A high-efficiency optical sectioning microscopic fluorescence imaging system and method

Abstract

The application discloses a kind of high-efficiency optical sectioning microscopic fluorescence imaging system and method.It includes: multi-wavelength beam combining laser module and single-mode optical fiber, collimating lens, first mirror, light splitting module, first optical path, second optical path, speckle generation module, fast switching module, second mirror, beam expander, third mirror, focusing lens, fluorescence module, tube lens, camera, objective lens, motorized displacement stage, condenser, illumination source and the like composition.For the current HiLo microscopic imaging speckle homogenization complex, uniform illumination and speckle illumination high-speed switching difficult, low light energy utilization efficiency and other technical problems;The application obtains the 2 images required for imaging by fast switching module and camera linkage efficiently;Compact structure design, effective utilization of system incident excitation light.The optical sectioning microscopic fluorescence imaging system of the application can realize the optical tomography of target sample efficiently and conveniently, and well expands the practical application range of HiLo in high-speed imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical microscopic imaging technology, in particular to a high-efficiency optical sectioning microscopic fluorescence imaging method and an imaging light path thereof. BACKGROUND

[0002] With the continuous development of science and technology, there are many methods for monitoring and diagnosing human life and health. Life science is getting more and more attention from people. However, in the current life science research process, one of the challenges of microscopic imaging is to perform deep imaging on complete organisms, organs, tissues, living cells, etc. With the gradual increase of sample depth, scattering and optical aberration distortion are also increasingly deteriorated, and the out-of-focus signal of depth greatly reduces the signal-to-noise ratio and resolution, ultimately greatly limiting the imaging depth.

[0003] At present, the existing confocal fluorescence imaging and multi-photon fluorescence imaging can well suppress the out-of-focus signal and improve the imaging depth and axial resolution. However, the single-point scanning imaging mode greatly restricts the high-speed dynamic imaging activity and has a great limitation on the imaging of living organisms and living cells. Another technology for limiting out-of-focus signal through optical sectioning (HiLo) has the advantages of flexibility and efficiency. The existing methods for realizing HiLo imaging include using a mixed excitation light unit composed of a coherent fluorescence excitation light source and an incoherent fluorescence excitation light source to obtain speckle images and uniform illumination images (for example, Chinese invention patent, application number 202010993369.2), using a high-speed moving scattering medium to obtain uniform illumination images (for example, literature DOI: 10.1117 / 1.3528656), and using a relatively complex laser speckle attenuator (for example, Chinese invention patent, application number 202210615568.9). The above methods have the problems of limited high-speed operation of light source switching electric control, possible reduction of service life due to long-time light source pulse switching, unstable power, and possible influence of high-speed moving scattering medium on the overall imaging speed. These deficiencies seriously restrict the application and popularization of HiLo optical sectioning technology. SUMMARY

[0004] To address the technical problems of current HiLo optical slice imaging technology, such as the time sacrificed in converting the scattering medium to uniform illumination, which affects the overall imaging speed, the relatively complex structure of the scattering medium, and low light energy utilization efficiency, this invention proposes a high-efficiency optical slice microscopic fluorescence imaging method and its imaging optical path. In this invention, the light source is a single source output for efficient beam splitting. There is no need to turn multiple light sources on and off or to process the scattering medium when switching between speckle illumination and uniform illumination, greatly improving the stability of optical power. Furthermore, the switching between speckle illumination and uniform illumination can achieve high-speed switching imaging at the kHz level through a high-speed switching device (e.g., a galvanometer), without requiring other constraints related to the scattering medium. Therefore, this invention has greater applicability and can achieve high-efficiency optical slice imaging. Moreover, the overall structure is relatively simple, the cost is low, and it is conducive to integration with other applications.

[0005] The objective of this invention is achieved by at least one of the following technical solutions.

[0006] A high-efficiency optical section microscopy fluorescence imaging system with multiple imaging modes, including: bright-field imaging, wide-field fluorescence imaging and optical section imaging;

[0007] A multi-wavelength beam combining laser module, used as a fluorescence imaging illumination source, can contain a visible / near-infrared laser source, which is coupled into a single-mode fiber via an internal reflector, dichroic mirror, coupling lens group and other devices.

