An extended field of view light sheet fluorescence microscopy imaging system, method and product

Abstract

This application provides an extended field-of-view light sheet fluorescence microscopy imaging system, method, and product, including a beam generation unit, a light sheet generation unit, a sample carrying unit, and a signal detection unit. The beam generation unit is configured to phase-modulate an incident Gaussian beam based on a preset phase pattern to obtain a target Bessel beam. The light sheet generation unit is configured to generate a Bessel light sheet based on the target Bessel beam. The sample carrying unit is configured to position the carried sample in the scanning area of ​​the Bessel light sheet. The signal detection unit is configured to collect the fluorescence signal generated by the sample in the scanning area. This application utilizes a long depth-of-focus, diffraction-free beam as the illumination light in light sheet fluorescence imaging, which overcomes the technical bottleneck of traditional Bessel beam light sheet imaging, which cannot guarantee resolution while improving the imaging field of view. This allows for covering a larger sample area in a single exposure, thus increasing imaging speed.

Description

Technical Field

[0001] This application relates to the field of fluorescence microscopy imaging technology, and in particular to a light-sheet fluorescence microscopy imaging system, method and product for extending the field of view. Background Technology

[0002] Light sheet fluorescence microscopy utilizes a thin beam of light to selectively excite a specific focal plane of a fluorescent sample, effectively avoiding non-specific excitation of fluorophores and endogenous molecules outside the focal plane. This significantly reduces photobleaching and phototoxicity effects, making it suitable for long-term, high-contrast three-dimensional imaging of live samples. In imaging systems, the field of view is the spatial extent of the sample acquired in a single acquisition. However, the field of view of a light sheet microscopy system is primarily limited by the penetrating power of the illumination light in the sample, essentially depending on the non-diffraction propagation distance of the beam.

[0003] Early light sheet imaging typically used Gaussian beams as illumination sources; however, their relatively short non-diffraction distance limited the imaging field of view. To expand the field of view, images often needed to be acquired from multiple angles and fused, which not only increased image acquisition time but also led to an increase in cumulative light dose, exacerbating photobleaching and photodamage problems. Bessel beams, as non-diffraction beams, not only possess a longer non-diffraction propagation distance than Gaussian beams but also exhibit unique "self-restoring" properties, maintaining their beam shape even in scattering media. Therefore, they demonstrate excellent applicability in biological sample imaging and have been widely used to extend the field of view and resolution of light sheet microscopy.

[0004] However, traditional Bessel beams still have a bottleneck: there is a constraint between their lateral resolution and diffraction-free distance. When using a lower numerical aperture objective lens, the diffraction-free distance can be extended, but the main lobe size of the beam increases, resulting in a decrease in lateral resolution; conversely, if a higher numerical aperture objective lens is used to improve resolution, the diffraction-free distance will be significantly shortened. Summary of the Invention

[0005] The purpose of this application is to provide a light sheet fluorescence microscopy imaging system, method and product that expands the field of view, which can at least solve the problems of small field of view and low resolution in related technologies under certain objective lens focusing conditions.

[0006] To address the aforementioned technical problems, the first aspect of this application provides an extended field-of-view light sheet fluorescence microscopy imaging system, comprising a beam generation unit, a light sheet generation unit, a sample carrying unit, and a signal detection unit; The beam generation unit is configured to perform phase modulation on the incident Gaussian beam based on a preset phase pattern to obtain a target Bessel beam; wherein, the target Bessel beam is composed of multiple segments of the original Bessel beam axially spliced ​​together, and the diffraction-free propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam; The light sheet generation unit is configured to generate a Bessel light sheet based on the target Bessel beam; The sample carrier unit is configured to position the carried sample to the scanning area of ​​the Bessel sheet; The signal detection unit is configured to acquire the fluorescence signal generated by the sample when it is stimulated in the scanning area.

[0007] In some embodiments, the beam generating unit includes a laser, an optical beam expander, a mirror, a phase diffraction unit, and an optical beam shrinker. The laser is configured to provide the incident Gaussian beam with a preset wavelength and power; The mirror is configured to reflect the incident Gaussian beam, which has been shaped and expanded by the optical beam expander, to the phase diffraction unit. The phase diffraction unit is configured to perform phase modulation on the shaped Gaussian beam based on a preset phase pattern to obtain the target Bessel beam.

[0008] In some embodiments, the expression for the preset phase pattern is: , in, It is an integer greater than 1. For the first Modulation parameters of amplitude and phase of the segmental Bessel beam. wave number and , The incident light wavelength, The objective lens convergence angle. For the first The axial position of the Bessel beam within the focal region of the illumination objective. For the first Phase control parameters of a segmented Bessel beam. This is a phase extraction function used to extract phase information; The transmittance function of a high-pass filter is expressed as: , in, This represents the radial coordinates within the plane containing the filter. This indicates the effective aperture radius of the filter, used to adjust the axial length of the generated single Bessel beam.

[0009] In some embodiments, the optical beam expander unit includes a first 4f system composed of a first cemented doublet lens and a second cemented doublet lens, and the optical beam shrinker unit includes a second 4f system composed of a third cemented doublet lens and a fourth cemented doublet lens.

[0010] In some embodiments, the light sheet generation unit includes an aperture stop, a galvanometer, a scanning lens, a first sleeve lens, and a first objective lens; The aperture stop is configured to filter out the unmodulated zero-order light in the target Bessel beam; The galvanometer is configured to scan the filtered target Bessel beam; The scanning lens is configured to perform angle correction on the scanned target Bessel beam; The first sleeve lens is configured to convert the corrected target Bessel beam into a collimated beam parallel to the optical axis; The first objective lens is configured to converge the collimated beam to form the Bessel plate.

