Single-objective lens sheet type three-dimensional fluorescence imaging system

Abstract

This single-objective optical three-dimensional fluorescence imaging system includes an illumination objective (1), a relay scanning lens group, a galvanometer mirror (4), an illumination module, and an imaging module. The scanning lens group includes a first scanning lens and a second scanning lens (3, 5), whose main optical axes are orthogonal and confocal. The common focal position of the first and second scanning lenses (3, 5) is located at the center of the galvanometer mirror (4). Fluorescence emitted from the sample excitation plane is collected by the illumination objective (1), then enters the scanning lens group in the opposite direction to the illumination light, and is imaged by the imaging module, resulting in a projection of the fluorescence on a plane orthogonal to the main optical axis of the illumination objective (1) at the sample excitation plane. The imaging module includes an area detector (8). By continuously acquiring the projection of the sample excitation plane on a plane perpendicular to the main optical axis of the illumination objective (1), it replaces the conventional perfect imaging of the sample excitation plane, eliminates the need for two remote imaging objectives for phase contrast calibration, removes the limitations on the optical parameters of the illumination objective (1), easily realizes in-situ magnification switching, and enables high-speed imaging.

Description

[Technical Field]

[0001] The present invention relates to the field of microscopic imaging technology, and more particularly to a single objective lens sheet-type three-dimensional fluorescence imaging system. [Background technology]

[0002] Conventional light sheet microscopes have orthogonal light sheet illumination and fluorescence detection optical paths, each composed of a detection objective and an illumination objective, making the overall optical path complex and difficult to construct. A light sheet microscope that combines the illumination and detection optical paths into a single path and uses a single objective to simultaneously illuminate and detect the sample is called a single-objective light sheet microscope.

[0003] However, when simultaneously illuminating and imaging using a single objective, the light sheet illumination optical path and the fluorescence detection optical path intersect obliquely, resulting in a phase difference on one side, forcing the use of a high-numerical aperture objective as the single objective on the other side. To correct for the phase difference and achieve perfect imaging, remote focusing using three objectives is currently used, but this system is complex and has limited applicability. Furthermore, the need to use a high-numerical aperture objective limits the field of view of single-objective light sheet microscopes, as high-numerical aperture objectives generally have high magnifications and narrow fields of view.

[0004] Previously, single-objective sheet-type fluorescence imaging systems lacked on-the-fly magnification switching, making it difficult to observe samples at different magnifications, significantly hindering the commercialization of single-objective sheet-type fluorescence microscopes. Chinese patent document CN115685515A proposes a single-objective sheet-type coaxial imaging system that overcomes the numerical aperture (NA) limitations of the illumination objective by using coaxially arranged second and third objective lenses. While this allows for on-the-fly magnification switching, this system sacrifices field of view and imaging speed. It achieves this by stacking two-dimensional images of the excitation plane within a scanning cycle, and three-dimensional imaging requires sample movement. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention addresses the drawbacks and demands of the prior art by providing a single-objective lens sheet-type three-dimensional fluorescence imaging system. The objective of the present invention is to obtain three-dimensional fluorescence data of a sample through three-dimensional reconstruction by continuously acquiring projections of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective in a single-objective lens imaging system, replacing the conventional complete imaging of the sample excitation plane. This eliminates the need for two remote imaging objectives for phase difference correction, overcomes the optical parameter limitations of the illumination objective, and facilitates on-the-fly magnification switching. The present invention solves the technical problems of the prior art, which require complete imaging data of the sample excitation plane, but which have strict limitations on the optical parameters of the three objective lenses, making on-the-fly magnification switching impossible or slowing down the imaging speed due to on-the-fly magnification switching through sequential exposure. [Means for solving the problem]

[0006] To achieve the above object, according to one aspect of the present invention, a single-objective microscope imaging system is provided, comprising an illumination objective, a relay scanning lens group, a galvanometer mirror, an illumination module, and an imaging module, and having the following features:

[0007] The scanning lens group includes a first scanning lens and a second scanning lens, the main optical axes of which are orthogonal to each other and arranged confocal, and the common focal position of the first scanning lens and the second scanning lens is located at the center of the galvanometer mirror.