[0008] The laser source output from the single-mode fiber is converted into a collimated beam by a collimating lens and then transmitted. The collimated beam is split by the first reflector and then enters the first optical path and the second optical path respectively.

[0009] The first optical path passes through the neutral density filter and is power-adjusted before being incident on the reflector, and then transmitted to the fast switching module via the reflector.

[0010] The incident light in the second optical path is redirected by two mirrors and then enters the speckle generation module. The incident light modulated by this module is integrated into the desired speckle distribution and transmitted into the fast switching module.

[0011] The fast switching module selectively outputs two beams of light to the second reflector by converting the incident light from the first and second optical paths at different angles; then the beam is transmitted into the beam expander; the collimated beam output from the beam expander is transmitted to the focusing lens via the third reflector, the focusing lens converges the beam and transmits it to the rear focal plane of the objective lens via the fluorescence module, the beam focused on the rear focal plane of the microscope objective lens in the microscopic imaging module is then converted into parallel light by the objective lens and illuminates the sample being observed;

[0012] The microscopic imaging module can be divided into a bright field illumination light source module, a fluorescence module and an imaging module. The bright field illumination module is composed of a wide spectrum light source, an aperture diaphragm, a field diaphragm, a compound eye lens and a condenser lens, adopts a Kohler illumination light path, and modulates the light emitted by the wide spectrum light source into uniform white light to illuminate the sample to be measured. The fluorescence module is composed of an incident light filter, a dichroic mirror and an exit fluorescence filter. The imaging module is composed of an electric displacement table, an objective lens, a tube lens and a camera. The signals collected by the camera are converted into digital images and transmitted to a PC end for display and storage.

[0013] The high-efficiency optical sectioning microscopic fluorescence imaging system specifically comprises a multi-wavelength beam combining laser module, a collimating lens, a first mirror, a light splitting module, a first light path first mirror, a first light path neutral density filter, a second light path first mirror, a second light path second mirror, a speckle generation module, a fast switching module, a second mirror, a beam expander, a third mirror, a focusing lens, a fluorescence module, a tube lens, a camera, an objective lens, an electric displacement table, a condenser lens and an illumination light source.

[0014] The light path of the multi-wavelength beam combining laser module and the single-mode optical fiber passes through the collimating lens, enters the light splitting module after the first mirror, and forms a first light path and a second light path. The first light path passes through the first light path first mirror and the first light path neutral density filter. The second light path passes through the second light path first mirror and the second light path second mirror, then passes through the speckle generation module, and then converges with the first light path after the fast switching module, the second mirror, the third mirror and the focusing lens, and enters the microscope imaging structure component.

[0015] Further, the multi-wavelength beam combining laser module is a coherent laser light source or a fiber output LED light source.

[0016] Further, the light splitting module is a depolarization light splitting prism or a combination of a 1 / 2 wave plate and a polarization light splitting prism.

[0017] Further, the incident light in the speckle generation module passes through an engineering scattering sheet with different particle sizes and angles, and is then collimated by a lens to convert into a collimated light beam with scattering distribution. Different speckle patterns can be switched according to sample requirements. In addition, the engineering scattering sheet can also be replaced by other scattering transparent media, such as phase separation materials controlled by critical temperature.

[0018] Further, the deflection angle of the mirror in the first light path is θ; the incidence is set to 0-45° according to actual needs; the spatial distance between the mirror in the first light path and the second light path needs to be considered in the actual design of the mechanical structure, and the appropriate sizes of L and D are determined; the relationship between them is D=L·tanθ; wherein L is the distance from the intersection of the first light path through the neutral density filter in the first light path and the second light path through the speckle generation module to the fast switching module; D is the distance from the intersection of the first light path through the neutral density filter in the first light path and the second light path through the speckle generation module to the mirror in the first light path.

[0019] Further, the fast switching module is a high-speed rotating device such as a micro-electromechanical system scanning mirror MEMS, a galvanometer, or a rotary motor; when switching the first light path or the second light path, the rotation angle is α, and the angle between the optical axis and the outgoing light of the first light path is

[0020] When the second light path is used for imaging, it is α0, the second light path is reflected through the fast switching module, the light beam reaches the second mirror, and then is used for imaging illumination; at this time, the incident light of the first light path is reflected through the fast switching module (10), the light beam reaches the light absorption device, and the light beam is absorbed to avoid stray light interference with imaging;

[0021] When the first light path is used for imaging, it is α1, that is, the angle between the optical axis and the outgoing light of the first light path is α=(90°-θ) / 2 is obtained; the first light path is reflected through the fast switching module, the light beam reaches the second mirror, and then is used for imaging illumination; at this time, the incident light of the second light path is reflected through the fast switching module, the light beam reaches the light absorption device, and the light beam is absorbed to avoid stray light interference with imaging.

