Large-view-field and high-temporal-spatial-resolution X-ray plate imaging system and method

Abstract

The invention provides a large-view-field and high-temporal-spatial-resolution light sheet imaging system and method, and relates to the field of optical microscope imaging, and the system comprises a laser which is used for generating Gaussian laser; the 1 / 2 wave plate is used for adjusting the polarization direction of the laser; the spatial light modulator is used for light field regulation and control, and the Gaussian laser is modulated into a bent light beam through the spatial light modulator; the scanning galvanometer is used for synchronously scanning the modulated exciting light and controlling the light beam to form area array imaging on the sample; the scanning lens is positioned behind the galvanometer and is used for collecting the laser beams subjected to area array scanning, so that the laser is focused to a target area of the sample; the tube lens I is matched with the illumination objective lens to form a microscope system; the sample pool is used for placing and fixing a sample; a four-dimensional displacement table, a detection objective lens, an optical filter and a camera. According to the invention, through LSFM imaging of the curved light beam with the axial focal length obviously stretched, high-flux three-dimensional imaging with large field of view and high spatial-temporal resolution is realized.

Description

Technical Field

[0001] The present invention relates to the field of optical microscope imaging, and in particular to a light sheet imaging system and method with a large field of view and high temporal and spatial resolution. Background Art

[0002] Optical microscopy has the advantages of non-contact, non-destructive and specific. It plays an important role in revealing cell morphology, dynamics, interactions and positioning. As an important means of observing the microscopic world, it has always been an indispensable tool in modern biomedical research. From the initial two-dimensional low-resolution observation of stained plant cells, it has evolved to high-resolution, multi-dimensional real-time observation of tissues, organs or living small animals with single-cell resolution. However, the existence of the diffraction limit of light limits its resolution ability for microscopic objects, so it is impossible to clearly distinguish biological structures below 200nm in size. At the same time, phototoxicity and photodrift make it difficult to perform long-term in vivo imaging. However, the visualization and quantitative analysis of biological processes in samples such as organisms, tissues, and cells requires a gentle microscopic observation method that can perform fast and high-resolution three-dimensional observations in a large field of view. Light-sheet fluorescence microscopy (LSFM) plays an increasingly important role in the field of modern biomedicine due to its high spatiotemporal resolution, low light damage, and suitability for long-term three-dimensional imaging of dynamic life processes of living samples.

[0003] Light sheet fluorescence microscopy (LSFM) is a wide-field three-dimensional microscopic imaging technique that arranges illumination and detection orthogonally. The sample is illuminated by a thin sheet of light field (called a light sheet) emitted by the excitation (illumination) objective lens, and the excited fluorescence is collected by the detection objective lens perpendicular to the light sheet. By changing the relative position of the light sheet and the sample, the camera takes a series of two-dimensional fluorescence images layer by layer and finally achieves three-dimensional microscopic imaging. The light sheet only illuminates the in-focus area of ​​the sample, and the out-of-focus area will not be excited to emit fluorescence signals. Therefore, LSFM has the advantages of tomographic imaging and low photodamage. In addition, the wide-field imaging method based on area array detection also guarantees the imaging speed advantage, making LSFM very suitable for long-term three-dimensional dynamic observation. Compared with wide-field fluorescence microscopy, LSFM has super tomographic capabilities and lower phototoxicity and photobleaching, so it can perform low-damage rapid three-dimensional imaging of samples. Compared with laser scanning confocal microscopy, its wide-field detection method is much higher than confocal imaging in terms of imaging flux, and its phototoxicity and photobleaching are also much lower than the latter. This extremely efficient illumination and detection method makes it possible to study fast life processes with fine structures. Its low phototoxicity and photobleaching properties make it possible to observe living samples for a long time, making it an irreplaceable role in three-dimensional high-speed imaging of tissues and organs.

