CN117608066B - Microscopic imaging device and method for positive and negative integrated scanning light field - Google Patents

Abstract

The present invention relates to the field of microscopic imaging technology, and in particular to an integrated forward and reverse scanning light field microscopic imaging device and method, wherein the device includes: a microscope, an excitation light path, a heavy-duty rotating stage, a microlens array, etc. A target biological sample is placed on the stage of the microscope; the heavy-duty rotating stage is used to adjust the position of the objective lens of the microscope according to the shooting requirements; a two-dimensional scanning system, a microlens array and a camera are used to process the fluorescence emitted by the target biological sample and generate a scanning light field image and a fluorescence image; a control system synchronously controls the two-dimensional scanning system and the camera to realize the integrated forward and reverse scanning light field microscopic imaging, and obtains the three-dimensional volume of the target biological sample based on the scanning light field image and the fluorescence image. Thus, the problems that the microscopic imaging system in the related art cannot meet the shooting requirements at different angles, and there are problems such as high cost and low intelligence.

Description

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

Technical Field

The present invention relates to the field of microscopic imaging technology, and in particular to a positive and negative integrated scanning light field microscopic imaging device and method.

Background technique

Light field microscopy is an important tool for observing the three-dimensional structure and function of living organisms. In recent years, a variety of light field microscopy variants have gradually emerged, including scanning optical microscopy technology, which has improved the spatial resolution of light field imaging.

The current three-dimensional fluorescence microscopy imaging system itself is expensive. In actual application, due to the characteristics of the sample itself, it needs to be placed upright or inverted for easy shooting, so two or more imaging systems may be required, which brings a large financial burden to the experimenter.

Summary of the invention

The present invention provides a positive and inverted integrated scanning light field microscopic imaging device and method to solve the problems that the microscopic imaging system in the related art cannot meet the shooting requirements at different angles and has high manufacturing cost and low intelligence.

The first aspect of the present invention provides an integrated positive 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 objective lens position 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 achieve integrated positive and inverted 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.

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

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

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

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.

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 rear focus of the cylindrical lens.

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

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, for performing 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.

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.

A second aspect of the present invention provides an integrated positive and inverted scanning light field microscopy imaging method, which uses an integrated positive 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 image plane of the fluorescence emitted by the target biological sample 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 achieve integrated positive and inverted scanning light field microscopy imaging, and obtaining the three-dimensional volume of the target biological sample based on the scanning light field image and the fluorescence image.

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

The embodiment of the present invention can use a heavy-duty rotating stage to control the direction of the objective lens of the microscope, realize upright, inverted and various angle shooting, excite the point light source of the target biological sample through the excitation light path, and perform a fast two-dimensional scan of the fluorescence emitted by the sample 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 by a microlens array, which is then collected by the camera as a fluorescence image, and 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 microscopic imaging system is improved, with a simple structure and low cost, and 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, which is more integrated and intelligent, and solves the problems that the microscopic imaging system in the related art cannot meet the shooting requirements of different angles, and has high cost and low intelligence.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 is a structural diagram of a forward and reverse integrated scanning light field microscopic imaging device provided according to an embodiment of the present invention;

FIG2 is a structural diagram of a forward and inverted integrated scanning light field microscopic imaging device provided according to an embodiment of the present invention;

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 ways

Embodiments of the present invention are described in detail below, 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 should not be construed as limiting the present invention.

The following describes the positive and inverted integrated scanning light field microscopic imaging device and method of the embodiment of the present invention with reference to the accompanying drawings. In view of the problems mentioned in the above background technology, the present invention provides a positive and inverted integrated scanning light field microscopic imaging device, which uses a heavy-duty rotating stage to control the direction of the objective lens of the microscope to achieve positive, inverted and various angle shooting, excites the point light source of the target biological sample through the excitation light path, and performs a fast two-dimensional scan of the fluorescence emitted by the sample 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 collected by the camera as a fluorescence image, and based on the multiple scanning 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 microscopic imaging system is improved, the structure is simple, the cost is low, the shooting requirements of different samples and different angles can be met, and the three-dimensional volume of the target biological sample is determined in combination with the three-dimensional fluorescence system, which is more integrated and intelligent, and solves the problems that the microscopic imaging system in the related technology cannot meet the shooting requirements of different angles, and there are high cost and low intelligence.

