Dual-mode region-of-interest three-dimensional fluorescence imaging system and method based on light field and light sheet

Abstract

The invention discloses a dual-mode region-of-interest three-dimensional fluorescence imaging system and method based on a light field and a light sheet. The system has a light field imaging mode and a light sheet imaging mode, and the illumination light path comprises a light field illumination light path and a light sheet illumination light path which share an imaging objective lens; the light field illumination light path and the light sheet illumination light path are adjustably coupled, and during working, the relative range of an area of interest in an illumination light field is determined in a light field imaging mode, so that the illumination light sheet performs scanning imaging in the relative range. Through adjustable coupling of the light field illumination light path and the light sheet illumination light path, position information conversion of a light field imaging mode and a light sheet imaging mode is realized, and rapid three-dimensional fluorescence imaging is performed on a sample in the light field imaging mode to position a region of interest. High-resolution imaging is only carried out on the region of interest in the X-ray imaging mode through hardware coupling, X-ray scanning does not need to be carried out on the non-region of interest, and therefore the time cost of high-resolution three-dimensional fluorescence imaging of the region of interest is greatly saved.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-imaging, and more specifically, relates to a three-dimensional fluorescence imaging system and method for regions of interest based on light field and light sheet dual modes. Background Art

[0002] In the field of bio-fluorescence imaging, it is often necessary to search for key information in a vast amount of sample information for imaging. In previous experiments, regions of interest were mostly manually selected, and after determining the three-dimensional imaging range, imaging was carried out, or the entire sample or culture dish was imaged as a whole, and then available information was manually selected. The former requires a large amount of manpower and is inefficient, while the latter will collect a lot of regions of no interest, resulting in a waste of time and storage space.

[0003] Light Sheet Fluorescence Microscopy (LSFM), also known as Selective Plane Illumination Microscopy (SPIM) or selective illumination technology, is an advanced three-dimensional microscopy imaging technology. Its core design concept is to only excite the fluorescence signal of a specific focal plane in the sample through a thin light sheet, thereby reducing phototoxicity and achieving high-resolution in vivo imaging, but the imaging speed is slow.

[0004] The core principle of light field fluorescence microscopy is to record the direction and intensity information of incident light through a microlens array or coded aperture, synchronously capture multi-angle light of the sample, and reconstruct the three-dimensional spatial structure using computational algorithms. Different from the layer-by-layer scanning of light sheet fluorescence microscopy, it can obtain full-field three-dimensional data with a single exposure, significantly improving the imaging speed to the millisecond level and reducing photobleaching and phototoxicity. However, the spatial resolution of light field fluorescence microscopy is limited by the density of the sensor and microlenses, and is lower than that of light sheet fluorescence microscopy; especially the axial resolution is relatively low. Summary of the Invention

[0005] Aiming at the above defects or improvement requirements of the prior art, the present invention provides a three-dimensional fluorescence imaging system for regions of interest based on light field and light sheet dual modes. Its purpose is to couple the light field imaging and light sheet imaging through hardware of the imaging range, so that the light sheet imaging mode scans within a specified space range of the imaging range of the light field imaging mode, thereby realizing the hardware switching of the position information of the region of interest in the light field imaging mode and the light sheet imaging mode, greatly saving the time cost of obtaining high-resolution three-dimensional fluorescence images of regions of interest in the sample, and particularly suitable for the high-resolution three-dimensional fluorescence imaging requirements where the region of interest occupies a small volume of the sample, thus solving the technical problem that high-resolution three-dimensional fluorescence imaging of regions of interest currently requires collecting a large amount of data of non-interested regions, resulting in a waste of time cost.

[0006] To achieve the above object, according to one aspect of the present invention, a three-dimensional fluorescence imaging system for a region of interest based on a light field and a light sheet dual mode is provided, which has a light field imaging mode and a light sheet imaging mode. The illumination optical path includes a light field illumination optical path and a light sheet illumination optical path, and the two share an imaging objective lens;

[0007] The light field illumination optical path generates an illumination light field; the light sheet illumination optical path generates an illumination light sheet;

[0008] The light field illumination optical path and the light sheet illumination optical path are adjustable and coupled, that is, the Rayleigh range of the illumination light sheet generated by the light sheet illumination optical path is at a preset position of the illumination light field generated by the light field illumination optical path and the preset position is adjustable;

[0009] During operation, in the light field imaging mode, the relative range of the region of interest in the illumination light field is determined, and the illumination light sheet is scanned and imaged within this relative range.

