Large field of view and high resolution light sheet illumination imaging system

Abstract

The present invention discloses a large-field-of-view, high-resolution light-sheet illumination imaging system, comprising: a light source module (S1), a first modulation module (S2), a first scanning module (S3), a second modulation module (S4), a second scanning module (S5), an excitation module (S6), and a detection module (S7). The present invention provides a large-field-of-view, high-resolution light-sheet illumination imaging system that can maintain subcellular resolution and effectively expand the field of view of the light-sheet illumination to several mm. The present invention utilizes birefringent crystals and phase modulation technology to generate multiple beams of light, and utilizes a spatial light modulator to independently control the multiple beams, thereby enabling one-time imaging of a large field of view of several millimeters in the sample space near the focal plane of the objective lens. While maintaining uniform high axial resolution, the imaging field of view is significantly expanded. The present invention can provide a technical foundation for high-resolution, high-throughput three-dimensional imaging of large-scale biological samples.

Description

Technical Field

[0001] The present invention relates to the technical field of optical microscopic imaging, and in particular to a large-field-of-view high-resolution light sheet illumination imaging system. Background Art

[0002] Individual life is a complex organism. Understanding and understanding individual life requires not only observing the information and response processes of different regions during their life activities, but also recording specific areas with high detail and resolution. Brain imaging, in particular, requires mapping the neuronal structure of the entire brain and tracking and imaging the dynamics of neuronal signals across regions spanning millimeters or even centimeters. This demands observation tools with a large field of view, high resolution, high throughput, and minimal damage. This is how light-sheet microscopy emerged. Light-sheet microscopy uses a thin laser to excite a fluorescently labeled sample and then detects and images the fluorescence at a perpendicular angle to the light sheet. Light-sheet scanning microscopy, with its advantages of high-speed 3D tomography and low light damage, is widely used for 3D imaging of transparent biological samples. However, due to the optical principle of conflict between the field of view and resolution of optical microscopy, subcellular resolution (approximately 1 to 5 microns) rarely exceeds 1 mm. To maintain resolution while increasing the field of view, the most common approach is to mount the biological sample on a 2D stage. After illuminating and imaging a specific small area with fluorescence, the sample is moved, imaged multiple times, and then stitched together to restore the large-field image. The disadvantages of this method are that it is slow, the stitching requires high precision, and it is very unfavorable for high-throughput imaging.

[0003] In order to maintain a high axial resolution and increase the field of view, some methods have been proposed in recent years. The article (Takanezawa, S., Saitou, T. & Imamura, T. Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination. Nat Commun 12, 2979 (2021)) generates a Bessel beam through a variable axis conical mirror, optimizes the light transmission efficiency, and combines it with two-photon nonlinear fluorescence imaging to achieve a 600-1000um field of view and an axial resolution of 2-3um illumination imaging. It uses a non-diffraction Bessel beam to generate many side lobes, and each side lobe receives almost equal energy. Although two-photon nonlinear excitation can effectively suppress the fluorescence effect of the side lobes, the main lobe energy density is low, and it is difficult to continue to increase the field of view.

[0004] Another way to resolve the contradiction between large field of view and axial resolution is to use a spatial light modulator to switch the phase within the exposure time of a camera to achieve time-sharing splicing of small field of view light sheets. This can achieve higher axial resolution and increase the field of view (Gao L. Extend the field of view of selective plan illumination microscopy bytiling the excitation light sheet [J]. Optics express, 2015, 23 (5): 6102-6111.). It performs three splicing operations under a 0.35 numerical aperture objective lens to achieve an axial resolution of 0.85um while increasing the field of view from 9.4um to 28um, a three-fold increase. Imaging a large field of view requires multiple adjustments to the position of the light sheet, and the excitation of light sheets in different areas is still time-sharing. The temporal resolution of observing biological activity reactions is limited by the response speed of the spatial light modulator. For a given spatial light modulator, large field of view and high spatiotemporal resolution imaging are in conflict.

[0005] Replacing the beam control device with a higher response speed can alleviate the above contradiction to a certain extent. The tunable acoustic gradient refractive index element has a fast response speed (up to 450 kHz). The article (Zong W, Zhao J, Chen X, et al. Large-field high-resolution two-photon digital scanned light-sheet microscopy [J]. Cell research, 2015, 25 (2): 254-257.) uses the axial zoom of the tunable acoustic gradient refractive index element (TAG) to quickly change the focal plane position of the beam focus. Combined with two-photon excitation, it can quickly obtain a 50-500um tunable imaging field of view while maintaining an axial resolution of 2-3um, alleviating the field of view and high spatiotemporal resolution problems. However, it fails to fundamentally solve the above problems. In addition, the refractive power of the tunable acoustic gradient refractive index element is limited, making it difficult to obtain a larger zoom range to achieve a larger field of view illumination.

[0006] Voigt FF, Kirschenbaum D, Platonova E, et al. The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue [J]. Nature methods, 2019, 16 (11): 1105-1108., using mesoSPIM light sheet microscope combined with transparency technology, achieved imaging with a field of view of 2.8mm to 28mm and an axial resolution of 6.5um. They used a zoom lens (ETL) to axially move the focal plane of the light sheet and only expose the corresponding row pixels of the camera to achieve high-resolution imaging over a large range. In essence, this method is still a single small field of view imaging, and then multiple stitching of single frames is used. The same stitching problem mentioned above exists. In addition, the linear response speed of the zoom lens is slow, and the liquid crystal inside is easily affected by gravity, becoming unstable and resulting in large aberrations.

[0007] The current methods for increasing the field of view can be divided into the following methods:

[0008] 1. Scanning a non-diffracting beam to generate light-sheet illumination technology. This non-diffracting beam includes Bessel and Airy beams. Increasing the field of view inevitably leads to an increase in sidelobes, diverting more laser energy from the main lobe to the sidelobes. Taking a Bessel beam light sheet as an example, it can be calculated that the sidelobe range is approximately proportional to the Bessel beam length (effective light-sheet field of view) (Liang and Gao. Optimization of the excitation light sheet in selective plane illumination microscopy. [J]. Biomedical Optics Express, 2015.). The effective field of view of a Bessel light sheet is defined by the effective length of the Bessel beam main lobe. Since each lobe of a Bessel beam carries approximately equal energy, this means that for a given light-sheet thickness (the primary parameter determining the axial resolution of a light-sheet microscope), expanding the field of view by a factor of N increases the sidelobe range by a factor of N, while reducing the main lobe energy by a factor of N. In single-photon imaging, excessive sidelobes can excite out-of-focus fluorescence, affecting image contrast, reducing axial resolution, and increasing phototoxicity. In the two-photon imaging mode, the fluorescence efficiency of the excitation is proportional to the square of the intensity of the excitation light. Under the same conditions, if the field of view increases N times, the fluorescence intensity will decrease to the original N. -2 , the field of view of the two-photon light sheet is difficult to further improve.

