An ultramicroscopic imaging system and method

Abstract

The application provides a kind of super-resolution light sheet microscopic imaging system and method, the system includes illumination light path and fluorescence light path, and based on the principle of structure light illumination of opposite interference, illumination light path and fluorescence light path are integrated in a set of upper and lower opposed objective lens architecture. By two-way opposite oblique light sheet interference occurs at sample, form high contrast, controllable period interference fringes, to excite sample to produce fluorescence. The method includes the basis of the system carries out multidirectional and multi-phase three-dimensional data acquisition, for each preset illumination direction, collect multiple interference images with fixed phase difference, and after sequentially completing complete three-dimensional data acquisition under multiple different illumination directions, finally, the super-resolution image that breaks through the diffraction limit in three-dimensional space is reconstructed using a special image reconstruction algorithm. The application can effectively break through the diffraction limit, realize high-speed, high-resolution, low phototoxicity super-resolution imaging of biological samples.

Description

Technical Field

[0001] This application relates to the field of microscopic imaging technology, and in particular to a super-resolution light sheet microscopic imaging system and method. Background Technology

[0002] Light-sheet fluorescence microscopy (LSFM) is a three-dimensional microscopic imaging technique. It involves selectively exciting a thin sheet of light within the sample and collecting the fluorescence signal using a probe objective orthogonal to the excitation beam. Due to its low phototoxicity, high imaging speed, and excellent three-dimensional imaging capabilities, this technique has applications in in vivo observation and large-sample imaging.

[0003] However, existing light-sheet fluorescence microscopy techniques still have several limitations. First, to obtain a uniform light sheet within a large field of view, a low numerical aperture excitation objective is typically required, resulting in a thicker light sheet and limiting the system's axial resolution. Second, due to the orthogonal geometry of the excitation and probe optical paths, it is often difficult to use a high numerical aperture objective at the probe end, causing the overall spatial resolution of the system to be lower than that of techniques such as confocal microscopy. Furthermore, this inherent optical path structure is difficult to directly accommodate the fringe rotation and phase modulation requirements of super-resolution imaging methods such as structured light illumination microscopy, thus hindering the realization of super-resolution observations. These factors, to some extent, restrict the further application of this technology in scenarios requiring high-resolution, high-precision observations.

[0004] Therefore, how to provide a super-resolution light sheet microscopy system and method that can further improve imaging resolution and meet its further application in high-precision observation scenarios has become one of the technical problems that urgently need to be solved by people in this field. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems in the related art.

[0006] Therefore, the first objective of this application is to propose a super-resolution light sheet microscopy system that can further improve imaging resolution.

[0007] To achieve the above objectives, a first aspect of this application provides a super-resolution light sheet microscopy imaging system, comprising:

[0008] The illumination optical path is used to generate two opposing tilted light plates, and to make the two tilted light plates interfere with each other on the excitation surface of the sample to form structured light fringes. The fluorescence optical path is used to collect the fluorescence signal generated by the sample being excited by the structured light stripes, and to detect and image the fluorescence signal.

[0009] The illumination optical path projects two tilted light plates onto the excitation surface of the sample through two vertically opposed objective lenses.

[0010] Optionally, the illumination light path and the fluorescence light path each include two illumination sub-light paths and two fluorescence sub-light paths corresponding to the two objective lenses that are positioned vertically opposite each other; wherein, the two illumination sub-light paths are used to generate two opposing tilted light plates; and the two fluorescence sub-light paths are used to collect the fluorescence signals generated on the sample excitation surface in different directions.

[0011] Optionally, the illumination optical path includes a light source unit, a light sheet generation unit, and a scanning unit; wherein, the light source unit is used to output a laser beam of single or multiple wavelengths, the light sheet generation unit is disposed downstream of the light source unit, and is used to receive the laser beam and convert the laser beam into a light sheet output; the scanning unit is disposed downstream of the light sheet generation unit, and is used to receive and adjust the output direction of the light sheet so that the light sheet passes through the objective lens and scans the excitation surface of the sample at a preset tilt angle.

[0012] Optionally, the light sheet generation unit is provided with at least a phase grating and a cylindrical lens in sequence; wherein, the phase grating is disposed in the downstream optical path of the light source unit and is composed of a spatial light modulator, a half-wave plate and a polarizing beam splitter, and is used to perform phase modulation on the incident laser beam; the cylindrical lens is disposed in the downstream optical path of the phase grating and is used to convert the modulated beam output by the phase grating into a light sheet output.