[0010] Preferably, in the three-dimensional fluorescence imaging system for a region of interest based on a light field and a light sheet dual mode, the Rayleigh range of the illumination light sheet is within the preset depth of field range of the illumination light field, and the illumination light sheet scans laterally within the field of view of the illumination light field. In a preferred embodiment, the height of the illumination light sheet is adapted to the size of the region of interest of the sample.

[0011] Preferably, in the three-dimensional fluorescence imaging system for a region of interest based on a light field and a light sheet dual mode, the working bands of the light field imaging mode and the light sheet imaging mode are the same or different.

[0012] Preferably, in the three-dimensional fluorescence imaging system for a region of interest based on a light field and a light sheet dual mode, the imaging optical paths of the light field imaging mode and the light sheet imaging mode are coupled or decoupled in space.

[0013] Preferably, in the three-dimensional fluorescence imaging system for a region of interest based on a light field and a light sheet dual mode, the imaging optical paths of the light field imaging mode and the light sheet imaging mode are coupled in space, and the fluorescence collected by the imaging optical paths of the light field imaging mode and the light sheet imaging mode is imaged through the same camera;

[0014] It includes a scanning lens group, and the light sheet illumination optical path forms an illumination light sheet through the scanning lens group;

[0015] The fluorescence collected by the objective lens passes back through the scanning lens group and is imaged on the camera;

[0016] The scanning lens group includes a first and a second scanning lens, the principal optical axes of the first and the second scanning lens are orthogonal and confocal; the common focus of the first and the second scanning lens falls on the center of the galvanometer mirror.

[0017] Preferably, in the three-dimensional fluorescence imaging system based on light field and light sheet dual-mode region of interest, the light field imaging optical path and the light sheet imaging optical path operate during their respective working time periods, that is, when the light field imaging mode operates, only the illumination light field enters the rear pupil plane of the imaging objective; when the light sheet imaging mode operates, only the illumination light sheet enters the rear pupil plane of the imaging objective.

[0018] When the light field imaging optical path operates, a microlens array is arranged between the camera and the imaging lens, and the photosensitive surface of the camera is arranged at the focal plane of the microlens array; when the light sheet imaging optical path operates, the excited fluorescence is projected onto the photosensitive surface of the camera through the imaging lens for imaging, and the photosensitive surface of the camera is arranged at the focal plane of the imaging lens.

[0019] Preferably, in the three-dimensional fluorescence imaging system based on light field and light sheet dual-mode region of interest, the imaging optical paths of the light field imaging mode and the light sheet imaging mode are decoupled in space, and the fluorescence collected by the imaging optical paths of the light field imaging mode and the light sheet imaging mode is respectively collected by the camera for imaging.

[0020] A flip mirror is arranged on the rear pupil plane of the objective lens for selecting the fluorescence to enter the light field imaging optical path or the light sheet imaging optical path.

[0021] The light field illumination optical path and the light sheet illumination optical path are provided with a selector that is mechanically associated or signal-associated with the flip mirror for selecting the illumination light field generated by the light field illumination optical path or the illumination light sheet generated by the light sheet illumination optical path to enter the rear pupil plane of the objective lens.

[0022] According to another aspect of the present invention, a three-dimensional fluorescence imaging method based on light field and light sheet dual-mode region of interest is provided, which uses the three-dimensional fluorescence imaging system based on light field and light sheet dual-mode region of interest provided by the present invention for imaging.

[0023] Preferably, the three-dimensional fluorescence imaging method based on light field and light sheet dual-mode region of interest includes the following steps:

[0024] Image the sample in the light field imaging mode to obtain a three-dimensional light field image of the sample; divide the region of interest in the three-dimensional light field image, and determine the coordinate range within the field of view and the depth range within the depth of field in the light field mode.

[0025] In the light sheet imaging mode, scan the Rayleigh range of the illumination light sheet to cover the corresponding field of view coordinate range and depth range of the light field mode, image the sample and reconstruct it into a three-dimensional image of the region of interest.

[0026] Preferably, in the three-dimensional fluorescence imaging method based on light field and light sheet dual-mode region of interest, the specific method of dividing the region of interest in the three-dimensional light field image is as follows:

[0027] Input the three-dimensional image into the trained and converged region of interest recognition model to automatically recognize the region of interest.