[0008] The illumination module generates an illumination laser, which passes through a scanning lens group and enters the rear pupil plane of the illumination objective, projecting an illumination sheet at an inclination angle α onto the excitation plane of the sample to excite fluorescence. The excitation plane intersects the main optical axis of the illumination objective at an oblique angle θ.

[0009] The fluorescence emitted from the sample excitation plane is collected by the illumination objective, passes through the scanning lens group in the opposite direction to the illumination light, and enters the imaging module where it is imaged, resulting in a projection of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective.

[0010] The imaging module includes an area detector.

[0011] More preferably, the single-objective microscope imaging system further includes a sample stage having an axial feeder for moving the sample in the axial direction of the illumination objective, the axial feeder varying the axial position stepwise or continuously, and the axial displacement L within the rotation period T of the galvanometer mirror satisfies the following equation: TIFF2025529009000002.tif1241 where s is the Rayleigh range of the sheet and θ is the tilt angle of the light sheet relative to the scanning direction.

[0012] More preferably, the single-objective microscope imaging system scans the excitation plane of the sample in a direction perpendicular to the main optical axis of the illumination objective lens by the action of a galvanometer mirror, and the fluorescence signal is then scanned in reverse by the galvanometer mirror, and projections of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective lens are obtained in chronological order at the same position on the surface detector of the imaging module, and a three-dimensional fluorescence image of the sample is obtained by reconstruction and stacking processing.

[0013] More preferably, in the single-objective microscope imaging system, the galvanometer mirror changes its angle stepwise or continuously, so that the excitation plane of the sample is scanned in a direction perpendicular to the main optical axis of the illumination objective, and the displacement d of the sample excitation plane corresponding to the adjacent projections in the scanning direction satisfies the following formula: TIFF2025529009000003.tif2272 where h is the sheet thickness and θ is the tilt angle of the light sheet relative to the scanning direction.

[0014] More preferably, in the single-objective microscope imaging system, a tubular lens unit is disposed between the scanning lens group and the illumination objective lens, and is fixed relative to the position of the scanning lens group to perform focusing correction of the fluorescence signal collected by the objective lens.

[0015] More preferably, in the single-objective microscope imaging system, the illumination module comprises a collimated laser and a modulator, the modulator comprises a mask, and the illumination laser is modulated by the modulator to form a predetermined sheet shape, which is combined with the fluorescence light path via a dichroic beam splitter and directed to a scanning lens group.

[0016] More preferably, in the single objective microscope imaging system, the sheet thickness is 0.3 μm to 5 μm.

[0017] More preferably, the single-objective microscope imaging system further comprises an imaging module, and an area detector is disposed at the focal plane of the lens, and the area detector acquires a projection of the sample excitation plane in a plane perpendicular to the illumination objective main optical axis according to a time sequence of the control signals.

[0018] More preferably, the single objective lens microscope imaging system includes a depth of field extension unit disposed between the scanning lens group and the lens, and the depth of field extension unit extends the depth of field of the sheet in the axial direction of the objective lens.

[0019] More preferably, the focal depth extending unit is a phase modulation mask, a spatial light modulator, an on-axis prism, or a prism group. [Effects of the Invention]

[0020] Compared with the prior art, the above technical solution of the present invention has the following advantageous effects:

[0021] The novelty of this invention lies in the acquisition of projection information by scanning with a galvanometer mirror, the projection of the sample excitation plane on a plane perpendicular to the main optical axis of the illumination objective, and the acquisition of 3D fluorescence data of the sample. This allows for sample illumination and signal acquisition using only a single illumination objective. Combined with a 3D reconstruction algorithm, this method enables rapid and easy acquisition of 3D structural information of biological samples. More importantly, sheet-type 3D fluorescence imaging using a single objective eliminates the need for two remote imaging objectives for phase contrast correction, and the optical path construction is no longer limited by the magnification of the illumination objective. Therefore, when magnification switching is required, the illumination objective can be simply replaced on the spot, enabling easy on-the-fly magnification switching without affecting imaging speed. The imaging speed is comparable to that of conventional sheet-type fluorescence microscopes and significantly faster than that of confocal microscopes, significantly expanding the application range of sheet-type fluorescence microscopes from research to commercial applications such as inspection and diagnostic imaging. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a diagram illustrating an optical system of a single objective lens sheet type three-dimensional fluorescence imaging system according to a first embodiment of the present invention. [Figure 2] FIG. 10 is an optical system diagram of a single objective lens sheet type three-dimensional fluorescence imaging system according to Example 2 of the present invention. [Figure 3] FIG. 3 is a control signal diagram used in an embodiment of the present invention.