[0004] LSFM generates light sheets in two forms: static light sheets and dynamic scanning light sheets. Static light sheets are directly generated by cylindrical mirrors to generate Gaussian light sheets. The system is simple and the imaging speed is fast, but there are problems such as the contradiction between field of view and resolution and uneven energy of static light sheets. Dynamic scanning light sheets, namely digitally scanned light-sheet microscopy (DSLM), use scanning galvanometers to generate light sheet fields. Compared with the light sheet generated by focusing with cylindrical mirrors, the intensity distribution of the light sheet obtained by scanning is more uniform and the energy utilization rate is higher. In addition, the scanning beam uses a variety of types of non-diffraction beams. The thickness of the light sheet can reach sub-micron level under the same field of view, providing higher axial resolution. At present, static LSFM uses Gaussian light sheet fields. The observation field of view and axial resolution are mutually restricted. Large field of view observation requires sacrificing axial resolution, and the lateral resolution is limited by the diffraction limit; dynamic LSFM mostly uses Bessel light sheet fields, which are formed by galvanometer scanning to form digital light sheets. The field of view and resolution are improved, but it is difficult to meet the needs of high-throughput imaging. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a light sheet imaging system and method with a large field of view and high spatiotemporal resolution. The imaging field of view is greatly improved without losing the axial resolution of the system; then the lateral resolution of the entire LSFM system is improved without changing the numerical aperture of the microscope objective lens, so as to achieve super-resolution imaging. The present invention realizes high-throughput three-dimensional imaging with a large field of view and high spatiotemporal resolution through LSFM imaging of a bent light beam with a significantly stretched axial focal length. First, based on a multiple energy oscillation mechanism, a "snake-shaped" light field with an arbitrarily modulated focal length is generated, which greatly improves the imaging field of view without sacrificing the axial resolution of the system. Then, two-photon absorption is used to effectively reduce the influence of the side lobes of the excitation beam on the image quality and near-infrared illumination is used to enhance the penetration of the light sheet. Finally, combined with the stochastic optical reconstruction super-resolution imaging microscope (STORM) technology, the lateral resolution of the two-photon LSFM system is improved, and finally a large field of view and high spatiotemporal resolution three-dimensional imaging is achieved, providing technical means for long-term, high spatiotemporal resolution, and three-dimensional imaging of large tissues, organs or animals, and further promoting the application of light sheet imaging technology in biomedical imaging.

[0006] Based on the above objectives, in a first aspect, the present invention provides a light sheet imaging system with a large field of view and high spatiotemporal resolution, the system comprising:

[0007] Laser: used to generate Gaussian laser, provide high-energy laser beam, and meet the high excitation power requirements of two-photon excitation. The laser can be a femtosecond pulse laser, which is used to generate femtosecond pulse laser. The wavelength of the laser is tunable (680-1300nm) to meet different experimental requirements.

[0008] 1 / 2 wave plate: Located in the optical path of the Gaussian laser generated by the laser, it is used to adjust the polarization direction of the laser to ensure that the laser is incident on the spatial light modulator (SLM) in the correct polarization state.

[0009] Spatial light modulator (SLM): used for light field control. Gaussian laser is modulated into a curved light beam by a spatial light modulator, wherein the Gaussian light field is converted into a curved "snake" light field, and the Gaussian light field is converted into a curved "snake" light field with an arbitrary focal length by loading a specific hologram, thereby significantly improving the imaging field of view without reducing the axial resolution. The spatial light modulator also modulates the Gaussian light field into a curved light beam by loading a hologram, and the modulated curved light beam passes through a one-dimensional scanning galvanometer, and then is conjugated to the entrance pupil of the excitation objective lens through a 4f system, and a "snake" light sheet field is generated at the focal plane of the detection objective lens through rapid scanning of the galvanometer.

[0010] Scanning galvanometer: It is used to synchronously scan the modulated excitation light and control the process of the light beam forming a planar array imaging on the sample. The rapid scanning of the scanning galvanometer can generate the required "snake-shaped" light sheet field on the focal plane of the detection objective.

[0011] Scanning lens: Located after the galvanometer, it is used to collect the laser beam after area scanning to ensure that the excitation light can be accurately focused on the target area of ​​the sample.