[0009] 2. Use zoom elements to change the focus position of the light beam to achieve a wider field of view. These zoom elements include spatial light modulators, tunable acoustic gradient refractive index elements, and zoom lenses. However, each of these elements has its own shortcomings. Spatial light modulators are slow; tunable acoustic gradient refractive index elements are fast but have limited refractive power; zoom lenses have intermediate speed and zoom power, but their working performance is unstable and prone to aberrations. These methods require post-processing of image stitching for large field of view imaging, and the instability of the scanning element directly affects the stitching effect.

[0010] In summary, light-sheet microscopy with a large field of view and high axial resolution is crucial for acquiring dynamic structural information from large biological samples in real time. The effective length of the light beam determines the effective imaging field of view of a light-sheet microscope, while the beam thickness determines its axial resolution. However, current methods struggle to achieve high-resolution, real-time imaging of fields of view on the order of several millimeters. To address these challenges, the present invention proposes a solution. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a large-field-of-view and high-resolution light sheet illumination imaging system in response to the above-mentioned deficiencies in the prior art.

[0012] To solve the above technical problems, the technical solution adopted by the present invention is: a large-field-of-view high-resolution light sheet illumination imaging system, comprising: a light source module, a first modulation module, a first scanning module, a second modulation module, a second scanning module, an excitation module, and a detection module;

[0013] The light beam emitted by the light source module is divided into two parts: a reflection part and a transmission part. The reflection part enters the first modulation module, and after modulation, enters the first scanning module. The generated first scanning light beam enters the excitation module to excite the sample to produce fluorescence; the transmission part enters the second modulation module, and after modulation, enters the second scanning module. The generated second scanning light beam enters the excitation module to excite the sample to produce fluorescence. The fluorescence emitted by the sample is collected and imaged by the detection module.

[0014] The first modulation module and the first scanning module constitute the first channel of the system, the second modulation module and the second scanning module constitute the second channel of the system, and the first scanning beam output by the first channel and the second scanning beam output by the second channel enter the excitation module in a mutually perpendicular manner.

[0015] Preferably, the light source module includes a laser, a collimating beam expander, an aperture, a half-wave plate, a light source birefringent crystal, a light source D-shaped half-wave plate and a light source beam splitter, which are sequentially arranged along the light path;

[0016] The laser light emitted by the laser passes through the collimating beam expander, the aperture, and the half-wave plate in sequence and then enters the light source birefringent crystal, and is divided into two beams of light: ordinary light o (002) and extraordinary light e (001). The extraordinary light e becomes horizontally polarized light e parallel to the ordinary light o after passing through the D-shaped half-wave plate of the light source. The ordinary light o and the horizontally polarized light e enter the light source spectroscope and are both divided into a transmitted light beam and a reflected light beam in equal proportions: the ordinary light o is divided into a transmitted light o (022) and a reflected light o (021) by the light source spectroscope, and the horizontally polarized light e is divided into a transmitted light e (012) and a reflected light e (011) by the light source spectroscope.

[0017] The reflected light o(021) and the reflected light e(011) enter the first modulation module, and the transmitted light o(022) and the transmitted light e(012) enter the second modulation module.

[0018] Preferably, the first modulation module includes a first modulation reflector, a first optical phase modulation device, a first spatial light modulator, a first lens, a first modulation D-shaped half-wave plate, a first modulation birefringent crystal and a second lens, which are sequentially arranged along the optical path;

[0019] The reflected light e(011) and the reflected light o(021) emitted by the light source module are reflected by the first modulating reflector and vertically incident on the first optical phase modulation device. The reflected light e is evenly divided into M beams, each beam has equal energy, and adjacent beams are separated by equal angles; the reflected light o is also evenly divided into M beams, each beam has equal energy, and adjacent beams are separated by equal angles; thereby forming a 2*M beam array and incident on the first spatial light modulator. The first spatial light modulator is divided into 2*M spatial regions, and each spatial region independently applies phase control to the corresponding beam in the 2*M beam array, so that each beam in the 2*M beam array contains a unique code, thereby obtaining a 2*M modulated beam.

[0020] After the 2*M modulated light beam passes through the first lens, the part of the modulated light beam originating from the reflected light e enters the first modulated D-shaped half-wave plate, changes from horizontal polarization to vertical polarization, and then enters the first modulated birefringent crystal together with the part of the modulated light beam originating from the reflected light o, and is combined into a 1*M modulated light beam, which enters the first scanning module after passing through the second lens.

[0021] Preferably, the first scanning module includes a first x-direction scanning galvanometer, a first scanning lens, a second scanning lens, a first z-direction scanning galvanometer, a third scanning lens and a fourth scanning lens, which are sequentially arranged along the optical path;

[0022] The 1*M modulated light beam output by the first modulation module is incident on the first x-direction scanning galvanometer, and then passes through the first scanning lens and the second scanning lens and is projected onto the first z-direction scanning galvanometer.

[0023] The first x-direction scanning galvanometer scans the light beam along the x-axis, and the first z-direction scanning galvanometer scans the light beam along the z-direction. The light beam emitted by the first z-direction scanning galvanometer passes through the third scanning lens and the fourth scanning lens (tube lens) in sequence to form a first scanning beam, and is incident on the excitation module.

[0024] Preferably, the second modulation module comprises a second modulation reflector, a second optical phase modulation device, a second spatial light modulator, a third lens, a second modulation D-shaped half-wave plate, a second modulation birefringent crystal and a fourth lens, which are sequentially arranged along the optical path;

[0025] The transmitted light o (022) and the transmitted light e (012) emitted by the light source module are reflected by the second modulating reflector and vertically incident on the second optical phase modulation device. The transmitted light e is evenly divided into M beams, each beam has equal energy, and adjacent beams are separated at equal angles. The transmitted light o is also evenly divided into M beams, each beam has equal energy, and adjacent beams are separated at equal angles. Thus, a 2*M beam array is formed and incident on the second spatial light modulator. The second spatial light modulator is divided into 2*M spatial regions, and each spatial region independently applies phase control to the corresponding beam in the 2*M beam array, so that each beam in the 2*M beam array contains a unique code, thereby obtaining a 2*M modulated beam.

[0026] After the 2*M modulated light beam passes through the third lens, the portion of the modulated light beam originating from the transmitted light e enters the second modulated D-shaped half-wave plate, changes from horizontal polarization to vertical polarization, and then enters the second modulated birefringent crystal together with the portion of the modulated light beam originating from the transmitted light o, and is combined into a 1*M modulated light beam, which enters the second scanning module after passing through the fourth lens.

[0027] Preferably, the second scanning module includes a second X-direction scanning galvanometer, a fifth scanning lens, a sixth scanning lens, a second Z-direction scanning galvanometer, a seventh scanning lens, a scanning reflector and an eighth scanning lens, which are sequentially arranged along the optical path;

[0028] The 1*M modulated light beam output by the second modulation module is incident on the second x-direction scanning galvanometer, and then passes through the fifth scanning lens and the sixth scanning lens and is projected onto the second z-direction scanning galvanometer;

[0029] The second x-direction scanning galvanometer scans the light beam along the x-axis, and the second z-direction scanning galvanometer scans the light beam along the z-direction. The light beam emitted by the second z-direction scanning galvanometer passes through the seventh scanning lens, the scanning reflector and the eighth scanning lens in sequence to form a second scanning beam, and is incident on the excitation module.