[0023] In all figures, the same reference numerals refer to the same elements or components, where 1 is the illumination objective lens, 2 is the tube lens, 3 is the first scan lens, 4 is the galvanometer mirror, 5 is the second scan lens, 6 is the lens, 7 is the condenser lens, 8 is the detector, 9 is the illumination laser, and 10 is the focal depth extension module. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in more detail below with reference to examples, in order to clarify the objectives, technical solutions, and advantages of the present invention. The examples described herein are for the purpose of illustrating the present invention, but are not intended to limit the present invention. Furthermore, the technical features of the present invention described in the following examples can be freely combined as long as they are not mutually contradictory.

[0025] The present invention provides a single-objective microscope imaging system that includes an illumination objective, a relay scan lens group, a galvanometer mirror, an illumination module, an imaging module, and a sample stage.

[0026] The scanning lens group includes a first scanning lens and a second scanning lens, the main optical axes of which are orthogonal to each other and are arranged confocal, and the common focal position of the first scanning lens and the second scanning lens is located at the center of the galvanometer mirror.

[0027] The illumination laser passes through the scanning lens group and enters the rear pupil plane of the illumination objective, projecting an illumination sheet at an inclination angle θ onto the excitation plane of the sample, exciting fluorescence. The excitation plane intersects the main optical axis of the illumination objective at an oblique angle equal to the sheet inclination angle θ.

[0028] The fluorescence emitted from the sample excitation plane is collected by the illumination objective, passes through the scanning lens group in the opposite direction to the illumination light, and enters the imaging module where it is imaged, resulting in a projection of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective.

[0029] The imaging module includes an area detector.

[0030] Due to the action of the galvanometer mirror, the excitation plane of the sample is scanned in a direction perpendicular to the main optical axis of the illumination objective lens, and the fluorescence signal is then scanned in reverse by the galvanometer mirror. After that, projections of the sample excitation plane on a plane perpendicular to the main optical axis of the illumination objective lens are acquired in chronological order at the same position on the planar detector of the imaging module, and a three-dimensional fluorescence image of the sample is obtained by reconstruction and stacking processing.

[0031] The galvanometer mirror changes its angle stepwise or continuously, scanning the excitation plane of the sample in a direction perpendicular to the main optical axis of the illumination objective. In this case, the displacement d of the excitation plane of the sample corresponding to the adjacent projection in the scanning direction satisfies the following equation: TIFF2025529009000004.tif2372Here, h is the sheet thickness and θ is the tilt angle of the light sheet relative to the scanning direction.

[0032] By matching the rotation speed of the galvanometer mirror with the imaging speed, the displacement d in the scanning direction of the sample excitation plane corresponding to adjacent projections can be controlled within an appropriate range, avoiding insufficient sampling due to excessively sparse sampling and crosstalk of fluorescence information due to excessively dense sampling, thereby improving the image quality after 3D reconstruction.

[0033] This system uses a scanning lens and galvanometer mirror to scan the excitation plane of the sample and acquire the projection data to directly form a 3D image. This eliminates the need for complete imaging of the sample excitation plane and the need for second and third objective lenses for phase contrast correction. This eliminates the limitation on the numerical aperture (NA) of the illumination objective, and allows for easy on-the-fly magnification switching by simply exchanging the illumination objective. When magnification switching is required, the illumination objective can be simply replaced with one of a different magnification; there is no need to replace or realign other components of the system or reconfigure the optical path.

[0034] However, because 3D reconstruction is based on projection rather than complete image data, crosstalk caused by projection must be avoided. If the projection shifts position on the detector as the excitation plane is scanned, crosstalk can cause a misalignment when restoring the sample excitation plane, resulting in a failure of 3D reconstruction. Our system uses a galvanometer mirror to act on both the illumination and detection optical paths. Because the excitation and detection optical paths pass in opposite directions on the galvanometer mirror, the imaging position on the detector is fixed during scanning and counter-scanning. Simply acquiring each frame of image in chronological order is sufficient; the detector image changes continuously, eliminating the need for synchronization with the galvanometer mirror phase. This effectively avoids 3D reconstruction failures due to crosstalk caused by projection. There is no need to avoid crosstalk by using complex galvanometer mirror control to perform multiple scans.

