Line-scan confocal scanning light-field microscopic imaging apparatus and method

Abstract

A line-scan confocal scanning light-field microscopic imaging apparatus (10) and method. Said apparatus comprises: a microscope (100), a target biological sample being placed on a stage of the microscope (100); an excitation optical path (200), which is used for outputting a line-shaped light source for exciting fluorescence of the target biological sample; a line-scan hardware optical path (300), which is used for performing multi-dimensional scanning of the line-shaped light source to obtain a scanned light-field image; a microlens array (400), which is used for light-field modulation of the line-shaped light source to obtain a line-shaped light field; a camera (500), which is used for performing three-dimensional imaging on the basis of the line-shaped light field to obtain a three-dimensional image; and a control system (600), which is used for controlling the line-scan hardware optical path (300) and the camera (500) to execute a line-scan confocal action, so that the number of pixel rows exposed simultaneously at a same moment is consistent with a sample region illuminated by the line-shaped light source, so as to separate a sample fluorescence signal and a background fluorescence signal of the target biological sample, and, on the basis of the scanned light-field image and the three-dimensional image, generate a three-dimensional microscopic image with background fluorescence removed for the target biological sample. Therefore, the problem of background fluorescence in a scanning light-field microscopy system is solved.

Description

Line scan confocal scanning light field microscopy device and method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410073855.0 and application date of January 18, 2024, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference. Technical Field

[0003] The present application relates to the field of microscopic imaging technology, and in particular to a line-scan confocal scanning light-field microscopic imaging device and method. Background Art

[0004] Light-field microscopy is a non-invasive technique for observing the three-dimensional structure and function of living organisms. It utilizes the propagation and focusing properties of light to capture information about the internal structure and function of organisms. In recent years, light-field microscopy has developed into various variants, such as scanning optical microscopy. These technologies enhance the spatial resolution of light-field imaging, improving the precision and accuracy of observation.

[0005] However, while light-field microscopy increases acquisition parallelism, it also faces challenges. Due to the complexity and uncertainty of the biological environment, traditional wide-field illumination and acquisition methods can easily lead to reduced imaging performance. The presence of significant autofluorescence and background fluorescence within organisms can obscure the structural signals of the specimen, compromising the accuracy and reliability of observations.

[0006] To address this issue, confocal light-field microscopy was invented. This technique achieves background removal by blocking out-of-focus background signals from light-field excitation using complex optical masks. However, confocal light-field microscopy systems are based on a Fourier transform light-field design, which cannot achieve diffraction-limited resolution with a synthetic aperture, thus hindering the observation of fine subcellular structures.

[0007] Summary of the Invention

[0008] The present application provides a line-scan confocal scanning light-field microscopy imaging device and method to solve problems such as background fluorescence in scanning light-field microscopy systems.

[0009] In a first aspect, an embodiment of the present application provides a line scan confocal scanning light field microscopy imaging device, comprising: a microscope, a target biological sample is placed on the stage of the microscope; an excitation light path, for outputting a linear light source that excites the fluorescence of the target biological sample; a line scan hardware light path, for multi-dimensionally scanning the linear light source to obtain a scanned light field image; a microlens array, for modulating the light field of the linear light source to obtain a linear light field; a camera, for performing three-dimensional imaging based on the linear light field to obtain a three-dimensional image; a control system, for controlling the line scan hardware light path and the camera to perform a line scan confocal action, so that the number of pixel rows exposed at the same time is consistent with the sample area illuminated by the linear light source, so as to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample, and generate a three-dimensional microscopic image of the target biological sample with the background fluorescence removed based on the scanned light field image and the three-dimensional image.

[0010] Optionally, the line scanning hardware optical path includes: a one-dimensional scanning system for one-dimensionally scanning the linear light source to obtain a first scanning light field image; and a two-dimensional scanning system for two-dimensionally scanning the sample fluorescence signal to obtain a second scanning light field image.

[0011] Optionally, the control system is also used to control the one-dimensional scanning system and the camera to scan simultaneously so that the center of the area of ​​the target biological sample remains consistent to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample, and to control the gap between the image frames captured by the camera by the two-dimensional scanning system, and to control the two-dimensional scanning system to scan to the next light field modulation position.