[0022] Further, the camera is an EMCCD or sCOMS camera; the camera acquisition is synchronized with the timing of the fast switching module through PC external triggering.

[0023] Further, in the actual high-speed three-dimensional imaging process, the dwell time T1 and the dwell time T2 of the switching device in the fast switching module need to be set; and the exposure time t1 and the exposure time t2 of the camera image acquisition; and the running period P in the linkage process.

[0024] A method of a high-efficiency optical sectioning microscopic fluorescence imaging system, the observed sample is placed on an electric displacement table, a suitable fluorescence module is selected, the camera, the laser light source are turned on; if only two-dimensional images are collected, the period P is associated with the XY axis of the electric displacement table to run, at the same position, one fluorescence image excited by speckle illumination and one fluorescence image excited by uniform illumination are collected respectively, and the HiLo optical sectioning algorithm is used to process the two images to obtain an optical sectioning image; the total time consumption of the final high-resolution single image is the period P time; the period P includes the exposure time t1, the exposure time t2 and the data export storage time;

[0025] The HiLo optical sectioning algorithm is: the high-frequency component of the focal plane of the imaged object is obtained by high-pass filtering the uniform illumination image; the proportion of the focal plane information in the uniform illumination image is obtained by calculating the speckle contrast of the difference image of the uniform illumination image and the speckle illumination image, and then the focal plane information in the uniform illumination image is extracted; the low-frequency component of the focal plane of the imaged object is obtained by low-pass filtering the extracted focal plane information in the uniform illumination image; finally, the focal plane information of the imaged object, i.e. the optical sectioning image, is obtained by fusing the high-frequency component and the low-frequency component.

[0026] If three-dimensional images are collected, the period P is associated with the Z axis of the electric displacement table to run, at the same Z axis focal plane, one fluorescence image excited by speckle illumination and one fluorescence image excited by uniform illumination are collected respectively, and the HiLo optical sectioning algorithm is used to process the two images to obtain an optical sectioning image; then each layer of optical sectioning image is obtained by moving the Z axis in turn; and then a three-dimensional high-resolution image is obtained by a three-dimensional reconstruction method.

[0027] Compared with the prior art, the advantages of the present application are:

[0028] (1) The system structure is simple, the cost is low, and the high-speed speckle illumination and uniform illumination switching can be realized without changing the incident light source switch.

[0029] (2) The system does not need to rotate the ground glass device, and the influence of imaging background flicker or the influence of speckle granular noise in high-speed imaging is reduced.

[0030] (3) The system has strong expandability, and the speckle generation module can also be designed to be changed to other structured light illumination modes to further realize multi-mode imaging illumination. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is an exemplary embodiment schematic diagram of a high-efficiency optical sectioning microscopic fluorescence imaging system.

[0032] Figure 2 is a schematic diagram of speckle illumination and uniform illumination switching and camera linkage running timing setting.

[0033] Figure 3 The actual imaging flow chart of the high-efficiency optical sectioning microscopic fluorescence imaging system.

[0034] Figure 4 The contrast chart of the uniform illumination of the measured sample and the optical sectioning two-dimensional image.

[0035] Figure 5 The contrast chart of the uniform illumination of the measured sample and the optical sectioning three-dimensional image.

[0036] The components in the figure are: a multi-wavelength beam combining laser module 1, a collimating lens 2, a first mirror 3, a light splitting module 4, a first light path first mirror 8, a first light path neutral density filter 11, a second light path first mirror 5, a second light path second mirror 6, a speckle generation module 7, an optical absorption device 9, a fast switching module 10, a second mirror 13, a beam expander 14, a third mirror 15, a focusing lens 16, a fluorescence module 17, a tube lens 18, a camera 19, an objective lens 20, an electric displacement table 21, a condenser lens 22, and an illumination light source 23. DETAILED DESCRIPTION

[0037] In the following description, the technical solutions are described in conjunction with specific drawings so as to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and similar extensions made by those of ordinary skill in the art without creative efforts are within the scope of protection of the present application.