[0011] In some embodiments, the signal detection unit includes a second objective lens, a filter, a second sleeve lens, and a data acquisition unit; The second objective lens is configured to acquire fluorescence signals generated by the sample in the scanning region upon stimulation; The filter is configured to filter the fluorescence signal; The second sleeve lens is configured to focus and image the filtered fluorescence signal; The acquisition unit is configured to receive the fluorescence signal focused and imaged by the second sleeve lens.

[0012] In some embodiments, the sample carrying unit includes a sample clamping unit and a displacement stage.

[0013] A second aspect of this application provides a light sheet fluorescence microscopy imaging method for extending the field of view, applied to a light sheet fluorescence microscopy imaging system for extending the field of view, including a beam generation unit, a light sheet generation unit, a sample carrying unit, and a signal detection unit; the method includes: The beam generation unit modulates the phase of the incident Gaussian beam based on a preset phase pattern to obtain the target Bessel beam; wherein, the target Bessel beam is composed of multiple segments of the original Bessel beam spliced ​​together axially, and the diffraction-free propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam; The light sheet generation unit generates a Bessel light sheet based on the target Bessel beam; The sample carrying unit positions the sample it carries to the scanning area of ​​the Bessel sheet; The signal detection unit acquires the fluorescence signal generated by the sample when it is stimulated in the scanning area.

[0014] A third aspect of this application provides an electronic device, including a memory and a processor, wherein the processor is configured to execute a computer program stored in the memory, and when the processor executes the computer program, it implements the steps of the extended field-of-view light sheet fluorescence microscopy imaging method described in the second aspect of the embodiments of this application.

[0015] The fourth aspect of this application provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the extended field-of-view light sheet fluorescence microscopy imaging method described in the second aspect of the embodiments of this application.

[0016] As described above, this application provides a light sheet fluorescence microscopy imaging system with an extended field of view, including a beam generation unit, a light sheet generation unit, a sample carrying unit, and a signal detection unit. The beam generation unit is configured to perform phase modulation on an incident Gaussian beam based on a preset phase pattern to obtain a target Bessel beam. The target Bessel beam is composed of multiple segments of original Bessel beams axially spliced ​​together, and the non-diffraction propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam. The light sheet generation unit is configured to generate a Bessel light sheet based on the target Bessel beam. The sample carrying unit is configured to position the carried sample in the scanning area of ​​the Bessel light sheet. The signal detection unit is configured to collect the fluorescence signal generated by the sample in the scanning area. This invention uses a laser with a Gaussian output mode as the excitation source, and the non-diffraction beam generation module generates a long-depth-of-focus Bessel beam with a non-diffraction distance many times that of traditional Bessel beams, thus improving the imaging field of view of light sheet fluorescence microscopy imaging while maintaining the same lateral resolution. By using a long focal depth non-diffraction beam as illumination light in light sheet fluorescence imaging, the technical bottleneck of not being able to guarantee resolution while improving the imaging field of view in traditional Bessel beam light sheet imaging can be overcome, enabling the coverage of a larger sample area in a single exposure and improving imaging speed.

[0017] It should be understood that the description in this section is not intended to identify key or important features of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0018] To more clearly illustrate the related technologies or the technical solutions in the embodiments of this application, the drawings used in the description of the related technologies or the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application, and not all embodiments. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1A schematic diagram of the structure of the light sheet fluorescence microscopy imaging system with extended field of view provided in the embodiments of this application; Figure 2 A detailed structural schematic diagram of the light sheet fluorescence microscopy imaging system with an extended field of view provided in an embodiment of this application; Figure 3 A detailed structural schematic diagram of the light sheet fluorescence microscopy imaging system with an extended field of view provided in an embodiment of this application; Figure 4 A detailed structural schematic diagram of the light sheet fluorescence microscopy imaging system with an extended field of view provided in an embodiment of this application; Figure 5 A schematic diagram of the specific structure of the light sheet fluorescence microscopy imaging system with extended field of view provided in the embodiments of this application; Figure 6 Comparison of axial cross-sectional light intensity distribution diagrams and axial light intensity curves for different light beams provided in the embodiments of this application; Figure 7 A schematic flowchart of the light sheet fluorescence microscopy imaging method for expanding the field of view provided in the embodiments of this application; Figure 8 A module block diagram of the electronic device provided in the embodiments of this application; Figure 9 A block diagram of a computer-readable storage medium provided in an embodiment of this application.

[0020] Explanation of reference numerals in the attached figures: 1. Laser; 2. First cemented doublet; 3. Second cemented doublet; 4. Mirror; 5. Phase diffraction device; 6. Third cemented doublet; 7. Fourth cemented doublet; 8. Aperture stop; 9. Galvanometer; 10. Scanning lens; 11. First sleeve lens; 12. First objective lens; 13. Three-axis translation stage; 14. Second objective lens; 15. Filter; 16. Second sleeve lens; 17. Camera; 18. Host computer; 19. Three-dimensional schematic diagram of the light sheet. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this application more apparent and understandable, this application will be clearly and completely described below in conjunction with its embodiments and accompanying drawings. Throughout, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. It should be understood that the various embodiments of this application described below are merely illustrative of this application and are not intended to limit this application. That is, all other embodiments obtained by those skilled in the art based on the various embodiments of this application without creative effort are within the scope of protection of this application. Furthermore, the technical features involved in the various embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0022] Light sheet fluorescence microscopy utilizes a thin beam of light to selectively excite a specific focal plane of a fluorescent sample, effectively avoiding non-specific excitation of fluorophores and endogenous molecules outside the focal plane. This significantly reduces photobleaching and phototoxicity effects, making it suitable for long-term, high-contrast three-dimensional imaging of live samples. In imaging systems, the field of view is the spatial extent of the sample acquired in a single acquisition. However, the field of view of a light sheet microscopy system is primarily limited by the penetrating power of the illumination light in the sample, essentially depending on the non-diffraction propagation distance of the beam.

