Upright and inverted integrated scanning light-field microscopic imaging device and method

Abstract

An upright and inverted integrated scanning light-field microscopic imaging device (10) and method. The device (10) comprises: a heavy-load rotary table (100), a microscope (200), an excitation light path (300), a two-dimensional scanning system (400), a micro-lens array (500), a camera (600), and a control system (700). A target biological sample is placed on a stage of the microscope (200); the heavy-load rotary table (100) is used for adjusting the position of an objective lens of the microscope (200) according to a photography requirement; the two-dimensional scanning system (400), the micro-lens array (500) and the camera (600) are used to process fluorescence emitted by the target biological sample and generate a scanning light-field image and a fluorescent image; and the control system (700) synchronously controls the two-dimensional scanning system (400) and the camera (600) to realize upright and inverted integrated scanning light-field microscopic imaging, and acquires the three-dimensional volume of the target biological sample on the basis of the scanning light-field image and the fluorescent image.

Description

Positive and inverted integrated scanning light field microscopy imaging device and method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410084873.9 and application date on January 19, 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 invention relates to the field of microscopic imaging technology, and in particular to a forward and reverse integrated scanning light field microscopic imaging device and method. Background Art

[0004] Light-field microscopy is an important tool for observing the three-dimensional structure and function of living organisms. In recent years, various variations of light-field microscopy have emerged, including scanning optical microscopy, which has improved the spatial resolution of light-field imaging.

[0005] Current three-dimensional fluorescence microscopy systems are expensive. In actual applications, due to the characteristics of the sample itself, it needs to be placed upright or inverted for easy shooting. Therefore, two or more imaging systems may be required, which brings a large financial burden to the experimenter.

[0006] Summary of the Invention

[0007] The present invention provides an integrated forward and inverted scanning light field microscopic imaging device and method to solve the problems of the microscopic imaging system in the related art that it cannot meet the shooting requirements of different angles and has high cost and low intelligence.

[0008] The first aspect of the present invention provides an integrated forward and inverted scanning light field microscopy imaging device, comprising the following steps: a microscope, a target biological sample is placed on the stage of the microscope; an excitation light path, used to output a point light source that excites the fluorescence of the target biological sample; a heavy-duty rotating stage, used to adjust the position of the objective lens of the microscope according to shooting requirements; a two-dimensional scanning system, used to perform two-dimensional scanning of the fluorescence emitted by the target biological sample at the image plane to obtain a scanned light field image; a microlens array, used to modulate the light beam of the point light source into a light field; a camera, used to generate a fluorescence image based on the light beam of the light field; a control system, used to synchronously control the two-dimensional scanning system and the camera to realize the integrated forward and inverted scanning light field microscopy imaging, and obtain the three-dimensional volume of the target biological sample based on the scanned light field image and the fluorescence image.

[0009] Optionally, in one embodiment of the present invention, the heavy-load rotating stage also controls the rotation of the excitation light path.

[0010] Optionally, in one embodiment of the present invention, the heavy-load rotating stage is perpendicular to the phase plane of the camera.

[0011] Optionally, in one embodiment of the present invention, the control system is further configured to control the two-dimensional scanning system to scan to the next light field modulation position in the interval between image frames captured by the camera.

[0012] Optionally, in one embodiment of the present invention, the microscope includes: a dichroic mirror for separating the fluorescence emitted by the point light source and the target biological sample; an objective lens and an imaging tube lens, wherein the objective lens and the tube lens cooperate to magnify the target biological sample.

[0013] Optionally, in one embodiment of the present invention, the excitation light path includes: a laser light source for outputting divergent circular laser light; and an illumination tube lens for converging the divergent circular laser light into a point shape at the back focus of a cylindrical lens.

[0014] Optionally, in one embodiment of the present invention, the positions of the objective lens, the illumination tube lens and the dichroic mirror are fixed.

[0015] Optionally, in one embodiment of the present invention, the two-dimensional scanning system includes: a front-stage lens, used to convert the fluorescence emitted by the target biological sample from the image plane to the frequency domain plane; a 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; and a rear-stage lens, used to convert the light from the frequency domain plane to the image plane.

