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 belongs to the field of microscope imaging technology, and more specifically, relates to a single objective lens sheet type three-dimensional fluorescence imaging system.

Background Art

[0002] Conventional light sheet microscopes have orthogonal light sheet illumination optical paths and fluorescence detection optical paths, which are respectively composed of a detection objective lens and an illumination objective lens. Therefore, the overall optical path is complex and difficult to construct. In a light sheet microscope, a device that combines the illumination optical path and the detection optical path into one path and simultaneously illuminates and detects a sample using a single objective lens is called a single objective lens light sheet microscope.

[0003] However, when simultaneously performing illumination and imaging using a single objective lens, since the light sheet illumination optical path and the fluorescence detection optical path are obliquely intersecting, on the one hand, a phase difference occurs, and on the other hand, an objective lens with a high numerical aperture has to be selected as the single objective lens. In order to correct the phase difference and achieve perfect imaging, at present, telecentric focusing using three objective lenses is used, but the system is complex and the applicability is low. Furthermore, since it is necessary to use an objective lens with a high numerical aperture, and an objective lens with a high numerical aperture generally has a high magnification and a narrow field of view, the field of view of the single objective lens light sheet microscope is limited.

[0004] Conventionally, single-objective-lens sheet type fluorescence imaging systems have been largely hindered from commercializing single-objective-lens sheet type fluorescence microscopes because they do not allow for in-situ magnification switching, making it difficult to observe samples at different magnifications. Chinese patent document CN115685515A provides a single-objective-lens sheet type coaxial imaging system that overcomes the limitation of the numerical aperture (NA) of the illumination objective lens by using a second and third objective lens arranged coaxially. Although this enables in-situ magnification switching, this system sacrifices field of view and imaging speed, achieving it by stacking two-dimensional images of the excitation plane within the scanning cycle, and sample movement is required for three-dimensional imaging. [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention provides a single-objective lens sheet type three-dimensional fluorescence imaging system to address the shortcomings or improvement requirements of the above-mentioned prior art. The object of the present invention is to acquire three-dimensional fluorescence data of a sample by three-dimensional reconstruction, instead of complete imaging of the sample excitation plane in the conventional method, by continuously acquiring the projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens in a single-objective lens imaging system. This eliminates the need for two remote imaging objective lenses for phase difference correction, resolves the limitations on the optical parameters of the illumination objective lens, and facilitates on-the-spot magnification switching. The present invention solves the technical problems of the prior art, where complete imaging data of the sample excitation plane is required, resulting in strict limitations on the optical parameters of the three objective lenses, making on-the-spot magnification switching impossible, or where on-the-spot magnification switching by sequential exposure slows down the imaging speed. [Means for solving the problem]

[0006] To achieve the above objective, according to one aspect of the present invention, a single-objective lens microscope imaging system is provided, comprising an illumination objective lens, 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 comprises a first scanning lens and a second scanning lens, the principal optical axes of the first and second scanning lenses being orthogonal to each other and confocally positioned. The common focal point of the first and second scanning lenses is located at the center of the galvanometer mirror.

[0008] The illumination module generates an illumination laser. The illumination laser passes through the scanning lens group and enters the posterior pupil surface of the illumination objective lens. Light tilted at an optical sheet tilt angle θ Try the sheet Encouragement It is projected onto the plane of origin to excite fluorescence. The optical sheet tilt angle θ is the tilt angle of the optical sheet with respect to the direction perpendicular to the principal optical axis of the illumination objective lens, and the sample The excitation plane intersects the principal optical axis of the illumination objective lens at an oblique angle, and the angle of intersection is light The seat inclination angle is θ.

[0009] Fluorescence emitted from the sample excitation plane is collected by the illumination objective lens, passes through the scanning lens group in the opposite direction to the illumination light, and enters the imaging module for imaging, obtaining a projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens.

[0010] The imaging module is equipped with a planar detector.

[0011] More preferably, the single-objective lens microscope imaging system further comprises a sample stage. The sample stage includes an axial feed device that moves the sample in the axial direction of the illumination objective lens. This axial feed device changes the axial position in steps or continuously, and within the rotation period T of the galvanometer mirror, the axial displacement L satisfies the following equation. TIFF0007842909000001.tif1149 Here, s is the Rayleigh range of the sheet. θ is Light sheet tilt angle θ That is the case.