[0023] Early light sheet imaging typically used Gaussian beams as illumination sources; however, their relatively short non-diffraction distance limited the imaging field of view. To expand the field of view, images often needed to be acquired from multiple angles and fused, which not only increased image acquisition time but also led to an increase in cumulative light dose, exacerbating photobleaching and photodamage problems. Bessel beams, as non-diffraction beams, not only possess a longer non-diffraction propagation distance than Gaussian beams but also exhibit unique "self-restoring" properties, maintaining their beam shape even in scattering media. Therefore, they demonstrate excellent applicability in biological sample imaging and have been widely used to extend the field of view and resolution of light sheet microscopy.

[0024] However, traditional Bessel beams still face a bottleneck: a constraint exists between their lateral resolution and diffraction-free distance. While using lower numerical aperture objectives can extend the diffraction-free distance, it increases the main lobe size, leading to a decrease in lateral resolution. Conversely, using higher numerical aperture objectives to improve resolution significantly shortens the diffraction-free distance. Therefore, overcoming the trade-off between field of view and resolution under certain objective focusing conditions to achieve high-resolution imaging with a larger field of view remains a key challenge for light-sheet fluorescence microscopy and a crucial prerequisite for precise observation of biological activities.

[0025] This application provides a light sheet fluorescence microscopy imaging system for extending the field of view. For details, please refer to... Figure 1 , Figure 1 This is a schematic diagram of the structure of the extended field-of-view light sheet fluorescence microscopy imaging system provided in this application embodiment. The extended field-of-view light sheet fluorescence microscopy imaging system includes a beam generation unit 101, a light sheet generation unit 102, a sample carrying unit 103, and a signal detection unit 104. The beam generation unit 101 is configured to perform phase modulation on the incident Gaussian beam based on a preset phase pattern to obtain a target Bessel beam. The target Bessel beam is composed of multiple segments of original Bessel beams axially spliced ​​together, and the non-diffraction propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam. The light sheet generation unit 102 is configured to generate a Bessel light sheet based on the target Bessel beam. The sample carrying unit 103 is configured to position the carried sample to the scanning area of ​​the Bessel light sheet. The signal detection unit 104 is configured to collect the fluorescence signal generated by the sample in the scanning area.

[0026] Specifically, the beam generation unit modulates the phase of the incident Gaussian beam based on a preset phase pattern, ultimately outputting a target Bessel beam. This target Bessel beam is composed of multiple segments of the original Bessel beam seamlessly spliced ​​together along the axial dimension. Its diffraction-free propagation distance is positively correlated with the number of segments of the original Bessel beam. By increasing the number of spliced ​​segments of the original Bessel beam, the diffraction-free propagation distance of the target Bessel beam can be significantly extended, generating a long-depth-of-focus Bessel beam 1.5 to 3 times longer than a traditional Bessel beam, which is then incident on the scanning microscope, providing a foundation for subsequent large-field-of-view imaging while maintaining the high lateral resolution characteristic inherent in the Bessel beam itself. The light sheet generation unit receives the target Bessel beam output from the beam generation unit and, based on the propagation characteristics and energy distribution of the target Bessel beam, converts it into a Bessel light sheet capable of covering a large sample area. The effective illumination range of the Bessel light sheet is adapted to the diffraction-free propagation distance of the target Bessel beam, ensuring a large-field-of-view illumination effect. The sample-carrying unit is used to carry the sample to be observed, including but not limited to cell samples, tissue sections, and live samples. According to preset imaging requirements, the sample is precisely positioned within the scanning area of ​​the Bessel sheet, ensuring that the area to be observed is fully covered and excited by the Bessel sheet. The signal detection unit is aligned with the scanning area of ​​the Bessel sheet. When the sample is excited by the Bessel sheet and generates a fluorescence signal, this unit collects the fluorescence signal in real time for subsequent imaging. In summary, the extended field-of-view light-sheet fluorescence microscopy imaging system provided by this invention can utilize Bessel sheets with long focal depths. Combined with the sample control module, it can achieve large field-of-view high-resolution volumetric imaging, capturing large field-of-view high-resolution optical sections of samples at different depths, thereby rapidly obtaining high-resolution structural information of the sample, and possessing high imaging speed.

[0027] Figure 2 A detailed structural schematic diagram of a light sheet fluorescence microscopy imaging system for expanding the field of view, provided in an embodiment of this application, is shown below. Figure 2 As shown, in some embodiments of this application, the beam generation unit 101 includes a laser 1011, an optical beam expander 1012, a reflector 1013, a phase diffraction unit 1014, and an optical beam shrinker 1015; the laser 1011 is configured to provide an incident Gaussian beam with a preset wavelength and power; the reflector 1013 is configured to reflect the incident Gaussian beam, which has been shaped and expanded by the optical beam expander 1012, to the phase diffraction unit 1014; the phase diffraction unit 1014 is configured to perform phase modulation on the shaped Gaussian beam based on a preset phase pattern to obtain a target Bessel beam.

[0028] Specifically, the laser serves as the emission source for the incident Gaussian beam, providing an incident Gaussian beam with a preset wavelength and power. This incident Gaussian beam provides the basic light source for subsequent phase modulation, and its wavelength and power can be adapted and adjusted according to actual imaging requirements (such as sample type, imaging depth, etc.) to ensure that the light source characteristics match the subsequent modulation and imaging needs. The laser's output mode is Gaussian and has power adjustment capabilities. Using near-infrared light as the excitation source can enhance the penetration ability into biological tissues and effectively reduce phototoxicity and photobleaching effects. Preferably, the Gaussian excitation light is femtosecond-level excitation light or near-infrared light.

