Variable-magnification single-objective light sheet microscopic system based on light field reconstruction

Abstract

The invention discloses a variable-magnification single-objective light sheet microscopic system based on light field reconstruction. An illumination light path and an imaging light path share a scanning lens group and an objective; the illumination light path is provided with a variable diaphragm; the illumination laser is shaped into laser sheet light through the iris diaphragm, and enters the objective lens through the scanning lens group to form an illumination light sheet to excite sample fluorescence; the imaging light path is provided with a micro-lens vertical column according to the height direction of the light sheet; sample fluorescence collected by the objective lens reversely passes through the scanning lens group and is exposed on the camera through the micro-lens vertical column for light field imaging; and when the objective lenses with different multiplying powers are switched, the variable diaphragm and / or the micro-lens vertical column are / is adjusted, so that the sample fluorescence is exposed on the camera through the micro-lens vertical column with the corresponding multiplying power. According to the invention, by adjusting the parameter of the variable diaphragm and / or the position of the micro-lens vertical column under different multiplying powers, the fluorescent narrow strip under different multiplying powers realizes the zoom of the single-objective light field fluorescence imaging system with selective illumination through the micro-lens vertical column with the parameter and multiplying power adaptive to each other.

Description

Technical Field

[0001] The present invention belongs to the field of microscopic imaging, and more specifically, relates to a variable magnification single objective light sheet microscope system based on light field reconstruction. Background Art

[0002] The development of modern molecular medicine and intelligent information technology requires biomedical imaging technology to provide higher resolution, higher sensitivity, and multi-modal imaging capabilities. Among them, light field imaging technology is an advanced imaging method. By simultaneously capturing light information at multiple angles, it not only captures the two-dimensional light intensity information of the image but also obtains the light direction information entering the sample from different angles. This enables light field microscopy to generate multi-dimensional data with depth perception without physically changing the focal length or moving the sample, providing richer three-dimensional (3D) structural information and breaking through the limitations of classical imaging. However, the light field imaging technology itself also has some limitations. Limited by the wide-field illumination mode, there are many out-of-focus signal interferences that affect the imaging quality, and the imaging quality deteriorates rapidly with increasing depth.

[0003] The single objective tilted light sheet microscope is an emerging microscopic modality that can use a single objective to complete the excitation and detection of signals while liberating the sample placement space. However, the image captured by the tilted light sheet single objective system is a projection of the fluorescence signal, which requires affine transformation for reconstruction and restoration. In addition, accurate measurement of the light sheet angle parameters is required, and the implementation is relatively complex.

[0004] Sara Madaan et al. designed a light field microscope system with single objective selective illumination, which suppresses the background fluorescence outside the region of interest through light sheet selective illumination, thereby improving the contrast of light field three-dimensional imaging. In this light field microscope system with such a structure, when switching magnifications, since the microlens array encoding the direction angle information is coupled with the magnification of the detection objective, it is impossible to achieve magnification switching of light field imaging. Summary of the Invention

[0005] Aiming at the above defects or improvement requirements of the prior art, the present invention provides a variable magnification single objective light sheet microscope system based on light field reconstruction. The purpose is to fix the position of the fluorescence narrow strip formed by the fluorescence of the sample excitation plane during selective illumination scanning imaging through a scanning lens group, and through the coordinated switching of the variable aperture and the vertical column of microlenses at different magnifications, enable the fluorescence narrow strip formed by the fluorescence collected by different magnification objectives to pass through the vertical column of microlenses adapted to the objective magnification, realizing in-situ magnification change of the light field microscope system with single objective selective illumination, thereby solving the technical problems of the existing light field microscope system with single objective selective illumination.

[0006] To achieve the above object, according to one aspect of the present invention, a variable magnification single objective light sheet microscopy system based on light field reconstruction is provided, in which the illumination optical path and the imaging optical path share a scanning lens group and an objective lens;

[0007] The illumination optical path is provided with a variable aperture; the illumination laser is shaped into a laser sheet light through the variable aperture, and enters the objective lens through the scanning lens group to form an illumination light sheet to excite the fluorescence of the sample;

[0008] The imaging optical path is provided with a vertical column of microlenses in the height direction of the light sheet; the fluorescence of the sample collected by the objective lens passes through the scanning lens group in the reverse direction, and is exposed on the camera through the vertical column of microlenses for light field imaging;

[0009] When switching to objective lenses with different magnifications, the variable aperture and / or the vertical column of microlenses are adjusted so that the fluorescence of the sample is exposed on the camera through the vertical column of microlenses with the corresponding magnification.

