet super-resolution imaging in a large field of view, high spatial-temporal resolution light sheet imaging system and method according to an embodiment of the invention.

Reference numerals:

1-laser, 2-1/2 wave plate, 3-spatial light modulator, 4-scanning galvanometer, 5-scanning lens, 6-tube mirror I, 7-illumination objective, 8-four-dimensional displacement table, 9-sample cell, 10-detection objective, 11-filter, 12-tube mirror II, and 13-camera.

Detailed Description

The present application will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.

In order to make the objects, technical solutions and advantages of the present application more apparent, the following embodiments of the present application will be described in further detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.

It should be noted that, in the embodiments of the present invention, all the expressions "first" and "second" are used to distinguish two non-identical entities with the same name or non-identical parameters, and it is noted that the "first" and "second" are only used for convenience of expression, and should not be construed as limiting the embodiments of the present invention. Furthermore, the terms "comprise" and "have," and any variations thereof, are intended to cover a non-exclusive inclusion, such as a process, method, system, article, or other step or unit that comprises a list of steps or units.

The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The flow diagrams depicted in the figures are merely illustrative and not necessarily all of the elements and operations/steps are included or performed in the order described. For example, some operations/steps may be further divided, combined, or partially combined, so that the order of actual execution may be changed according to actual situations.

Some embodiments of the present application are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.

Referring to fig. 1, the embodiment of the invention provides a large-view-field high-space-time resolution optical imaging system, which is used for imaging large-view-field high-space-time resolution LSFM capable of breaking through the optical diffraction limit, greatly improving the imaging view field on the premise of not losing the axial resolution of the system, and then improving the transverse resolution of the whole LSFM system under the condition of not changing the numerical aperture of a microscope objective lens to realize super-resolution imaging. Provides technical means for long-time, high space-time resolution and three-dimensional imaging of large tissues, organs or animals, and further promotes the application of the optical sheet imaging technology in biomedical imaging.

Referring to fig. 1, the optical sheet imaging system includes:

a laser 1 for generating Gaussian laser light;

The 1/2 wave plate 2 is used for adjusting the polarization direction of the laser;

the spatial light modulator 3 is used for regulating and controlling an optical field, and Gaussian laser is modulated into a curved beam through the spatial light modulator 3;

The scanning galvanometer 4 is used for synchronously scanning the modulated excitation light and controlling the light beam to form an area array imaging on the sample;

The scanning lens 5 is positioned behind the galvanometer and used for collecting laser beams scanned by the area array so as to focus the laser to a target area of the sample;

the tube lens I6 is matched with the illumination objective lens 7 to form a microscope system;

An illumination objective lens 7 for focusing laser to a focal plane and forming a digital light sheet by matching with the scanning galvanometer 4;

The sample cell 9 is used for maintaining the water environment, and placing and fixing a sample;

The four-dimensional displacement table 8 is used for moving the sample and realizing three-dimensional imaging by matching with the scanning galvanometer 4;

a probe objective lens 10 for collecting fluorescent signals emitted from the sample;

the optical filter 11 is used for removing stray light outside the fluorescent signals collected by the detection objective lens 10;

The tube lens II12 is used for receiving and focusing light rays and is matched with the detection objective lens 10 to amplify object images with different multiplying powers;

camera 13 for acquiring and recording fluorescence images.

In this embodiment, the laser 1 is a femtosecond pulse laser for generating a femtosecond pulse laser, wherein the wavelength tunable range of the femtosecond pulse laser source of the laser 1 is 680-1300nm.

In this embodiment, the 1/2 wave plate 2 is located on the optical path of the gaussian laser generated by the laser 1, and adjusts the polarization direction of the laser light to be incident on the spatial light modulator 3 in a polarized state. The 1/2 wave plate 2 is located between the laser 1 and the spatial light modulator 3, and is used for enabling Gaussian laser generated by the laser 1 to be incident on the spatial light modulator 3 in horizontal polarized light after being regulated by the 1/2 wave plate 2 after being emitted.