[0013] Optionally, the light sheet generation unit further includes a mask disposed in the optical path downstream of the cylindrical lens, the mask being used to selectively transmit light sheets of a preset diffraction order.

[0014] Optionally, the scanning unit includes a first galvanometer and a lens group located between the first galvanometer and the objective lens; wherein the first galvanometer is positioned conjugate to the rear pupil plane of the objective lens, and the lens group is arranged between the first galvanometer and the objective lens in a 4f relay configuration.

[0015] Optionally, the fluorescence optical path includes a depth-of-field extension unit and an imaging unit; wherein, The depth-of-field extension unit is disposed between the objective lens and the imaging unit, and is used to project the fluorescence signal collected by the objective lens onto the imaging unit and extend the imaging depth of the imaging unit; the depth-of-field extension unit includes one of the following: phase modulation type, amplitude modulation type, multi-focal surface beam splitting type depth-of-field extension device or diffractive optical element type depth-of-field extension device.

[0016] Optionally, the fluorescence optical path includes an image rotation unit and an imaging unit; wherein, The image rotation unit is disposed between the objective lens and the imaging unit and is located in the image space conjugate with the sample. It includes at least one second galvanometer and multiple reflecting mirrors, and is used to rotate the imaging direction of the fluorescence signal collected by the objective lens. The imaging unit is disposed in the downstream optical path of the image rotation unit, and the imaging surface maintains a preset tilt angle with the optical axis of the objective lens, and is used to perform imaging detection on the image after it has been rotated by the image rotation unit.

[0017] Optionally, it also includes a sample module, which includes a three-dimensional precision displacement stage for carrying the sample and a temperature and humidity control device for maintaining the sample environment.

[0018] To achieve the above objectives, a second aspect of this application provides a super-resolution light sheet microscopy imaging method, comprising: Two opposing tilted light plates are generated using the illumination optical path, and the two tilted light plates interfere on the sample excitation surface to form structured light fringes; The fluorescence signal generated by the structured light stripes excited on the sample excitation surface is collected using two objective lenses positioned vertically opposite each other. The imaging optical path is used to expand the imaging depth of field and detect the fluorescence signals collected by the two objectives, thereby obtaining a super-resolution image of the sample excitation surface. The step of generating two opposing tilted light plates using the illumination optical path includes generating multiple two opposing tilted light plates in different directions using the illumination optical path, and the two opposing tilted light plates generated in each direction interfere with each other on the sample excitation surface to form structured light stripes including structured light stripes with multiple different phases.

[0019] The super-resolution light plate microscopy imaging system and method provided in this application have at least the following beneficial effects: This application provides a super-resolution light-sheet microscopy system, comprising an illumination path and a fluorescence path. Based on the principle of structured light illumination with opposing interference, this application ingeniously integrates the illumination and fluorescence paths into a single, vertically opposed objective lens architecture. Interference occurs between the two opposing, tilted light sheets at the sample, forming high-contrast, controllable-period interference fringes to excite fluorescence in the sample. This design lays the physical foundation for achieving light-sheet super-resolution imaging. Combined with subsequent computational reconstruction algorithms, this system can effectively overcome the diffraction limit, achieving high-speed, high-resolution, and low-phototoxicity super-resolution imaging of biological samples.

[0020] Furthermore, this application also provides a super-resolution light sheet microscopy imaging method. This method involves first generating two opposing tilted light sheets using symmetrically arranged upper and lower illumination paths, causing them to interfere at the sample to form high-contrast structured light fringes, thereby exciting fluorescence signals. To achieve super-resolution reconstruction, this method systematically implements multi-directional and multi-phase three-dimensional data acquisition. For each preset illumination direction, at least three interference images with a fixed phase difference are acquired. After completing the full three-dimensional data acquisition under at least three different illumination directions, a dedicated image reconstruction algorithm is used to process the acquired multi-directional, multi-phase, and multi-layered original image data, thereby reconstructing a super-resolution image that breaks the diffraction limit in three-dimensional space. This method combines the super-resolution capability of structured light illumination with the high-speed and low-phototoxicity advantages of light sheet microscopy, providing a complete and efficient technical solution for achieving high spatiotemporal resolution three-dimensional imaging of living samples.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a structural block diagram of a super-resolution optical sheet microscopy system according to an embodiment of this application.