[0016] Optionally, in one embodiment of the present invention, the scanning step length of the two-dimensional galvanometer is smaller than the diameter of the microlens array.

[0017] A second aspect of the present invention provides an integrated forward and inverted scanning light field microscopy imaging method, which uses the integrated forward and inverted scanning light field microscopy imaging device as described in any one of the above embodiments for imaging, and includes the following steps: based on shooting requirements, using a heavy-loaded rotating stage to adjust the objective lens position of the microscope; using an excitation light path to output a point light source that excites the fluorescence of a target biological sample; using a two-dimensional scanning system to perform two-dimensional scanning on the fluorescence emitted by the target biological sample at the image plane to obtain a scanned light field image; modulating the light beam of the point light source into a light field through a microlens array, and using a camera to generate a fluorescence image based on the light beam of the light field; synchronously controlling the two-dimensional scanning system and the camera to realize integrated forward and inverted scanning light field microscopy imaging, and obtaining the three-dimensional volume of the target biological sample based on the scanned light field image and the fluorescence image.

[0018] Therefore, the present invention has at least the following beneficial effects:

[0019] The embodiment of the present invention can use a heavy-duty rotating stage to control the direction of the objective lens of the microscope, realizing upright, inverted and various angle shooting. The point light source of the target biological sample is excited by the excitation light path, and the fluorescence emitted by the sample is quickly scanned in two dimensions at the image plane to increase the spatial resolution. At the same time, the light beam of the point light source is modulated into a light field using a microlens array, which is then captured by the camera as a fluorescence image. Based on the multiple scanned light field images scanned by the two-dimensional scanning system, the pixel rearrangement algorithm and the three-dimensional reconstruction algorithm are used to process and obtain the three-dimensional volume of the target biological sample. Therefore, by adding a heavy-duty rotating stage, the scanning light field microscopy imaging system is improved, which has a simple structure and low cost, can meet the shooting requirements of different samples and different angles, and is combined with a three-dimensional fluorescence system to determine the three-dimensional volume of the target biological sample. It is more integrated and intelligent, solving the problems that the microscopy imaging system in the related art cannot meet the shooting requirements of different angles, and has high cost and low intelligence.

[0020] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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:

[0022] FIG1 is a structural diagram of a forward and reverse integrated scanning light field microscopy imaging device according to an embodiment of the present invention;

[0023] FIG2 is a structural diagram of a forward and reverse integrated scanning light field microscopy imaging device according to one embodiment of the present invention;

[0024] FIG3 is a flow chart of a forward and inverted integrated scanning light field microscopy imaging method provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0026] The following describes an embodiment of the present invention, an integrated scanning light field microscopy imaging device and method for upright and inverted imaging, with reference to the accompanying drawings. To address the problems mentioned in the background art, the present invention provides an integrated scanning light field microscopy imaging device for upright and inverted imaging. A heavy-duty rotating stage is used to control the direction of the microscope's objective lens, enabling imaging in upright, inverted, and at various angles. A point-like light source is excited by an excitation optical path, and the fluorescence emitted by the sample is rapidly scanned two-dimensionally at the image plane to increase spatial resolution. A microlens array is used to modulate the light beam from the point-like light source into a light field, which is then captured by a camera as a fluorescence image. Based on the multiple scanned light field images scanned by the two-dimensional scanning system, a pixel rearrangement algorithm and a three-dimensional reconstruction algorithm are used to process and obtain the three-dimensional volume of the target biological sample. Thus, by adding a heavy-duty rotating stage, the scanning light field microscopy imaging system is improved, with a simple structure and low cost. It can meet the imaging requirements of different samples and different angles, and is combined with a three-dimensional fluorescence system to determine the three-dimensional volume of the target biological sample, making it more integrated and intelligent. This solves the problems of related microscopy imaging systems that cannot meet the imaging requirements of different angles, are high in cost, and have low intelligence.

[0027] Specifically, FIG1 is a block diagram of a forward and reverse integrated scanning light field microscopy imaging device provided by an embodiment of the present invention.