[0010] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, the illumination light sheet intersects obliquely with the principal optical axis of the objective lens.

[0011] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, the width of the illumination light sheet matches the width of the vertical column of microlenses, satisfying the following conditions:

[0012]

[0013] where θ is the light sheet inclination angle, that is, the angle between the light sheet and the objective lens plane, λ is the wavelength of the illumination laser, NA_real is the effective numerical aperture of the system, which is used to measure the light collection ability of the system, n is the refractive index of the medium, and NA is the numerical aperture of the objective lens.

[0014] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, the position of the variable aperture is adjustable in the transverse direction, and the variable aperture has a slit with adjustable position; preferably, it is a row integrated mask plate configured with a two-axis displacement stage. Different thickness slits are etched on different rows in the height direction of the row integrated mask plate to match objective lenses with different magnifications. When the magnification is switched, the two-axis displacement stage controls the row integrated mask plate to move along the height direction to select the corresponding slit and controls the row integrated mask plate to move along the transverse direction to adjust the slit position to match different vertical columns of microlenses.

[0015] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, vertical columns of microlenses with different magnifications are integrated to form a microlens array, which is arranged in front of the camera; when the magnification is switched, the variable aperture adjusts the position of the illumination light sheet so that the fluorescence of the sample is exposed on the camera through the vertical column of microlenses with the corresponding magnification.

[0016] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, the microlenses with different magnifications are vertically arranged in corresponding gears of the switching mechanism; the switching mechanism is mechanically moved to make the corresponding gear in the imaging optical path and thus in the working state.

[0017] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, it uses an electric control signal to keep the switching of the microlens columns synchronized with the objective switching. Multiple microlens columns are loaded on the electric control switching mechanism, and the electric control switching mechanism includes: an electric control objective turntable or an electric control displacement stage.

[0018] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, the scanning lens group includes a first and a second scanning lens, and the principal optical axes of the first and second scanning lenses are orthogonal and confocal; the common focus of the first and second scanning lenses falls on the center of the scanning galvanometer.

[0019] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, under the action of the scanning galvanometer, the excitation plane objective of the sample scans along a direction orthogonal to the principal optical axis of the illumination objective. The fluorescence signal is anti-scanned by the scanning galvanometer and then a light field image of the sample excitation plane is obtained at the same position of the camera in the imaging module according to the time sequence. After reconstruction and stacking, a three-dimensional fluorescence image of the sample is obtained.

[0020] Preferably, in the variable magnification single objective light sheet microscopy system based on light field reconstruction, the scanning galvanometer of the scanning lens group performs a fixed rate scan.

[0021] Generally speaking, compared with the prior art by the above technical solution conceived by the present invention, the following beneficial effects can be achieved:

[0022] The present invention uses a light sheet to achieve selective illumination and uses light field reconstruction to achieve three-dimensional fluorescence image reconstruction. While combining the advantages of the two imaging methods, the scanning lens group is used based on the scanning-anti-scanning principle to make the fluorescence emitted from different sample excitation planes excited by the scanning light sheet fall at the same position of the camera after passing through the imaging optical path at a fixed magnification, realizing the controllability of the position of the fluorescence narrow strip at a fixed magnification. At different magnifications, by adjusting the parameters of the variable aperture and / or the position of the microlens columns, the fluorescence narrow strip at different magnifications passes through the microlens columns with parameters adapted to the magnification, and light field imaging is performed at different magnifications, realizing the variable magnification of the selective illumination single objective light field fluorescence imaging system.