[0035] A lens (preferably a tubular lens) is disposed between the scanning lens group and the illumination objective, and is fixed relative to the position of the scanning lens group to provide focusing correction of the fluorescence signal collected by the objective, thereby improving compatibility with objectives of various magnifications and improving image quality under various objectives.

[0036] The illumination module generates an illumination laser. Preferably, the illumination module includes a collimated laser and a modulator, the modulator including a mask. The illumination laser is modulated by the modulator to form a predetermined sheet shape, which is then combined with the fluorescent light path via a dichroic beam splitter and guided to a scanning lens group. The sheet thickness is 0.3 μm to 5 μm. If the sheet is too thick, projection crosstalk becomes significant, while if it is too thin, it is disadvantageous for stacking three-dimensional imaging. Furthermore, the thinner the sheet, the shorter the Rayleigh range, which increases the cost of axial scanning. When designing, it is necessary to consider the relationship between the Rayleigh range of the sheet and the focal depth of the objective lens.

[0037] The imaging module includes a lens, and an area detector is disposed at the focal plane of the lens, and the area detector acquires a projection of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective lens according to the timing of the control signal, and the lens is preferably a tubular lens.

[0038] The imaging device preferably further includes a depth of field extension unit, which is disposed between the scanning lens group and the lens and can be a phase modulation mask, a spatial light modulator, an on-axis prism, or a prism group. The depth of field extension unit extends the depth of field of the sheet in the axial direction of the objective lens, thereby expanding the field of view of a single frame image and obtaining more depth information in a single exposure, thereby increasing the axial movement step width of the objective lens and reducing the number of axial scans.

[0039] The sample stage is equipped with an axial feeder that moves the sample in the axial direction of the illumination objective lens. This axial feeder changes the axial position stepwise or continuously, and the axial displacement L within the rotation period T of the galvanometer mirror satisfies the following equation: TIFF2025529009000005.tif1146Here, s is the Rayleigh range of the sheet and θ is the tilt angle of the light sheet relative to the scanning direction.

[0040] An example is shown below. [Example]

[0041] As shown in FIG. 1, the single-objective microscope imaging system according to this embodiment includes an illumination objective, a relay scanning lens group, a galvanometer mirror, an illumination module, an imaging module, and a sample stage.

[0042] Illumination module: The laser light emitted from the laser is collimated and expanded by a beam expander, and then emitted parallel as an illumination laser 9, reflected by a dichroic mirror, and guided to a scanning lens group.

[0043] Scanning lens group: Light entering the second scanning lens 5 is focused on the galvanometer mirror 4. The focal position on the galvanometer mirror is imaged on the back pupil plane of the illumination objective lens 1 via the first scanning lens 3 and lens 2. A sheet is formed on the sample surface via the illumination objective lens 1, exciting sample fluorescence. The galvanometer mirror and the back pupil plane of the objective lens are conjugate, and the sheet is scanned laterally on the sample surface as the galvanometer mirror 4 vibrates. The sheet parameters are determined by the lens group 2. The laser-excited fluorescence is collected by the same illumination objective lens 1, passes through the first scanning lens 3, and is then scanned back by the galvanometer mirror 4, entering the scanning lens 5 as stable diverging light. After passing through the scanning lens 5, it passes through the dichroic mirror as parallel light.

[0044] Imaging module: The dichroic mirror reflects the short wavelength laser light and transmits the long wavelength fluorescence. The parallel light is collected on the light receiving surface of the area detector 8 via the lens 7 made of a tubular lens.

[0045] The sample stage (not shown) is equipped with an axial (Z-direction) feeder that moves the sample in the axial direction of the illumination objective lens. This axial feeder changes the axial position stepwise or continuously, and the axial displacement L within the rotation period T of the galvanometer mirror satisfies the following equation: TIFF2025529009000006.tif1249Here, s is the Rayleigh range of the sheet and θ is the tilt angle of the light sheet relative to the scanning direction.

[0046] In addition to the Z-direction feeder, the sample stage is equipped with a two-dimensional moving stage, enabling rapid search of the field of view.