[0028] As shown in FIG1 , the integrated forward and inverted scanning light field microscopy imaging device 10 includes: a heavy-duty rotating stage 100 , a microscope 200 , an excitation light path 300 , a two-dimensional scanning system 400 , a microlens array 500 , a camera 600 and a control system 700 .

[0029] Among them, a target biological sample is placed on the stage of the microscope 200; the excitation light path 300 is used to output a point light source that excites the fluorescence of the target biological sample; the heavy-loaded rotating stage 100 is used to adjust the objective lens position of the microscope 200 according to shooting requirements; the two-dimensional scanning system 400 is used to perform two-dimensional scanning of the fluorescence emitted by the target biological sample at the image plane to obtain a scanned light field image; the microlens array 500 is used to modulate the light beam of the point light source into a light field; the camera 600 is used to generate a fluorescence image based on the light beam of the light field; the control system 700 is used to synchronously control the two-dimensional scanning system 400 and the camera 600 to realize positive and inverted integrated scanning light field microscopy imaging, and obtain the three-dimensional volume of the target biological sample based on the scanning light field image and the fluorescence image.

[0030] It is understood that the embodiment of the present invention can use a heavy-duty rotating stage 100 to control the direction of the objective lens of the microscope 200, enabling upright, inverted, and various angled photography. A point-like light source is excited by the excitation light path 300, and the fluorescence emitted by the sample is rapidly scanned in two dimensions at the image plane to increase spatial resolution. Simultaneously, a microlens array 500 is used to modulate the light beam of the point-like light source into a light field, which is then captured by the camera 600 as a fluorescence image. Based on the multiple scanned light field images scanned by the two-dimensional scanning system 400, a pixel rearrangement algorithm and a three-dimensional reconstruction algorithm are used to process and obtain the three-dimensional volume of the target biological sample. Thus, by adding a heavy-duty rotating stage, the scanning light field microscopy imaging system is improved, with a simple structure and low cost, capable of meeting the photography requirements of different samples and different angles. Combined with a three-dimensional fluorescence system, the three-dimensional volume of the target biological sample is determined, making it more integrated and intelligent.

[0031] The camera may be a scientific complementary metal oxide semiconductor transistor, a monochrome sensor, a charge coupled device, or a complementary metal oxide semiconductor transistor, etc., without specific limitation.

[0032] Furthermore, the heavy-duty rotating stage 100 of the embodiment of the present invention also controls the rotation of the excitation light path 300, which can better meet the shooting requirements, and the rotation axis of the heavy-duty rotating stage 100 is perpendicular to the phase plane of the camera 600, so no matter at any angle, it does not affect the imaging of the camera.

[0033] In one embodiment of the present invention, the control system 700 is further configured to control the two-dimensional scanning system 400 to scan to the next light field modulation position during the intervals between image frames captured by the camera 600 .

[0034] It can be understood that, as shown in Figure 2, the control system 700 of the embodiment of the present invention can include a hardware program 710, a controller 720, and a connecting wire 730, which can accurately control the two-dimensional scanning system 400 to scan to the next light field modulation position in the gap between frames.

[0035] In actual implementation, hardware program 710 generates a timing voltage signal curve for controlling the camera and two-dimensional scanning system 400, meeting the specific requirements of scanning the light field. Controller 720 physically implements the logic of hardware program 710, outputting multi-channel analog and digital voltages. Connecting wires 730 connect the two-dimensional scanning system 400 and camera 600, transmitting the output voltage of controller 720 to related devices for synchronous control. This control process can be implemented on a hardware system such as a standard personal computer or workstation.

[0036] In one embodiment of the present invention, as shown in FIG. 2 , the excitation optical path 300 includes: a laser light source 310 and an illumination tube lens 320 ; the microscope 200 includes: a dichroic mirror 210 , an objective lens 220 and an imaging tube lens 230 .