[0038] The materials, reagents, etc. used in the following examples of the present application, without special instructions, can be obtained from commercial channels.

[0039] The present embodiment provides a high-efficiency optical sectioning microscopic fluorescence imaging system, which has multiple imaging modes, including: bright field imaging, wide field fluorescence imaging and optical sectioning imaging; as shown in the figure. Figure 1

[0040] The multi-wavelength beam combining laser module serves as a fluorescence imaging illumination light source, and can contain a visible / near-infrared laser source inside the module, which is coupled into a single-mode optical fiber through internal mirrors, dichroic mirrors, coupling lens groups and other devices;

[0041] The laser light source output by the single-mode optical fiber is converted into a collimated light beam by a collimating lens; the collimated light beam enters the light splitting module after the first mirror and is split into the first light path and the second light path;

[0042] The first light path passes through the neutral density filter 11 for power adjustment and is incident to the mirror, which transmits the light to the fast switching module;

[0043] ​The second light path incident light is converted in direction by two mirrors and then incident to the speckle generation module. The modulated incident light is integrated into the required speckle distribution and transmitted into the fast switching module;

[0044] The fast switching module converts the incident light of the first light path and the second light path into different angle selective light and then emits the light to the second mirror. The light is transmitted into the beam expander. The collimated light beam output by the beam expander is transmitted to the focusing lens through the third mirror. The focusing lens converges the light beam to the back focal plane of the objective lens through the fluorescence module. The light beam focused to the back focal plane of the microscope objective lens is converted into parallel light by the objective lens and then irradiated to the observed sample.

[0045] The microscopic imaging module can be divided into a bright field illumination light source module, a fluorescence module and an imaging module. The bright field illumination module is composed of a wide spectrum light source, an aperture diaphragm, a field diaphragm, a compound eye lens and a condenser. The wide spectrum light source emits light which is modulated into uniform white light to illuminate the sample to be measured by using Kohler illumination light path. The fluorescence module is composed of an incident light filter, a dichroic mirror and an exit fluorescence filter. The imaging module is composed of an electric displacement table, an objective lens, a tube lens and a camera. The signals collected by the camera are converted into digital images and then transmitted to the PC end for display and storage.

[0046] The embodiment specifically includes a multi-wavelength beam combining laser module 1, a collimating lens 2, a first mirror 3, a light splitting module 4, a first light path first mirror 8, a first light path neutral density filter 11, a second light path first mirror 5, a second light path second mirror 6, a speckle generation module 7, a fast switching module 10, a second mirror 13, a beam expander 14, a third mirror 15, a focusing lens 16, a fluorescence module 17, a tube lens 18, a camera 19, an objective lens 20, an electric displacement table 21, a condenser 22 and an illumination light source 23.

[0047] The light path of the multi-wavelength beam combining laser module and the single-mode optical fiber 1 passes through the collimating lens 2, enters the light splitting module 4 after the first mirror 3, and forms a first light path and a second light path; wherein the first light path passes through the first light path first mirror 8 and the first light path neutral density filter 11; the second light path passes through the second light path first mirror 5 and the second light path second mirror 6, and then passes through the speckle generation module 7 and converges with the first light path, and then passes through the fast switching module 10, the second mirror 13, the third mirror 15, and the focusing lens 16 to enter the microscope imaging structure component. The multi-wavelength beam combining laser module 1 is a coherent laser light source or a fiber output LED light source. The light splitting module is a depolarization light splitting prism or a 1 / 2 wave plate combined with a polarization light splitting prism. The incident light in the speckle generation module 7 passes through engineering scattering sheets of different particle sizes and angles, and then is converted into a scattered collimated light beam through a lens; wherein different speckle patterns are switched according to sample requirements. The deflection angle of the mirror 8 in the first light path is θ; the incidence is set to 0-45° according to actual needs; the spatial distance between the first light path mirror 8 and the second light path determines the appropriate L and D sizes; the relationship between them is D=L·tanθ; wherein L is the distance from the intersection of the first light path through the first light path neutral density filter 11 and the second light path through the second light path speckle generation module 7 to the fast switching module 10; D is the distance from the intersection of the first light path through the first light path neutral density filter 11 and the second light path through the second light path speckle generation module 7 to the mirror 8 in the first light path. The fast switching module 10 is a high-speed rotating device such as a micro-electro-mechanical system scanning mirror MEMS, a galvanometer, a rotary motor, etc.; when switching the first light path or the second light path, the rotation angle is α, and the angle between the optical axis and the first light path exit light is