[0030] Preferably, the excitation module includes an excitation spectrometer, a fifth lens, a first reflector, a second reflector, a third reflector, a sixth lens, a fourth reflector and a first excitation objective lens arranged in sequence along the optical path of the first side of the excitation spectrometer, and a seventh lens, a fifth reflector, an eighth lens, a sixth reflector and a second excitation objective lens arranged in sequence along the optical path of the first side of the excitation spectrometer; the sample to be measured is arranged between the first excitation objective lens and the second excitation objective lens.

[0031] Preferably, the first scanning light beam output by the first scanning module is incident on the excitation spectroscope and is equally divided into a first scanning reflected light and a first scanning transmitted light. The first scanning reflected light is transmitted along the optical path of the first side of the excitation spectroscope, passes through the fifth lens, the first reflector, the second reflector, the third reflector, the sixth lens, the fourth reflector and the first excitation objective lens in sequence, and is then converged by the first excitation objective lens onto the first side of the sample.

[0032] The first scanning transmitted light is transmitted along the optical path of the second side of the excitation beam splitter, passes through the seventh lens, the fifth reflector, the eighth lens, the sixth reflector and the second excitation objective lens in sequence, and then the second excitation objective lens converges the light beam to the second side of the sample;

[0033] The second scanning beam output by the second scanning module, which is perpendicular to the first scanning beam, is incident on the excitation spectroscope and is equally divided into a second scanning reflected light and a second scanning transmitted light. The second scanning transmitted light is transmitted along the optical path on the first side of the excitation spectroscope, passes through the fifth lens, the first reflector, the second reflector, the third reflector, the sixth lens, the fourth reflector and the first excitation objective lens in sequence, and is then converged by the first excitation objective lens onto the first side of the sample.

[0034] The second scanning reflected light is transmitted along the optical path of the second side of the excitation spectrometer, and after passing through the seventh lens, the fifth reflector, the eighth lens, the sixth reflector and the second excitation objective lens in sequence, the second excitation objective lens converges the light beam to the second side of the sample.

[0035] Preferably, the detection module includes a large-field-of-view objective lens, a condenser, a filter and a camera arranged in sequence along the light path. The large-field-of-view objective lens is located above the sample to collect fluorescence emitted by the sample. The fluorescence collected by the large-field-of-view objective lens passes through the condenser and the filter and is collected by the camera to form an image.

[0036] Preferably, the first optical phase modulation device and the second optical phase modulation device are both spatial light modulators or diffractive optical elements.

[0037] The beneficial effects of the present invention are:

[0038] (1) The present invention provides a large-field, high-resolution light-sheet illumination imaging system that can maintain subcellular resolution and effectively expand the field of view of the light-sheet illumination to several mm. The present invention utilizes birefringent crystals and phase modulation technology to generate multiple beams of light, and utilizes a spatial light modulator to independently control the multiple beams, thereby enabling one-time large-field imaging of several millimeters of sample space near the focal plane of the objective lens. While maintaining uniform high axial resolution, the imaging field of view is significantly expanded. The present invention can provide a technical basis for high-resolution, high-throughput three-dimensional imaging of large-scale biological samples.

[0039] (2) Current methods of expanding the field of view using non-diffracting beams, such as Bessel beams, will introduce more side lobes in order to maintain high axial resolution and expand the field of view, which will reduce the energy density of the main lobe, leading to fluorescence excitation by side lobes in non-focal planes, reducing imaging contrast, and increasing photobleaching. The present invention uses optical phase elements to modulate the light beam to produce multi-beam illumination with adjustable lateral distances, thus avoiding fluorescence crosstalk between out-of-focus planes or between different focal points, and preventing photodamage and photobleaching of biological samples by the side lobes of the non-diffracting beam, thereby achieving higher resolution, higher contrast, and large-field imaging.

[0040] (3) For the diffraction-free beam light sheet in the multi-photon nonlinear excitation mode, increasing the field of view and enhancing the sidelobe effect causes more laser energy to be dispersed into the sidelobes; while maintaining the same main lobe of the beam, the effective field of view increases by N times, and the mainlobe energy for effective nonlinear excitation is only 1 / N of the total incident energy. Taking two-photon nonlinear excitation as an example, the fluorescence excitation efficiency is proportional to the square of the light intensity, resulting in a decrease in the effective two-photon excitation efficiency by the order of N^2. The sidelobe effect makes it difficult to excite fluorescence in nonlinear multiphoton mode under a large field of view. The present invention utilizes multi-beam control technology to generate multiple Gaussian light sheets (which can also be Bessel light sheets, Airy light sheets or other types of light sheets with few sidelobes) in the focal space of the objective lens, effectively improving the energy utilization rate of the excitation light in multi-photon nonlinear imaging, and avoiding or reducing the damage to biological samples caused by the thermal effect of the sidelobes of the diffraction-free light sheet in nonlinear excitation. Large-field, high-resolution, non-destructive, high-throughput three-dimensional multiphoton imaging of large-scale biological samples such as mouse brain and organoids is achieved.

[0041] (4) Another existing technology for maintaining high resolution and large field of view imaging uses a thin and short light beam to scan to form a light sheet, which completes the structural information collection of large-scale samples in small areas multiple times in a time-sharing manner, and restores the large-field three-dimensional image through post-image splicing processing. For large-scale samples of several millimeters or even centimeters, it often takes a long time, which is very unfavorable for capturing dynamic processes. The present invention uses multiple light sheets for simultaneous illumination, and can obtain dynamic field information of several millimeters or even centimeters through a one-time scan, which is conducive to observing the interaction between different functional areas of large-scale samples. It is conducive to high-throughput three-dimensional imaging of whole-brain neural structure information in brain science, conducive to studying the transmission, release, and reception process of signals between neurons in the whole brain, and conducive to drawing three-dimensional maps of brain structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the structure of the large-field-of-view and high-resolution light sheet illumination imaging system of the present invention;

[0043] Figure 2 A schematic diagram of the structure of the system of the present invention including channel 1;

[0044] Figure 3 This is a schematic diagram of the structure of the system of the present invention including channel 2;

[0045] Figure 4 This is a schematic diagram of the light source birefringent crystal splitting of the present invention;

[0046] Figure 5 Schematic diagram of the light source spectroscope of the present invention;

[0047] Figure 6 Schematic diagram of the light splitting principle of the first and second optical phase modulation devices of the present invention;

[0048] Figure 7 This is the beam array distribution diagram of the present invention;

[0049] Figure 8 Schematic diagram of the first and second spatial light modulators modulating light beams in different regions according to the present invention;

[0050] Figure 9 Schematic diagram of the formation of the first and second excitation objective rear light sheets of the present invention.