[0023] Figure 2 This is a schematic diagram of the objective lens placement and the interference plate structure at the sample location, according to an embodiment of this application.

[0024] Figure 3 This is an enlarged three-dimensional structural schematic diagram of the interference plate according to an embodiment of this application.

[0025] Figure 4 This is a schematic diagram of the optical path structure of a super-resolution optical sheet microscopy system according to an embodiment of this application.

[0026] Figure 5 This is a schematic diagram of the structure of a mask according to an embodiment of this application.

[0027] Figure 6 This is a three-dimensional schematic diagram of the light distribution at the rear pupil of an objective lens and the structured light sheet corresponding to the sample plane, according to an embodiment of this application.

[0028] 100 light source units; 200 light sheet generation units; 300 scanning units; 400 objective lenses; 500 depth-of-field extension units; 600 imaging units; 700 sample modules. Detailed Implementation

[0029] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0030] According to one aspect of this application, a super-resolution light-sheet microscopy imaging system is provided, such as... Figures 1-4 As shown, the system includes an illumination path and a fluorescence path. The illumination path generates two opposing tilted light plates, which interfere on the excitation surface of the sample to form structured light fringes. The fluorescence path collects the fluorescence signal generated by the sample excited by the structured light fringes and performs super-resolution light plate microscopy based on the collected fluorescence signal. The illumination and fluorescence paths share two opposing objectives 400, and the illumination path projects the two tilted light plates onto the excitation surface of the sample through the two objectives 400.

[0031] It is understood that the super-resolution light-sheet microscopy system provided in this application is a fluorescence microscopy technique based on the fact that a light-sheet formed by the binding of a fluorescent label with a specific biomolecule emits a fluorescence signal under the excitation of structured light fringes formed by mutual interference.

[0032] Two opposing light plates, incident at an angle, interfere with each other at the sample (excitation surface) after passing through an objective lens at 40°, thus producing interference fringes (structured light fringes) with high contrast and controllable period. This interference-based structured light illumination method, combined with subsequent super-resolution image reconstruction algorithms, can break through the diffraction resolution limit of traditional light plate microscopy and achieve super-resolution imaging.

[0033] In some embodiments, the illumination light path and the fluorescence light path each include two illumination sub-paths and two fluorescence sub-paths corresponding to the two opposing objective lenses 400. The two illumination sub-paths are used to generate two opposing tilted light plates; the two fluorescence sub-paths are used to collect fluorescence signals generated by the sample excitation surface in different directions.

[0034] Understandably, employing two independent illumination sub-paths and fluorescence sub-paths, arranged symmetrically in space, aims to achieve a bidirectional illumination and bidirectional collection imaging mode, thereby enhancing the integrity of fluorescence signal collection and providing assurance for super-resolution imaging.

[0035] Specifically, each of the two illumination sub-paths independently generates a tilted light plate, which is then projected onto the sample excitation surface through the corresponding upper / lower objective lenses 400. This independent design enables precise matching and separate control of parameters such as the angle, phase, and light intensity of the two light plates, ensuring that the two light plates form high-contrast interference fringes on the excitation surface.

[0036] Simultaneously, the two fluorescent sub-paths rely on the opposing objectives 400 to collect fluorescence signals from different directions. The design of the opposing detection paths can significantly broaden the collection efficiency and signal-to-noise ratio of fluorescence signals. Especially for thick biological samples, the upper and lower objectives 400 can capture the fluorescence emitted from the upper and lower regions of the sample respectively, reducing signal attenuation caused by sample scattering and absorption.

[0037] As an example, the illumination optical path includes a light source unit 100, a light sheet generation unit 200, and a scanning unit 300.

[0038] The light source unit 100 is used to output a laser beam of one or more wavelengths. The light sheet generation unit 200 is disposed in the downstream optical path of the light source unit 100 and is used to receive the laser beam and convert the laser beam into a light sheet output. The scanning unit 300 is disposed in the downstream optical path of the light sheet generation unit 200 and is used to receive and adjust the output direction of the light sheet so that the light sheet passes through the objective lens 400 and is scanned on the excitation surface of the sample at a preset tilt angle.

[0039] The light source unit 100 includes at least one light source composed of one or more lasers, as well as a lens group, a reflector, and a dichroic mirror adapted to the light source. The lasers may include lasers with wavelengths of 405nm, 445nm, 488nm, 561nm, and 640nm, etc.