[0028] Generally speaking, compared with the prior art, the above technical solution conceived by the present invention can achieve the following beneficial effects:

[0029] The three-dimensional fluorescence imaging system and method for the region of interest based on the light field and light sheet dual-mode provided by the present invention realize the conversion of the position information between the light field imaging mode and the light sheet imaging mode through the adjustable coupling of the light field illumination optical path and the light sheet illumination optical path. Perform fast three-dimensional fluorescence imaging on the sample in the light field imaging mode, locate the coordinate range of the region of interest, and convert the coordinate range of the region of interest into the position information in the light sheet imaging mode. Through hardware coupling, only the region of interest is imaged with high resolution in the light sheet imaging mode, and there is no need to perform light sheet scanning on the regions of no interest, thereby greatly saving the time cost of high-resolution three-dimensional fluorescence imaging of the region of interest.

[0030] The three-dimensional fluorescence imaging system for the region of interest provided by the present invention integrates two imaging modes of the light sheet and the light field. The advantages and disadvantages of the light field and the light sheet complement each other. The respective advantages of the two are taken, and the disadvantages of the two are discarded. Avoid the problem of poor light field resolution, use the light field as an extremely time-consuming sensor, input the image, and output the coordinate information. Also, because the two imaging optical paths share one objective lens and their coordinates are the same, the position information obtained by the light field can be directly used to switch to the light sheet imaging mode for imaging. In this way, the advantages of the fast speed of the light field are used to greatly simplify the search and positioning links that originally required a lot of time in the light sheet mode, and then the light sheet mode is used for high-resolution imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the optical path diagram of the light sheet imaging mode of the three-dimensional fluorescence imaging system for the region of interest based on the light field and light sheet dual-mode provided in Embodiment 1 of the present invention;

[0032] Figure 2 is the optical path diagram of the light field imaging mode of the three-dimensional fluorescence imaging system for the region of interest based on the light field and light sheet dual-mode provided in Embodiment 1 of the present invention;

[0033] Figure 3 is the schematic diagram of the process of three-dimensional fluorescence imaging of the region of interest by the three-dimensional fluorescence imaging system for the region of interest based on the light field and light sheet dual-mode provided in Embodiment 1 of the present invention;

[0034] Figure 4 is the optical path diagram of the light sheet imaging mode of the three-dimensional fluorescence imaging system for the region of interest based on the light field and light sheet dual-mode provided in Embodiment 2 of the present invention;

[0035] Figure 5It is the optical path diagram of the light sheet imaging mode of the three-dimensional fluorescence imaging system for regions of interest based on light field and light sheet dual modes provided in Embodiment 3 of the present invention;

[0036] Figure 6 It is the optical path diagram of the three-dimensional fluorescence imaging system for regions of interest based on light field and light sheet dual modes provided in Embodiment 4 of the present invention.

[0037] In all the drawings, the same reference numerals are used to represent the same elements or structures, where: 1 is an imaging objective lens, 2 is a filter color block, 3 is a mirror, 4 is a sleeve lens, 5 is an electrically controlled flip mirror, 6 is a first scanning lens, 7 is a one-dimensional galvanometer, 8 is a second scanning lens, 9 is a dichroic mirror, 10 is a camera, 11 is a slit, 12 is a cylindrical lens, 13 is a first doublet lens, 14 is a second doublet lens, 15 is a collimating lens, 16 is a laser, 17 is a microlens array, 18 is an LED, 19 is a first correction objective lens, 20 is a second correction objective lens, and 21 is a second dichroic mirror. Detailed implementation manners

[0038] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0039] In order to shorten the imaging time of the region of interest and improve the imaging quality, the strategy of the present invention is to identify the region of interest through high-speed three-dimensional imaging with low resolution and large range, and then perform high-resolution fine scanning imaging on the region of interest, thereby saving the overall imaging time, extracting the information of the region of interest, and reducing the time and storage space consumed by imaging the regions of no interest.