[0012] Optionally, the one-dimensional scanning system includes: a first front-stage lens, used to convert the light of the linear light source from the image plane to the frequency domain plane; a first driving board, used to drive the one-dimensional galvanometer to deflect to the target position according to the control voltage of the control system; a one-dimensional galvanometer, placed on the frequency domain plane, used to perform one-dimensional angle scanning on the light; a first rear-stage lens, used to convert the light from the frequency domain plane to the image plane.

[0013] Optionally, the two-dimensional scanning system includes: a second front-stage lens, used to convert the light of the sample fluorescence signal from the image plane to the frequency domain plane; a second driving board, used to drive the two-dimensional galvanometer to deflect to the target position according to the control voltage of the control system; a two-dimensional galvanometer, placed on the frequency domain plane, used to perform two-dimensional angular scanning of the light, wherein the scanning step size of the two-dimensional galvanometer is smaller than the diameter of the microlens array; a second rear-stage lens, used to convert the light from the frequency domain plane to the image plane.

[0014] Optionally, the excitation light path includes: a light source for outputting laser light; and a lens for converging the laser light into a line at a rear focus of the lens to form a linear light source.

[0015] Optionally, the microscope includes: a dichroic mirror for separating a sample fluorescence signal and a background fluorescence signal of a target biological sample; an objective lens and a tube lens, wherein the objective lens and the tube lens cooperate to magnify the target biological sample.

[0016] Optionally, the window width of the camera is a preset multiple of the diffraction-limited resolution.

[0017] Optionally, it further includes: an optical slit, which is placed on a conjugate image plane of the excitation light path and is used to control the width of the linear light source to be a target width.

[0018] A second aspect of the present application provides a line scan confocal scanning light field microscopy imaging method, which uses the line scan confocal scanning light field microscopy imaging device as described above to perform imaging, wherein the method includes the following steps: placing a target biological sample on the stage of a microscope; outputting a linear light source that excites the fluorescence of the target biological sample through an excitation light path; obtaining a timing voltage signal curve that meets the line scan confocal function, and using the timing voltage signal curve to control the line scan hardware light path and the camera to perform a line scan confocal action, so that the number of pixel rows exposed at the same time is consistent with the sample area illuminated by the linear light source, so as to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample; multi-dimensionally scanning the linear light source through the line scan hardware light path to obtain a scanned light field image, collecting the linear light field of the sample fluorescence signal through the camera to obtain a three-dimensional image, and generating a three-dimensional microscopic image of the target biological sample with the background fluorescence removed based on the scanned light field image and the three-dimensional image.

[0019] Therefore, this application has at least the following beneficial effects:

[0020] The embodiments of this application improve the widefield excitation acquisition mode of a scanning light-field microscopy system by utilizing the line-scanning hardware optical path, camera, and control system. This effectively separates the sample signal from background fluorescence, mitigates the impact of background fluorescence on 3D microscopy, and improves 3D imaging results in living organisms with strong backgrounds. This resolves technical issues such as background fluorescence that exist in scanning light-field microscopy systems.

[0021] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0023] FIG1 is an exemplary diagram of a line scan confocal scanning light field microscopy imaging device according to an embodiment of the present application;

[0024] FIG2 is an exemplary diagram of a line scan confocal scanning light field microscopy imaging device according to one embodiment of the present application;

[0025] FIG3 is a flow chart of a line scan confocal scanning light field microscopy imaging method according to an embodiment of the present application;

[0026] FIG4 is a flow chart of a line scan confocal scanning light field microscopy imaging method provided according to one embodiment of the present application. DETAILED DESCRIPTION

[0027] The following describes the line scan confocal scanning light field microscopy imaging device and method of the embodiment of the present application with reference to the accompanying drawings. In response to the problem of background removal in three-dimensional fluorescence imaging mentioned in the above background technology, the present application provides a line scan confocal scanning light field microscopy imaging device. Through the line scan hardware optical path, camera and control system, the wide field excitation acquisition mode of the scanning light field microscopy imaging system is improved, the sample signal and background fluorescence are effectively separated, the influence of background fluorescence on three-dimensional microscopy imaging is alleviated, and the three-dimensional imaging results in a strong background environment of the living body are improved. In this way, the background fluorescence and other problems existing in the scanning light field microscopy system are solved.