[0040] For example, the light source may include lasers Laser1, Laser2, and Laser3. These three lasers, Laser1, Laser2, and Laser3, can emit laser light of different wavelengths. These three lasers, Laser1, Laser2, and Laser3, are respectively equipped with lens groups L1 and L2, L3 and L4, and L5 and L6, so that the laser light emitted from the corresponding laser can be expanded into parallel light of equal diameter by the corresponding lens group. The reflector M1 and the dichroic mirrors (or beam splitters) DM1 and DM2 combine the parallel light of equal diameter from the different lasers into a single laser beam.

[0041] For example, the light source unit 100 may further include an acousto-optic tunable filter AOTF, which is configured in the laser optical path downstream of the dichroic mirror DM2, so that the integrated laser beam can be incident on the acousto-optic tunable filter AOTF. The function of the acousto-optic tunable filter AOTF is to selectively ensure the passage of light of a specific wavelength and / or to control the power of the light output through it.

[0042] The light-generating unit 200 includes at least a beam expander group, a phase grating, and a cylindrical lens arranged sequentially. The beam expander group can consist of a pair of circular lenses arranged in a 4F system, and the phase grating consists of a spatial light modulator, a half-wave plate, and a polarizing beam splitter prism.

[0043] For example, the light sheet generation unit 200 specifically includes a set of beam expanders L1 and L2, a binarized phase grating (composed of a half-wave plate HWP2, a polarizing beam splitter PBS2, and an amplitude-type spatial light modulator SLM), a cylindrical lens CL3, and a mask located on the focal plane of the cylindrical lens CL3.

[0044] Circular lenses L1 and L2 are used to expand the parallel light output from the light source unit 100 from a small aperture to a larger aperture. The pinhole is placed at the focal plane between the two lenses to act as a spatial filter, filtering out stray light and thus improving beam quality. After the beam is expanded, it is incident on the binarized phase grating and diffracted. The output light is then shaped into a light sheet after passing through the cylindrical lens CL3. After passing through the mask, only the +1 or -1 order light sheet is allowed to pass through.

[0045] It should be noted that the binarized phase grating here can also be replaced with a phase-type spatial light modulator, loaded with a specially designed blazed grating, so that the energy of the diffracted beam is concentrated on a specific order, such as the +1st or -1st order, to maximize light utilization. The mask is placed at the focal plane of CL3 and is specially designed to match light sheets of different directions.

[0046] For example, the mask can be three radially arranged slits on a circular sheet, with the three slits equidistant from the center and an angular interval of 60° between them. Specifically, a schematic diagram of the mask structure that can be used for the upper and lower illumination sub-paths is shown below. Figure 5 As shown.

[0047] It should be noted that the specific dimensions of the mask can be flexibly set according to system requirements to limit the thickness of the incident light sheet and ensure that the light sheet emitted to the scanning unit 300 meets specific application requirements.

[0048] The scanning unit 300 includes at least a first galvanometer and a lens group located between the first galvanometer and the objective lens 400. The first galvanometer is positioned conjugate to the rear pupil of the objective lens 400, and the lens group is arranged in a 4F system between the first galvanometer and the objective lens 400.

[0049] For example, the scanning unit 300 may specifically include a set of lenses L4 and L5 arranged in a 4F system, a first galvanometer Z Galvo, and lenses L6 and L7 located between the first galvanometer Z Galvo and the reflector M4 and arranged in a 4F system.

[0050] Lenses L4 and L5, arranged in a 4F system, can relay and image the output light sheet of the upstream light sheet generation unit 200 onto the reflective surface of the first galvanometer Z Galvo without distortion. Subsequently, lenses L6 and L7, also arranged in a 4F system, image the reflective surface of the first galvanometer onto the rear pupil of the objective lens. According to the Fourier principle of optics, the position of the light spot on the rear pupil of the objective lens 400 directly determines the incident angle of the light beam entering the objective lens 400. Therefore, a slight rotation of the first galvanometer around its axis will change the angle of the reflected light beam, and this angular change is ultimately converted into the scanning of the tilted light sheet in the sample space.