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

[0013] More preferably, in the single objective lens microscope imaging system, the galvanometer mirror changes its angle in a stepwise or continuously manner. Encouragement The projection plane is scanned in a direction perpendicular to the principal optical axis of the illumination objective lens. At this time, the displacement d of the sample excitation plane corresponding to the adjacent projection in the scanning direction satisfies the following equation. TIFF0007842909000002.tif2374 Here, h is the sheet thickness. θ is Light sheet tilt angle θ That is the case.

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

[0015] More preferably, in the single objective lens microscope imaging system, the illumination module comprises a collimating laser and a modulator, the modulator comprising a mask, the illumination laser being modulated by the modulator to form a preset sheet shape, combined with a fluorescence path via a dichroic beam splitter, and guided to a scanning lens group.

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

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

[0018] More preferably, the single objective lens microscope imaging system includes a depth of field expansion unit, which is positioned between the scanning lens group and the lens, and the depth of field expansion unit expands the depth of field of the sheet in the axial direction of the objective lens.

[0019] More preferably, the depth of field extension unit is a phase modulation mask, a spatial light modulator, an axial prism, or a group of prisms. [Effects of the Invention]

[0020] The above-mentioned technical solution according to the present invention offers the following advantageous effects compared to the prior art.

[0021] The novelty of this invention lies in acquiring projection information by scanning with a galvanometer mirror, performing three-dimensional reconstruction using the projection of the sample excitation plane in a plane orthogonal to the principal optical axis of the illumination objective lens, and obtaining three-dimensional fluorescence data of the sample. This allows for both illumination and signal acquisition of the sample using only one illumination objective lens. Combined with a three-dimensional reconstruction algorithm, this enables rapid and easy acquisition of three-dimensional structural information of biological samples. More importantly, in sheet-type three-dimensional fluorescence imaging using a single objective lens, two remote imaging objective lenses for phase contrast correction are unnecessary. Since the optical path construction is no longer limited by the magnification of the illumination objective lens, if magnification switching is required, it is simply a matter of changing the illumination objective lens on the spot, enabling easy on-the-spot magnification switching without affecting the imaging speed. The imaging speed is equivalent to that of conventional sheet-type fluorescence microscopes and significantly faster than confocal microscopes, greatly expanding the application range of sheet-type fluorescence microscopes from research to commercial applications such as inspection and diagnostic imaging. [Brief explanation of the drawing]

[0022] [Figure 1] This is an optical system diagram of a single objective lens sheet type three-dimensional fluorescence imaging system according to Embodiment 1 of the present invention. [Figure 2] This is an optical system diagram of a single objective lens sheet type three-dimensional fluorescence imaging system according to Embodiment 2 of the present invention. [Figure 3] This is a control signal diagram used in the embodiments of the present invention.

[0023] In all the figures, the same reference numerals indicate the same elements or components. Here, 1 is an illumination objective lens, 2 is a tube lens, 3 is a first scanning lens, 4 is a galvanometer mirror, 5 is a second scanning lens, 6 is a lens, 7 is a condenser lens, 8 is a detector, 9 is an illumination laser, and 10 indicates a depth of focus expansion module.

Mode for Carrying Out the Invention

[0024] Hereinafter, the present invention will be described in more detail with reference to embodiments. This is to make the object, technical solution and advantages of the present invention clearer. The embodiments described here are for explaining the present invention and do not limit the present invention. Furthermore, each technical feature of the present invention described in the following embodiments can be freely combined as long as they do not conflict with each other.

[0025] The present invention provides a single objective lens microscope imaging system including an illumination objective lens, a scanning lens group for relay, a galvanometer mirror, an illumination module, an imaging module, and a sample stage.

[0026] Here, the scanning lens group includes a first scanning lens and a second scanning lens. The principal optical axes of the first scanning lens and the second scanning lens are orthogonal to each other and are arranged confocal. The common focal position of the first scanning lens and the second scanning lens is located at the center of the galvanometer mirror. <000011l> The illumination laser passes through the scanning lens group, enters the rear pupil plane of the illumination objective lens, and has an inclination angle of θ light The sheet is projected onto the excitation plane of the sample to excite fluorescence. The excitation plane is oblique to the principal optical axis of the illumination objective lens, and the angle of intersection is light The seat inclination angle is θ.

[0028] Fluorescence emitted from the sample excitation plane is collected by the illumination objective lens, passes through the scanning lens group in the opposite direction to the illumination light, and enters the imaging module for imaging, obtaining a projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens.