[0028] Specifically, FIG1 is a block diagram of a line scan confocal scanning light field microscopy imaging device provided in an embodiment of the present application.

[0029] As shown in FIG1 , the line scan confocal scanning light field microscopy imaging device 10 includes: a microscope 100 , an excitation light path 200 , a line scan hardware light path 300 , a microlens array 400 , a camera 500 and a control system 600 .

[0030] Among them, a target biological sample is placed on the stage of the microscope 100; the excitation light path 200 is used to output a linear light source that excites the fluorescence of the target biological sample; the line scanning hardware light path 300 is used to multi-dimensionally scan the linear light source to obtain a scanned light field image; the microlens array 400 is used to modulate the light field of the linear light source to obtain a linear light field; the camera 500 is used to perform three-dimensional imaging based on the linear light field to obtain a three-dimensional image; the control system 600 is used to control the line scanning hardware light path 300 and the camera 500 to perform a line scanning confocal action, so that the number of pixel rows exposed at the same time is consistent with the sample area illuminated by the linear light source, so as to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample, and generate a three-dimensional microscopic image of the target biological sample with the background fluorescence removed based on the scanned light field image and the three-dimensional image.

[0031] It can be understood that the embodiments of the present application can improve the wide-field excitation acquisition mode of the scanning light field microscopy system through the line scanning hardware optical path, camera and control system, effectively separate the sample signal and background fluorescence, alleviate the impact of background fluorescence on three-dimensional microscopy imaging, and improve the three-dimensional imaging results in a strong background environment of the living body.

[0032] In the embodiment of the present application, as shown in FIG. 2 , the microscope 100 includes a dichroic mirror 110 , an objective lens 120 and a tube lens 130 .

[0033] The dichroic mirror 110 is used to separate the sample fluorescence signal of the target biological sample from the background fluorescence signal; the objective lens 120 and the tube lens 130 cooperate to amplify the target biological sample.

[0034] It will be appreciated that the dichroic mirror in the embodiments of this application has two different refractive indices, allowing it to separate light of different wavelengths. By separating the sample fluorescence signal from the background fluorescence signal, the structure and function of the target biological sample can be better identified and analyzed. Through the coordination of the objective lens and the tube lens, the microscope in the embodiments of this application can amplify the image of the target biological sample, transmitting the image focused by the objective lens to the observer.

[0035] In the embodiment of the present application, the excitation light path 200 includes: a light source 210 and a lens 220;

[0036] The light source 210 is used to output laser light; the lens 220 is used to converge the laser light into a line at the rear focus of the lens 220 to form a linear light source.

[0037] It can be understood that in the embodiment of the present application, the laser light source is used for subsequent fluorescence excitation of biological samples, and the lens is used to converge the circular laser output by the laser light source into a line at the rear focus of the lens, thereby forming a linear light source.

[0038] In the embodiment of the present application, the device 10 of the embodiment of the present application further includes: an optical slit 230 .

[0039] The optical slit 230 is placed on the conjugate image plane of the excitation light path 200 to control the width of the linear light source to a target width. The conjugate image plane can be the location of the target sample after imaging by the optical system.

[0040] It is understandable that the embodiments of the present application can achieve precise control of the width of the linear light source by adjusting the size and shape of the optical slit, thereby affecting the shape and brightness distribution of the light beam irradiated on the target sample.

[0041] In the embodiment of the present application, as shown in FIG2 , the line scanning hardware optical path 300 includes: a one-dimensional scanning system 310 and a two-dimensional scanning system 320 .

[0042] The one-dimensional scanning system 310 is used for one-dimensionally scanning a linear light source to obtain a first scanning light field image; the two-dimensional scanning system 320 is used for two-dimensionally scanning a sample fluorescence signal to obtain a second scanning light field image.