[0037] The laser light source 310 is used to output a diverging circular laser beam; the illumination tube lens 320 is used to converge the diverging circular laser beam into a point shape at the back focus of the cylindrical lens. The dichroic mirror 210 is used to separate the point light source from the fluorescence emitted by the target biological sample; the objective lens 220 and the imaging tube lens 230 cooperate to magnify the target biological sample.

[0038] Furthermore, the positions of the objective lens 220, the illumination tube lens 320 and the dichroic mirror 210 are fixed. The illumination tube lens 320 can converge the divergent circular laser output by the laser light source into a point shape at the rear focus of the cylindrical lens, pass through the dichroic mirror 210, and then pass through the objective lens 220 to reach the target biological sample.

[0039] In one embodiment of the present invention, as shown in FIG2 , a two-dimensional scanning system 400 includes: a front-stage lens 410 , a driving board 420 , a two-dimensional galvanometer mirror 430 , a rear-stage lens 440 , and a power supply.

[0040] The front-stage lens 410 is used to convert the fluorescence emitted by the target biological sample from the image plane to the frequency domain plane. The driver board 420 is used to drive the two-dimensional galvanometer mirror 430 to the target position according to the control voltage of the control system 700. The two-dimensional galvanometer mirror 430 is placed on the frequency domain plane, and a coordinate system is established in the frequency domain plane. The two-dimensional galvanometer mirror 430 is further used to scan light at high speed along the x-axis and y-axis directions. The rear-stage lens 440 is used to convert the light from the frequency domain plane to the image plane. The scanning step size of the two-dimensional galvanometer mirror 430 is smaller than the diameter of the microlens array 500.

[0041] The integrated upright and inverted scanning light-field microscopy imaging device proposed in an embodiment of the present invention combines light-field microscopy with scanning technology. By adding a heavy-duty rotating stage for adjusting the microscope's objective lens position, it can meet the needs of capturing images of different samples and angles, making the three-dimensional fluorescence system more integrated and intelligent. This achieves integrated upright and inverted scanning light-field microscopy, and offers the advantages of low cost, high speed, and suitability for in vivo microscopic observation. This solves the problems of related microscopy systems, such as their inability to meet the needs of capturing images at different angles, high cost, and low intelligence.

[0042] Next, the forward and inverted integrated scanning light field microscopy imaging method proposed in an embodiment of the present invention will be described with reference to the accompanying drawings.

[0043] FIG3 is a flow chart of a forward and inverted integrated scanning light field microscopy imaging method according to an embodiment of the present invention.

[0044] As shown in FIG3 , the integrated forward and inverted scanning light field microscopy imaging method includes the following steps:

[0045] In step S101 , based on shooting requirements, a heavy-duty rotating stage is used to adjust the position of the objective lens of a microscope.

[0046] In step S102 , an excitation light path is used to output a point light source that excites fluorescence of a target biological sample.

[0047] In step S103 , a two-dimensional scanning system is used to perform two-dimensional scanning on the image plane of the fluorescence emitted by the target biological sample to obtain a scanned light field image.

[0048] In step S104 , the light beam of the point light source is modulated into a light field by a microlens array, and a camera is used to generate a fluorescent image according to the light beam of the light field.

[0049] In step S105 , the two-dimensional scanning system and the camera are synchronously controlled to realize forward and inverted integrated scanning light field microscopy imaging, and the three-dimensional volume of the target biological sample is acquired based on the scanning light field image and the fluorescence image.

[0050] It should be noted that the upright and inverted integrated scanning light field microscopy imaging method of an embodiment of the present invention utilizes the upright and inverted integrated scanning light field microscopy imaging device of the above-mentioned embodiment for imaging. The above-mentioned explanation of the upright and inverted integrated scanning light field microscopy imaging device is also applicable to the upright and inverted integrated scanning light field microscopy imaging method of this embodiment, and will not be repeated here.

[0051] The integrated upright and inverted scanning light-field microscopy method proposed in an embodiment of the present invention combines light-field microscopy with scanning technology. By adding a heavy-duty rotating stage to adjust the microscope's objective lens position, it can meet the needs of capturing images of different samples and angles, making the three-dimensional fluorescence system more integrated and intelligent. This method achieves integrated upright and inverted scanning light-field microscopy, offering the advantages of low cost, rapidity, and suitability for in vivo microscopic observation. This overcomes the problems of related microscopy systems, such as their inability to meet the needs of capturing images at different angles, high cost, and low intelligence.