[0040] The present invention utilizes the imaging characteristics of high speed of light field three-dimensional imaging and high resolution of light sheet fluorescence imaging. Based on the light field and light sheet dual modes, it quickly identifies the region of interest for high-precision imaging, and provides a three-dimensional fluorescence imaging system for regions of interest based on light field and light sheet dual modes, which has a light field imaging mode and a light sheet imaging mode. The imaging ranges of the light field imaging mode and the light sheet imaging mode are coupled, that is, the illumination light sheet in the light sheet imaging mode can be scanned controllably within the field of view range and depth of field range of the light field imaging, and is achieved through the following methods:

[0041] In the light field mode package, the light field illumination optical path and the light field imaging optical path are in working states; the light field illumination optical path is used to generate an illumination light field, and the illumination light field falls on the rear pupil surface of the imaging objective lens to illuminate and excite the sample to generate fluorescence; the fluorescence generated by the excitation of the sample is collected by the imaging objective lens and imaged on the camera through the light field imaging optical path to obtain a light field three-dimensional image;

[0042] In the light sheet mode, the light sheet illumination optical path and the light sheet imaging optical path are in the working state. The light sheet illumination optical path is used to generate an illumination light sheet. The illumination light sheet passes through the scanning mechanism and forms a light sheet illumination through the pupil plane of the imaging objective to excite the fluorescence of the sample. The fluorescence generated by the sample is collected by the imaging objective and imaged on the camera through the light sheet imaging optical path to obtain a three-dimensional light sheet image.

[0043] The light field illumination optical path and the light sheet illumination optical path are adjustable and coupled, that is, the Rayleigh range of the illumination light sheet generated by the light sheet illumination optical path is at a preset position of the illumination light field generated by the light field illumination optical path and the preset position is adjustable, so as to realize the coupling of the imaging ranges of the light field imaging mode and the light sheet imaging mode. More easily achieved, the Rayleigh range of the illumination light sheet is within the preset depth of field range of the illumination light field, and the illumination light sheet scans laterally within the field of view of the illumination light field. In a preferred embodiment, the height of the illumination light sheet is adapted to the size of the region of interest of the sample. Specifically, the sample plane is the XY plane, the principal optical axis is the Z axis, the scanning direction is the X axis, and the height of the light sheet on the Y axis is adjustable, for example, by shaping the light source to adjust the height of the light sheet. When the height of the light sheet is not adjustable, high-quality imaging of the region of interest can also be achieved by cropping the imaging area.

[0044] The coupling of the light field illumination optical path and the light sheet illumination optical path ensures the rapid and accurate communication of the position information of the region of interest between the light field mode and the light sheet mode, without the need to perform conversion between the multi-modal imaging ranges through spatial coordinates, and realizes the rapid and accurate switching of the high-resolution region of interest.

[0045] During operation, in the light field imaging mode, the relative range of the region of interest in the illumination light field is determined, and the illumination light sheet scans and images within this relative range.

[0046] For the light field mode and the light sheet mode, illumination light of the same wavelength can be used for imaging with the same wavelength. In the case of imaging with the same wavelength, usually the light field mode and the light sheet mode need to work alternately, and the imaging optical paths of the light field imaging mode and the light sheet imaging mode are coupled or decoupled in space. The imaging optical paths are coupled and multiplexed to reduce the volume of the dual-mode microscope, and the multiplexing of the camera effectively reduces the cost of the dual-mode microscope.

[0047] The imaging optical paths of its light field imaging mode and light sheet imaging mode are spatially coupled, that is, the imaging optical paths of the light field imaging mode and the light sheet imaging mode collect fluorescence and are imaged by the same camera; the light sheet illumination scanning mechanism uses a scanning lens group, and the light sheet illumination optical path forms an illumination light sheet through the scanning lens group; the fluorescence collected by the objective lens passes back through the scanning lens group and is imaged on the camera; that is, the scanning - anti - scanning mechanism. The scanning lens group includes a first and a second scanning lens, the principal optical axes of the first and second scanning lenses are orthogonal and confocal; the common focus of the first and second scanning lenses falls on the center of the galvanometer mirror.

[0048] The application of the scanning - anti - scanning mechanism makes the image of each frame fall on the same position of the camera. When the light sheet imaging mode is working, the area illuminated by the light sheet projects as a long and narrow rectangle on the camera. When the coordinate range of the region of interest is anisotropic, using fine selective illumination light sheet scanning of the region of interest can further avoid the regions of no interest, thereby shortening the time for three - dimensional light sheet fluorescence imaging of the region of interest.