[0043] It is understood that in the embodiment of the present application, the one-dimensional scanning system rapidly scans the linear light source along the short side, which is accomplished by a mechanical or electronic system. The position and movement of the light source can be precisely controlled to generate a first scanned light field image. In the two-dimensional scanning system, the scanning step size is smaller than the diameter of the microlens and is accomplished by controlling the movement of the camera and optical system. This can capture the fluorescence signal of the sample at different positions and angles to generate a second scanned light field image. By precisely controlling the movement of the one-dimensional and two-dimensional scanning systems, the embodiment of the present application can ensure that the number of pixel rows exposed simultaneously at each moment is consistent with the sample area illuminated by the linear light source, thereby obtaining a high-quality fluorescence image.

[0044] In the embodiment of the present application, as shown in FIG2 , the one-dimensional scanning system 310 includes: a first front-stage lens 311 , a first driving plate 312 , a one-dimensional galvanometer 313 , a first rear-stage lens 314 and a first power supply 315 .

[0045] Among them, the first front lens 311 is used to convert the light of the linear light source from the image plane to the frequency domain plane; the first driving board 312 is used to drive the one-dimensional galvanometer 313 to deflect to the target position according to the control voltage of the control system; the one-dimensional galvanometer 313 is placed on the frequency domain plane for one-dimensional angle scanning of the light; the first rear lens 314 is used to convert the light from the frequency domain plane to the image plane.

[0046] It can be understood that the embodiment of the present application can ensure accurate scanning of light on the frequency domain plane by controlling the first driving plate and the one-dimensional galvanometer, wherein a coordinate system is established on the frequency domain plane, and through the conversion of the first front lens and the first rear lens, a high-quality image is finally formed on the image plane.

[0047] It should be noted that the first front-stage lens and the first rear-stage lens form a 4f system, which plays a relay role. Among them, the 4f system can be an optical system with two lenses and two plane mirrors. Specifically, the first front-stage lens performs preliminary focusing and collimation on the input light, and then transmits the light to the first reflector. The first reflector reflects the light to the first rear-stage lens, further focuses and collimates the light, and then transmits the light to the second reflector. The second reflector reflects the light to the target position, realizing the relay transmission of the light.

[0048] In the embodiment of the present application, as shown in FIG2 , the two-dimensional scanning system 320 includes: a second front-stage lens 321 , a second driving plate 322 , a two-dimensional galvanometer mirror 323 , a second rear-stage lens 324 and a second power supply 325 .

[0049] Among them, the second front-stage lens 321 is used to convert the light of the sample fluorescence signal from the image plane to the frequency domain plane; the second driving plate 322 is used to drive the two-dimensional galvanometer 323 to deflect to the target position according to the control voltage of the control system 600; the two-dimensional galvanometer 323 is placed on the frequency domain plane, and is used to perform two-dimensional angle scanning on the light, wherein the scanning step size of the two-dimensional galvanometer 323 is smaller than the diameter of the microlens array; the second rear-stage lens 324 is used to convert the light from the frequency domain plane to the image plane.

[0050] It is understood that by precisely controlling the two-dimensional galvanometer to scan the light beam at high speed along both the x-axis and y-axis, the embodiments of the present application ensure that the center of the target sample region remains within the camera's field of view in each frame captured by the camera, thereby obtaining high-quality fluorescence images. Furthermore, by controlling the scanning step size of the two-dimensional galvanometer and the diameter of the microlens array, precise control of light field modulation can be achieved.

[0051] In the embodiment of the present application, the microlens array 400 is used to modulate the input light beam into a light field form, wherein the fluorescent signal in a wide field form is converted into a light field form.

[0052] In the embodiment of the present application, the window width of the camera 500 is a preset multiple of the diffraction-limited resolution.

[0053] Among them, the preset times diffraction limit resolution can be specifically calibrated, such as 10 times the diffraction limit resolution size.

[0054] It can be understood that in the embodiment of the present application, the camera is used to capture linear light field fluorescence images and quickly scan at a preset diffraction limit resolution to cover the full field of view image.