[0012] Tube lens I: Combined with the illumination objective lens to form a microscope system.

[0013] Illumination objective lens: used to focus the laser onto the focal plane and cooperate with the scanning galvanometer to form a digital light sheet to achieve precise light sheet illumination.

[0014] Sample pool: used to maintain the water environment, place and fix the sample, and ensure the stability of the sample and the imaging effect during the imaging process.

[0015] Four-dimensional translation stage: used to move samples and cooperate with the scanning galvanometer to achieve three-dimensional imaging, so that the imaging system can scan and capture samples from multiple angles and in all directions.

[0016] Detection objective: used to collect the fluorescence signal emitted by the sample to ensure efficient fluorescence collection and imaging quality.

[0017] Optical filters: used to remove stray light and selectively filter specific wavelengths of light to improve the signal-to-noise ratio and quality of the image.

[0018] Tube lens II: Located between the detection objective lens and the camera, it is used to receive and focus light, and cooperate with the detection objective lens to magnify the image at different magnifications.

[0019] Camera (sCMOS): used to collect and record fluorescence image data, with high sensitivity and high-speed imaging capabilities, suitable for three-dimensional imaging with high temporal and spatial resolution.

[0020] Through the coordinated work of the above-mentioned components, the light sheet imaging system of the present invention can greatly improve the imaging field of view without sacrificing the axial resolution of the system, and realize three-dimensional imaging with high temporal and spatial resolution, which is particularly suitable for long-term, high temporal and spatial resolution imaging of large tissues, organs or animals.

[0021] In a second aspect, the present invention further provides a light sheet imaging method with a large field of view and high spatiotemporal resolution, the method comprising the following steps:

[0022] (1) Light field generation: A laser beam of Gaussian laser is generated by a laser. After the polarization direction of the laser is adjusted by a 1 / 2 wave plate, it is modulated by a spatial light modulator to generate a curved beam, namely a "snake-shaped" light field.

[0023] (2) Light field modulation: A hologram is loaded onto a spatial light modulator to convert a Gaussian laser beam into a “snake-shaped” light field, thereby significantly expanding the imaging field of view. This “snake-shaped” light field can significantly increase the imaging field of view without sacrificing axial resolution.

[0024] (3) Sample excitation: The modulated “snake-shaped” light field is focused onto the focal plane through the scanning galvanometer, tube lens I and illumination objective lens to achieve efficient light sheet excitation, and the influence of the excitation beam side lobes on the image quality is reduced by two-photon absorption technology.

[0025] (4) Two-dimensional fluorescence acquisition: The fluorescence signal emitted by the sample under the excitation of the “snake-shaped” light sheet field is collected through the detection objective lens, and the fluorescence signal is recorded by the camera to generate light sheet two-dimensional imaging data;

[0026] (5) Three-dimensional fluorescence acquisition: Based on two-dimensional fluorescence acquisition, the relative position of the sample and the detection surface is moved by a four-dimensional translation stage to generate light sheet image data at different positions, and then the three-dimensional data is reconstructed.

[0027] The method of the present invention can also process the acquired image data and use the STORM technology to accurately locate the fluorescent molecules to improve the lateral resolution of the system and break through the optical diffraction limit. More specifically: using a spontaneous fluorescence scintillation probe, within an open loop time, a galvanometer is used to quickly scan in one dimension to achieve wide-field illumination of a large field of view light sheet, while synchronizing the camera exposure time, such as Figure 2As shown on the right side of d, the compatibility between the light sheet system and STORM technology is solved, and finally, super-resolution imaging is achieved by using the single-molecule localization algorithm. This significantly improves the lateral resolution of the light sheet system.

[0028] Compared with the prior art, the light sheet imaging system and method with a large field of view and high temporal and spatial resolution proposed in the present invention have the following beneficial effects:

[0029] 1. Large field of view imaging: This invention effectively expands the field of view of the imaging system (field of view 500×500μm) by introducing a "snake-shaped" light sheet design. 2 ). Without changing the numerical aperture of the microscope objective, this design can cover a larger imaging area, thereby achieving three-dimensional imaging of large tissues, organs or whole animals. This feature is particularly suitable for biomedical research that requires high-throughput data acquisition, such as neural network imaging and developmental biology research.