[0029] The imaging module is equipped with a planar detector.

[0030] The galvanometer mirror scans the excitation plane of the sample in a direction perpendicular to the principal optical axis of the illumination objective lens. The fluorescence signal is then scanned in reverse by the galvanometer mirror, and the projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens is acquired in chronological order at the same position on the planar detector of the imaging module. A three-dimensional fluorescence image of the sample is obtained through reconstruction and stacking.

[0031] The galvanometer mirror changes its angle in steps or continuously, scanning the excitation plane of the sample in a direction perpendicular to the principal optical axis of the illumination objective lens. At this time, the displacement d of the sample excitation plane in the scanning direction corresponding to the adjacent projection satisfies the following equation. TIFF0007842909000003.tif2372 Here, h is the sheet thickness and θ is the tilt angle of the optical sheet with respect to the scanning direction.

[0032] By matching the rotation speed of the galvanometer mirror with the imaging speed, the displacement d of the sample excitation plane in the scanning direction corresponding to adjacent projections is controlled within an appropriate range. This avoids under-sampling due to excessively sparse sampling and crosstalk of fluorescence information due to excessively dense sampling, thereby improving the image quality after three-dimensional reconstruction.

[0033] This system directly forms a three-dimensional image by scanning the excitation plane of the sample using a scanning lens and a galvanometer mirror and acquiring projection data. 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. As a result, the limitations on the numerical aperture (NA) of the illumination objective lens are removed, and magnification can be easily switched on the spot by changing the illumination objective lens. When magnification switching is required, it is only necessary to replace the illumination objective lens with one of a different magnification; there is no need to replace, readjust, or reconfigure other components of the system or the optical path.

[0034] However, since this system uses projection-based three-dimensional reconstruction rather than complete image-based three-dimensional reconstruction, it is necessary to avoid projection-induced crosstalk. If the projection shifts position on the detector as the excitation plane is scanned, crosstalk will cause a shift when reconstructing the sample excitation plane, resulting in failure of three-dimensional reconstruction. This system uses galvanometer mirrors on both the illumination and detection light paths. Since the excitation and detection light paths pass in opposite directions through the galvanometer mirror, the imaging position on the detector is fixed by scanning and reverse scanning. It is only necessary to acquire images for each frame in chronological order, the detector imaging changes continuously, and synchronization with the phase of the galvanometer mirror is not required, effectively avoiding failure of three-dimensional reconstruction due to projection-induced crosstalk. There is no need to avoid crosstalk by using multiple scans with complex galvanometer mirror control.

[0035] A lens (preferably a tubular lens) is positioned between the scanning lens group and the illumination objective lens, fixed relative to the position of the scanning lens group, and performs focusing correction of the fluorescence signal collected by the objective lens. This improves compatibility with objective lenses of various magnifications and enhances image quality with various objective lenses.

[0036] The illumination module generates an illumination laser. Preferably, the illumination module comprises a collimating laser and a modulator, the modulator comprising a mask. The illumination laser is modulated by the modulator to form a preset sheet shape, combined with a 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, and if it is too thin, it is disadvantageous for stacking three-dimensional imaging. Also, the thinner the sheet, the shorter the Rayleigh range, increasing the cost of axial scanning. In design, it is necessary to consider the relationship between the Rayleigh range of the sheet and the depth of focus of the objective lens.

[0037] The imaging module includes a lens, and a planar detector is positioned at the focal plane of the lens. The planar detector acquires a projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens, according to the timing of the control signal. Preferably, the lens is a tubular lens.

[0038] Furthermore, it is preferable to include a depth of field expansion unit. This depth of field expansion unit is positioned between the scanning lens group and the lens and can be a phase modulation mask, a spatial light modulator, an axial prism, or a group of prisms. The depth of field expansion unit expands the depth of field of the sheet in the axial direction of the objective lens, expanding the field of view of the single-frame image and allowing more depth information to be acquired in a single exposure. This allows for an increase in the axial movement step width of the objective lens and a reduction in the number of axial scans.

[0039] The sample holder is equipped with an axial feed device that moves the sample in the axial direction of the illumination objective lens. This axial feed device changes the axial position in steps or continuously, and within the rotation period T of the galvanometer mirror, the axial displacement L satisfies the following equation. TIFF0007842909000004.tif1146 Here, s is the Rayleigh range of the sheet and θ is the tilt angle of the optical sheet with respect to the scanning direction.