[0051] As an example, the fluorescence optical path includes at least a depth-of-field extension unit 500 and an imaging unit 600, as well as a lens group disposed between the objective lens 400 and the imaging unit 600. The depth-of-field extension unit 500 includes one of a phase-modulated type, an amplitude-modulated type, a multi-focal-plane beam-splitting type depth-of-field extension device, or a diffractive optical element type depth-of-field extension device.

[0052] Because the depth-of-field extension unit 500 is positioned between the objective lens 400 and the imaging unit 600, it can modulate the incident fluorescence signal, thereby significantly extending the depth of field of fluorescence imaging. This detection scheme eliminates the need for subsequent volumetric imaging using remote focusing, greatly simplifying the optical path complexity, reducing light flux loss, and improving fluorescence detection efficiency. The imaging unit 600 detects the fluorescence signal projected by the depth-of-field extension unit 500 and acquires a super-resolution image of the sample excitation surface based on the detection results.

[0053] For example, the fluorescence optical path specifically includes lenses L8 and L9 arranged in a 4F system, a depth-of-field extension element (eDOF), lens L10, and an imaging element (sCMOS) located at the focal plane of lens L10. The eDOF is located at the focal plane of lens L9 and is arranged in a conjugate configuration with the rear pupil of objective lens 400. Lenses L10 and L9 are also arranged in a 4F system, and the sCMOS imaging element is positioned at the focal plane of lens L10 for detection.

[0054] Furthermore, the beam splitter DM3 is positioned between the lens L8 and the objective lens OBJ, and is also located between the mirror M4 and the objective lens OBJ. It can transmit light emitted from the mirror M4 to the objective lens OBJ, and reflect the fluorescence signal emitted from the rear pupil of the objective lens OBJ to the depth-of-field extension element eDOF.

[0055] It should be noted that the use of an eDOF (extensible direct current device) to detect fluorescence signals in the above examples is merely illustrative and should not be considered a technical limitation on the fluorescence signal detection scheme of this application. In other words, this application does not impose specific limitations on the specific detection and imaging scheme for fluorescence signals. For example, in other embodiments, the fluorescence optical path may also include an image rotation unit and an imaging unit; wherein the image rotation unit is disposed between the objective lens and the imaging unit, and is located in the image space conjugate with the sample, including at least one second galvanometer and multiple reflecting mirrors, for rotating the imaging direction of the fluorescence signal collected by the objective lens; the imaging unit is disposed downstream of the image rotation unit, and the imaging surface maintains a preset tilt angle with the optical axis of the objective lens, for imaging and detecting the image after the rotation direction of the image rotation unit.

[0056] Furthermore, the two illumination sub-paths within the illumination path and the two fluorescence sub-paths within the fluorescence path are arranged in the same manner. Further details will not be provided here.

[0057] Meanwhile, the laser beam output from the light source unit 100 can have its optical power distribution controlled by the half-wave plates HWP1 and PBS1, which are input to the two illumination sub-paths. Normally, the optical power of the two illumination sub-paths is evenly distributed.

[0058] In some embodiments, the system further includes a sample module 700, which includes a three-dimensional precision displacement stage for carrying the sample and a temperature and humidity control device for maintaining the sample environment.

[0059] In summary, the first aspect of this application provides a super-resolution light-sheet microscopy system, which includes an illumination path and a fluorescence path. Based on the principle of structured light illumination with opposing interference, this application integrates the illumination and fluorescence paths into a single, vertically opposed objective lens architecture. Interference occurs between the two opposing, tilted light sheets at the sample's focal plane, forming high-contrast, controllable-period interference fringes to excite fluorescence in the sample. This design lays the physical foundation for achieving super-resolution light-sheet imaging. Combined with subsequent computational reconstruction algorithms, this system can effectively overcome the diffraction limit, achieving high-speed, high-resolution, and low-phototoxicity super-resolution imaging of biological samples.

[0060] According to a second aspect of this application, a super-resolution light-sheet microscopy method is also provided. This method employs the super-resolution light-sheet microscopy system described in the first aspect of the embodiment and further includes the following steps: S1, using the illumination optical path to generate two opposing tilted light plates, and causing the two tilted light plates to interfere on the sample excitation surface to form structured light fringes; S2, using two objective lenses positioned vertically opposite each other to collect the fluorescence signal generated by the structured light stripes on the sample excitation surface; S3 utilizes the imaging optical path to extend the imaging depth of field and detect the fluorescence signals collected by the two objectives, thereby acquiring a super-resolution image of the sample excitation surface.