[0030] 2. High spatiotemporal resolution: This method combines STORM technology to significantly improve the lateral resolution of the system (lateral resolution 30nm). Through the super-resolution imaging capability of STORM, this system can distinguish the structural details within the cell at the nanometer level. At the same time, due to the use of femtosecond pulsed lasers and efficient fluorescence acquisition strategies, the system also maintains a high level of temporal resolution, which is suitable for studying fast dynamic processes.

[0031] 3. Reduced phototoxicity and photobleaching: The present invention uses two-photon excitation technology to increase imaging depth and reduce phototoxicity and photobleaching effects, thereby ensuring the stability of long-term imaging. This is particularly important for continuous observation and long-term experiments of living samples.

[0032] 4. Flexible light field control: The present invention uses a spatial light modulator (SLM) to control the "snake-shaped" light sheet field, so that the focal length of the light sheet field can be adjusted according to experimental requirements. This flexibility allows users to optimize imaging parameters according to different sample types and experimental requirements, thereby improving imaging efficiency and data quality.

[0033] 5. Light sheet super-resolution: This invention combines STORM technology, and the system breaks through the diffraction limit of traditional optical microscopes and achieves super-resolution imaging. This enables researchers to observe microscopic structures that cannot be resolved by traditional optical microscopy, especially clear observation of substructures and molecular levels within cells.

[0034] In summary, the light sheet imaging system and method with large field of view and high spatiotemporal resolution of the present invention reduces system complexity and cost. Compared with the traditional multi-beam or multi-lens solution, the present system can achieve diversified imaging requirements using a single optical path configuration and SLM control, thereby reducing equipment investment and maintenance costs. The present invention realizes super-resolution imaging under a large field of view, greatly improves the speed of three-dimensional imaging, and provides a powerful tool for high-throughput three-dimensional imaging.

[0035] These and other aspects of the present application will be more concise and understandable in the following description of the embodiments. It should be understood that the above general description and the following detailed description are only exemplary and explanatory and cannot limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other embodiments can be obtained based on these drawings without paying creative work.

[0037] In the figure:

[0038] Figure 1 1 is a light path diagram of a light sheet imaging system with a large field of view and high spatiotemporal resolution according to an embodiment of the present invention.

[0039] Figure 2 This is a schematic diagram of the principle of light field control and light sheet super-resolution imaging in a light sheet imaging system and method with a large field of view and high spatiotemporal resolution according to an embodiment of the present invention.

[0040] Reference numerals:

[0041] 1-laser, 2-1 / 2 wave plate, 3-spatial light modulator, 4-scanning galvanometer, 5-scanning lens, 6-tube lens I, 7-illumination objective lens, 8-four-dimensional translation stage, 9-sample cell, 10-detection objective lens, 11-filter, 12-tube lens II, 13-camera. DETAILED DESCRIPTION

[0042] Below, the present application is further described in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in combination with specific embodiments and with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0044] It should be noted that all expressions using "first" and "second" in the embodiments of the present invention are intended to distinguish two non-identical entities or non-identical parameters with the same name. It can be seen that "first" and "second" are only for the convenience of expression and should not be understood as limitations on the embodiments of the present invention. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, other steps or units inherent to a process, method, system, product or device that includes a series of steps or units.

[0045] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0046] The flowcharts shown in the accompanying drawings are only examples and do not necessarily include all the contents and operations / steps, nor must they be executed in the order described. For example, some operations / steps may also be decomposed, combined or partially merged, so the actual execution order may change according to actual conditions.