[0047] In the single-objective microscope imaging system of this example, when a 60x 1.3 NA objective lens 1 is used, the sheet width incident on the illumination objective lens 1 is 3.54 mm, and the sheet is offset by 2.13 mm from the center of the rear pupil of the illumination objective lens 1. The tilt angle θ between the sheet and the scanning direction is designed to be 54°, and the sheet thickness is 407 nm. The galvanometer mirror changes its angle stepwise or continuously, and the speed is limited by h and θ. [Example]

[0048] As shown in Figure 2, the single-objective microscope imaging system of this embodiment includes an illumination module, a scanning / reverse scanning module, a sample mounting module, a focal length extension module, and an imaging module. The illumination module and the scanning / reverse scanning module are the same as those of the first embodiment, but unlike the first embodiment, after passing through the scanning mirror 5, parallel light is emitted from the lens 6, passes through the dichromatic mirror, is focused by the lens 7, and passes through the focal length extension module 10 before the detector 9. The focal length extension module 10 can include, but is not limited to, a conical lens, a diffractive optical element, a spectroscopic prism, etc.

[0049] In this example, when using a 60x 1.3 NA objective lens 1, the addition of a depth-of-field extension module increases the detection-side sheet thickness from 216 nm to 439 nm, expanding the field of view of a single frame image by approximately 7 times to 4080 nm. The galvanometer mirror changes angle in a stepwise or continuous manner, with the speed limited by h and θ. Expanding the field of view of a single frame increases the scanning step width of the galvanometer mirror, improving the scanning speed of the galvanometer mirror. At the same time, the axial movement step width of the objective lens increases, reducing the number of axial scans. [Example]

[0050] In the microscope imaging systems according to the first and second embodiments, the control timing of the galvanometer mirror, the sample stage, and the area detector is shown in FIG.

[0051] In control method 1, the galvanometer mirror changes its angle stepwise to perform horizontal scanning.

[0052] The area detector synchronizes with the galvanometer mirror to acquire projections of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective lens, where the displacement d of the sample excitation plane in the scanning direction corresponding to adjacent projections satisfies the following equation: TIFF2025529009000007.tif2372Here, h is the sheet thickness and θ is the tilt angle of the light sheet relative to the scanning direction.

[0053] The sample stage moves in steps in the Z direction to perform axial scanning. Each time the galvanometer mirror completes one rotation cycle, the sample stage moves one step in the Z direction.

[0054] In control method 2, the galvanometer mirror performs horizontal scanning by continuously changing the angle.

[0055] The area detector acquires projections of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective in a specific time sequence, where the displacement d of the sample excitation plane in the scanning direction corresponding to adjacent projections satisfies the following equation: TIFF2025529009000008.tif2172Here, h is the sheet thickness and θ is the tilt angle of the light sheet relative to the scanning direction.

[0056] The sample stage moves in steps in the Z direction, scanning the axis. Each time the galvanometer mirror completes one rotation, the sample stage moves one step in the Z direction.

[0057] In control method 3, the galvanometer mirror performs horizontal scanning by continuously changing the angle.

[0058] The area detector acquires projections of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective in a specific time sequence, where the displacement d of the sample excitation plane in the scanning direction corresponding to adjacent projections satisfies the following equation: TIFF2025529009000009.tif2272Here, h is the sheet thickness and θ is the tilt angle of the light sheet relative to the scanning direction.

[0059] The sample stage moves continuously in the Z direction, performing axial scanning. Each time the galvanometer mirror completes one rotation cycle, the sample stage moves in the Z direction by a displacement L equivalent to the focal depth. L satisfies the following formula: TIFF2025529009000010.tif1246, where s is the Rayleigh range of the sheet and θ is the tilt angle of the light sheet relative to the scanning direction.