[0061] In step S1, the step of generating two opposing tilted light sheets using the illumination optical path includes: generating multiple two opposing tilted light sheets in different directions using the illumination optical path, and the two opposing tilted light sheets generated in each direction interfere with each other on the sample excitation surface and form structured light stripes including multiple structured light stripes with different phases.

[0062] Furthermore, fluorescence signal acquisition requires at least the acquisition of fluorescence signals generated by two opposing tilted light plates in three different directions excited on the sample excitation surface. Simultaneously, for two opposing tilted light plates generated in the same direction, the interference they produce on the sample excitation surface and the resulting structured light fringes also require at least three different phases of structured light fringes. The generation of light plates in different directions is determined by the fringe gratings loaded on the SLM and SLM' with different directional gratings.

[0063] As an example, such as Figure 6 As shown, a specific data acquisition and processing workflow of this application includes the following process: First, three-dimensional data is acquired under the first illumination direction (e.g., direction one). At this time, the spatial light modulator (SLM) above loads the phase fringe corresponding to direction one, and its output beam, after passing through the light sheet formed by the subsequent illumination optical path, appears as follows at the rear pupil of objective lens OBJ1: Figure 6 The specific light field distribution is shown in (a). Simultaneously, a light field distribution is formed below at the rear pupil of objective lens OBJ2, as shown in (a). Figure 6 (b) shows the light field distribution. The two opposing tilted light plates from the upper and lower paths interfere with each other at the sample, and combine to form the light field distribution. Figure 6 (c) shows the alternating light and dark structured light stripes that provide illumination for a specific layer of the sample.

[0064] Secondly, to obtain complete super-resolution information for this layer, the system also needs to acquire multiple frames of interferometric images with a fixed phase difference. After completing the multi-phase image acquisition for this layer, the system moves the illumination plate to the next layer and repeats the above multi-phase acquisition process. This cycle continues until the entire three-dimensional volume of the target in the specified direction has been scanned.

[0065] Subsequently, the system switches to the next illumination direction (e.g., direction two). At this time, the upper SLM and the lower SLM' respectively switch to load the phase fringes corresponding to direction two, and their light field distributions at the back pupil are as follows: Figure 6 As shown in (d) and (e), the interference forms as follows Figure 6 The stripes are shown in (f). The system completes the entire 3D volume data acquisition in direction two following the exact same procedure as in direction one. The data acquisition procedure for direction three is also the same.

[0066] Finally, after completing the full set of data acquisitions in three different directions, at each axial level, and for each phase state, a series of raw images will be obtained. Ultimately, the multi-directional, multi-phase raw data is processed using a dedicated image reconstruction algorithm, and after image registration and 3D reconstruction, the final super-resolution image can be obtained.

[0067] It should be noted that for details not disclosed in the super-resolution light sheet microscopy imaging method provided in this embodiment, please refer to the super-resolution light sheet microscopy imaging system provided in the first aspect, which will not be described in detail here.

[0068] In summary, the second aspect of this application provides a super-resolution light sheet microscopy imaging method. This method involves first generating two opposing tilted light sheets using symmetrically arranged upper and lower illumination paths, causing them to interfere at the sample to form high-contrast structured light fringes, thereby exciting fluorescence signals. To achieve super-resolution reconstruction, this method systematically implements multi-directional and multi-phase three-dimensional data acquisition. For each preset illumination direction, at least three interference images with a fixed phase difference are acquired. After completing the full three-dimensional data acquisition under at least three different illumination directions, a dedicated image reconstruction algorithm is used to process the acquired multi-directional, multi-phase, and multi-layered original image data, thereby reconstructing a super-resolution image that breaks the diffraction limit. This method combines the super-resolution capability of structured light illumination with the high-speed and low-phototoxicity advantages of light sheet microscopy, providing a complete and efficient technical solution for achieving high spatiotemporal resolution three-dimensional imaging of living samples.

[0069] In the foregoing descriptions of the embodiments, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0070] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

Claims

1. A super-resolution light-sheet microscopy imaging system, characterized in that, include: The illumination optical path is used to generate two opposing tilted light plates, and to make the two tilted light plates interfere with each other on the excitation surface of the sample to form structured light fringes. A fluorescence optical path is used to collect the fluorescence signal generated by the sample being excited by the structured light stripes, and to detect and image the fluorescence signal; The illumination optical path projects two tilted light plates onto the excitation surface of the sample through two objective lenses positioned vertically opposite each other.