[0047] In conjunction with the accompanying drawings, some embodiments of the present application are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0048] See also Figure 1 As shown, an embodiment of the present invention provides a light sheet imaging system with a large field of view and high spatiotemporal resolution. The system is a large field of view, high spatiotemporal resolution LSFM imaging that can break through the optical diffraction limit, greatly improving the imaging field of view without losing the axial resolution of the system; then, the lateral resolution of the entire LSFM system is improved without changing the numerical aperture of the microscope objective lens, thereby achieving super-resolution imaging. It provides technical means for long-term, high spatiotemporal resolution, and three-dimensional imaging of large tissues, organs, or animals, and further promotes the application of light sheet imaging technology in biomedical imaging.

[0049] See also Figure 1 As shown, the light sheet imaging system includes:

[0050] Laser 1: used to generate Gaussian laser;

[0051] 1 / 2 wave plate 2: used to adjust the polarization direction of the laser;

[0052] Spatial light modulator 3: used for light field control. Gaussian laser is modulated into a curved light beam by spatial light modulator 3.

[0053] Scanning galvanometer 4: used to synchronously scan the modulated excitation light and control the light beam to form a planar array imaging on the sample;

[0054] Scanning lens 5: located behind the galvanometer, used to collect the laser beam after the area array scanning and focus the laser on the target area of ​​the sample;

[0055] Tube lens I 6: It forms a microscope system together with the illumination objective lens 7;

[0056] Illumination objective lens 7: used to focus the laser to the focal plane and cooperate with the scanning galvanometer 4 to form a digital light sheet;

[0057] Sample pool 9: used to maintain water environment, place and fix samples;

[0058] A four-dimensional translation stage 8, used to move the sample and cooperate with the scanning galvanometer 4 to achieve three-dimensional imaging;

[0059] A detection objective lens 10 is used to collect the fluorescent signal emitted by the sample;

[0060] Filter 11: used to remove stray light other than the fluorescent signal collected by the detection lens 10;

[0061] The tube lens II 12 is used to receive and focus light, and cooperates with the detection objective lens 10 to magnify the image at different magnifications;

[0062] Camera 13: used to collect and record fluorescence images.

[0063] In this embodiment, the laser 1 is a femtosecond pulse laser, which is used to generate femtosecond pulse laser, wherein the wavelength tunable range of the femtosecond pulse laser light source of the laser 1 is 680-1300nm.

[0064] 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 so that it is 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 adjusting the Gaussian laser generated by the laser 1 after it is emitted, and then incident on the spatial light modulator 3 as horizontally polarized light after being adjusted by the 1 / 2 wave plate 2.

[0065] In this embodiment, the spatial light modulator 3 also modulates the Gaussian light field into a curved light beam by loading a hologram. The modulated curved light beam passes through the one-dimensional scanning galvanometer 4 and then conjugates to the entrance pupil of the excitation objective lens through the 4f system. The galvanometer quickly scans the light beam to generate a "snake-shaped" light sheet field on the focal plane of the detection objective lens 10.

[0066] The tube lens I6 is located between the scanning lens 5 and the illumination objective lens 7, and is used to focus the excitation light onto the focal plane 10. The filter 11 is located between the detection objective lens 10 and the camera 13. The tube lens II 12 is arranged between the filter 11 and the camera 13.

[0067] The light sheet imaging system with large field of view and high spatiotemporal resolution of the present invention has only one femtosecond pulsed laser light source with tunable wavelength (680-1300nm) to better meet the actual excitation wavelength requirements. After the Gaussian laser is emitted, it passes through a 1 / 2 wave plate 2 to ensure that the horizontally polarized light is incident on the spatial light modulator 3 (SLM). The Gaussian beam is modulated into a "snake-shaped" beam by loading a hologram on the SLM. The modulated beam passes through a one-dimensional scanning galvanometer and then conjugates to the entrance pupil of the excitation objective lens through a 4f system. The required "snake-shaped" light sheet field is generated at the focal plane of the detection objective lens 10 by rapid scanning with the scanning galvanometer 4.