[0048] When the second light path is used for imaging, α0, the second light path is reflected by the fast switching module 10, the light beam reaches the second mirror 13, and then is used for imaging illumination; at this time, the incident light of the first light path is reflected by the fast switching module 10, the light beam reaches the light absorption device 9, and the light beam is absorbed to avoid stray light interference with imaging;

[0049] When the first light path is used for imaging, α1, that is, the angle between the optical axis and the first light path exit light is α=(90°-θ) / 2 is obtained; the first light path is reflected by the fast switching module 10, the light beam reaches the second mirror 13, and then is used for imaging illumination; at this time, the incident light of the second light path is reflected by the fast switching module 10, the light beam reaches the light absorption device 12, and the light beam is absorbed to avoid stray light interference with imaging.

[0050] The camera 19 is an EMCCD or sCOMS camera; the camera acquisition is synchronized with the fast switching module 10 through PC external triggering and timing synchronization linkage.

[0051] In the actual high-speed three-dimensional imaging process, the dwell time T1 and the dwell time T2 of the switching device in the fast switching module 10 need to be set, and the exposure time t1 and the exposure time t2 of the camera image acquisition need to be set, and the running period P in the linkage process.

[0052] As shown in Figure 2 , the linkage, acquisition timing and time setting of the imaging method are shown. In the total time P of a single image acquisition, the switching device is connected and synchronously controlled by an external acquisition card to realize high-speed switching of the first light path and the second light path, and trigger control of the imaging exposure time t1 and t2 in the two light paths.

[0053] As shown in Figure 3 , the embodiment also provides a method of a high-efficiency optical sectioning microscopic fluorescence imaging system. The observed sample is placed on the electric displacement table 21, the appropriate fluorescence module 17 is selected, and the camera and the laser light source are turned on. If only two-dimensional images are acquired, the period P is associated with the XY axis of the electric displacement table 21 for running, and one fluorescence image excited by the speckle illumination and one fluorescence image excited by the uniform illumination need to be acquired at the same position. The HiLo optical sectioning algorithm is used to process the two images to obtain an optical sectioning image. The total time consumption of the final high-resolution single image is the period P time. The period P includes the exposure time t1, the exposure time t2 and the data export and storage time.

[0054] The HiLo optical sectioning algorithm is: the high-frequency component of the focal plane of the imaged object is obtained by high-pass filtering the uniform illumination image; the proportion of the focal plane information in the uniform illumination image is obtained by calculating the speckle contrast of the difference image between the uniform illumination image and the speckle illumination image, and then the focal plane information in the uniform illumination image is extracted; the low-frequency component of the focal plane of the imaged object is obtained by low-pass filtering the extracted focal plane information in the uniform illumination image; and finally, the focal plane information of the imaged object, i.e. the optical sectioning image, is obtained by fusing the high-frequency component and the low-frequency component.

[0055] If three-dimensional images are acquired, the period P is associated with the Z axis of the electric displacement table 21 for running, and one fluorescence image excited by the speckle illumination and one fluorescence image excited by the uniform illumination need to be acquired at the same Z axis focal plane. The HiLo optical sectioning algorithm is used to process the two images to obtain an optical sectioning image. Then, each layer of optical sectioning image is obtained by moving the Z axis in turn. Then, a three-dimensional high-resolution image is obtained by a three-dimensional reconstruction method.

[0056] As shown in Figure 4The image shows two-dimensional fluorescence imaging of oleander leaf samples using this imaging method. The scale bar is 10 micrometers. The experiment used a 40X microscope objective, NA=0.75, a light source of 525nm wavelength, and a single-image exposure time of 0.05 seconds for the CMOS camera. The left image is the XY fluorescence image of the sample obtained using ordinary wide-field uniform illumination. The right image is the XY fluorescence image of the sample obtained using this imaging method. The comparison shows that the fluorescence image obtained by this imaging method has a higher signal-to-noise ratio and higher contrast than the ordinary wide-field fluorescence image.