[0040] Examples are shown below. [Examples]

[0041] As shown in Figure 1, the single-objective lens microscope imaging system according to this embodiment comprises an illumination objective lens, a set of relay scanning lenses, a galvanometer mirror, an illumination module, an imaging module, and a sample mounting stage.

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

[0043] Scanning lens group: Light incident on the second scanning lens 5 is focused on the galvanometer mirror 4. The focal position on the galvanometer mirror is imaged onto the rear pupil 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 the sample fluorescence. The galvanometer mirror and the rear pupil of the objective lens are conjugate, and the sheet is scanned laterally on the sample surface as the galvanometer mirror 4 vibrates. The sheet parameter is determined by lens group 2. The laser-excited fluorescence is collected by the same illumination objective lens 1, passes through the first scanning lens 3, is then scanned in reverse by the galvanometer mirror 4, enters the scanning lens 5 as stable divergent light, passes through the scanning lens 5, and is transmitted through the dichroic mirror as parallel light.

[0044] Imaging module: The dichroic mirror reflects short-wavelength laser light and transmits long-wavelength fluorescence. Parallel light is focused onto the light-receiving surface of the planar detector 8 via a lens 7 made of tubular lenses.

[0045] The sample holder (not shown) is equipped with an axial (Z-direction) feed device that moves the sample in the axial direction of the illumination objective lens. This axial feed device changes the axial position in steps or continuously, and within the rotation period T of the galvanometer mirror, the axial displacement L satisfies the following equation. TIFF0007842909000005.tif1249 Here, s is the Rayleigh range of the sheet and θ is the tilt angle of the optical sheet with respect to the scanning direction.

[0046] In addition to a feed mechanism in the Z direction, the sample mounting stage is equipped with a two-dimensional movement stage, enabling rapid exploration of the field of view.

[0047] In this embodiment, when using a 60x 1.3NA objective lens 1, the sheet width incident on the illumination objective lens 1 is 3.54 mm, and the sheet is offset 2.13 mm from the posterior pupil center of the illumination objective lens 1. The tilt angle θ between the sheet and the scanning direction is 54°, and the sheet thickness is designed to be 407 nm. The galvanometer mirror changes angle in steps or continuously, and its speed is limited by h and θ. [Examples]

[0048] The single-objective lens microscope imaging system according to this embodiment, as shown in Figure 2, comprises an illumination module, a scanning / reverse scanning module, a sample mounting module, a depth-of-focus expansion module, and an imaging module. The illumination module and the scanning / reverse scanning module are the same as in Embodiment 1, but unlike Embodiment 1, the light emitted parallel from the lens 6 after passing through the scanning telescope 5 passes through a bidirectional chromatic mirror, is focused by the lens 7, and passes through the depth-of-focus expansion module 10 before the probe 9. The depth-of-focus expansion module 10 includes, but is not limited to, conical lenses, diffractive optical elements, and spectral prisms.

[0049] In this embodiment, when using a 60x 1.3NA objective lens 1, the single-objective lens imaging system, with the addition of a depth-of-focus expansion module, increases the detection sheet thickness from 216 nm to 439 nm, expanding the field of view of a single-frame image by approximately seven times to 4080 nm. The galvanometer mirror changes angle in steps or continuously, and its speed is 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, and the number of axial scans decreases. [Examples]

[0050] In the microscope imaging systems according to Examples 1 and 2, the control timing of the galvanometer mirror, sample stage, and planar detector is shown in Figure 3.

[0051] In control method 1, lateral scanning is performed by changing the angle of the galvanometer mirror in a step-like manner.

[0052] The planar detector, in synchronization with the galvanometer mirror, acquires a projection of the sample excitation plane on a plane perpendicular to the principal optical axis of the illumination objective lens. At this time, the displacement d in the scanning direction of the sample excitation plane corresponding to an adjacent projection satisfies the following equation. TIFF0007842909000006.tif2372 Here, h is the sheet thickness and θ is the tilt angle of the optical sheet with respect to the scanning direction.

[0053] The sample stage moves in steps in the Z direction, performing 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, lateral scanning is performed by continuously changing the angle of the galvanometer mirror.

[0055] The planar detector acquires a projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens, according to an inherent time order. At this time, the displacement d in the scanning direction of the sample excitation plane corresponding to an adjacent projection satisfies the following equation. TIFF0007842909000007.tif2172 Here, h is the sheet thickness and θ is the tilt angle of the optical sheet with respect to the scanning direction.