[0055] It should be noted that the camera 500 may be a scientific complementary metal oxide semiconductor transistor (SCMOS), a monochrome sensor, a charge coupled device (CCD), or a complementary metal oxide semiconductor transistor (CMOS), and may have a rolling shutter exposure function.

[0056] In the embodiment of the present application, as shown in FIG. 2 , the control system 600 includes: a hardware program 610 , a controller 620 and a connecting wire 630 .

[0057] In an embodiment of the present application, the control system 600 is also used to control the one-dimensional scanning system 310 and the camera 500 to scan simultaneously so that the center of the area of ​​the target biological sample remains consistent to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample, and to control the gap between the image frames captured by the two-dimensional scanning system 320 by the camera 500, and to control the two-dimensional scanning system 320 to scan to the next light field modulation position.

[0058] It will be appreciated that the embodiments of the present application can ensure that the center of the target sample region remains at the center of the camera's field of view during the scanning process, thereby obtaining high-quality fluorescence images. Furthermore, by precisely controlling the gaps between image frames captured by the two-dimensional scanning system, precise control of light field modulation can be achieved.

[0059] The line-scan confocal scanning light-field microscopy device proposed in the present application improves the widefield excitation acquisition mode of the scanning light-field microscopy system through the line-scan hardware optical path, camera, and control system. This effectively separates the sample signal from background fluorescence, mitigates the impact of background fluorescence on 3D microscopic imaging, and improves 3D imaging results in strong background environments. This resolves the background fluorescence and other issues that often plague scanning light-field microscopy systems.

[0060] In summary, the embodiments of the present application can achieve 3D imaging of a sample with just a few shots, while also eliminating background signals. Furthermore, the confocal portion is achieved in conjunction with the camera's own rolling shutter, without reducing imaging speed. This system is faster than traditional confocal imaging systems, making it more suitable for imaging real-world scenes.

[0061] Next, the line scan confocal scanning light field microscopy imaging method proposed according to the embodiment of the present application is described with reference to the accompanying drawings.

[0062] Specifically, FIG3 is a flow chart of a line scan confocal scanning light field microscopy imaging method provided in an embodiment of the present application.

[0063] As shown in FIG3 , the line scan confocal scanning light field microscopy imaging method uses a line scan confocal scanning light field microscopy imaging device for imaging. The line scan confocal scanning light field microscopy imaging method includes the following steps:

[0064] In step S101 , a target biological sample is placed on a stage of a microscope.

[0065] It can be understood that when placing the target biological sample, the embodiment of the present application ensures that the surface of the stage is clean and dry. The stage can accurately locate and fix the position of the target biological sample to ensure that the sample does not move or slip during observation, while adjusting the observation angle to improve observation accuracy and stability.

[0066] In step S102 , a linear light source that excites fluorescence of a target biological sample is output through an excitation light path.

[0067] It can be understood that the embodiment of the present application can generate high-brightness, good uniformity, and excitation light of a specific wavelength through the excitation effect of a linear light source, thereby exciting the target biological sample to emit fluorescence.

[0068] In step S103, a timing voltage signal curve that satisfies the line scan confocal function is obtained, and the timing voltage signal curve is used to control the line scan hardware optical path and the camera to perform the line scan confocal action, so that the number of pixel rows exposed at the same time is consistent with the sample area illuminated by the linear light source, so as to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample.

[0069] It can be understood that the embodiments of the present application can use the acquired timing voltage signal curve to control the movement of the line scanning hardware optical path, and the exposed camera is consistent with the sample area illuminated by the linear light source, effectively separating the sample fluorescence signal and the background fluorescence signal of the target biological sample, avoiding interference, and improving the accuracy and reliability of observation.

[0070] In step S104, a scanning light field image is obtained by multi-dimensionally scanning a linear light source through a line scanning hardware optical path, a three-dimensional image is obtained by collecting the linear light field of the sample fluorescence signal through a camera, and a three-dimensional microscopic image of the target biological sample with background fluorescence removed is generated based on the scanning light field image and the three-dimensional image.

[0071] The three-dimensional image may include an image of the three-dimensional morphology of the target biological sample under fluorescence.