[0049] At the same time, in the scanning - anti - scanning mode, only dozens of rows of pixels in the center of the camera are opened for exposure to complete image acquisition, which greatly improves the acquisition speed of the light sheet mode. Moreover, a single image contains different depth information, and an image stack can be obtained after single - layer reconstruction, further improving the imaging throughput of the light sheet imaging mode.

[0050] The light field imaging optical path and the light sheet imaging optical path work in their respective working time periods, that is, when the light field imaging mode is working, only the illumination light field enters the rear pupil plane of the imaging objective lens; when the light sheet imaging mode is working, only the illumination light sheet enters the rear pupil plane of the imaging objective lens.

[0051] When the light field imaging optical path is working, a microlens array is arranged between the camera and the imaging lens, and the photosensitive surface of the camera is arranged on the focal plane of the microlens array; when the light sheet imaging optical path is working, the excited fluorescence is projected onto the photosensitive surface of the camera through the imaging lens for imaging, and the photosensitive surface of the camera is arranged on the focal plane of the imaging lens.

[0052] In the case where the imaging optical paths of the light field imaging mode and the light sheet imaging mode are spatially decoupled, the imaging optical paths of the light field imaging mode and the light sheet imaging mode collect fluorescence and are respectively collected and imaged by the camera; in some embodiments, a flip mirror is provided on the rear pupil plane of the objective lens for selecting fluorescence to enter the light field imaging optical path or the light sheet imaging optical path; the light field illumination optical path and the light sheet illumination optical path have a selector mechanically or signal - associated with the flip mirror for selecting the illumination light field generated by the light field illumination optical path or the illumination light sheet generated by the light sheet illumination optical path to enter the rear pupil plane of the objective lens.

[0053] The working bands of the light field imaging mode and the light sheet imaging mode are the same, which can accurately determine the region of interest, and thus perform high-resolution light sheet scanning imaging on it precisely. The working bands of the light field imaging mode and the light sheet imaging mode can also adopt different wavelengths for dual-wavelength imaging; when performing dual-wavelength imaging, the excited fluorescence can also be imaged separately by wavelength, so as to achieve simultaneous imaging. Continuously observe the region of interest in the light field mode, and perform high-resolution three-dimensional scanning imaging of the light sheet in real time, eliminating the imaging time difference between the light field imaging mode and the light sheet imaging mode. For in vivo sample imaging, it can accurately capture the biological processes carried out by the in vivo sample and determine its biological state.

[0054] The three-dimensional fluorescence imaging method for the region of interest based on the light field and light sheet dual-mode provided by the present invention uses the three-dimensional fluorescence imaging system for the region of interest based on the light field and light sheet dual-mode to perform imaging, including the following steps:

[0055] Image the sample in the light field imaging mode to obtain a three-dimensional light field image of the sample; divide the region of interest in the three-dimensional light field image, and determine the coordinate range within the field of view in the light field mode and the depth range within the depth of field of the region of interest;

[0056] In the light sheet imaging mode, make the Rayleigh range of the illumination light sheet scan to cover the corresponding field of view coordinate range and depth range of the light field mode, image the sample and reconstruct it into a three-dimensional image of the region of interest.

[0057] Dividing the region of interest in the three-dimensional light field image can be manually marked or automatically marked. The automatic marking is specifically:

[0058] Input the three-dimensional image into a trained and converged region of interest recognition model to automatically identify the region of interest.

[0059] The following are examples:

[0060] Example 1

[0061] The three-dimensional fluorescence imaging system for the region of interest based on the light field and light sheet dual-mode provided in this example has a light field imaging mode and a light sheet imaging mode:

[0062] In the light sheet imaging mode, as Figure 1As shown in the figure, the light emitted by the laser 16 that forms the illumination beam first passes through the collimating objective lens 15 for collimation, and then passes through the second doublet lens 14 with f = 50 and the first doublet lens 13 with f = 125. The beam diameter is enlarged by 2.5 times. After passing through the cylindrical lens 12, the light in one dimension is compressed, and the light in the other perpendicular dimension remains parallel. The combination of the two dimensions forms a light sheet. A rectangular slit 11 is placed at the waist of the light sheet, allowing only some marginal light rays to pass through. The purpose is to form an inclined illumination light sheet behind the imaging objective lens 1. Then the light passes through the sleeve lens 4 with f = 180, is reflected by the dichroic mirror 9, and passes through the scanning lens group for lateral scanning composed of the first scanning lens 6, the one-dimensional galvanometer 7, and the second scanning lens 8. After being reflected by the electronically controlled flip mirror 5, it enters the frame. Inside the frame, it passes through the mirror 3, the sleeve lens 4 with f = 180, the mirror 3, and the filter color block 2 (containing a dichroic mirror with 405, 488, 561, and 637 four-way communication) to reach the imaging objective lens 1, forming inclined light sheet illumination behind the imaging objective lens 1. On the detection path, the fluorescence emitted by the sample is collected by the imaging objective lens 1, passes through a part of the common optical path for illumination and detection to the dichroic mirror 9, passes through the dichroic mirror 9, and then reaches the camera 10 for detection and imaging via the sleeve lens 4 with f = 180, the mirror 3, and the sleeve lens 4.

[0063] In the light field imaging mode, as Figure 2 shown, the illumination light is emitted by the LED 18, passes through the filter color block 2 after reflection, and forms wide-field illumination through the objective lens. The fluorescence emitted by the sample is collected by the imaging objective lens 1, passes through the filter color block 2, passes through the mirror 3, the sleeve lens 4 with f = 180, and the mirror 3 and then exits the frame. At this time, the electronically controlled flip mirror 5 flips out of the optical path, and the detection light continues to pass through the optical relay system composed of three mirrors 3 and four sleeve lenses 4 with f = 180 and then reaches the microlens array 17, and then is detected and imaged by the camera 10.

[0064] The switching between the light field imaging mode and the light sheet imaging mode is achieved by controlling the electronically controlled flip mirror 5. It flips into the optical path in the light sheet imaging mode and flips out of the optical path in the light field imaging mode. At the same time, the coupling of the light field imaging mode and the light sheet imaging mode is realized: the position of the electronically controlled flip mirror 5 is determined by the imaging optical path in the light field imaging mode, so that when the electronically controlled flip mirror 5 flips in, it is exactly in the light field imaging optical path. The common section of the light sheet illumination optical path and the light sheet imaging optical path is calibrated with the flip mirror. The illumination light sheet falls on the corresponding area of the light field imaging optical path on the flip mirror, and the scanning range of the light sheet is calibrated with this area, so that the Rayleigh range of the illumination light sheet generated by the light sheet illumination optical path is at a preset position of the illumination light field generated by the light field illumination optical path and the preset position is adjustable, realizing the imaging range coupling of the light field imaging mode and the light sheet imaging mode. The Rayleigh range of the illumination light sheet is within the preset depth of field range of the illumination light field, and the illumination light sheet scans laterally within the field of view of the illumination light field.

[0065] During the actual imaging process, such as Figure 3 shown, first, the information within the field of view is observed in real-time in the light field imaging mode. Then, the trained target recognition network is used to identify specific biological events and output the spatial position information of the event occurrence area, including the lateral xy area position and the z-direction range. Subsequently, the light sheet imaging mode is switched, and through the coupling relationship between the light sheet imaging mode and the light field imaging mode, the spatial position information in the light field mode is converted into the scanning area in the light sheet imaging mode, and high-resolution imaging is performed on the event occurrence area.

[0066] Embodiment 2

[0067] The three-dimensional fluorescence imaging system for regions of interest based on the dual-mode of light field and light sheet provided in this embodiment has a light field imaging mode and a light sheet imaging mode:

[0068] In the light sheet imaging mode, as Figure 4 shown, the illumination optical path is the same as that in Embodiment 1. On the detection path, the fluorescence emitted by the sample is collected by the objective lens and then passes through a part of the optical path shared by illumination and detection to the dichroic mirror 9. After passing through the dichroic mirror 9, it is imaged in space via the sleeve lens 4 with f = 180 and the first correction objective lens 19. Then, the second correction objective lens 20 performs oblique plane correction secondary detection on the image formed by the first correction objective lens 19, and then reaches the camera for imaging through the sleeve lens 4. The use of oblique plane correction secondary detection to achieve single-objective light sheet imaging can be adapted to other light sheet scanning mechanisms to replace the scanning lens group composed of the first scanning lens 6, the one-dimensional galvanometer 7, and the second scanning lens 8.

[0069] The light field imaging mode is the same as that in Embodiment 1.