[0057] like Figure 5 The image shows a three-dimensional fluorescence image of an oleander leaf sample obtained using this imaging method. The image size is 99*127*8 micrometers. A 40X microscope objective with NA=0.75 was used, the light source was a wavelength of 525nm, and the single-image exposure time of the CMOS camera was 0.05 seconds. The left image is the three-dimensional fluorescence image of the sample obtained using ordinary wide-field uniform illumination. The right image is the three-dimensional fluorescence image of the sample obtained using this imaging method. The comparison shows that the fluorescence image obtained by this imaging method has a higher signal-to-noise ratio, less background noise interference, and higher contrast than the ordinary wide-field fluorescence image.

Claims

1. A high-efficiency optical sectioning fluorescence imaging system with multiple imaging modes, including: Bright-field imaging, wide-field fluorescence imaging, and optical slice imaging are characterized by, A multi-wavelength beam combining laser module, used as a fluorescence imaging illumination source, contains a visible / near-infrared laser source, which is coupled into a single-mode optical fiber via an internal reflector and dichroic mirror and a coupling lens group. The laser source output from the single-mode fiber is converted into a collimated beam by a collimating lens and then transmitted. The collimated beam is split by the first reflector and then enters the first optical path and the second optical path respectively. The first optical path passes through the neutral density filter (11) and is incident on the reflector after power adjustment, and is transmitted to the fast switching module through the reflector; The incident light in the second optical path is redirected by two mirrors and then enters the speckle generation module. The incident light modulated by this module is integrated into the desired speckle distribution and transmitted into the fast switching module. The fast switching module selectively outputs two beams of light to the second reflector by converting the incident light from the first and second optical paths at different angles; then the beam is transmitted into the beam expander; the collimated beam output from the beam expander is transmitted to the focusing lens via the third reflector, the focusing lens converges the beam and transmits it to the rear focal plane of the objective lens via the fluorescence module, the beam focused on the rear focal plane of the microscope objective lens in the microscopic imaging module is then converted into parallel light by the objective lens and illuminates the sample being observed; The microscopic imaging module is divided into a bright-field illumination module, a fluorescence module, and an imaging module. The bright-field illumination module consists of a broadband light source, an aperture stop, a field stop, a compound eye lens, and a condenser lens. It uses a Kohler illumination optical path to modulate the light emitted from the broadband light source into uniform white light to illuminate the sample under test. The fluorescence module consists of an incident light filter, a dichroic mirror, and an exit fluorescence filter. The imaging module consists of a motorized stage, objective lens, tube lens, and camera. The signal acquired by the camera is converted into a digital image and transmitted to a PC for display and storage.

2. The high-efficiency optical sectioning fluorescence imaging system according to claim 1, characterized in that: Specifically, it includes a multi-wavelength beam combining laser module (1), collimating lens (2), first reflector (3), beam splitting module (4), first optical path first reflector (8), first optical path neutral density filter (11), second optical path first reflector (5), second optical path second reflector (6), speckle generation module (7), fast switching module (10), second reflector (13), beam expander (14), third reflector (15), focusing lens (16), fluorescence module (17), tube lens (18), camera (19), objective lens (20), motorized stage (21), condenser lens (22), and illumination source (23); The optical paths of the multi-wavelength beam combining laser module (1) and single-mode fiber pass through the collimating lens (2), the first reflecting mirror (3), and then enter the beam splitting module (4) to form the first optical path and the second optical path. The first optical path passes through the first reflecting mirror (8) and the neutral density filter (11). The second optical path passes through the first reflecting mirror (5) and the second reflecting mirror (6), then passes through the speckle generation module (7) and merges with the first optical path. After passing through the fast switching module (10), the second reflecting mirror (13), the third reflecting mirror (15), and the focusing lens (16), it enters the microscope imaging structure component.

3. The high-efficiency optical sectioning fluorescence imaging system according to claim 2, characterized in that: The multi-wavelength beam combining laser module (1) is a coherent laser source or an optical fiber output LED source.

4. The high-efficiency optical sectioning fluorescence imaging system according to claim 2, characterized in that: The beam splitting module is a depolarizing beam splitter prism or a combination of a half-wave plate and a polarizing beam splitter prism.