[0029] The optical beam expander receives the incident Gaussian beam from the laser and performs beam shaping and expansion. Shaping optimizes the beam's spot uniformity and optical field distribution, preventing subsequent phase modulation distortion due to poor beam quality. Expanding adjusts the beam size to match the modulation requirements of the phase diffraction unit, providing conditions for precise phase modulation and ultimately outputting a well-shaped Gaussian beam.

[0030] A reflector is positioned between the optical beam expander and the phase diffraction unit to change the transmission direction of the shaped Gaussian beam. This ensures that the shaped Gaussian beam, after being processed by the optical beam expander, is accurately reflected to the incident end of the phase diffraction unit, guaranteeing that the beam can enter the modulation stage according to the preset optical path.

[0031] The phase diffraction unit is the core modulation component of the beam generation unit, configured to perform wavefront phase modulation on the incident shaped Gaussian beam based on a preset phase pattern. This modulation process achieves axial splicing of multiple segments of the original Bessel beam by directionally controlling the phase distribution of the shaped Gaussian beam, ultimately generating a target Bessel beam whose diffraction-free propagation distance is positively correlated with the number of segments of the original Bessel beam.

[0032] The optical beam-shrinking unit receives the target Bessel beam output from the phase diffraction unit, performs beam-shrinking processing on it, and conjugates the beam-shrinking target Bessel beam to the light sheet generating unit, so that the size of the target Bessel beam matches the incident parameters of the light sheet generating unit.

[0033] In some embodiments of this application, the expression for the preset phase pattern is: , in, It is an integer greater than 1. For the first Modulation parameters of amplitude and phase of the segmental Bessel beam. wave number and , The incident light wavelength, The objective lens convergence angle. For the first The axial position of the Bessel beam within the focal region of the illumination objective. For the first Phase control parameters of a segmented Bessel beam. This is a phase extraction function used to extract phase information; The transmittance function of a high-pass filter is expressed as: , in, This represents the radial coordinates within the plane containing the filter. This indicates the effective aperture radius of the filter, used to adjust the axial length of the generated single Bessel beam.

[0034] Specifically, phase modulation mainly modulates the incident beam by processing a phase plate or loading a phase pattern onto a spatial light modulator, connecting N segments of the Bessel beam axially to ultimately form a Bessel beam with a long depth of focus. It can be understood that the formula... It is the natural exponential function, representing exponential operations with the natural constant e (the natural constant, with a value of approximately 2.71828) as the base. The imaginary unit is used to construct the phase factor in complex form.

[0035] In some embodiments of this application, the optical beam expander 1012 includes a first 4f system composed of a first cemented doublet lens and a second cemented doublet lens, and the optical beam reducer 1014 includes a second 4f system composed of a third cemented doublet lens and a fourth cemented doublet lens.

[0036] Specifically, the optical beam expanding unit includes a first cemented doublet and a second cemented doublet, which are arranged sequentially along the optical path to form a first 4f system. The core function of this system is to shape and expand the incident Gaussian beam output from the laser: the focal length parameters of the first and second cemented doublets are matched, and the spacing is fixed according to the optical path design requirements of the 4f system (the spacing is equal to the sum of the focal lengths of the two lenses) to ensure that the beam can be transmitted along a preset optical path; the incident Gaussian beam is initially shaped by the first cemented doublet to correct the wavefront distortion of the beam and optimize the uniformity of the beam spot; then the shaped beam is expanded by the second cemented doublet to adjust the beam size to a range that matches the modulation aperture of the phase diffraction unit.

[0037] Similarly, the optical beam-shrinking unit includes a third cemented doublet and a fourth cemented doublet, which are arranged sequentially along the optical path to form a second 4f system. The core function of this system is to shrink and conjugate the target Bessel beam output from the phase diffraction unit: the focal length parameters of the third and fourth cemented doublets are matched according to a preset beam-shrinking ratio, and the optical path design specifications of the 4f system are also followed to ensure the stability and repeatability of beam transmission; the target Bessel beam is shrunken to a preset size (preferably shrunken to one-quarter of the original spot size) through this second 4f system, and the shrunken target Bessel beam is conjugated to the galvanometer surface of the sheet generation unit, so that the incident size of the beam is precisely matched with the effective scanning area of ​​the galvanometer and the incident aperture parameters of the first objective lens in the sheet generation unit, avoiding sheet distortion or energy loss caused by beam size incompatibility, and providing suitable beam conditions for the stable generation of subsequent Bessel sheets.

[0038] Figure 3 A detailed structural schematic diagram of a light sheet fluorescence microscopy imaging system for expanding the field of view, provided in an embodiment of this application, is shown below. Figure 3 As shown, in some embodiments of this application, the light sheet generation unit 102 includes an aperture stop 1021, a galvanometer 1022, a scanning lens 1023, a first sleeve lens 1024, and a first objective lens 1025; the aperture stop 1021 is configured to filter out the unmodulated zero-order light in the target Bessel beam; the galvanometer 1022 is configured to scan the filtered target Bessel beam; the scanning lens 1023 is configured to perform angle correction on the scanned target Bessel beam; the first sleeve lens 1024 is configured to convert the corrected target Bessel beam into a collimated beam parallel to the optical axis; and the first objective lens 1025 is configured to converge the collimated beam to form a Bessel light sheet.