[0023] The present invention simultaneously has the characteristics of a simple installation process and excellent imaging performance: by selectively illuminating the relevant sub-volumes of the sample, the background fluorescence in the external area of the sample is reduced, thereby improving the image contrast and effective resolution; and the added microlens array can capture the direction and angle information of light, and can directly restore the original image during the light field reconstruction process without algorithmic or optical oblique plane correction, thereby reducing the computational complexity and implementation difficulty. This technology not only improves the imaging quality, but also provides a more accurate tool for biomedical research, especially in the fields of neuroscience and cell biology, which is of great significance for observing and recording cell dynamics in complex biological processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of the imaging principle of the variable magnification single objective light sheet microscopy system based on light field reconstruction provided by the present invention;

[0025] Figure 2 is a schematic diagram of the structure of the variable aperture of the present invention;

[0026] Figure 3 is a design diagram of the row integrated mask used for the variable aperture provided in the embodiment of the present invention;

[0027] Figure 4 is a schematic diagram of the structure of the variable magnification single objective light sheet microscopy system imaging based on light field reconstruction provided in Embodiment 1 of the present invention;

[0028] Figure 5 is a schematic diagram of the vertical structure of the microlens array used for the variable magnification single objective light sheet microscopy system imaging provided in Embodiment 2 of the present invention.

[0029] In all the drawings, the same reference numerals are used to represent the same elements or structures, where: 1 is the objective lens, 2 is the scanning lens group, 21 is the first tube lens, 22 is the second tube lens, 23 is the first scanning lens, 24 is the second scanning lens, 25 is the scanning galvanometer, 3 is the laser, 4 is the collimation system, 5 is the beam expander group, 6 is the cylindrical lens, 7 is the variable aperture, 8 is the dichroic mirror, 9 is the tube lens, 10 is the vertical column of microlenses, and 11 is the camera. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] The variable magnification single objective light sheet microscopy system based on light field reconstruction provided by the present invention includes an illumination optical path and an imaging optical path. The illumination optical path and the imaging optical path share a scanning lens group and an objective lens. The specific settings are as follows:

[0032] The illumination optical path is provided with a variable aperture. The illumination laser is shaped into a laser sheet by the variable aperture and enters the objective lens through the scanning lens group to form an illumination light sheet to excite the fluorescence of the sample. The illumination light sheet is obliquely intersecting with the principal optical axis of the objective lens, which is a feature of the single objective light sheet fluorescence microscope.

[0033] The imaging optical path is provided with a vertical column of microlenses in the height direction of the light sheet. The fluorescence of the sample collected by the objective lens passes through the scanning lens group in the reverse direction and is exposed on the camera through the vertical column of microlenses. Utilizing the principle of light field imaging in three-dimensional imaging applications, a microlens array is used to collect depth direction information, restore the three-dimensional fluorescence signal of the sample excitation plane, and directly stack it into a three-dimensional image of the sample, which has a high contrast and improves the three-dimensional imaging quality and speed.

[0034] During operation, the scanning galvanometer of the scanning lens group performs horizontal fixed-rate scanning, and cooperates with the sample displacement stage to achieve three-dimensional fluorescence imaging of the sample.

[0035] However, directly using a traditional microlens array also limits the magnification switching of the microscopy system. The theoretical resolution of the light field is mainly determined by the spacing and diameter of the microlens array, and the relationship is as follows:

[0036]

[0037]

[0038] Wherein, U is the spacing of the microlens array, M is the magnification, and N u is the angular resolution of light field detection, and is determined by N u =NA×D / 0.47λM, and NA is the numerical aperture of the objective lens. In summary, a microlens array with a single distribution spacing only matches the corresponding magnification M. Since the microlens array encoding the direction angle information is coupled with the magnification of the detection objective lens, when the objective lens switches the magnification in situ, the microlens array cannot match, which limits the magnification switching of the microscopy system.

[0039] In order to break through the magnification switching limitation of the light field microscopy system with objective selective illumination, the present invention introduces a scanning lens group into the optical path to make the position of the fluorescence of the sample excitation plane adjustable and fixed relative to the camera plane. A vertical column of microlenses is arranged in the height direction of the light sheet instead of a microlens array, and the magnification switching is achieved by controlling the fluorescence of the excitation plane at different magnifications to pass through the corresponding microlens columns. Specifically, when switching the objective lenses with different magnifications, the variable aperture and / or the vertical column of microlenses are adjusted to make the sample fluorescence pass through the corresponding vertical column of microlenses with the corresponding magnification and be exposed on the camera.