[0051] Description of reference numerals:

[0052] S1—Light source module

[0053] 01—Laser; 02—Collimating beam expander; 03—Aperture stop; 04—Half-wave plate; 05—Light source birefringent crystal; 06—Light source D-shaped half-wave plate; 07—Light source beam splitter;

[0054] S2—first modulation module

[0055] 100—first modulating reflector; 101—first optical phase modulator; 102—first spatial light modulator; 103—first lens; 104—first modulating D-shaped half-wave plate; 105—first modulating birefringent crystal; 106—second lens;

[0056] S3—First Scanning Module

[0057] 107—first X-direction scanning galvanometer; 108—first scanning lens; 109—second scanning lens; 110—first Z-direction scanning galvanometer; 111—third scanning lens; 112—fourth scanning lens;

[0058] S4—Second modulation module

[0059] 200—second modulating reflector; 201—second optical phase modulator; 202—second spatial light modulator; 203—third lens; 204—second modulating D-shaped half-wave plate; 205—second modulating birefringent crystal; 206—fourth lens;

[0060] S5—Second Scanning Module

[0061] 207 — second X-direction scanning galvanometer; 208 — fifth scanning lens; 209 — sixth scanning lens; 210 — second Z-direction scanning galvanometer; 211 — seventh scanning lens; 212 — scanning reflector; 213 — eighth scanning lens;

[0062] S6—Excitation Module

[0063] 300—excitation spectroscope; 301—fifth lens; 302—first reflector; 303—second reflector; 304—third reflector; 305—sixth lens; 306—fourth reflector; 307—first excitation objective lens;

[0064] 310—seventh lens; 311—fifth reflector; 312—eighth lens; 313—sixth reflector; 314—second excitation objective lens; 315—sample;

[0065] S7-Detection Module

[0066] 401—Wide field of view objective lens; 402—Condenser; 403—Filter; 404—Camera. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0068] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0069] Reference Figure 1-9 The present invention provides a large-field-of-view high-resolution light sheet illumination imaging system, comprising: a light source module S1, a first modulation module S2, a first scanning module S3, a second modulation module S4, a second scanning module S5, an excitation module S6, and a detection module S7;

[0070] The light beam emitted by the light source module S1 is divided into two parts: a reflection part and a transmission part. The reflection part enters the first modulation module S2, and after modulation, enters the first scanning module S3 to achieve beam scanning. The generated first scanning beam enters the excitation module S6 to excite the sample 315 to produce fluorescence. The transmission part enters the second modulation module S4, and after modulation, enters the second scanning module S5 to achieve beam scanning. The generated second scanning beam enters the excitation module S6 to excite the sample 315 to produce fluorescence. The fluorescence emitted by the sample 315 is collected and imaged by the detection module S7.

[0071] The first modulation module S2 and the first scanning module S3 constitute the first channel of the system, the second modulation module S4 and the second scanning module S5 constitute the second channel of the system, and the first scanning beam output by the first channel and the second scanning beam output by the second channel enter the excitation module S6 in a mutually perpendicular manner.

[0072] Reference Figure 1-3 In the present invention, the light source module S1 includes a laser 01, a collimating beam expander 02, an aperture 03, a half-wave plate 04, a light source birefringent crystal 05, a light source D-shaped half-wave plate 06 and a light source beam splitter 07, which are sequentially arranged along the light path;

[0073] The laser light emitted by laser 01 passes through collimating beam expander 02, aperture 03, and half-wave plate 04 in sequence and then enters light source birefringent crystal 05, where it is split into two beams: ordinary light o002 and extraordinary light e001. Extraordinary light e passes through light source D-shaped half-wave plate 06 and becomes horizontally polarized light e, which is parallel to ordinary light o. Ordinary light o and horizontally polarized light e enter light source beam splitter 07 and are equally split into transmitted light and reflected light beams: ordinary light o is split into transmitted light o022 and reflected light o021 by light source beam splitter 07, and horizontally polarized light e is split into transmitted light e012 and reflected light e011 by light source beam splitter 07.

[0074] The reflected light o021 and the reflected light e011 enter the first modulation module S2, and the transmitted light o022 and the transmitted light e012 enter the second modulation module S4.

[0075] In the present invention, the first modulation module S2 includes a first modulation reflector 100, a first optical phase modulation device 101, a first spatial light modulator 102, a first lens 103, a first modulation D-shaped half-wave plate 104, a first modulation birefringent crystal 105 and a second lens 106, which are sequentially arranged along the optical path;

[0076] The reflected light e011 and reflected light o021 emitted by the light source module S1 are reflected by the first modulating reflector 100 and vertically incident on the first optical phase modulator 101. The reflected light e is evenly divided into M beams, each with equal energy, and adjacent beams are separated by equal angles. The reflected light o is also evenly divided into M beams, each with equal energy, and adjacent beams are separated by equal angles. Thus, a 2*M beam array is formed and incident on the first spatial light modulator 102. The first spatial light modulator 102 is divided into 2*M spatial regions. Each spatial region independently applies phase control to the corresponding beam in the 2*M beam array, so that each beam in the 2*M beam array contains a unique code, thereby obtaining a 2*M modulated beam.

[0077] After the 2*M modulated light beam passes through the first lens 103, the portion of the modulated light beam originating from the reflected light e enters the first modulated D-shaped half-wave plate 104, where it is converted from horizontal polarization to vertical polarization. It then enters the first modulated birefringent crystal 105 together with the portion of the modulated light beam originating from the reflected light o, and is combined into a 1*M modulated light beam. After passing through the second lens 106, it enters the first scanning module S3.

[0078] In the present invention, the first scanning module S3 includes a first x-direction scanning galvanometer 107, a first scanning lens 108, a second scanning lens 109, a first z-direction scanning galvanometer 110, a third scanning lens 111 and a fourth scanning lens 112, which are sequentially arranged along the optical path;

[0079] The 1*M modulated light beam output by the first modulation module S2 is incident on the first x-direction scanning galvanometer 107, and then passes through the first scanning lens 108 and the second scanning lens 109 and is projected onto the first z-direction scanning galvanometer 110.

[0080] The first x-direction scanning galvanometer 107 scans the light beam along the x-axis, and the first z-direction scanning galvanometer 110 scans the light beam along the z-direction. The light beam emitted by the first z-direction scanning galvanometer 110 passes through the third scanning lens 111 and the fourth scanning lens 112 in sequence to form a first scanning beam, and is incident on the excitation module S6.

[0081] In the present invention, the second modulation module S4 includes a second modulation reflector 200, a second optical phase modulation device 201, a second spatial light modulator 202, a third lens 203, a second modulation D-shaped half-wave plate 204, a second modulation birefringent crystal 205 and a fourth lens 206 arranged in sequence along the optical path;

[0082] The transmitted light o022 and transmitted light e012 emitted by the light source module S1 are reflected by the second modulating reflector 200 and vertically incident on the second optical phase modulation device 201. The transmitted light e is evenly divided into M beams, each with equal energy, and adjacent beams are separated by equal angles. The transmitted light o is also evenly divided into M beams, each with equal energy, and adjacent beams are separated by equal angles. Thus, a 2*M beam array is formed and incident on the second spatial light modulator 202. The second spatial light modulator 202 is divided into 2*M spatial regions. Each spatial region independently applies phase control to the corresponding beam in the 2*M beam array, so that each beam in the 2*M beam array contains a unique code, thereby obtaining a 2*M modulated beam.