5. The high-efficiency optical sectioning fluorescence imaging system according to claim 2, characterized in that: In the speckle generation module (7), the incident light passes through engineering scattering sheets of different particle sizes and angles, and is then collimated by a lens into a collimated beam with a scattering distribution. Different speckle patterns are switched according to the sample requirements.

6. The high-efficiency optical sectioning fluorescence imaging system according to claim 2, characterized in that: The deflection angle in the first reflector (8) of the first optical path is θ; Its incident angle is set to 0-45° according to actual needs; the spatial distance between the first reflector (8) of the first optical path and the second optical path determines the appropriate L and D dimensions; the relationship between the two is as follows: θ; where L is the distance between the intersection of the first optical path through the first optical path neutral density filter (11) and the second optical path through the second optical path speckle generation module (7) and the fast switching module (10); D is the distance between the intersection of the first optical path through the first optical path neutral density filter (11) and the second optical path through the second optical path speckle generation module (7) and the first optical path first reflector (8).

7. The high-efficiency optical sectioning fluorescence imaging system according to claim 2, characterized in that: The fast switching module (10) is a high-speed rotating device for a microelectromechanical system (MEMS) scanning mirror / galvanometer / rotary motor; when switching between the first and second optical paths, the rotation angle is α, and the angle between the optical axis and the emitted light from the first optical path is... ; When the second optical path is used for imaging, it is α0. The second optical path is reflected by the fast switching module (10), and the beam reaches the second reflecting mirror (13), and is then used for imaging illumination. At this time, the incident light of the first optical path is reflected by the fast switching module (10), and the beam reaches the first light-absorbing device. The beam is absorbed to avoid stray light interfering with imaging. When the first optical path is used for imaging, it is α1, that is, the angle between the optical axis and the outgoing light of the first optical path is α1. Seeking ; The first optical path is reflected by the fast switching module (10), and the beam reaches the second reflector (13) and is then used for imaging illumination; at this time, the incident light of the second optical path is reflected by the fast switching module (10), and the beam reaches the second light-absorbing device, and the beam is absorbed to avoid stray light interfering with imaging.

8. The high-efficiency optical sectioning fluorescence imaging system according to claim 2, characterized in that: The camera (19) is an EMCCD or sCOMS camera; camera acquisition is performed in a time-synchronized manner through external triggering on the PC and fast switching module (10).

9. The high-efficiency optical sectioning fluorescence imaging system according to claim 2, characterized in that: In the actual high-speed 3D imaging process, it is necessary to set the dwell time T1 and dwell time T2 of the switching device in the fast switching module (10); the exposure time t1 and exposure time t2 of the camera image acquisition; and the running cycle P of the linkage process.

10. The method of a high-efficiency optical sectioning fluorescence imaging system according to any one of claims 1-9, characterized in that: The sample to be observed is placed on the electric displacement stage (21), a suitable fluorescence module (17) is selected, and the camera and laser light source are turned on. If only two-dimensional images are acquired, the period P is associated with the XY axis of the electric displacement stage (21). At the same position, one fluorescence image excited by speckle illumination and one fluorescence image excited by uniform illumination are acquired. The HiLo optical slicing algorithm is used to process the two images to obtain the optical slice image. The total time for the final high-resolution single image is the period P time. The period P includes exposure time t1, exposure time t2, and data export and storage time; The HiLo optical slicing algorithm is as follows: High-pass filtering is applied to the uniform illumination image to obtain the high-frequency components of the focal plane of the imaging object; the speckle contrast is calculated by comparing the difference image of the uniform illumination image and the speckle illumination image to obtain the proportion of focal plane information in the uniform illumination image, thereby extracting the focal plane information from the uniform illumination image; then, low-pass filtering is applied to the extracted focal plane information from the uniform illumination image to obtain the low-frequency components of the focal plane of the imaging object; finally, the high-frequency and low-frequency components are fused to obtain the focal plane information of the imaging object, i.e., the optical slice image. To acquire three-dimensional images, the period P is associated with the Z-axis of the electric displacement stage (21). For the same Z-axis focal plane, one fluorescence image excited by speckle illumination and one fluorescence image excited by uniform illumination are acquired. The HiLo optical slicing algorithm is used to process the two images to obtain optical slice images. Then, the Z-axis is moved to acquire each layer of optical slice images in sequence. Then, a three-dimensional high-resolution image is obtained through the three-dimensional reconstruction method.

Images