[0060] The examples described herein are intended to illustrate preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalents, improvements, etc. made within the spirit and scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. A single-objective microscope imaging system comprising: an illumination objective; a relay scan lens group; a galvanometer mirror; an illumination module; and an imaging module; the scanning lens group includes a first scanning lens and a second scanning lens, the main optical axes of the first scanning lens and the second scanning lens are orthogonal to each other and are arranged confocally, and the common focal position of the first scanning lens and the second scanning lens is located at the center of the galvanometer mirror; the illumination module generates an illumination laser, which passes through the scanning lens group and enters the rear pupil plane of the illumination objective lens, and projects an illumination sheet with an inclination angle α onto an excitation plane of the sample to excite fluorescence, the excitation plane obliquely intersecting with the main optical axis of the illumination objective lens at an angle of inclination θ of the light sheet; the fluorescence emitted from the sample excitation plane is collected by the illumination objective, passes through the scanning lens group in a direction opposite to the illumination light, and is incident on the imaging module and imaged, thereby obtaining a projection of the sample excitation plane in a plane perpendicular to a main optical axis of the illumination objective; A single objective lens sheet type three-dimensional fluorescence imaging system, characterized in that the imaging module is equipped with a surface detector.

2. 2. A single-objective lens sheet-type three-dimensional fluorescence imaging system, characterized in that the single-objective lens microscope imaging system described in claim 1 further comprises a sample mounting stage, the sample mounting stage having an axial feed device for moving the sample in the axial direction of the illumination objective lens, the axial feed device changing the axial position in a stepwise or continuous manner, and the axial displacement L within the rotation period T of the galvanometer mirror satisfies the following formula: where s is the Rayleigh range of the sheet and θ is the tilt angle of the light sheet relative to the scanning direction.

3. 3. A single-objective lens sheet-type three-dimensional fluorescence imaging system, as described in claim 1 or 2, characterized in that, by the action of the galvanometer mirror, the excitation plane objective lens of the sample is scanned in a direction perpendicular to the main optical axis of the illumination objective lens, the fluorescence signal is reverse-scanned by the galvanometer mirror, and then a projection of the sample excitation plane on a plane perpendicular to the main optical axis of the illumination objective lens is acquired in chronological order by a surface detector of the imaging module, and a three-dimensional fluorescence image of the sample is obtained by reconstructing and stacking the projection data.

4. 4. The single-objective lens sheet-type three-dimensional fluorescence imaging system of claim 3, characterized in that the galvanometer mirror changes its angle stepwise or continuously, thereby scanning the excitation plane of the sample in a direction perpendicular to the main optical axis of the illumination objective lens, and the displacement d of the sample excitation plane in the scanning direction corresponding to adjacent projections satisfies the following equation: where h is the thickness of the light sheet and θ is the tilt angle of the light sheet relative to the scanning direction.

5. 3. A single-objective lens sheet-type three-dimensional fluorescence imaging system, characterized in that in the single-objective lens microscope imaging system described in claim 1 or 2, a tube mirror assembly is arranged between the scanning lens group and the illumination objective lens, and the first lens is fixed relatively to the position of the scanning lens group to perform focusing correction of the fluorescence signal collected by the objective lens.

6. 3. A single-objective lens sheet-type three-dimensional fluorescence imaging system according to claim 1 or 2, characterized in that the illumination module comprises a collimated laser and a modulator equipped with a mask, the illumination laser is modulated by the modulator to form a sheet of a predetermined shape, and the sheet is combined with the fluorescence light path via a dichroic beam splitter and then guided to the scanning lens group.

7. 7. The single-objective lens sheet type three-dimensional fluorescence imaging system according to claim 6, wherein the thickness of the illumination sheet is 0.3 μm to 5 μm.

8. 3. A single-objective lens sheet-type three-dimensional fluorescence imaging system, characterized in that, in the single-objective lens microscope imaging system described in claim 1 or 2, the imaging module comprises a focusing lens and a planar detector, the planar detector is arranged on the focal plane of the focusing lens, and the planar detector acquires a projection of the sample excitation plane in a plane perpendicular to the main optical axis of the illumination objective lens according to the time sequence of control signals.

9. A single-objective lens sheet-type three-dimensional fluorescence imaging system, characterized in that the single-objective lens microscope imaging system described in claim 1 or 2 is provided with a focal depth extension unit, which is arranged between the scanning lens group and the focusing lens, and which extends the focal depth of the sheet in the axial direction of the objective lens by the focal depth extension unit.

10. 10. A single-objective lens sheet type three-dimensional fluorescence imaging system, characterized in that in the single-objective lens microscope imaging system described in claim 9, the focal depth extension unit is a phase modulation mask, a spatial light modulator, an on-axis prism, or a prism group.

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