[0052] 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 invention. 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 the features of different embodiments or examples without contradiction.

[0053] 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 the present invention, "N" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0054] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code comprising one or N executable instructions for implementing a custom logical function or step of a process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may be performed out of the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present invention pertain.

[0055] It should be understood that various parts of the present invention 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 logic gate circuits for implementing logical functions on data signals, an application-specific integrated circuit having suitable combinational logic gate circuits, a programmable gate array, a field programmable gate array, etc.

[0056] 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.

[0057] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An upright and inverted integrated 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 point light source that excites the fluorescence of the target biological sample; A heavy-duty rotary stage for adjusting the position of the objective lens of the microscope according to the shooting requirements; A two-dimensional scanning system for two-dimensionally scanning the fluorescence emitted by the target biological sample at the image plane to obtain a scanned light field image; A microlens array for modulating the light beam of the point light source into a light field; A camera for generating a fluorescence image according to the light beam of the light field; A control system for synchronously controlling the two-dimensional scanning system and the camera to achieve positive and negative integrated scanned light field microscopy imaging, and obtaining the three-dimensional volume of the target biological sample based on the scanned light field image and the fluorescence image.

2. The integrated upright and inverted scanning light field microscopy imaging device according to claim 1, wherein The heavy-duty rotary stage also controls the rotation of the excitation optical path.

3. The integrated forward and reverse scanning light field microscopic imaging device according to claim 1, wherein The heavy-duty rotary stage is perpendicular to the image plane of the camera.

4. The integrated upright and inverted scanning light field microscopy imaging device according to claim 1, characterized in that, The control system is further configured to 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.

5. The integrated forward and inverted scanning light field microscopic imaging device according to claim 1, wherein The microscope includes: A dichroic mirror for separating the point light source from the fluorescence emitted by the target biological sample; An objective lens and an imaging tube lens, which cooperate to magnify the target biological sample.

6. The integrated forward and inverted scanning light field microscopy imaging device according to claim 5, characterized in that, The excitation optical path includes: A laser light source for outputting a divergent circular laser; An illumination tube lens for converging the divergent circular laser into a point at the rear focal point of the cylindrical lens.

7. The integrated forward and inverted scanning light field microscopy imaging device according to claim 6, characterized in that, The positions of the objective lens, the illumination tube lens and the dichroic mirror are fixed relative to each other.

8. The integrated upright and inverted scanning light field microscopy imaging device according to claim 1, wherein The two-dimensional scanning system includes: A pre-stage lens for converting the fluorescence emitted by the target biological sample from the image plane to the frequency domain plane; A drive board for driving 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 for two-dimensionally scanning the light; A post-stage lens for converting the light from the frequency domain plane to the image plane.

9. The integrated forward and inverted scanning light field microscopy imaging device according to claim 8, wherein The scanning step of the two-dimensional galvanometer is smaller than the diameter of the microlens array.

10. A method for positive and inverted integrated scanning light field microscopic imaging, characterized in that, The method uses the positive and negative integrated scanned light field microscopy imaging device according to any one of claims 1-9 for imaging, wherein the method includes the following steps: Based on the shooting requirements, use the heavy-duty rotary stage to adjust the position of the objective lens of the microscope; Use the excitation optical path to output a point light source that excites the fluorescence of the target biological sample; Use the two-dimensional scanning system to two-dimensionally scan the fluorescence emitted by the target biological sample at the image plane to obtain a scanned light field image; Modulate the light beam of the point light source into a light field through the microlens array, and use the camera to generate a fluorescence image according to the light beam of the light field; Synchronously control the two-dimensional scanning system and the camera to achieve positive and negative integrated scanned light field microscopy imaging, and obtain the three-dimensional volume of the target biological sample based on the scanned light field image and the fluorescence image.

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