[0083] After the 2*M modulated light beam passes through the third lens 203, the portion of the modulated light beam originating from the transmitted light e enters the second modulated D-shaped half-wave plate 204, where it is converted from horizontal polarization to vertical polarization. It then enters the second modulated birefringent crystal 205 together with the portion of the modulated light beam originating from the transmitted light o, and is combined into a 1*M modulated light beam. After passing through the fourth lens 206, it enters the second scanning module S5.

[0084] In the present invention, the second scanning module S5 includes a second x-direction scanning galvanometer 207, a fifth scanning lens 208, a sixth scanning lens 209, a second z-direction scanning galvanometer 210, a seventh scanning lens 211, a scanning reflector 212, and an eighth scanning lens 213, which are sequentially arranged along the optical path.

[0085] The 1*M modulated light beam output by the second modulation module S4 is incident on the second x-direction scanning galvanometer 207 , and then passes through the fifth scanning lens 208 and the sixth scanning lens 209 and is projected onto the second z-direction scanning galvanometer 210 ;

[0086] The second x-direction scanning galvanometer 207 scans the light beam along the x-axis, and the second z-direction scanning galvanometer 210 scans the light beam along the z-direction. The light beam emitted by the second z-direction scanning galvanometer 210 passes through the seventh scanning lens 211, the scanning reflector 212 and the eighth scanning lens 213 in sequence to form a second scanning beam, and is incident on the excitation module S6.

[0087] In the present invention, the excitation module S6 includes an excitation spectroscope 300, a fifth lens 301, a first reflector 302, a second reflector 303, a third reflector 304, a sixth lens 305, a fourth reflector 306, and a first excitation objective lens 307, which are sequentially arranged along the optical path of the first side of the excitation spectroscope 300, and a seventh lens 310, a fifth reflector 311, an eighth lens 312, a sixth reflector 313, and a second excitation objective lens 314, which are sequentially arranged along the optical path of the first side of the excitation spectroscope 300; a sample 315 to be measured is arranged between the first excitation objective lens 307 and the second excitation objective lens 314;

[0088] The first scanning beam output by the first scanning module S3 is incident on the excitation spectroscope 300 and is equally divided into a first scanning reflected light and a first scanning transmitted light. The first scanning reflected light is transmitted along the optical path of the first side of the excitation spectroscope 300, and passes through the fifth lens 301, the first reflector 302, the second reflector 303, the third reflector 304, the sixth lens 305, the fourth reflector 306 and the first excitation objective lens 307 in sequence. The first excitation objective lens 307 then converges the light beam onto the first side of the sample 315.

[0089] The first scanning transmission light is transmitted along the optical path of the second side of the excitation beam splitter 300, passes through the seventh lens 310, the fifth reflector 311, the eighth lens 312, the sixth reflector 313 and the second excitation objective lens 314 in sequence, and then is focused by the second excitation objective lens 314 onto the second side of the sample 315;

[0090] The second scanning beam output by the second scanning module S5, which is perpendicular to the first scanning beam, is incident on the excitation spectroscope 300 and is equally divided into a second scanning reflected light and a second scanning transmitted light. The second scanning transmitted light is transmitted along the optical path of the first side of the excitation spectroscope 300, and passes through the fifth lens 301, the first reflector 302, the second reflector 303, the third reflector 304, the sixth lens 305, the fourth reflector 306 and the first excitation objective lens 307 in sequence. The first excitation objective lens 307 then converges the light beam onto the first side of the sample 315.

[0091] The second scanning reflected light is transmitted along the optical path of the second side of the excitation spectrometer 300, and after passing through the seventh lens 310, the fifth reflector 311, the eighth lens 312, the sixth reflector 313 and the second excitation objective lens 314 in sequence, the second excitation objective lens 314 converges the light beam to the second side of the sample 315.

[0092] In the present invention, the detection module S7 includes a large-field-of-view objective lens (also known as a mesoscopic objective lens, which is a microscope objective lens with high resolution and a large field of view. The field of view of the large-field-of-view objective lens in the present invention is greater than 8 mm, the numerical aperture is 0.5, and the working band is 400 nm to 1000 nm) 401, a condenser 402, a filter 403 and a camera 404 arranged in sequence along the optical path. The large-field-of-view objective lens 401 is located above the sample 315 to collect the fluorescence emitted by the sample 315. The fluorescence collected by the large-field-of-view objective lens 401 passes through the condenser 402 and the filter 403 and is collected by the camera 404 for imaging.

[0093] In a preferred embodiment, the first optical phase modulation device 101 and the second optical phase modulation device 201 are both spatial light modulators or diffractive optical elements.

[0094] In a preferred embodiment, the second scanning lens 109 , the fourth scanning lens 112 , the sixth scanning lens 209 , and the eighth scanning lens 213 are all tube lenses.

[0095] In a preferred embodiment, the condenser lens 402 is a tube lens.

[0096] The optical path principle of the system of the present invention is described in detail below using the optical paths including channel 1 and channel 2 to facilitate understanding of the present invention.

[0097] 1. System optical path including channel 1

[0098] (1)Reference Figure 2 The laser 01 emits a continuous linearly polarized laser with a wavelength of 488 nm. This laser source is used as an example in this invention, but it can also be a pulsed laser or laser of other wavelengths. It is collimated by the collimating beam expander 02, the aperture 03 with adjustable light aperture, and the half-wave plate 04 before entering the light source birefringent crystal 05.

[0099] Reference Figure 4 , rotate the fast axis direction of the half-wave plate 04 so that the polarization of the outgoing light forms a 45-degree angle with the horizontal ground. The light beam 000 is divided into two beams: horizontally polarized ordinary light o002 and vertically polarized extraordinary light e001. The birefringent crystal here divides the incident light into two parallel beams with mutually orthogonal polarization directions due to the birefringence effect. The separation distance is determined by the length of the birefringent crystal. The extraordinary light e becomes horizontally polarized light e parallel to the ordinary light o after passing through the light source D-shaped half-wave plate 06. The ordinary light o and the horizontally polarized light e enter the light source beam splitter 07 and are equally divided into a transmitted beam and a reflected beam. Figure 5 : Let ordinary light o be divided into transmitted light o022 and reflected light o021 by light source beam splitter 07, and horizontal polarized light e be divided into transmitted light e012 and reflected light e011 by light source beam splitter 07;

[0100] The reflected light o021 and the reflected light e011 enter the first modulation module S2, and the transmitted light o022 and the transmitted light e012 enter the second modulation module S4.