2. The super-resolution light-sheet microscopy system according to claim 1, characterized in that, The illumination light path and the fluorescence light path each include two illumination sub-light paths and two fluorescence sub-light paths corresponding to the two objective lenses that are positioned vertically opposite each other; wherein, the two illumination sub-light paths are used to generate two opposing tilted light plates; and the two fluorescence sub-light paths are used to collect the fluorescence signals generated on the sample excitation surface in different directions.

3. The super-resolution light-sheet microscopy system according to claim 1, characterized in that, The illumination optical path includes a light source unit, a light sheet generation unit, and a scanning unit; wherein, the light source unit is used to output a laser beam of single or multiple wavelengths, the light sheet generation unit is disposed downstream of the light source unit, and is used to receive the laser beam and convert the laser beam into a light sheet output; the scanning unit is disposed downstream of the light sheet generation unit, and is used to receive and adjust the output direction of the light sheet, so that the light sheet passes through the objective lens and scans the excitation surface of the sample at a preset tilt angle.

4. The super-resolution light-sheet microscopy system according to claim 3, characterized in that, The light sheet generation unit is provided with at least a phase grating and a cylindrical lens in sequence; wherein, the phase grating is disposed in the downstream optical path of the light source unit and is composed of a spatial light modulator, a half-wave plate and a polarizing beam splitter, and is used to perform phase modulation on the incident laser beam; the cylindrical lens is disposed in the downstream optical path of the phase grating and is used to convert the modulated beam output by the phase grating into a light sheet output.

5. The super-resolution light-sheet microscopy system according to claim 4, characterized in that, The light sheet generation unit also includes a mask disposed in the optical path downstream of the cylindrical lens, the mask being used to selectively transmit light sheets of a preset diffraction order.

6. The super-resolution light-sheet microscopy system according to claim 3, characterized in that, The scanning unit includes a first galvanometer and a lens group located between the first galvanometer and the objective lens; wherein the first galvanometer is positioned conjugate to the rear pupil plane of the objective lens, and the lens group is arranged between the first galvanometer and the objective lens in a 4f relay configuration.

7. The super-resolution light-sheet microscopy system according to claim 1, characterized in that, The fluorescence optical path includes a depth-of-field extension unit and an imaging unit; wherein... The depth-of-field extension unit is disposed between the objective lens and the imaging unit, and is used to project the fluorescence signal collected by the objective lens onto the imaging unit and extend the imaging depth of the imaging unit; the depth-of-field extension unit includes one of the following: phase modulation type, amplitude modulation type, multi-focal surface beam splitting type depth-of-field extension device or diffractive optical element type depth-of-field extension device.

8. The super-resolution light-sheet microscopy system according to claim 1, characterized in that, The fluorescence optical path includes an image rotation unit and an imaging unit; wherein... The image rotation unit is disposed between the objective lens and the imaging unit and is located in the image space conjugate with the sample. It includes at least one second galvanometer and multiple reflecting mirrors, and is used to rotate the imaging direction of the fluorescence signal collected by the objective lens. The imaging unit is disposed in the downstream optical path of the image rotation unit, and the imaging surface maintains a preset tilt angle with the optical axis of the objective lens, and is used to perform imaging detection on the image after it has been rotated by the image rotation unit.

9. The super-resolution light-sheet microscopy system according to any one of claims 1 to 8, characterized in that, It also includes a sample module, which includes a three-dimensional precision displacement stage for carrying the sample and a temperature and humidity control device for maintaining the sample environment.

10. A super-resolution light-sheet microscopy imaging method, characterized in that, include: Two opposing tilted light plates are generated using the illumination optical path, and the two tilted light plates interfere on the sample excitation surface to form structured light fringes; The fluorescence signal generated by the structured light stripes excited on the sample excitation surface is collected using two objective lenses positioned vertically opposite each other. The imaging optical path is used to expand the imaging depth of field and detect the fluorescence signals collected by the two objectives, thereby obtaining a super-resolution image of the sample excitation surface. The step of generating two opposing tilted light plates using the illumination optical path includes generating multiple two opposing tilted light plates in different directions using the illumination optical path, and the two opposing tilted light plates generated in each direction interfere with each other on the sample excitation surface to form structured light stripes including structured light stripes with multiple different phases.

Images