[0039] Specifically, the aperture stop, as a pre-filter component of the beam generation unit, is used to filter out the unmodulated zero-order light in the target Bessel beam. Since a small amount of unmodulated zero-order light is generated during the phase modulation process of the beam generation unit, and this zero-order light does not possess long depth-of-focus characteristics, its direct participation in beam generation would lead to a decrease in beam quality and increased stray light interference. Therefore, the aperture stop filters out only the phase-modulated first-order light.

[0040] The galvanometer is positioned between the aperture stop and the first objective lens to scan the filtered target Bessel beam. Through scanning motion along a preset trajectory, the target Bessel beam, which has line focusing characteristics, is expanded into a beam with area illumination capability. The scanning rhythm can be adapted to the imaging requirements of the system to ensure that the light sheet formed subsequently can cover the area of ​​the sample to be observed.

[0041] The scanning lens is configured to receive the offset beam after scanning by the galvanometer. Its core function is to perform angle pre-correction and optical path straightening on the offset beam. To address the beam deviation angle caused by the galvanometer scanning, the scanning lens adjusts the beam propagation direction using its own optical properties, initially offsetting part of the deviation and adjusting the divergent offset beam into a nearly parallel beam state, thus preventing the accumulation of deviation from increasing the difficulty of subsequent collimation.

[0042] The sleeve lens and scanning lens are the core components for solving the aberration problem of the optical sheet. The key advantage of this combined design is that, through precise matching of the focal length parameters of the scanning lens and the sleeve lens, the beam offset caused by the galvanometer scanning can be completely canceled out. Even if the beam deviates from the optical axis due to scanning, it can be converted into a collimated beam completely parallel to the optical axis after the synergistic effect of the two. This design completely solves the problems of optical sheet blurring and excessive aberration caused by the direct combination of the galvanometer and objective lens in traditional methods, making the optical path when the beam enters the first objective lens more regular.

[0043] The first objective lens, as the core converging component for light sheet generation, is configured to precisely converge the collimated beam, corrected by the scanning lens and the sleeve lens, ultimately forming a Bessel light sheet with minimal aberrations, regular shape, and sharp edges. The optical parameters of the first objective lens are precisely matched with the characteristics of the collimated beam, enabling stable focusing of the collimated beam into a thin-film illumination field. This Bessel light sheet not only inherits the large field of view and high resolution characteristics of the target Bessel beam, but also possesses higher illumination uniformity and signal excitation efficiency due to effective aberration suppression. After incident on the sample surface positioned by the sample carrier unit, it can effectively excite the sample to generate a high signal-to-noise ratio fluorescence signal.

[0044] Figure 4 A detailed structural schematic diagram of a light sheet fluorescence microscopy imaging system for expanding the field of view, provided in an embodiment of this application, is shown below. Figure 4 As shown, in some embodiments of this application, the signal detection unit 104 includes a second objective lens 1041, a filter 1042, a second sleeve lens 1043, and a acquisition unit 1044; the second objective lens 1041 is configured to acquire the fluorescence signal generated by the sample stimulated in the scanning area; the filter 1042 is configured to filter the fluorescence signal; the second sleeve lens 1043 is configured to focus the filtered fluorescence signal; and the acquisition unit 1044 is configured to receive the fluorescence signal focused by the second sleeve lens.

[0045] Specifically, the second objective lens is used to accurately acquire the fluorescence signal generated by the sample excited by the Bessel plate within the scanning area. This second objective lens is positioned above the sample, and its optical parameters are adapted to the illumination range of the Bessel plate and the characteristics of the sample, enabling it to efficiently capture fluorescence signals generated in different regions of the sample.

[0046] A filter is positioned between the second objective lens and the second sleeve lens to filter the fluorescence signal acquired by the second objective lens. Since the raw acquired signal may contain interference signals such as excitation light not absorbed by the sample and ambient stray light, the filter, through specific spectral filtering characteristics, allows only the target fluorescence signal to pass through, filtering out excitation light and other irrelevant stray light. This purifies the fluorescence signal and prevents interference signals from affecting subsequent imaging quality.

[0047] The second sleeve lens is configured to focus and image the fluorescence signal purified by the filter. Through the optical focusing effect of the second sleeve lens, the divergent fluorescence signal is converged into a well-shaped and energy-concentrated imaging spot, optimizing the spatial distribution of the signal and the image sharpness.

[0048] The acquisition unit is configured to receive the fluorescence signal after it has been focused and imaged by the second sleeve lens, and to convert the focused optical fluorescence signal into an electrical or digital signal that can be used for subsequent processing. The receiving end of the acquisition unit is precisely aligned with the imaging plane of the second sleeve lens to ensure complete capture of the focused fluorescence signal. The converted signal data can be directly used for image reconstruction in the system.

[0049] In some embodiments of this application, the sample carrying unit 103 includes a sample clamping unit and a displacement stage.

[0050] Specifically, the sample clamping unit is configured to fix the sample to be observed, including but not limited to different types of observation objects such as cell samples, tissue sections, and live samples. The stable clamping structure ensures that the sample maintains a fixed spatial orientation during imaging, preventing deviation of the scanning area or distortion of fluorescence signal acquisition due to sample displacement or shaking. The displacement stage is configured to move the sample to adjust its spatial position, precisely positioning the area to be observed within the scanning area of ​​the Bessel sheet. The movement direction of the displacement stage is adapted to the scanning range of the Bessel sheet, allowing adjustment of the sample's spatial position according to imaging requirements. This enables different areas of the sample to sequentially enter the scanning area, achieving orderly observation of the entire sample area. Simultaneously, its positioning accuracy matches the scanning accuracy of the sheet generation unit and the acquisition rhythm of the signal detection unit, ensuring the coordination of sample positioning, sheet scanning, and signal acquisition, guaranteeing the continuity and integrity of the imaging.