[0040] Scanning lens group, including a first and a second scanning lens, the principal optical axes of the first and second scanning lenses are orthogonal and confocal; the common focus of the first and second scanning lenses falls on the center of the scanning galvanometer. Under the action of the scanning galvanometer, the excitation plane objective lens of the sample scans along a direction orthogonal to the principal optical axis of the illumination objective lens. The fluorescence signal is anti-scanned by the scanning galvanometer and the light field image of the sample excitation plane is obtained at the same position of the camera in the imaging module in sequence, and the three-dimensional fluorescence image of the sample is obtained by reconstruction and stacking.

[0041] In a light sheet fluorescence microscope, this mechanism of the scanning lens group is used to solve the problem of camera photosensitive crosstalk caused by the imaging of the sample excitation plane at different positions and different times in the camera during oblique light sheet scanning, ensuring that the camera signal at the same moment is the fluorescence signal emitted by the same sample excitation plane. For example, in Chinese patent document CN116107076A, in the single objective light sheet microscopy system provided by the present invention, the scanning lens group is used to project the fluorescence of the sample excitation plane at a fixed magnification onto a vertical column of microlenses at a fixed position. When the magnification is switched, the vertical column of microlenses or the image position is adjusted accordingly to match the parameters of the vertical column of microlenses with the magnification, realizing in-situ magnification switching.

[0042] Based on the above principle, the difference between the vertical column of microlenses and the microlens array lies in the following two points: First, the vertical column of microlenses has directionality. At the same magnification, the position of the fluorescence emitted by the sample excitation plane on the camera remains unchanged. When the magnification is switched, it still maintains in the light sheet height direction, and may change in the width direction according to the imaging conditions and magnification switching strategy. Therefore, the height direction of the vertical column of microlenses matches the light sheet height direction, so that the "narrow strip" of fluorescence collected through the imaging optical path passes through the microlens array with a matching direction and falls on the corresponding position of the camera.

[0043] Specifically, as Figure 1 shown, the illumination light sheet irradiates the sample at a light sheet inclination angle θ, forming a sample excitation plane within its Rayleigh range. The width d of the fluorescence narrow strip entering the vertical column of microlenses is:

[0044]

[0045] where θ is the light sheet inclination angle, that is, the angle between the light sheet and the objective plane, h is the light sheet thickness, λ is the illumination laser wavelength, NA_real is the system effective numerical aperture. Since the beam in this system does not enter at the full aperture and the rear pupil of the objective lens is not fully utilized, that is, the system effective numerical aperture measuring the light collection ability of the system is smaller than the objective lens numerical aperture, NA is the numerical aperture of the objective lens, n is the refractive index of the objective lens medium. For example, for a 20x 0.7 air objective lens, its medium is air, n = 1.0; for a 100x 1.5 oil immersion objective lens, its medium is oil, n = 1.51.

[0046] To ensure complete light collection, the width D of the vertical column of microlenses should be greater than or equal to the width of the narrow fluorescence strip, that is

[0047]

[0048] where θ is the tilt angle of the light sheet, that is, the angle between the light sheet and the objective plane, h is the thickness of the light sheet, λ is the wavelength of the illumination laser, NA_real is the effective numerical aperture of the system, which is used to measure the light collection ability of the system, n is the refractive index of the objective medium, and NA is the numerical aperture of the objective.

[0049] There are multiple implementation schemes for exposing the fluorescence of the sample through the vertical column of microlenses with corresponding magnification on the camera: on the one hand, the position of the narrow fluorescence strip can be adjusted so that it falls on different vertical columns of microlenses, and on the other hand, in-situ switching of the vertical column of microlenses can be performed.