In this embodiment, the spatial light modulator 3 further modulates the gaussian light field into a curved light beam by loading a hologram, the modulated curved light beam is conjugated to the entrance pupil of the excitation objective lens through a 4f system after passing through the one-dimensional scanning galvanometer 4, and a "snake-shaped" light sheet field is generated on the focal plane of the detection objective lens 10 by fast scanning through the galvanometer.

Wherein the tube lens I6 is located between the scanning lens 5 and the illumination objective 7 for focusing the excitation light to the focal plane 10, the optical filter 11 is located between the detection objective 10 and the camera 13, and the tube lens II12 is arranged between the optical filter 11 and the camera 13.

The large-view-field high-space-time resolution optical sheet imaging system has only one femtosecond pulse laser source, and the wavelength is tunable (680-1300 nm) to better meet the actual excitation wavelength requirement. The Gaussian laser is emitted and then passes through a 1/2 wave plate 2 to ensure that horizontal polarized light is incident on a spatial light modulator 3 (SLM), the Gaussian beam is modulated into a 'snake-shaped' beam by loading a hologram on the SLM, and the modulated beam is conjugated to the entrance pupil of an excitation objective lens through a 4f system after passing through a one-dimensional scanning galvanometer. The fast scan by the scanning galvanometer 4 creates the desired "serpentine" field of light at the focal plane of the detection objective 10.

The fluorescence emitted by the sample in the sample cell 9 is collected by the detection objective 10 and imaged onto a camera 13 (sCMOS). The 1/2 wave plate 2 is used for adjusting the polarization state of the light beam, the sample is fixed on the four-dimensional displacement table 8, the optical filter 11 is used for filtering stray light except collecting fluorescence, and the type and parameters of the microscope system light sheet can be controlled by a software program. Three-dimensional fluorescence imaging can be realized by scanning the galvanometer 4 in one dimension, matching with sample movement and synchronizing the exposure time of the camera 13. In addition, (1) fig. 1 illustrates two-photon excitation only, and the invention is not limited to two-photon excitation, single-photon excitation and multiphoton excitation, and (2) light beam modulation is not limited to SLM, optical diffraction element and digital micromirror device.

In one embodiment of the present invention, the embodiment of the present invention further provides a large field-of-view, high spatial-temporal resolution optical sheet imaging method, the method comprising the steps of:

(1) The light field is generated by generating a laser beam of Gaussian laser by the laser 1, adjusting the polarization direction of the laser beam by the 1/2 wave plate 2, and bending the laser beam by the spatial light modulator 3.

(2) Light field modulation, namely loading a hologram by using a spatial light modulator 3, converting Gaussian laser beams into a 'snake-shaped' light field, and adjusting focal length to realize remarkable expansion of an imaging view field, wherein the 'snake-shaped' light field can greatly improve the imaging view field without sacrificing axial resolution.

(3) And (3) sample excitation, namely focusing the modulated snake-shaped optical field onto a focal plane 10 through a scanning galvanometer 4, realizing efficient light sheet excitation, and reducing the influence of an excitation light beam sidelobe on image quality through a two-photon absorption technology.

(4) Two-dimensional fluorescence acquisition, the fluorescence signal emitted by the sample under excitation of the "snake-shaped" light sheet field is collected by the detection objective lens 10, and image data generated by the fluorescence signal is recorded by the camera 13.

The invention effectively expands the field of view of an imaging system by introducing a serpentine light sheet field design. The design can cover a larger imaging area without changing the numerical aperture of the microscope objective, thereby realizing three-dimensional imaging of large tissues, organs or whole animals. This feature is particularly suitable for biomedical research requiring high throughput data acquisition, such as neural network imaging and developmental biology research.

The method combines STORM technology, and the transverse resolution of the system is obviously improved. By virtue of the super-resolution imaging capability of STORM, the present system is able to resolve structural details within cells at the nanometer level. Meanwhile, due to the use of the femtosecond pulse laser and the efficient fluorescence acquisition strategy, the system also maintains a higher level in terms of time resolution, and is suitable for researching a rapid dynamic process.

The invention improves the imaging depth and reduces phototoxicity and photobleaching effect by a two-photon excitation technology, thereby ensuring the stability of long-time imaging. This is particularly important for continuous observation and long-term experiments of living samples.