Specifically, FIG1 is a block diagram of a positive and inverted integrated scanning light field microscopic imaging device provided by an embodiment of the present invention.

As shown in FIG. 1 , 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 .

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-load rotating stage 100 is used to adjust the objective lens position of the microscope 200 according to the shooting requirements; the two-dimensional scanning system 400 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; 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.

It is understandable that the embodiment of the present invention can use the heavy-duty rotating stage 100 to control the direction of the objective lens of the microscope 200, realize upright, inverted and various angle shooting, excite the point light source of the target biological sample through the excitation light path 300, and perform a fast two-dimensional scan of the fluorescence emitted by the sample at the image plane to increase the spatial resolution. At the same time, the microlens array 500 is used to modulate the light beam of the point light source into a light field, which is then collected by the camera 600 as a fluorescence image, and based on the multiple scanned light field images scanned by the two-dimensional scanning system 400, 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 microscopic 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, which is more integrated and intelligent.

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

Furthermore, the heavy-duty rotating table 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 table 100 is perpendicular to the phase plane of the camera 600, so no matter at any angle, it will not affect the imaging of the camera.

In one embodiment of the present invention, the control system 700 is further used to control the two-dimensional scanning system 400 to scan to the next light field modulation position in the interval between image frames captured by the camera 600 .

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

In the actual execution process, the hardware program 710 is used to generate a timing voltage signal curve for controlling the camera and the two-dimensional scanning system 400 to meet the specific requirements of scanning the light field; the controller 720 is used to physically implement the logic of the hardware program 710 and output multi-channel analog and digital voltages; the connecting wire 730 is used to connect the two-dimensional scanning system 400 and the camera 600, and transmit the output voltage of the controller 720 to related devices for synchronous control. Its control process can be implemented on a hardware system such as an ordinary personal computer or workstation.

In one embodiment of the present invention, as shown in FIG. 2 , the excitation light 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 .

The laser light source 310 is used to output a divergent circular laser; the illumination tube lens 320 is used to converge the divergent circular laser into a point shape at the rear focus of the cylindrical lens. The dichroic mirror 210 is used to separate the point light source and the fluorescence emitted by the target biological sample; the objective lens 220 cooperates with the imaging tube lens 230 to magnify the target biological sample.

Furthermore, the positions of the objective lens 220, the illumination tube lens 320 and the dichroic mirror 210 are fixed, and 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 reach the target biological sample through the objective lens 220.

In one embodiment of the present invention, as shown in FIG. 2 , 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.

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

The positive and inverted integrated scanning light field microscopic imaging device proposed in the embodiment of the present invention combines light field microscopy and scanning technology, and realizes the shooting requirements of different samples and different angles by adding a heavy-duty rotating stage for adjusting the position of the microscope objective lens, making the three-dimensional fluorescence system more integrated and intelligent, thereby realizing positive and inverted integrated scanning light field microscopic imaging, and having the advantages of low cost, fast speed, and suitability for in vivo microscopic observation. Thus, the problems that the microscopic imaging system in the related art cannot meet the shooting requirements of different angles, and has high cost and low intelligence are solved.

Next, the positive and inverted integrated scanning light field microscopic imaging method proposed according to an embodiment of the present invention is described with reference to the accompanying drawings.

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.

As shown in FIG3 , the forward and inverted integrated scanning light field microscopy imaging method comprises the following steps:

In step S101 , based on the shooting requirements, the position of the objective lens of the microscope is adjusted using a heavy-duty rotating stage.

In step S102, a point light source that excites fluorescence of a target biological sample is outputted using an excitation light path.

In step S103, the fluorescence emitted by the target biological sample is two-dimensionally scanned at the image plane using a two-dimensional scanning system to obtain a scanned light field image.

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.

In step S105, the two-dimensional scanning system and the camera are synchronously controlled to realize positive 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.

It should be noted that the upright and inverted integrated scanning light field microscopy imaging method of the 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 aforementioned 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.