[0068] The fluorescence emitted by the sample in the sample pool 9 is collected by the detection objective lens 10 and imaged onto the camera 13 (sCMOS). Among them, the 1 / 2 wave plate 2 is used to adjust the polarization state of the light beam, the sample is fixed on the four-dimensional translation stage 8, and the filter 11 is used to filter out stray light other than collected fluorescence. The type and parameters of the microscope system light sheet can be controlled by the software program. Three-dimensional fluorescence imaging can be achieved by scanning the scanning galvanometer 4 in one dimension, coordinating the movement of the sample, and synchronizing the exposure time of the camera 13. In addition, it should be noted that: (1) Figure 1 Taking two-photon excitation as an example, this invention is not limited to two-photon, but is also applicable to single-photon and multi-photon excitation; (2) beam modulation is not limited to SLM, but is also applicable to optical diffraction elements and digital micromirror devices.

[0069] Through the coordinated work of the above-mentioned components, the light sheet imaging system of the present invention can greatly improve the imaging field of view without sacrificing the axial resolution of the system, and realize three-dimensional imaging with high temporal and spatial resolution, which is particularly suitable for long-term, high temporal and spatial resolution imaging of large tissues, organs or animals.

[0070] In one embodiment of the present invention, the present invention also provides a light sheet imaging method with a large field of view and high temporal and spatial resolution, the method comprising the following steps:

[0071] (1) Light field generation: A laser beam of Gaussian type laser is generated by laser 1, and after the polarization direction of the laser is adjusted by 1 / 2 wave plate 2, it is bent by spatial light modulator 3.

[0072] (2) Light field modulation: The hologram is loaded using the spatial light modulator 3 to convert the Gaussian laser beam into a "snake-shaped" light field, and the focal length is adjusted to achieve a significant expansion of the imaging field of view. The "snake-shaped" light field can greatly improve the imaging field of view without sacrificing the axial resolution.

[0073] (3) Sample excitation: The modulated “snake-shaped” light field is focused onto the focal plane 10 through the scanning galvanometer 4 to achieve efficient light sheet excitation, and the influence of the excitation beam side lobes on the image quality is reduced through two-photon absorption technology.

[0074] (4) Two-dimensional fluorescence acquisition: The fluorescence signal emitted by the sample under the excitation of the “snake-shaped” light sheet field is collected through the detection objective lens 10, and the image data generated by the fluorescence signal is recorded through the camera 13.

[0075] The present invention effectively expands the field of view of the imaging system by introducing a "snake-shaped" light sheet design. Without changing the numerical aperture of the microscope objective, this design can cover a larger imaging area, thereby achieving three-dimensional imaging of large tissues, organs or whole animals. This feature is particularly suitable for biomedical research that requires high-throughput data acquisition, such as neural network imaging and developmental biology research.

[0076] This method combines STORM technology to significantly improve the lateral resolution of the system. Through the super-resolution imaging capability of STORM, this system can distinguish the structural details within the cell at the nanometer level. At the same time, due to the use of femtosecond pulsed lasers and efficient fluorescence acquisition strategies, the system also maintains a high level of temporal resolution, which is suitable for studying fast dynamic processes.

[0077] The present invention improves imaging depth and reduces phototoxicity and photobleaching effects through two-photon excitation technology, thereby ensuring the stability of long-term imaging, which is particularly important for continuous observation and long-term experiments of living samples.

[0078] The present invention uses a spatial light modulator 3 (SLM) to regulate the "snake-shaped" light sheet field, so that the focal length of the light sheet field can be adjusted according to experimental requirements. This flexibility allows users to optimize imaging parameters according to different sample types and experimental requirements, thereby improving imaging efficiency and data quality.

[0079] The present invention combines STORM technology, and the system breaks through the diffraction limit of traditional optical microscopes and achieves super-resolution imaging. This allows researchers to observe microscopic structures that cannot be resolved by traditional optical microscopy, especially clear observation of substructures and molecular levels within cells.