[0051] Figure 5 A schematic diagram of a light sheet fluorescence microscopy imaging system for extending the field of view, provided in an embodiment of this application, is shown below. Figure 5 As shown, in this embodiment, the beam generating unit 101 includes: Laser 1, with a Gaussian output mode and power adjustment capability, utilizes near-infrared light as the excitation source to enhance penetration into biological tissues and effectively reduce phototoxicity and photobleaching effects. This provides a crucial technological foundation for long-term, high-contrast three-dimensional observation and pathological diagnosis of live samples. The Gaussian excitation light is preferably femtosecond-level or near-infrared light.

[0052] The first cemented doublet lens 2 is used in conjunction with the second cemented doublet lens 3 to form a 4f system, which shapes and expands the light spot.

[0053] The first reflecting mirror 4 is used to reflect the shaped excitation light into the phase diffraction device 5; wherein the phase diffraction device is preferably a transmission-type processed phase plate.

[0054] Phase diffraction device 5 is used to phase modulate the excitation light to generate a long focal depth Bessel beam.

[0055] Phase modulation is mainly achieved by processing a phase plate or loading a phase pattern onto a spatial light modulator to modulate the incident beam, connecting N segments of the Bessel beam axially to ultimately form a Bessel beam with a long focal depth. Its phase pattern is represented as follows: , in, It is an integer greater than 1. For the first Modulation parameters of amplitude and phase of the segmental Bessel beam. wave number and , The incident light wavelength, The objective lens convergence angle. For the first The axial position of the Bessel beam within the focal region of the illumination objective. For the first Phase control parameters of a segmented Bessel beam. This is a phase extraction function used to extract phase information; The transmittance function of a high-pass filter is expressed as: , in, This represents the radial coordinates within the plane containing the filter. This indicates the effective aperture radius of the filter, used to adjust the axial length of the generated single Bessel beam.

[0056] The third cemented doublet lens 6 is used in conjunction with the fourth cemented doublet lens 7 to form a 4f system, which reduces the light spot and conjugates it to the galvanometer of the light sheet generating unit 102, wherein preferably the light spot is reduced to one-quarter of its original size.

[0057] In this embodiment, the light sheet generation unit 102 includes an aperture stop 8, a galvanometer 9, a scanning lens 10, a first sleeve lens 11, and a first objective lens 12; the excitation light output by the beam generation unit 101 passes sequentially through the aperture stop 8, the galvanometer 9, the scanning lens 10, the first sleeve lens 11, and the first objective lens 12 to generate a long focal depth Bessel beam which is then directed to the sample on the three-axis translation stage 13.

[0058] In this embodiment, the sample carrying unit 103 includes a sample holder and an XYZ triaxial displacement stage. The sample holder is used to fix cells, slices and samples, and the XYZ triaxial displacement stage is used to move the sample manually or electrically and to scan and excite fluorescence in different areas of the sample.

[0059] In this embodiment, the signal detection unit 104 includes a second objective lens 14, a filter 15, a second sleeve lens 16, and a camera 17; the filter 15 is placed between the second objective lens 14 and the second sleeve lens 16 to filter out signals other than the excitation light; the camera 17 is located on the Fourier surface of the second sleeve lens 16 to detect the fluorescence signal emitted by the sample. In this embodiment, the excitation beam generated by the beam generation unit 101 excites the sample in the sample carrier unit 103 via the light sheet generation unit 102. The generated fluorescence signal is collected by the second objective lens 14, filtered out by the filter 15 to remove information other than the fluorescence signal, then focused by the second sleeve lens 16, and converted into an electrical signal by the camera 17, which is then processed by the computer.

[0060] In some specific embodiments, the incident light is linearly polarized light with a diameter of 8 mm and a wavelength of 1040 nm; other wavelengths can be used in specific implementations. The numerical aperture of the objective lens 12 is 0.9, and the number of Bessel focal points N = 3; other values ​​for the numerical aperture and the number of focal points can be used in specific implementations. This system includes, but is not limited to, diffraction-free beam volumetric imaging and two-photon excitation fluorescence, wherein the diffraction-free beam includes, but is not limited to, a Bessel beam.

[0061] like Figure 6 The diagram shows the axial cross-sectional light intensity distribution and axial light intensity curve comparison diagrams of different beams provided in this embodiment. In this example, the objective lens has a numerical aperture of 0.9, and the incident light is linearly polarized light with a diameter of 8 mm and a wavelength of 1040 nm. After being modulated by phase plates with different phase distributions, the beams emitted from the objective lens are a Gaussian beam, a conventional Bessel beam, and a long focal depth Bessel beam generated by axial splicing. Figure 6 (a) shows the axial cross-sectional light intensity distribution and axial light intensity curve of the Gaussian beam under this condition; Figure 6 (b) shows the axial cross-sectional light intensity distribution and axial light intensity curve of a conventional Bessel beam under this condition; Figure 6 (c) to Figure 6 (f) shows the axial cross-sectional intensity distribution and axial intensity curves of long-focus Bessel beams with different focal numbers under this condition. Figure 6 (c) is a long focal depth Bessel beam with two Bessel focal points, whose focal positions are (-5.75, 5.75). Figure 6 (d) is a long focal depth Bessel beam with three Bessel focal points, and the focal positions are (-12.31, 0, 12.31). Figure 6 (e) is a long focal depth Bezier beam with four Bezier focal points, whose focal positions are (-14.55, -4.52, 4.52, 14.55). Figure 6 (f) shows a long-depth Bessel beam with five Bessel focal points, located at (-19.155, -8, 0, 8, 19.155). The upper part of the figure shows the two-dimensional intensity distribution in the YZ plane, while the lower part shows the one-dimensional normalized intensity distribution along the optical axis (z-axis). Other numerical aperture values ​​and beams of other wavelengths can be used in specific implementations. As can be seen from the figure, the intensity distribution of the long-depth Bessel beam (cf) obtained in this embodiment is significantly wider than that of the Gaussian beam (a) and the conventional Bessel beam (b). Under certain objective focusing conditions, it can achieve a significant increase in field of view and resolution.