The positive and inverted integrated scanning light field microscopy imaging method proposed in the embodiment of the present invention combines light field microscopy and scanning technology, and realizes the shooting requirements of different samples and different angles by adding a heavy-duty rotating stage for adjusting the position of the microscope objective lens, making the three-dimensional fluorescence system more integrated and intelligent, thereby realizing positive and inverted integrated scanning light field microscopy imaging, and having the advantages of low cost, fast speed, and suitability for in vivo microscopic observation. Therefore, the problems that the microscopic imaging system in the related art cannot meet the shooting requirements of different angles, and has high cost and low intelligence are solved.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. 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 are not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or N embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "N" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

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 the steps of a custom logical function or process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may not be performed in the order shown or discussed, including performing functions in a substantially simultaneous manner or in reverse order depending on the functions involved, which should be understood by technicians in the technical field to which the embodiments of the present invention belong.

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

A person skilled in the art may understand that all or part of the steps in the method for implementing the above-mentioned embodiment may be completed by instructing related hardware through a program, and the program may be stored in a computer-readable storage medium, which, when executed, includes one or a combination of the steps of the method embodiment.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (7)

1. A positive and inverted integrated scanning light field microscopic imaging device, characterized in that it comprises: A microscope, wherein a target biological sample is placed on a stage of the microscope; An excitation light path, used to output a point light source for exciting 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, to achieve upright, inverted and various angle shooting, and to control the rotation of the excitation light path; A two-dimensional scanning system, used for performing two-dimensional scanning on the image plane of the fluorescence emitted by the target biological sample to obtain a scanning light field image; A microlens array, used for modulating the light beam of the point light source into a light field; a camera for generating a fluorescent image according to the light beam of the light field, wherein the heavy-duty rotating stage is perpendicular to the phase plane of the camera; A control system is used to synchronously control the two-dimensional scanning system and the camera to realize positive and inverted integrated scanning light field microscopy imaging, obtain the three-dimensional volume of the target biological sample based on the scanning light field image and the fluorescent image, and control the two-dimensional scanning system to scan to the next light field modulation position through a hardware program in the gap between the image frames collected by the camera. 2. The integrated forward and inverted scanning light field microscopic imaging device according to claim 1, characterized in that the microscope comprises: a dichroic mirror, used to separate the fluorescence emitted by the point light source and the target biological sample; The objective lens and the imaging tube lens cooperate with each other to magnify the target biological sample. 3. The forward and inverted integrated scanning light field microscopic imaging device according to claim 2, characterized in that the excitation light path comprises: A laser light source, used for outputting divergent circular laser; The lighting tube lens is used to converge the divergent circular laser into a point shape at the rear focus of the cylindrical lens. 4. The upright and inverted integrated scanning light field microscopic imaging device according to claim 3, characterized in that the positions of the objective lens, the illumination tube lens and the dichroic mirror are fixed. 5. The integrated forward and inverted scanning light field microscopic imaging device according to claim 1, characterized in that the two-dimensional scanning system comprises: A front-stage lens, used for converting the fluorescence emitted by the target biological sample from an image plane to a frequency domain plane; A driving board, used for driving the two-dimensional galvanometer to deflect to a target position according to a control voltage of the control system; A two-dimensional galvanometer is placed on the frequency domain plane to perform two-dimensional angular scanning of light; The subsequent lens is used to convert the light from the frequency domain plane to the image plane. 6. The upright and inverted integrated scanning light field microscopic imaging device according to claim 5, characterized in that the scanning step length of the two-dimensional galvanometer is smaller than the diameter of the microlens array. 7. A method for integrated forward and inverted scanning light field microscopy imaging, characterized in that the method uses the integrated forward and inverted scanning light field microscopy imaging device according to any one of claims 1 to 6 for imaging, wherein the method comprises the following steps: Based on the shooting requirements, use the heavy-duty rotating stage to adjust the microscope objective position; A point light source is outputted by using an excitation light path to excite the fluorescence of a target biological sample; Using a two-dimensional scanning system to perform two-dimensional scanning on the image plane of the fluorescence emitted by the target biological sample to obtain a scanning light field image; Modulating the light beam of the point light source into a light field through a microlens array, and generating a fluorescent image according to the light beam of the light field using a camera; The two-dimensional scanning system and the camera are synchronously controlled to realize positive and inverted integrated scanning light field microscopic imaging, and the three-dimensional volume of the target biological sample is acquired based on the scanning light field image and the fluorescent image.