[0072] It can be understood that the embodiments of the present application can obtain the distribution of linear light sources in space, the camera performs exposure under precise control, captures the fluorescence signals of the sample at different positions and angles, and then generates a three-dimensional image through image processing technology. Based on the information of the scanned light field image and the three-dimensional image, the fluorescence signal of the target biological sample and the background fluorescence signal can be identified and distinguished.

[0073] It should be noted that the aforementioned explanation of the embodiment of the line-scan confocal scanning light-field microscopy imaging device is also applicable to the line-scan confocal scanning light-field microscopy imaging method of this embodiment, and will not be repeated here.

[0074] The line-scan confocal scanning light-field microscopy method proposed in the present application improves the widefield excitation acquisition mode of the scanning light-field microscopy system through the line-scan hardware optical path, camera, and control system. This effectively separates the sample signal from background fluorescence, mitigates the impact of background fluorescence on 3D microscopic imaging, and improves 3D imaging results in strong background environments. This resolves the background fluorescence and other issues that often plague scanning light-field microscopy systems.

[0075] The line scan confocal scanning light field microscopy imaging method will be further described below with reference to FIG4 , including the following steps:

[0076] In step S201, a cylindrical lens and an optical slit are used to convert the laser light emitted by the light source into a linear form with a width of about 10 times the diffraction limit resolution to ensure that the axial direction does not diverge rapidly and has a certain depth of field;

[0077] In step S202, a linear light source is scanned along the short side by a one-dimensional scanning galvanometer, and a dichroic mirror and an objective lens are used to cover the square sample field of view;

[0078] In step S203, the excited fluorescent light beam passes through a dichroic mirror, a tube lens, and a microlens array and is collected by a camera. A two-dimensional galvanometer is added to the optical path to perform sub-microlens scale scanning to increase spatial resolution.

[0079] In step S204, the camera rolling shutter and the one-dimensional scanning galvanometer are synchronously controlled so that the row of pixels under exposure coincides with the sample area illuminated by the linear light source. The width of the rolling shutter window is set to about 10 times the width of the diffraction-limited resolution, which can block the fluorescence signal from the out-of-focus layer.

[0080] In step S205 , a plurality of light field modulated images are recorded by an image sensor, and a pixel rearrangement algorithm and a three-dimensional reconstruction algorithm are used to process the plurality of scanned light field images to obtain a three-dimensional volume with background removed.

[0081] In summary, the embodiments of the present application combine light-field microscopy and scanning technology with line-scan confocal technology. By synchronously controlling the linear laser and rolling shutter, the background fluorescence signal is effectively removed while maintaining high resolution. This can be achieved by using an ordinary computer for data processing. It has a simple structure and has the advantages of low cost, high speed, and suitability for in vivo microscopic observation.

[0082] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or N embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0083] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Thus, a feature specified as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of this application, "N" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0084] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, fragment or portion of code comprising one or N executable instructions for implementing a custom logical function or process step, and the scope of the preferred embodiments of the present application includes alternative implementations in which functions may be performed in a different order than shown or discussed, including performing functions in a substantially simultaneous manner or in a reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present application pertain.

[0085] It should be understood that various parts of the present application can be implemented using hardware, software, firmware, or a combination thereof. In the above embodiment, the N steps or methods can be implemented using software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented using hardware, as in another embodiment, any one of the following technologies known in the art or a combination thereof can be used: a discrete logic circuit having a logic gate circuit for implementing a logic function on a data signal, an application-specific integrated circuit having a suitable combination of logic gate circuits, a programmable gate array, a field programmable gate array, etc.

[0086] Those skilled in the art will understand that all or part of the steps in the method of the above embodiment can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. When the program is executed, it includes one or a combination of the steps of the method embodiment.

[0087] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A line-scanning confocal scanning light field microscopy imaging device, characterized in that Comprising: A microscope, on the stage of which a target biological sample is placed; An excitation optical path for outputting a linear light source that excites the fluorescence of the target biological sample; A line-scanning hardware optical path for multi-dimensionally scanning the linear light source to obtain a scanned light field image; A microlens array for modulating the light field of the linear light source to obtain a linear light field; A camera for performing three-dimensional imaging based on the linear light field to obtain a three-dimensional image; A control system for controlling the line-scanning hardware optical path and the camera to perform a line-scanning confocal action, such that the number of pixel rows simultaneously exposed at the same moment is consistent with the sample area illuminated by the linear light source, so as to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample, and generating a three-dimensional microscopic image with the background fluorescence of the target biological sample removed based on the scanned light field image and the three-dimensional image.