[0062] In summary, after the laser 1 in the beam generation unit 101 outputs excitation light, it passes through the first cemented doublet lens 2, which, in conjunction with the second cemented doublet lens 3, expands the beam spot. The beam then passes through the first reflecting mirror 4 and is incident on the phase diffraction device 5. The third cemented doublet lens 6 and the fourth cemented doublet lens 7 form a 4f system, conjugating the beam spot emitted from the phase diffraction device 6 onto the scanning galvanometer in the scanning device 8. The beam then passes through the aperture stop 8 to filter out other orders of light, retaining only the first order. The galvanometer 9, scanning lens 10, first sleeve lens 11, and objective lens 12 generate a long-deep-focal-length Bessel optical sheet, which is then sent to the sample control module. The fluorescence signal excited by the sample sequentially passes through the objective lens 14, filter 15, and second sleeve lens 16 to the camera 17, acquiring optical slices at different positions of the sample and reconstructing them to achieve high-resolution, large-field-of-view optical sheet fluorescence microscopy imaging.

[0063] In summary, this application provides an extended field-of-view light sheet fluorescence microscopy imaging system, comprising a beam generation unit, a light sheet generation unit, a sample carrying unit, and a signal detection unit. The beam generation unit is configured to phase-modulate an incident Gaussian beam based on a preset phase pattern to obtain a target Bessel beam. The target Bessel beam is composed of multiple segments of original Bessel beams axially spliced ​​together, and the non-diffraction propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam. The light sheet generation unit is configured to generate a Bessel light sheet based on the target Bessel beam. The sample carrying unit is configured to position the carried sample within the scanning area of ​​the Bessel light sheet. The signal detection unit is configured to acquire the fluorescence signal generated by the sample in the scanning area. This invention uses a laser with a Gaussian output mode as the excitation source, and the non-diffraction beam generation module generates a long-depth-of-focus Bessel beam with a non-diffraction distance many times that of a traditional Bessel beam, thereby improving the imaging field of view of light sheet fluorescence microscopy imaging while maintaining the same lateral resolution. By using a long focal depth non-diffraction beam as illumination light in light sheet fluorescence imaging, the technical bottleneck of not being able to guarantee resolution while improving the imaging field of view in traditional Bessel beam light sheet imaging can be overcome, enabling the coverage of a larger sample area in a single exposure and improving imaging speed.

[0064] Figure 7 A schematic flowchart of the extended field-of-view light sheet fluorescence microscopy imaging method provided in the third aspect of this application is shown. The extended field-of-view light sheet fluorescence microscopy imaging method is applied to an extended field-of-view light sheet fluorescence microscopy imaging system, including a beam generation unit, a light sheet generation unit, a sample carrying unit, and a signal detection unit; the method includes steps 701 to 704: Step 701: The beam generation unit modulates the phase of the incident Gaussian beam based on a preset phase pattern to obtain the target Bessel beam; wherein, the target Bessel beam is composed of multiple segments of the original Bessel beam axially spliced ​​together, and the diffraction-free propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam. Step 702: The sheet generation unit generates a Bessel sheet based on the target Bessel beam; Step 703: The sample carrying unit positions the sample it carries to the scanning area of ​​the Bessel sheet; Step 704: The signal detection unit collects the fluorescence signal generated by the sample in the scanning area when it is stimulated.

[0065] Please see Figure 8 , Figure 8 A block diagram of an electronic device provided in an embodiment of this application.

[0066] like Figure 8As shown, this application embodiment also provides an electronic device that can be used to implement the extended field-of-view light sheet fluorescence microscopy imaging method in the foregoing embodiments. The electronic device includes a memory 801 and at least one processor 802. The memory 801 is used to store at least one program, and when the at least one program is executed by the at least one processor 802, the at least one processor 802 executes the extended field-of-view light sheet fluorescence microscopy imaging method provided in this application embodiment.

[0067] Please see Figure 9 , Figure 9 A block diagram of a computer-readable storage medium provided in an embodiment of this application.

[0068] like Figure 9 As shown, this application embodiment also provides a computer-readable storage medium 900, on which executable instructions 910 are stored. When the executable instructions 910 are executed, they perform the extended field-of-view light sheet fluorescence microscopy imaging method provided in this application embodiment.

[0069] The steps of the methods or algorithms described in conjunction with the embodiments disclosed herein can be implemented directly by hardware, a software module executed by a processor, or a combination of both. The software module can be located in random access memory (RAM), main memory, read-only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, removable disk, compact disc read-only memory (CD-ROM), or any other form of storage medium known in the art.

[0070] In the above embodiments, implementation can be achieved, in whole or in part, through software, hardware, firmware, or any combination thereof. When implemented in software, it can be implemented, in whole or in part, as a computer program product. A computer program product includes one or more computer instructions. When the computer program instructions are loaded and executed on a computer, all or part of the processes or functions described in this application are generated. The computer can be a general-purpose computer, a special-purpose computer, a computer network, or other programmable device. The computer instructions can be stored in a computer-readable storage medium or transmitted from one computer-readable storage medium to another. For example, computer instructions can be transmitted from one website, computer, server, or data center to another website, computer, server, or data center via wired (e.g., coaxial cable, fiber optic, digital subscriber line) or wireless (e.g., infrared, wireless, microwave, etc.) means. The computer-readable storage medium can be any available medium that a computer can access or a data storage device such as a server or data center that integrates one or more available media. The available medium can be a magnetic medium (e.g., floppy disk, hard disk, magnetic tape), an optical medium (e.g., Digital Video Disk, DVD), or a semiconductor medium (e.g., Solid State Disk).