The invention uses the spatial light modulator 3 (SLM) to regulate and control the 'S-shaped' light sheet field, so that the focal length of the light sheet field can be adjusted according to experimental requirements. This flexibility allows the user to optimize imaging parameters according to different sample types and experimental requirements, thereby improving imaging efficiency and data quality.

The invention combines STORM technology, and the system breaks through the diffraction limit of the traditional optical microscope to realize super-resolution imaging. This allows researchers to observe microstructures that are not distinguishable by conventional optical microscopy techniques, especially for clear observation of intracellular substructures and molecular levels.

Referring to fig. 2, fig. 2 is a basic schematic diagram of the implementation of the present invention for realizing large-field-of-view and super-resolution optical imaging based on the optical field regulation technology and the super-resolution technology. As shown in fig. 2a, the present invention is derived from the energy oscillation mechanism, i.e. the energy charging and discharging. The amplitude, phase and position of the light beam are controlled by the light field regulation and control technology (figure 2 b), so that the light beam generation of different oscillation times is realized (figure 2 c), the focal length of the light beam is obviously increased, and a light film field with large field of view illumination is finally generated on the focal plane of the illumination objective lens by matching with the scanning of the galvanometer. The novel light sheet fluorescence microscope based on the light field regulation technology can realize large-field imaging, but the transverse resolution is limited by the numerical aperture and diffraction limit of the detection objective lens. In order to improve the transverse resolution without changing the numerical aperture, a STORM technology is introduced, as shown in a schematic diagram of a wide-field STORM super-resolution imaging principle on the left side of fig. 2d, long-wavelength and short-wavelength lasers are utilized to regulate the fluorescence molecules to randomly generate the mutual transition between a fluorescence quenching state (dark state) and a fluorescence state, most of fluorescence probe molecules in a visual field are converted into the dark state in each cycle, only a small part of fluorescence molecules are randomly activated to emit fluorescence, images of the sparse luminescence fluorescence molecules are not overlapped with each other, highly accurate positioning information of the part of fluorescence molecules can be obtained through subsequent image processing, the accurate positioning of a large number of fluorescence molecules can be realized through repeated random lighting of the sparse fluorescence molecules, and the acquired sparse molecular luminescence image is integrated and reconstructed to obtain a super-resolution microscopic image. Therefore, an autofluorescence scintillation probe is adopted, a galvanometer is utilized to rapidly perform one-dimensional scanning to realize wide-field illumination of a large-field light sheet in an open-loop time, and meanwhile, the exposure time of a camera is synchronized, as shown on the right side of fig. 2d, so that compatibility of a light sheet system and a STORM technology is further solved, and finally, super-resolution imaging is realized after a single-molecule positioning algorithm is utilized. Thereby significantly improving the lateral resolution of the light sheet system.

The foregoing is an exemplary embodiment of the present disclosure, but it should be noted that various changes and modifications could be made herein without departing from the scope of the disclosure as defined by the appended claims. The functions, steps and/or actions of the method claims in accordance with the disclosed embodiments described herein need not be performed in any particular order. Furthermore, although elements of the disclosed embodiments may be described or claimed in the singular, the plural is contemplated unless limitation to the singular is explicitly stated.

It should be understood that as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly supports the exception. It should also be understood that "and/or" as used herein is meant to include any and all possible combinations of one or more of the associated listed items. The foregoing embodiment of the present invention has been disclosed with reference to the number of embodiments for the purpose of description only, and does not represent the advantages or disadvantages of the embodiments.

It will be appreciated by persons skilled in the art that the foregoing discussion of any embodiment is merely exemplary and is not intended to imply that the scope of the disclosure of embodiments of the invention, including the claims, is limited to such examples, that technical features of the above embodiments or different embodiments may be combined and that many other variations of the different aspects of the embodiments of the invention as described above exist within the spirit of the embodiments of the invention, which are not provided in detail for clarity. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the embodiments should be included in the protection scope of the embodiments of the present invention.