[0080] See also Figure 2 As shown, Figure 2 This is a basic principle diagram of the invention's implementation of large field of view, super-resolution light sheet imaging based on the organic combination of light field control technology and super-resolution technology. Figure 2 As shown in a, the present invention originates from the energy oscillation mechanism, namely, impulse energy and release energy. The amplitude, phase, and position of the light beam are controlled by light field control technology ( Figure 2 b), thereby realizing the generation of beams with different oscillation times ( Figure 2 c), thereby significantly increasing the focal length of the light beam, and cooperating with the galvanometer scanning, ultimately generating a light sheet field with a large field of view at the focal plane of the illumination objective. The new light sheet fluorescence microscope based on light field control technology can achieve large field of view imaging, but its lateral resolution is limited by the numerical aperture and diffraction limit of the detection objective. In order to improve the lateral resolution without changing the numerical aperture, the STORM technology is introduced, such as Figure 2 d The left side is a schematic diagram of the principle of wide-field STORM super-resolution imaging. Long-wavelength and short-wavelength lasers are used to regulate the random transition between fluorescence quenching state (dark state) and fluorescence state of fluorescent molecules. In each cycle, most of the fluorescent probe molecules in the field of view are converted to dark state, and only a small part of the fluorescent molecules undergo randomly activated fluorescence emission. The images of these sparsely luminous fluorescent molecules will not overlap with each other. Through subsequent image processing, highly accurate positioning information of these fluorescent molecules can be obtained. After repeated random lighting of sparse fluorescent molecules, a large number of fluorescent molecules can be accurately positioned, and the collected sparse molecular luminescence images are integrated and reconstructed to obtain super-resolution microscopic images. To this end, a spontaneous fluorescence scintillation probe is used. Within an open-loop time, a galvanometer is used to quickly scan in one dimension to achieve wide-field illumination of a large field of view light sheet, while synchronizing the camera exposure time, such as Figure 2 As shown on the right side of d, the compatibility between the light sheet system and STORM technology is solved, and finally, super-resolution imaging is achieved by using the single-molecule localization algorithm, thereby significantly improving the lateral resolution of the light sheet system.

[0081] In summary, the light sheet imaging system and method with large field of view and high spatiotemporal resolution of the present invention reduces system complexity and cost. Compared with the traditional multi-beam or multi-lens solution, the present system can achieve diversified imaging requirements using a single optical path configuration and SLM control, thereby reducing equipment investment and maintenance costs. The present invention realizes super-resolution imaging under a large field of view, greatly improves the speed of three-dimensional imaging, and provides a powerful tool for high-throughput three-dimensional imaging.

[0082] The above are exemplary embodiments disclosed in the present invention, but it should be noted that various changes and modifications may be made without departing from the scope disclosed in the embodiments of the present invention as defined in the claims. The functions, steps and / or actions of the method claims according to the disclosed embodiments described herein do not need to be performed in any particular order. In addition, although the elements disclosed in the embodiments of the present invention may be described or required in individual form, they may also be understood as multiple unless explicitly limited to the singular.

[0083] It should be understood that, as used herein, the singular form "a" or "an" is intended to include the plural form as well, unless the context clearly supports an exception. It should also be understood that, as used herein, "and / or" refers to any and all possible combinations of one or more of the items listed in association. The serial numbers of the embodiments disclosed in the above embodiments of the present invention are for description only and do not represent the advantages and disadvantages of the embodiments.

[0084] A person skilled in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the disclosure of the embodiments of the present invention (including the claims) is limited to these examples; under the concept of the embodiments of the present invention, the technical features in the above embodiments or different embodiments can also be combined, and there are many other changes in different aspects of the embodiments of the present invention as above, which are not provided in detail for the sake of simplicity. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present invention should be included in the protection scope of the embodiments of the present invention.