[0071] It should be noted that the various embodiments in this application are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For product-related embodiments, since they are similar to method-related embodiments, the descriptions are relatively simple, and relevant parts can be referred to the descriptions of the method-related embodiments.

[0072] It should also be noted that, in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0073] The above description of the disclosed embodiments enables those skilled in the art to implement or use the content of this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined in this application may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A light-sheet fluorescence microscopy imaging system for extending the field of view, characterized in that, It includes a beam generation unit, a light sheet generation unit, a sample carrying unit, and a signal detection unit; The beam generation unit is configured to perform phase modulation on the incident Gaussian beam based on a preset phase pattern to obtain a target Bessel beam; wherein, the target Bessel beam is composed of multiple segments of the original Bessel beam axially spliced ​​together, and the diffraction-free propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam; The light sheet generation unit is configured to generate a Bessel light sheet based on the target Bessel beam; The sample carrier unit is configured to position the carried sample to the scanning area of ​​the Bessel sheet; The signal detection unit is configured to acquire the fluorescence signal generated by the sample when it is stimulated in the scanning area.

2. The extended field-of-view light sheet fluorescence microscopy imaging system according to claim 1, characterized in that, The beam generation unit includes a laser, an optical beam expander, a mirror, a phase diffraction unit, and an optical beam shrinker. The laser is configured to provide the incident Gaussian beam with a preset wavelength and power; The mirror is configured to reflect the incident Gaussian beam, which has been shaped and expanded by the optical beam expander, to the phase diffraction unit. The phase diffraction unit is configured to perform phase modulation on the shaped Gaussian beam based on a preset phase pattern to obtain the target Bessel beam.

3. The extended field-of-view light sheet fluorescence microscopy imaging system according to claim 2, characterized in that, The expression for the preset phase pattern is: ， in, It is an integer greater than 1. For the first Modulation parameters of amplitude and phase of the segmental Bessel beam. wave number and , The incident light wavelength, The objective lens convergence angle. For the first The axial position of the Bessel beam within the focal region of the illumination objective. For the first Phase control parameters of a segmented Bessel beam. This is a phase extraction function used to extract phase information; The transmittance function of a high-pass filter is expressed as: ， in, This represents the radial coordinates within the plane containing the filter. This indicates the effective aperture radius of the filter, used to adjust the axial length of the generated single Bessel beam.

4. The extended field-of-view light sheet fluorescence microscopy imaging system according to claim 2, characterized in that, The optical beam expander unit includes a first 4f system composed of a first cemented doublet lens and a second cemented doublet lens, and the optical beam shrinker unit includes a second 4f system composed of a third cemented doublet lens and a fourth cemented doublet lens.

5. The extended field-of-view light sheet fluorescence microscopy imaging system according to claim 1, characterized in that, The light sheet generation unit includes an aperture stop, a galvanometer, a scanning lens, a first sleeve lens, and a first objective lens; The aperture stop is configured to filter out the unmodulated zero-order light in the target Bessel beam; The galvanometer is configured to scan the filtered target Bessel beam; The scanning lens is configured to perform angle correction on the scanned target Bessel beam; The first sleeve lens is configured to convert the corrected target Bessel beam into a collimated beam parallel to the optical axis; The first objective lens is configured to converge the collimated beam to form the Bessel plate.

6. The extended field-of-view light sheet fluorescence microscopy imaging system according to claim 1, characterized in that, The signal detection unit includes a second objective lens, a filter, a second sleeve lens, and a data acquisition unit; The second objective lens is configured to acquire fluorescence signals generated by the sample in the scanning region upon stimulation; The filter is configured to filter the fluorescence signal; The second sleeve lens is configured to focus and image the filtered fluorescence signal; The acquisition unit is configured to receive the fluorescence signal focused and imaged by the second sleeve lens.

7. The extended field-of-view light sheet fluorescence microscopy imaging system according to claim 1, characterized in that, The sample carrying unit includes a sample clamping unit and a displacement stage.

8. A light-sheet fluorescence microscopy imaging method for expanding the field of view, characterized in that, A light-sheet fluorescence microscopy imaging system for expanding the field of view includes a beam generation unit, a light-sheet generation unit, a sample carrying unit, and a signal detection unit; the method includes: The beam generation unit modulates the phase of the incident Gaussian beam based on a preset phase pattern to obtain the target Bessel beam; wherein, the target Bessel beam is composed of multiple segments of the original Bessel beam spliced ​​together axially, and the diffraction-free propagation distance of the target Bessel beam is positively correlated with the number of segments of the original Bessel beam; The light sheet generation unit generates a Bessel light sheet based on the target Bessel beam; The sample carrying unit positions the sample it carries to the scanning area of ​​the Bessel sheet; The signal detection unit acquires the fluorescence signal generated by the sample when it is stimulated in the scanning area.

9. An electronic device, characterized in that, Includes memory and processor, of which: The processor is used to execute computer programs stored in the memory; When the processor executes the computer program, it implements the steps in the extended field-of-view light sheet fluorescence microscopy imaging method of claim 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by the processor, it implements the steps in the extended field-of-view light sheet fluorescence microscopy imaging method of claim 8.

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