[0101] (2) Here we describe the optical path after entering the first modulation module S2:

[0102] The reflected light e011 and the reflected light o021 emitted by the light source module S1 are reflected by the first modulation reflector 100 and then vertically incident on the first optical phase modulation device 101. The optical phase modulation device has a specific phase distribution, which can be a phase set by a spatial light modulator, or an optical diffraction element manufactured according to a specific distribution, such as Figure 6As shown, the reflected light e is evenly divided into 6 beams, that is, M=6. Here, only 6 is used as an example for explanation, and it is not limited to 6 beams, namely beam 0111, beam 0112, beam 0113, beam 0114, beam 0115, and beam 0116. The energy of each beam is equal, and the angles of separation of adjacent beams are equal; the reflected light o is also evenly divided into 6 beams, namely beam 0211, beam 0212, beam 0213, beam 0214, beam 0215, and beam 0216. The energy of each beam is equal, and the angles of separation of adjacent beams are equal; refer to Figure 7 ;

[0103] Thus, a 2*6 beam array is formed and incident on the first spatial light modulator 102. The polarization direction of the modulated light is consistent with the incident light and is oriented in the horizontal direction. Figure 8 The first spatial light modulator 102 is divided into 2*6 spatial regions, and each spatial region independently applies phase control to the corresponding light beam in the 2*6 light beam array, so that each light beam in the 2*6 light beam array contains a unique code, thereby obtaining a 2*6 modulated light beam;

[0104] After the 2*6 modulated light beam passes through the first lens 103, the partial beams 0111, 0112, 0113, 0114, 0115, and 0116 of the modulated light beam originating from the reflected light e enter the first modulated D-shaped half-wave plate 104. The fast axis direction of the half-wave plate 04 is 45 degrees to the horizontal direction, and the polarization direction changes from horizontal polarization to vertical polarization. Then, together with the portion of the modulated light beam originating from the reflected light o, they enter the first modulated birefringent crystal 105. The light beams are spatially overlapped and combined into a 1*6 modulated light beam. This process is similar to Figure 4 The light splitting process is an inverse process, and after passing through the second lens 106 , the light enters the first scanning module S3 .

[0105] (3) The 1*6 modulated light beam output by the first modulation module S2 is incident on the first x-direction horizontal scanning galvanometer, and then passes through the first scanning lens 108 and the second scanning lens 109 to form a relay lens group and is projected onto the first z-direction vertical scanning galvanometer.

[0106] The first x-direction scanning galvanometer 107 scans the light beam along the x-axis, and the first z-direction scanning galvanometer 110 scans the light beam along the z-direction. The light beam emitted by the first z-direction scanning galvanometer 110 passes through the third scanning lens 111 and the fourth scanning lens 112 in sequence. The third scanning lens 111 and the fourth scanning lens 112 form a relay lens group to form a first scanning light beam, which is incident on the excitation module S6.

[0107] (4) The first scanning light beam output by the first scanning module S3 is incident on the excitation spectroscope 300 and is equally divided into a first scanning reflected light and a first scanning transmitted light. The first scanning reflected light is transmitted along the optical path of the first side of the excitation spectroscope 300, and passes through the fifth lens 301, the first reflector 302, the second reflector 303, the third reflector 304, the sixth lens 305, the fourth reflector 306 and the first excitation objective lens 307 in sequence. The first excitation objective lens 307 converges the light beam to the first side of the sample 315;

[0108] like Figure 9 As shown, light beams with different phase encoding are focused at different distances from the plane of the first excitation objective lens 307. The lateral distance of the focus position is determined by the beam separation angle and the focal length of the objective lens. The flexible change of the separation angle can make the lateral distance of the focus randomly changeable. The distance of the focus position from the focal plane can be controlled by the phase applied by the first spatial light modulator 102. The first x-direction scanning galvanometer 107 scans the light beam along the x-direction to form D1 to Dn light sheets to excite the sample. The number of light sheets is equal to the number of beams, n = M = 6.

[0109] Similarly, the first scanning transmitted light is transmitted along the optical path of the second side of the excitation spectrometer 300, and passes through the seventh lens 310, the fifth reflector 311, the eighth lens 312, the sixth reflector 313 and the second excitation objective lens 314 in sequence. Then, the second excitation objective lens 314 converges the array light beam with the separation angle to the second side of the sample 315; the light beams with different phase encoding are focused at different distances from the plane of the second excitation objective lens 314, and the lateral distance of the focus position is determined by the beam separation angle and the focal length of the objective lens. The flexible change of the separation angle can make the lateral distance of the focus change arbitrarily; the distance of the focus position from the focal plane can be controlled by the phase applied by the first spatial light modulator 102, and the first x-axis scanning galvanometer 107 scans the light beam along the x-direction to form D1 to Dn light sheets to excite the sample. The number of light sheets is equal to the number of beams, n=M=6; in channel one, the double-sided illumination doubles the field of view of the light sheet illumination, which can expand the field of view of the light sheet to 24 times that of a traditional single-beam light sheet fluorescence microscope.

[0110] (5) The wide-field objective lens 401 is located above the sample 315. The fluorescence emitted by the sample 315 is collected by the wide-field objective lens 401, and then filtered out by the condenser 402 and the filter 403 to remove stray light, and then collected by the camera 404 and imaged.

[0111] 2. System optical path including channel 2

[0112] (1) The light source module S1 is the same as the optical path of the system including channel 1, and will not be described in detail.

[0113] (2) The transmitted light o022 and the transmitted light e012 emitted by the light source module S1 are reflected by the second modulating reflector 200 and vertically incident on the second optical phase modulator 201. The second optical phase modulator 201 has a specific phase distribution, which can be a phase set by a spatial light modulator, or an optical diffraction element manufactured according to a specific distribution. The transmitted light e is evenly divided into 6 beams, that is, M=6. Here, only 6 is used as an example for explanation, and it is not limited to 6 beams, namely, beam 0121, beam 0122, beam 0123, beam 0124, beam 0125, and beam 0126. The energy of each beam is equal, and the angles of separation of adjacent beams are equal;

[0114] The transmitted light o is also equally divided into 6 beams, namely beam 0221, beam 0222, beam 0223, beam 0224, beam 0225, and beam 0226. Each beam has equal energy, and adjacent beams are separated by equal angles. This forms a 2*6 beam array, which is incident on the second spatial light modulator 202. The modulated polarization direction is consistent with the incident light and is oriented horizontally. The second spatial light modulator 202 is divided into 2*6 spatial regions. Each spatial region independently applies phase control to the corresponding beam in the 2*6 beam array, so that each beam in the 2*6 beam array contains a unique code, thereby obtaining a 2*6 modulated beam.

[0115] After the 2*6 modulated light beam passes through the third lens 203, the parts of the modulated light beam originating from the transmitted light e, namely, beam 0121, beam 0122, beam 0123, beam 0124, beam 0125, and beam 0126, enter the second modulated D-shaped half-wave plate 204. The fast axis direction of the half-wave plate 04 is 45 degrees to the horizontal direction, and the horizontal polarization is changed to vertical polarization. Then, together with the part of the modulated light beam originating from the transmitted light o, it enters the second modulated birefringent crystal 205. The light beams are spatially overlapped and combined into a 1*6 modulated light beam. This process is similar to Figure 4 The light splitting process is an inverse process, and after passing through the fourth lens 206 , the light enters the second scanning module S5 .

[0116] (3) The 1*6 modulated light beam output by the second modulation module S4 is incident on the second x-axis horizontal scanning galvanometer, and then passes through the fifth scanning lens 208 and the sixth scanning lens 209 to form a relay lens and is projected onto the second z-axis vertical scanning galvanometer;

[0117] The second x-direction scanning galvanometer 207 scans the light beam along the x-axis, and the second z-direction scanning galvanometer 210 scans the light beam along the z-direction. The light beam emitted by the second z-direction scanning galvanometer 210 passes through the seventh scanning lens 211, the scanning reflector 212 and the eighth scanning lens 213 in sequence to form a second scanning beam, and is incident on the excitation module S6.