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

[0057] In control method 3, lateral scanning is performed by continuously changing the angle of the galvanometer mirror.

[0058] The planar detector acquires a projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens, according to an inherent time order. At this time, the displacement d in the scanning direction of the sample excitation plane corresponding to an adjacent projection satisfies the following equation. TIFF0007842909000008.tif2272 Here, h is the sheet thickness and θ is the tilt angle of the optical sheet with respect to the scanning direction.

[0059] The sample stage moves continuously in the Z direction, performing axial scanning. Each time the galvanometer mirror completes one rotational period, the sample stage moves in the Z direction by a displacement L corresponding to the depth of focus. L satisfies the following equation. TIFF0007842909000009.tif1246, where s is the Rayleigh range of the sheet and θ is the tilt angle of the optical sheet with respect to the scanning direction.

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

Claims

1. A single-objective-lens sheet type three-dimensional fluorescence imaging system comprising an illumination objective lens, a relay scanning lens group, a galvanometer mirror, an illumination module, an imaging module, and a depth-of-focus expansion unit, The scanning lens group comprises a first scanning lens and a second scanning lens, the principal optical axes of the first and second scanning lenses are orthogonal to each other and confocally positioned, and the common focal point of the first and second scanning lenses 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 posterior pupil surface of the illumination objective lens, projecting an optical sheet tilted at an optical sheet tilt angle θ onto the sample excitation plane to excite fluorescence, wherein the optical sheet tilt angle θ is the tilt angle of the optical sheet with respect to the direction perpendicular to the principal optical axis of the illumination objective lens, and the sample excitation plane intersects the principal optical axis of the illumination objective lens at an oblique angle, with the angle of intersection being the optical sheet tilt angle θ. The fluorescence emitted from the sample excitation plane is collected by the illumination objective lens, passes through the scanning lens group in the opposite direction to the illumination light, and enters the imaging module for imaging, thereby obtaining a projection of the sample excitation plane in a plane perpendicular to the principal optical axis of the illumination objective lens. The imaging module includes a focusing lens and a planar detector. A single objective lens sheet type three-dimensional fluorescence imaging system characterized in that the planar detector is positioned at the focal plane of the condensing lens, the depth of focus expansion unit is positioned between the scanning lens group and the condensing lens, and the depth of focus expansion unit expands the depth of focus of the sheet in the axial direction of the objective lens.

2. A single objective lens sheet type three-dimensional fluorescence imaging system according to claim 1, further comprising a sample mounting stage, the sample mounting stage comprising 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 step-like manner or continuously, and within the rotation period T of the galvanometer mirror, the axial displacement L satisfies the following equation, characterized in that the single objective lens sheet type three-dimensional fluorescence imaging system. Here, s is the Rayleigh range of the optical sheet, and θ is the tilt angle θ of the optical sheet.

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

4. A single objective lens sheet type three-dimensional fluorescence imaging system according to claim 3, wherein the galvano mirror changes its angle in a stepwise or continuously manner so that the sample excitation plane is scanned in a direction perpendicular to the principal optical axis of the illumination objective lens, and the displacement d in the scanning direction of the sample excitation plane corresponding to an adjacent projection satisfies the following equation. Here, h is the thickness of the optical sheet, and θ is the optical sheet tilt angle θ.

5. A single objective lens sheet type three-dimensional fluorescence imaging system according to claim 1 or 2, characterized in that a tubular lens unit is arranged between the scanning lens group and the illumination objective lens, the tubular lens unit is fixed relative to the position of the scanning lens group, and performs focusing correction of the fluorescence signal collected by the objective lens.

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

7. A single objective lens sheet type three-dimensional fluorescence imaging system according to claim 6, characterized in that the thickness of the light sheet is 0.3 μm to 5 μm.

8. A single objective lens sheet type three-dimensional fluorescence imaging system according to claim 1 or 2, wherein the planar detector acquires the projection of the sample excitation plane on a plane perpendicular to the principal optical axis of the illumination objective lens in accordance with the time sequence of the control signals.

9. A single objective lens sheet type three-dimensional fluorescence imaging system according to claim 1, characterized in that the depth of field expansion unit is a phase modulation mask, a spatial light modulator, an axial prism, or a group of prisms.

Images