Claims

1. A light sheet imaging system with a large field of view and high temporal and spatial resolution, characterized in that: The system includes: Laser: used to generate Gaussian laser; 1 / 2 wave plate: used to adjust the polarization direction of the laser; Spatial light modulator: used for light field control. Gaussian laser is modulated into a curved beam by the spatial light modulator. Scanning galvanometer: used to synchronously scan the modulated excitation light and control the light beam to form a planar array imaging on the sample; Scanning lens: located behind the galvanometer, used to collect the laser beam after area array scanning and focus the laser on the target area of ​​the sample; Tube lens I: Combined with the illumination objective lens to form a microscope system; Illumination objective lens: used to focus the laser to the focal plane and cooperate with the scanning galvanometer to form a digital light sheet; Sample pool: used to maintain water environment, place and fix samples; The four-dimensional translation stage is used to move samples and realize three-dimensional imaging in conjunction with the scanning galvanometer; A detection objective lens, used to collect the fluorescence signal emitted by the sample; Filter: used to remove stray light other than the fluorescent signal collected by the detection objective; Camera: used to collect and record fluorescence images.

2. The light sheet imaging system with large field of view and high spatiotemporal resolution according to claim 1, characterized in that: The laser is a femtosecond pulse laser, which is used to generate femtosecond pulse laser.

3. The light sheet imaging system with large field of view and high spatiotemporal resolution according to claim 2, characterized in that: The wavelength tunable range of the femtosecond pulse laser light source of the laser is 680-1300nm.

4. The light sheet imaging system with large field of view and high spatiotemporal resolution according to claim 3, characterized in that: The 1 / 2 wave plate is located on the optical path of the Gaussian laser generated by the laser and is used to adjust the polarization direction of the laser so that the polarization direction of the Gaussian light is horizontally parallel to the liquid crystal surface and is incident on the spatial light modulator.

5. The light sheet imaging system with large field of view and high spatiotemporal resolution according to claim 4, characterized in that: The 1 / 2 wave plate is located between the laser and the spatial light modulator, and is used to adjust the Gaussian laser generated by the laser after it is emitted, and then incident on the spatial light modulator as horizontally polarized light after being adjusted by the 1 / 2 wave plate.

6. The light sheet imaging system with large field of view and high spatiotemporal resolution according to claim 5, characterized in that: The spatial light modulator also modulates the Gaussian beam into a curved beam by loading a hologram. The modulated curved beam passes through a one-dimensional scanning galvanometer and then conjugates to the entrance pupil of the excitation objective lens through a 4f system. The galvanometer quickly scans the beam to generate a "snake-shaped" light sheet field on the focal plane of the detection objective lens.

7. The light sheet imaging system with large field of view and high spatiotemporal resolution according to claim 6, characterized in that: The tube lens I is located between the scanning lens and the illumination objective lens, and is used to focus the excitation light onto the focal plane.

8. The light sheet imaging system with large field of view and high spatiotemporal resolution according to claim 7, characterized in that: The filter is located between the detection lens and the camera. A tube lens II is also provided between the filter and the camera. The tube lens II is used to receive and focus light and cooperate with the detection lens to magnify the image at different magnifications.

9. A light sheet imaging method with large field of view and high temporal and spatial resolution, characterized in that: The method is performed based on the light sheet imaging system with large field of view and high spatiotemporal resolution as claimed in claim 8, and the light sheet imaging method comprises the following steps: Step 1), light field generation: a laser beam of Gaussian laser is generated by a laser, the polarization direction of the laser is adjusted by a 1 / 2 wave plate, and then modulated by a spatial light modulator to generate a curved beam, i.e., a "snake-shaped" light field; Step 2) Light field modulation: Use a spatial light modulator to load the hologram, convert the Gaussian laser beam into a "snake-shaped" light field, and adjust the focal length; Step 3), sample excitation: the modulated "snake-shaped" light field is focused onto the focal plane through the scanning galvanometer, tube lens I and illumination objective lens, and a "snake-shaped" light sheet field is generated on the focal plane; Step 4), two-dimensional fluorescence acquisition: collect the fluorescence signal emitted by the sample under the excitation of the "snake-shaped" light sheet field through the detection objective lens, and record the fluorescence signal through the camera to generate light sheet two-dimensional imaging data; Step 5), three-dimensional fluorescence acquisition: Based on the two-dimensional fluorescence acquisition, the relative position of the sample and the detection surface is moved by a four-dimensional translation stage to generate light sheet image data at different positions, and then the three-dimensional data is reconstructed.

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