Claims

1. A large field-of-view, high spatial-temporal resolution optical sheet imaging system, comprising:

A laser for generating Gaussian laser light;

The 1/2 wave plate is used for adjusting the polarization direction of the laser;

the spatial light modulator is used for regulating and controlling the light field, and Gaussian laser is modulated into a curved beam through the spatial light modulator;

the scanning galvanometer is used for synchronously scanning the modulated excitation light and controlling the light beam to form an area array imaging on the sample;

The scanning lens is positioned behind the galvanometer and used for collecting laser beams scanned by the area array so as to focus the laser to a target area of the sample;

the tube lens I is matched with the illumination objective lens to form a microscope system;

the illumination objective lens is used for focusing laser to a focal plane and forming a digital light sheet by matching with the scanning galvanometer;

the sample cell is used for maintaining the water environment, and placing and fixing the sample;

the four-dimensional displacement table is used for moving the sample and realizing three-dimensional imaging by matching with the scanning galvanometer;

a detection objective lens for collecting fluorescent signals emitted by the sample;

The optical filter is used for removing stray light outside the fluorescent signals collected by the detection objective lens;

And the camera is used for acquiring and recording fluorescent images.

2. The large field of view, high spatial-temporal resolution optical sheet imaging system of claim 1, wherein the laser is a femtosecond pulsed laser for generating femtosecond pulsed laser.

3. The large field of view, high spatial-temporal resolution optical sheet imaging system of claim 2, wherein the wavelength tunable range of the femtosecond pulsed laser source of the laser is 680-1300nm.

4. The large field of view, high spatial and temporal resolution optical imaging system of claim 3 wherein the 1/2 wave plate is positioned in the path of the gaussian laser light produced by the laser for adjusting the polarization of the laser light such that the gaussian light polarization is incident on the spatial light modulator horizontally parallel to the liquid crystal plane.

5. The large field of view, high spatial and temporal resolution optical imaging system of claim 4 wherein the 1/2 wave plate is positioned between the laser and the spatial light modulator for allowing the gaussian laser light generated by the laser to be modulated by the 1/2 wave plate and then incident on the spatial light modulator as horizontally polarized light.

6. The large field of view, high spatial and temporal resolution optical imaging system of claim 5 wherein the spatial light modulator further modulates the gaussian beam into a curved beam by loading a hologram, the modulated curved beam is passed through a one-dimensional scanning galvanometer and then conjugated to the entrance pupil of the excitation objective lens via a 4f system, and the fast scanning by the galvanometer produces a "serpentine" optical field at the focal plane of the detection objective lens.

7. The large field of view, high spatial and temporal resolution optical imaging system of claim 6, wherein the tube lens I is positioned between the scanning lens and the illumination objective lens for focusing the excitation light to the focal plane.

8. The large field of view, high spatial and temporal resolution optical imaging system of claim 7 wherein the optical filter is positioned between the detection objective and the camera, and a tube lens II is further positioned between the optical filter and the camera, the tube lens II being adapted to receive and focus light and to cooperate with the detection objective to amplify the object images at different magnifications.

9. A large field of view, high spatial-temporal resolution optical sheet imaging method, wherein the method is performed based on the large field of view, high spatial-temporal resolution optical sheet imaging system of claim 8, the optical sheet imaging method comprising the steps of:

Step 1), generating a light field, namely generating a laser beam of Gaussian laser through a laser, adjusting the polarization direction of the laser through a 1/2 wave plate, and generating a bending light beam after modulating through a spatial light modulator, namely a snake-shaped light field;

Step 2), light field modulation, namely loading a hologram by using a spatial light modulator, converting Gaussian laser beams into a 'snake-shaped' light field, and adjusting focal length;

step 3), sample excitation, namely focusing the modulated S-shaped optical field on a focal plane through a scanning galvanometer, a tube mirror I and an illumination objective lens, and generating a S-shaped optical sheet field on the focal plane;

Step 4), two-dimensional fluorescence acquisition, namely collecting fluorescent signals emitted by a sample under excitation of a snake-shaped light sheet field through a detection objective lens, recording the fluorescent signals through a camera, and generating light sheet two-dimensional imaging data;

And 5) three-dimensional fluorescence acquisition, namely moving the relative positions of the sample and the detection surface through a four-dimensional displacement table on the basis of two-dimensional fluorescence acquisition to generate light sheet image data of different positions, and reconstructing three-dimensional data.