[0050] For the scheme of adjusting the position of the narrow fluorescence strip: we use the lateral distance Δx of the center of the slit relative to the principal optical axis to characterize the position of the narrow fluorescence strip, as Figure 2 、 3 shown, as follows:

[0051]

[0052] where θ is the tilt angle of the light sheet, which is determined by the width and position of the aperture, and l is the working distance of the objective. Therefore, by adjusting the width and position of the aperture, the lateral (such as Figure 2 shown, in the x-axis direction) position of the narrow fluorescence strip at different magnifications can be adjusted, so as to set the vertical column of microlenses with corresponding parameters at different lateral positions and achieve magnification switching. In this way, the magnification switching speed is fast and the mechanical stability of the overall system is strong. At the same time of magnification switching, the "switching" of the microlenses is completed. Under the characteristic of variable magnification of a single objective light sheet, the advantages of light field imaging are combined without loss. The automatic control of the position of the narrow light sheet strip enables the possibility of an integrated microlens array. This integrated component can greatly improve the utilization rate and reduce the cost brought by processing multiple arrays. Specifically, an electrically controlled displacement stage can be used to make the position of the aperture adjustable laterally.

[0053] Variable apertures can be selected: adjustable slits, such as masks; modifiable optical elements, such as spatial light modulators, digital micromirrors; diffractive elements, such as gratings, etc. In some embodiments, in order to reduce mechanical movement and improve system integration, a row-integrated mask plate is used as the aperture, and a biaxial displacement stage is used to control it as a variable aperture. The row-integrated mask plate, such as Figure 3As shown, slits with different thicknesses are etched on different rows in the height direction (y-direction) to match objective lenses with different magnifications. When the magnification is switched, the biaxial displacement stage controls the row-integrated mask plate to move along the height direction to select the corresponding slit and controls the row-integrated mask plate to move laterally to adjust the slit position to match different vertical columns of microlenses: By adjusting the lateral position (x-axis position) of the mask, the inclination angle θ of the light sheet can be changed, thereby changing the position of the fluorescence narrow strip and entering different vertical columns of microlenses; By adjusting the longitudinal position y of the mask, the slits of objective lenses with different magnifications can be selected.

[0054] The row-integrated mask plate can simultaneously realize the selection of different types of light sheets. The mask pattern is as Figure 3 shown in the example. Different masks are designed for objective lenses with different magnifications. For objective lenses with the same magnification, Gaussian / Bessel masks are also available for selection. The appropriate type of light sheet can be selected by moving in the y-direction.

[0055] In the scheme of adjusting the position of the fluorescence narrow strip, vertical columns of microlenses with different magnifications can be integrated to form a microlens array, which is arranged in front of the camera to improve the integration and system stability; When the magnification is switched, the variable aperture adjusts the position of the illumination light sheet so that the sample fluorescence is exposed on the camera through the vertical columns of microlenses with the corresponding magnification.

[0056] For the scheme of in-situ switching of vertical columns of microlenses, vertical columns of microlenses with different magnifications are arranged in the corresponding gears of the switching mechanism; The switching mechanism makes the corresponding gear in the imaging optical path and thus in the working state through mechanical movement. In a preferred scheme, an electric control signal is used to keep the switching of the vertical columns of microlenses synchronized with the switching of the objective lens. Multiple vertical columns of microlenses are loaded on the electric control switching mechanism, and the electric control switching mechanism is, for example: an electric control objective lens turntable, an electric control displacement stage. The electric control objective lens turntable rotates to switch to the target vertical column of microlenses, and the biaxial displacement stage that holds the mask moves to the target microlens array in the plane.

[0057] The following are examples:

[0058] Example 1

[0059] The variable-magnification single-objective light-sheet microscopy system based on light field reconstruction provided in this example, as Figure 4 shown, includes an illumination optical path and an imaging optical path; The illumination optical path endows the characteristics of the light sheet, and the imaging optical path endows the characteristics of the light field. The two share the frame and the scanning lens group 2 to form a single-objective system with de-scan. Among them:

[0060] An objective lens 1 is provided in the frame; a scanning lens group 2, including a first and a second tube lens 21, 22, a first and a second scanning lens 23, 24, the principal optical axes of the first and second scanning lenses 23, 24 are orthogonal and confocal, a tube lens for correction is arranged upstream and downstream of the scanning lens, the first tube lens 21 and the first scanning lens 23 form an optical 4f system, the first scanning lens 23 and the second scanning lens 24 form an optical 4f system, and the second tube lens 22 and the second scanning lens 24 form an optical 4f system; the common focus of the first and second scanning lenses 23, 24 falls on the center of the scanning galvanometer 25. Under the action of the scanning galvanometer 25, the excitation plane objective lens of the sample scans in a direction orthogonal to the principal optical axis of the illumination objective lens. Tube lenses 21, 22: TTL180 - A, THORLABS, the first and second scanning lenses 23, 24: scanning lenses 1, 2: CLS - SL, THORLABS, scanning galvanometer 25: GVS212, THORLABS.

[0061] In the illumination optical path, the laser emitted by the laser 3 enters the collimation system 4, the collimated laser light source passes through the beam expander group 5 composed of two lenses, and is focused by the cylindrical lens 6 (CY107R12, Lubang Optoelectronics) to form a light sheet. The light sheet is shaped by the variable aperture, and the variable aperture includes a customized 3 - inch line - integrated mask plate 7 and a biaxial displacement stage, and enters the scanning system 2 to realize the lateral scanning of the light sheet with small aberration and distortion; finally, the light sheet is focused by the Olympus objective lens 1 to form an illumination light sheet at the sample.

[0062] In the imaging optical path, the fluorescence signal excited at the sample is collected by the objective lens 1, passes through the scanning system in the reverse direction, the narrow strip remains in the same position after reverse scanning, is separated by the dichroic mirror 8, passes through the tube lens 9, and is encoded by the micro - lens array 10, and finally is focused on the plane of the camera 11. The micro - lens array 10 is customized by LEBTEK, and the camera 11 uses Hamamatsu C11440.

[0063] The wavelength of the excitation light is 488 nm, and the wavelength of the fluorescence is 510 nm. In order to accurately separate the excitation and imaging optical paths, a dichroic mirror (T505lpxr, Chroma) is used to selectively transmit / reflect the two light beams.

[0064] By controlling the scanning of the scanning galvanometer, the light sheet obtains the information in an xy plane, and is restored to a rectangular block area with a certain thickness through the light field reconstruction algorithm; control the z - axis displacement stage to enter the next depth position, repeat the above process, and finally obtain multiple block signals, which are stitched into the overall sample signal.

[0065] The specific reconstruction process is as follows:

[0066] 1. Light field image reconstruction. The fluorescence signal of the sample enters the camera through the encoding of the light field. Each narrow strip of the obtained image corresponds to the information of the oblique plane within a certain depth range, as well as the corresponding angle and direction encoding. Through the light field image reconstruction algorithm, the three-dimensional volume information within the current voxel range is restored (without the need for three-dimensional affine transformation);

[0067] 2. Three-dimensional stack superposition. By processing a series of tilted plane images P1, P2, P3, …, Pk, the corresponding three-dimensional volumes Z1, Z2, …, Zk are obtained. Through axial splicing, the three-dimensional stack of the entire sample can be obtained.

[0068] When switching magnifications: In order to match the corresponding vertical columns of microlenses, the corresponding vertical columns of microlenses need to be customized according to the objective lens to be switched, and the corresponding switching is performed when the system zooms. As Figure 4 shown, the microlens array is located at the front end of the camera. By installing multiple microlens arrays, rapid control can be achieved by a sliding or rotating multi-hole mount in cooperation with a piezoelectric motor. When the system switches the objective lens, the switching objective lens hole position is read, and a signal is sent to the motor. The motor reads the signal and performs the corresponding switching to achieve variable magnification light sheet-light field imaging.

[0069] Embodiment 2

[0070] The variable magnification single objective lens light sheet microscopy system based on light field reconstruction provided in this embodiment is only different from Embodiment 1 in that: the vertical column of microlenses is an integrated vertical column of microlenses, and the vertical columns of microlenses matching different magnifications are spliced together horizontally.