[0118] (4) The second scanning beam output by the second scanning module S5, which is perpendicular to the first scanning beam, is incident on the excitation spectroscope 300 and is equally divided into a second scanning reflected light and a second scanning transmitted light. The second scanning transmitted light is transmitted along the optical path of the first side of the excitation spectroscope 300, and passes through the fifth lens 301, the first reflector 302, the second reflector 303, the third reflector 304, the sixth lens 305, the fourth reflector 306 and the first excitation objective lens 307 in sequence. The first excitation objective lens 307 converges the light beam to the first side of the sample 315.

[0119] like Figure 9 As shown, light beams with different phase encoding are focused at different distances from the plane of the first excitation objective lens 307. The lateral distance of the focus position is determined by the beam separation angle and the focal length of the objective lens. The flexible change of the separation angle can make the lateral distance of the focus change arbitrarily. The distance of the focus position from the focal plane can be controlled by the phase applied by the second spatial light modulator 202. The second x-direction scanning galvanometer 207 scans the light beam along the x-direction to form D1 to Dn light sheets to excite the sample. The number of light sheets is equal to the number of beams, n = M = 6.

[0120] Similarly, the second scanned reflected light is transmitted along the optical path of the second side of the excitation spectrometer 300, and passes through the seventh lens 310, the fifth reflector 311, the eighth lens 312, the sixth reflector 313 and the second excitation objective lens 314 in sequence. Then, the second excitation objective lens 314 converges the array light beam with the separation angle to the second side of the sample 315; the light beams with different phase encoding are focused at different distances from the plane of the second excitation objective lens 314, and the lateral distance of the focus position is determined by the beam separation angle and the focal length of the objective lens. The flexible change of the separation angle can make the lateral distance of the focus change at will; the distance of the focus position from the focal plane can be controlled by the phase applied by the second spatial light modulator 202, and the second x-direction scanning galvanometer 207 scans the light beam along the x-direction to form D1 to Dn light sheets to excite the sample. The number of light sheets is equal to the number of beams, n=M=6; in channel two, the double-sided illumination doubles the field of view of the light sheet illumination, which can expand the field of view of the light sheet to 24 times that of a traditional single-beam light sheet fluorescence microscope.

[0121] (5) The wide-field objective lens 401 is located above the sample 315. The fluorescence emitted by the sample 315 is collected by the wide-field objective lens 401, and then filtered out by the condenser 402 and the filter 403 to remove stray light, and then collected by the camera 404 and imaged.

[0122] It can be seen that in the above examples of the present invention, the dual-channel dual-side illumination can expand the field of view of the single-beam light sheet by 48 times.

[0123] In one embodiment, the present invention is described by taking the following parameters as an example, but it should be understood that the present invention is not limited to the following parameters.

[0124] In laser 01, the beam diameter of the laser after collimation is 2 mm, and the magnification of the first modulation module S2 and the second modulation module S4 is 1. The magnification of the first scanning module S3 and the second scanning module S5 is 4. In the excitation module S6, the magnification of the relay group composed of the fifth lens 301 and the sixth lens 305 is 1, and the magnification of the relay group composed of the seventh lens 310 and the eighth lens 312 is 1. Then the magnification of the entrance pupil of the first excitation objective lens 307 and the second excitation objective lens 314 from the light source module S1 to the excitation module S6 is 4 times. Taking the Mitutyo 5x objective lens with NA = 0.14 and f = 40mm as an example, if a light sheet field of view of 8mm and an axial resolution of 4μm are required, the maximum distance between the focus point and the focal plane of the objective lens is 8mm. The effective numerical aperture of the light beam passing through the objective lens is NAeff = 2mm * 4 / (2 * 48mm) = 0.083, the beam focus waist radius is approximately 0.61 * 0.488μm / NAeff = 3.59μm, and the effective length of the light sheet is approximately: 2 * π / 0.488 * 3.59^2 * 48 = 7.965mm, which is basically consistent with the desired target field of view. The present invention uses a Gaussian beam as an example, but is not limited to Gaussian beams. Non-diffracting beams such as Bessel beams and Airy beams can also be used.

[0125] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A large-field-of-view, high-resolution light sheet illumination imaging system, characterized in that: include: A light source module (S1), a first modulation module (S2), a first scanning module (S3), a second modulation module (S4), a second scanning module (S5), an excitation module (S6), and a detection module (S7); The light beam emitted by the light source module (S1) is divided into two parts: a reflection part and a transmission part. The reflection part enters the first modulation module (S2), and after being modulated, enters the first scanning module (S3). The generated first scanning light beam enters the excitation module (S6) to excite the sample (315) to generate fluorescence; the transmission part enters the second modulation module (S4), and after being modulated, enters the second scanning module (S5). The generated second scanning light beam enters the excitation module (S6) to excite the sample (315) to generate fluorescence; the fluorescence emitted by the sample (315) is collected and imaged by the detection module (S7); The first modulation module (S2) and the first scanning module (S3) constitute the first channel of the system, the second modulation module (S4) and the second scanning module (S5) constitute the second channel of the system, and the first scanning light beam output by the first channel and the second scanning light beam output by the second channel enter the excitation module (S6) in a mutually perpendicular manner.

2. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 1, characterized in that: The light source module (S1) comprises a laser (01), a collimating beam expander (02), an aperture (03), a half-wave plate (04), a light source birefringent crystal (05), a light source D-shaped half-wave plate (06), and a light source spectroscope (07) arranged in sequence along the light path; The laser light emitted by the laser (01) passes through the collimating beam expander (02), the aperture (03), and the half-wave plate (04) in sequence and then enters the light source birefringent crystal (05), where it is divided into two beams of light: ordinary light o and extraordinary light e. The extraordinary light e passes through the light source D-shaped half-wave plate (06) and becomes a horizontally polarized light e parallel to the ordinary light o. The ordinary light o and the horizontally polarized light e enter the light source beam splitter (07) and are both divided into a transmitted light beam and a reflected light beam in equal proportions: the ordinary light o is divided into a transmitted light o and a reflected light o by the light source beam splitter (07), and the horizontally polarized light e is divided into a transmitted light e and a reflected light e by the light source beam splitter (07); The reflected light o and the reflected light e enter the first modulation module (S2), and the transmitted light o and the transmitted light e enter the second modulation module (S4).

3. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 2, characterized in that: The first modulation module (S2) comprises a first modulation reflector (100), a first optical phase modulation device (101), a first spatial light modulator (102), a first lens (103), a first modulation D-shaped half-wave plate (104), a first modulation birefringent crystal (105), and a second lens (106), which are sequentially arranged along the optical path; The reflected light e and the reflected light o emitted by the light source module (S1) are reflected by the first modulating reflector (100) and vertically incident on the first optical phase modulation device (101). The reflected light e is evenly divided into M beams, each beam has equal energy, and adjacent beams are separated at equal angles. The reflected light o is also evenly divided into M beams, each beam has equal energy, and adjacent beams are separated at equal angles. Thus, a 2*M beam array is formed and incident on the first spatial light modulator (102). The first spatial light modulator (102) is divided into 2*M spatial regions, and each spatial region independently applies phase control to the corresponding beam in the 2*M beam array, so that each beam in the 2*M beam array contains a unique code, thereby obtaining a 2*M modulated beam. After the 2*M modulated light beam passes through the first lens (103), the portion of the modulated light beam originating from the reflected light e enters the first modulated D-shaped half-wave plate (104), changes from horizontal polarization to vertical polarization, and then enters the first modulated birefringent crystal (105) together with the portion of the modulated light beam originating from the reflected light o, and is combined into a 1*M modulated light beam, which enters the first scanning module (S3) after passing through the second lens (106).

4. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 3, wherein: The first scanning module (S3) includes A first X-direction scanning galvanometer (107), a first scanning lens (108), a second scanning lens (109), a first Z-direction scanning galvanometer (110), a third scanning lens (111), and a fourth scanning lens (112) are sequentially arranged in the optical path; The 1*M modulated light beam output by the first modulation module (S2) is incident on the first x-direction scanning galvanometer (107), and then passes through the first scanning lens (108) and the second scanning lens (109) before being projected onto the first z-direction scanning galvanometer (110). The first x-direction scanning galvanometer (107) scans the light beam along the x-axis, and the first z-direction scanning galvanometer (110) scans the light beam along the z-direction. The light beam emitted by the first z-direction scanning galvanometer (110) passes through the third scanning lens (111) and the fourth scanning lens (112) in sequence to form a first scanning light beam, which is incident on the excitation module (S6).

5. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 4, characterized in that: The second modulation module (S4) comprises a second modulation reflector (200), a second optical phase modulation device (201), a second spatial light modulator (202), a third lens (203), a second modulation D-shaped half-wave plate (204), a second modulation birefringent crystal (205), and a fourth lens (206) which are sequentially arranged along the optical path; The transmitted light o and the transmitted light e emitted by the light source module (S1) are reflected by the second modulating reflector (200) and vertically incident on the second optical phase modulation device (201). The transmitted light e is evenly divided into M beams, each beam has equal energy, and adjacent beams are separated at equal angles. The transmitted light o is also evenly divided into M beams, each beam has equal energy, and adjacent beams are separated at equal angles. Thus, a 2*M beam array is formed and incident on the second spatial light modulator (202). The second spatial light modulator (202) is divided into 2*M spatial regions, and each spatial region independently applies phase control to the corresponding beam in the 2*M beam array, so that each beam in the 2*M beam array contains a unique code, thereby obtaining a 2*M modulated beam. After the 2*M modulated light beam passes through the third lens (203), the portion of the modulated light beam originating from the transmitted light e enters the second modulated D-shaped half-wave plate (204), changes from horizontal polarization to vertical polarization, and then enters the second modulated birefringent crystal (205) together with the portion of the modulated light beam originating from the transmitted light o, and is combined into a 1*M modulated light beam, which enters the second scanning module (S5) after passing through a fourth lens (206).

6. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 5, characterized in that: The second scanning module (S5) comprises a second X-direction scanning galvanometer (207), a fifth scanning lens (208), a sixth scanning lens (209), a second Z-direction scanning galvanometer (210), a seventh scanning lens (211), a scanning reflector (212), and an eighth scanning lens (213), which are sequentially arranged along the optical path; The 1*M modulated light beam output by the second modulation module (S4) is incident on the second x-direction scanning galvanometer (207), and then passes through the fifth scanning lens (208) and the sixth scanning lens (209) before being projected onto the second z-direction scanning galvanometer (210); The second x-direction scanning galvanometer (207) scans the light beam along the x-axis, and the second z-direction scanning galvanometer (210) scans the light beam along the z-direction. The light beam emitted by the second z-direction scanning galvanometer (210) passes through the seventh scanning lens (211), the scanning reflector (212), and the eighth scanning lens (213) in sequence to form a second scanning light beam, which is incident on the excitation module (S6).

7. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 6, characterized in that: The excitation module (S6) includes an excitation spectroscope (300), a fifth lens (301), a first reflector (302), a second reflector (303), a third reflector (304), a sixth lens (305), a fourth reflector (306) and a first excitation objective lens (307) arranged in sequence along the optical path of the first side of the excitation spectroscope (300), and a seventh lens (310), a fifth reflector (311), an eighth lens (312), a sixth reflector (313) and a second excitation objective lens (314) arranged in sequence along the optical path of the first side of the excitation spectroscope (300); a sample (315) to be measured is arranged between the first excitation objective lens (307) and the second excitation objective lens (314).

8. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 7, characterized in that: The first scanning light beam output by the first scanning module (S3) is incident on the excitation spectroscope (300) and is equally divided into a first scanning reflected light and a first scanning transmitted light. The first scanning reflected light is transmitted along the optical path of the first side of the excitation spectroscope (300), and passes through the fifth lens (301), the first reflector (302), the second reflector (303), the third reflector (304), the sixth lens (305), the fourth reflector (306) and the first excitation objective lens (307) in sequence. The first excitation objective lens (307) converges the light beam to the first side of the sample (315); The first scanning transmission light is transmitted along the optical path of the second side of the excitation spectroscope (300), and after passing through the seventh lens (310), the fifth reflector (311), the eighth lens (312), the sixth reflector (313) and the second excitation objective lens (314) in sequence, the second excitation objective lens (314) converges the light beam to the second side of the sample (315); The second scanning light output by the second scanning module (S5) and perpendicular to the first scanning light beam is incident on the excitation spectroscope (300) and is equally divided into a second scanning reflected light and a second scanning transmitted light. The second scanning transmitted light is transmitted along the optical path of the first side of the excitation spectroscope (300), passes through the fifth lens (301), the first reflector (302), the second reflector (303), the third reflector (304), the sixth lens (305), the fourth reflector (306) and the first excitation objective lens (307) in sequence, and then the first excitation objective lens (307) converges the light beam to the first side of the sample (315); The second scanning reflected light is transmitted along the optical path of the second side of the excitation spectrometer (300), and after passing through the seventh lens (310), the fifth reflector (311), the eighth lens (312), the sixth reflector (313) and the second excitation objective lens (314) in sequence, the second excitation objective lens (314) converges the light beam to the second side of the sample (315).

9. The large-field-of-view, high-resolution light sheet illumination imaging system according to claim 8, characterized in that: The detection module (S7) comprises a large-field-of-view objective lens (401), a condenser lens (402), a filter (403) and a camera (404) arranged in sequence along the light path direction. The large-field-of-view objective lens (401) is located above the sample (315) and is used to collect fluorescence emitted by the sample (315). The fluorescence collected by the large-field-of-view objective lens (401) passes through the condenser lens (402) and the filter (403) and is then collected by the camera (404) to form an image.

10. The large-field-of-view, high-resolution light sheet illumination imaging system according to any one of claims 1 to 9, characterized in that: The first optical phase modulation device (101) and the second optical phase modulation device (201) are both spatial light modulators or diffractive optical elements.

Images