2. The line-scanning confocal scanning light field microscopy imaging device according to claim 1, characterized in that, The line-scanning hardware optical path includes: A one-dimensional scanning system for one-dimensionally scanning the linear light source to obtain a first scanned light field image; A two-dimensional scanning system for two-dimensionally scanning the sample fluorescence signal to obtain a second scanned light field image.

3. The line-scanning confocal scanning light field microscopic imaging device according to claim 2, wherein The control system is further used to control the one-dimensional scanning system and the camera to scan simultaneously, such that the region center of the target biological sample remains consistent, so as to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample, and control the two-dimensional scanning system to scan to the next light field modulation position during the gap between the image frames acquired by the camera.

4. The line-scanning confocal scanning light field microscopy imaging device according to claim 2, characterized in that, The one-dimensional scanning system includes: A first pre-stage lens for converting the light of the linear light source from the image plane to the frequency domain plane; A first driving board for driving a one-dimensional galvanometer to deflect to a target position according to the control voltage of the control system; A one-dimensional galvanometer placed on the frequency domain plane for performing one-dimensional angular scanning on the light; A first post-stage lens for converting the light from the frequency domain plane to the image plane.

5. The line-scanning confocal scanning light field microscopy imaging device according to claim 2, wherein The two-dimensional scanning system includes: A second pre-stage lens for converting the light of the sample fluorescence signal from the image plane to the frequency domain plane; A second driving board for driving a two-dimensional galvanometer to deflect to a target position according to the control voltage of the control system; A two-dimensional galvanometer placed on the frequency domain plane for performing two-dimensional angular scanning on the light, wherein the scanning step of the two-dimensional galvanometer is smaller than the diameter of the microlens array; A second post-stage lens for converting the light from the frequency domain plane to the image plane.

6. The line-scanning confocal scanning light field microscopic imaging device according to claim 1, characterized in that The excitation optical path includes: A light source for outputting a laser; A lens for converging the laser into a line at the rear focal point of the lens to form a linear light source.

7. The line-scanning confocal scanning light field microscopy imaging device according to claim 1, characterized in that, The microscope includes: A dichroic mirror for separating the sample fluorescence signal and the background fluorescence signal of the target biological sample; An objective lens and a tube lens, which cooperate to magnify the target biological sample.

8. The line-scanning confocal scanning light field microscopy imaging device according to claim 1, wherein The window width of the camera is a preset multiple of the diffraction-limited resolution.

9. The line-scanning confocal scanning light field microscopic imaging device according to claim 1, wherein It further includes: An optical slit, which is placed on the conjugate image plane of the excitation optical path for controlling the width of the linear light source to be a target width.

10. A line-scanning confocal scanning light field microscopy imaging method, characterized in that, The method uses the line-scanning confocal scanning light field microscopic imaging device according to any one of claims 1-9 for imaging, wherein the method includes the following steps: A target biological sample is placed on the stage of a microscope; A linear light source for exciting the fluorescence of the target biological sample is output through an excitation optical path; A timing voltage signal curve that meets the line-scanning confocal function is obtained, and the timing voltage signal curve is used to control the line-scanning hardware optical path and the camera to perform a line-scanning confocal action, so that the number of pixel rows exposed simultaneously at the same moment is consistent with the sample area illuminated by the linear light source, in order to separate the sample fluorescence signal and the background fluorescence signal of the target biological sample; The linear light source is multi-dimensionally scanned through the line-scanning hardware optical path to obtain a scanned light field image, the linear light field of the sample fluorescence signal is collected by the camera to obtain a three-dimensional image, and a three-dimensional microscopic image with the background fluorescence of the target biological sample removed is generated according to the scanned light field image and the three-dimensional image.

Images