[0071] Due to the combined action of the tilted light sheet and reverse scanning, the signal received by the final detection end is a narrow strip with an unchanged position, that is, only the signal within a narrow strip range passes through the microlens array, and only the microlenses within this area range will produce a modulation effect, while the other microlenses in the array will be wasted. In order to utilize the space of the entire array and reduce costs at the same time, the array can be considered to be processed into different plates and spliced into an integrated microlens array to match objective lenses with different magnifications. As shown in Embodiment 2, the entire array is processed into three plates, corresponding to three objective lenses with different magnifications respectively. The array is clamped on an electrically controlled displacement stage and horizontally moved to the corresponding position when the system switches magnifications. That is, a variable magnification light field with efficient utilization of the array space without switching can be achieved. Also, through the variable aperture 7, while switching magnifications, the position of the light sheet in the x direction can be changed to adjust the position of the narrow strip so that it falls on the vertical column of lenses with the appropriate magnification.

[0072] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A variable-magnification single-objective light sheet microscopy system based on light field reconstruction, characterized in that: Its illumination optical path and imaging optical path share the scanning lens group and objective lens; The illumination light path is equipped with a variable aperture. The illumination laser is shaped into a laser sheet light by the variable aperture, and then enters the objective lens through the scanning lens group to form an illumination light sheet to excite the sample fluorescence. The imaging optical path is provided with vertical rows of microlenses in the direction of the light sheet height; the sample fluorescence collected by the objective lens passes through the scanning lens group in the reverse direction, and is exposed on the camera through the vertical rows of microlenses to perform light field imaging; When switching between objective lenses of different magnifications, the variable aperture and / or the microlens column are adjusted so that the sample fluorescence is exposed to the camera through the microlens column of the corresponding magnification.

2. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 1, characterized in that: The illumination light sheet is oblique to the principal optical axis of the objective lens.

3. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 1, characterized in that: The width of the illumination light sheet matches the width of the microlens column, satisfying the following conditions: Among them, θ is the light sheet inclination angle, that is, the angle between the light sheet and the objective lens plane, λ is the illumination laser wavelength, and NA_real is the system effective numerical aperture, which is used to measure the system's light collection ability. n is the refractive index of the medium, and NA is the numerical aperture of the objective lens.

4. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 1, characterized in that: The variable iris has an adjustable iris position in the transverse direction, and has a position-adjustable slit; preferably, a row-integrated mask plate is configured with a biaxial translation stage, and the row-integrated mask plate has slits of different thicknesses etched on different rows in the height direction to match objective lenses of different magnifications. When the magnification is switched, the biaxial translation stage controls the row-integrated mask plate to move in the height direction to select the corresponding slit and controls the row-integrated mask plate to move in the transverse direction to adjust the slit position to match different vertical columns of microlenses.

5. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 3, characterized in that: Vertical rows of microlenses with different magnifications are integrated to form a microlens array, which is arranged in front of the camera; when the magnification is switched, the variable iris adjusts the position of the illumination light sheet so that the sample fluorescence is exposed on the camera through the vertical rows of microlenses with corresponding magnifications.

6. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 1, characterized in that: Microlenses of different magnifications are arranged in vertical rows at corresponding gears of the switching mechanism; the switching mechanism places the corresponding gears in the imaging light path through mechanical activities so as to be in a working state.

7. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 7, characterized in that: An electric control signal is used to keep the switching of microlens columns synchronous with the switching of objective lenses. Multiple microlens columns are loaded on an electric control switching mechanism, which includes an electric control objective lens turntable or an electric control displacement stage.

8. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 1, characterized in that: 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 and confocal. The common focus of the first scanning lens and the second scanning lens falls on the center of the scanning galvanometer.

9. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 8, characterized in that: Under the action of the scanning galvanometer, the excitation plane objective of the sample is scanned in a direction orthogonal to the main optical axis of the illumination objective. After the fluorescence signal is back-scanned by the scanning galvanometer, a light field image of the sample excitation plane is obtained in a time sequence at the same position of the camera of the imaging module, and a three-dimensional fluorescence image of the sample is obtained by reconstruction and stacking.

10. The variable-magnification single-objective light sheet microscopy system based on light field reconstruction according to claim 8, characterized in that: The scanning galvanometer of the scanning lens group performs scanning at a fixed rate.

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