[0070] Coupling of the same-band illumination optical path in Embodiment 3

[0071] The three-dimensional fluorescence imaging system for regions of interest based on the dual-mode of light field and light sheet provided in this embodiment, as Figure 5 shown, has a light field imaging mode and a light sheet imaging mode:

[0072] The illumination optical path and the detection optical path in the light sheet imaging mode are the same as those in Embodiment 1

[0073] The illumination optical path in the light field imaging mode is the same as that in Embodiment 1, and the detection optical path is shared with the detection optical path in the light sheet mode. However, an electrically controlled switching device is set on the microlens array 17 (switched into the optical path in the light field imaging mode and switched out of the optical path in the light sheet mode). At the same time, an electric displacement stage is set below the camera to match the image plane positions of the light sheet mode and the light field imaging mode. This embodiment will slightly increase the modal switching time, but the dual-mode shares the imaging optical path, significantly reducing the hardware cost.

[0074] Dual-wavelength imaging in Embodiment 4

[0075] The three-dimensional fluorescence imaging system for regions of interest based on light field and light sheet dual modes provided in this embodiment, as Figure 6 shown, has a light field imaging mode and a light sheet imaging mode:

[0076] The difference from Embodiment 1 is that the flip mirror 5 in Embodiment 1 is replaced by a second dichroic mirror 21, so that when the light sheet and the light field are excited by lasers of different wavelengths, simultaneous imaging can be achieved. The time for mode switching is eliminated, but the sample is labeled with dual-band fluorescence.

[0077] During the specific imaging process, the state of the sample can be observed simultaneously in a dual-channel and dual-mode manner. Sometimes, multiple channels are labeled in biological imaging, and one of the channels is labeled with an event indicator, that is, when fluorescence appears in this channel, it represents the occurrence of an event of interest, and the other channel contains information to be observed. The channel of the event indicator is observed in the light field mode to clearly determine the position of the region of interest, and then the light sheet mode is used to perform high-resolution imaging on the channel to be observed.

[0078] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A three-dimensional fluorescence imaging system based on a light field light sheet dual-mode region of interest, characterized in that: It has a light field imaging mode and a light sheet imaging mode, and the illumination optical path includes a light field illumination optical path and a light sheet illumination optical path, and the two share an imaging objective lens; The light field illumination light path generates an illumination light field; the light sheet illumination light path generates an illumination light sheet; The light field illumination optical path is adjustably coupled to the light sheet illumination optical path, that is, the Rayleigh range of the illumination light sheet generated by the light sheet illumination optical path is at a preset position of the illumination light field generated by the light field illumination optical path and the preset position is adjustable; During operation, the relative range of the region of interest in the illumination light field is determined in the light field imaging mode, so that the illumination light sheet is scanned and imaged within the relative range.

2. The light field light sheet dual-mode region of interest three-dimensional fluorescence imaging system according to claim 1, characterized in that: The Rayleigh range of the illumination light sheet is within the preset depth of field range of the illumination light field, and the illumination light sheet is scanned horizontally within the field of view of the illumination light field. In a preferred solution, the height of the illumination light sheet is adapted to the size of the sample region of interest.

3. The light field light sheet dual-mode region of interest three-dimensional fluorescence imaging system according to claim 1, characterized in that: The light field imaging mode and the light sheet imaging mode have the same or different working bands.

4. The light field light sheet dual-mode region of interest three-dimensional fluorescence imaging system according to claim 1, characterized in that: The imaging optical paths of its light field imaging mode and light sheet imaging mode are spatially coupled or decoupled.

5. The light field light sheet dual-mode region of interest three-dimensional fluorescence imaging system according to claim 4, characterized in that: The imaging optical paths of the light field imaging mode and the light sheet imaging mode are spatially coupled, and the imaging optical paths of the light field imaging mode and the light sheet imaging mode collect fluorescence through the same camera for imaging; It includes a scanning lens group, and the light sheet illumination light path passes through the scanning lens group to form an illumination light sheet; The fluorescence collected by the objective lens passes through the scanning lens group in reverse and forms an image on the camera; The scanning lens group includes a first scanning lens and a second scanning lens. The main optical axes of the first scanning lens and the second scanning lens are orthogonal and confocal. The common focus of the first scanning lens and the second scanning lens falls on the center of the galvanometer.

6. The light field light sheet dual-mode region of interest three-dimensional fluorescence imaging system according to claim 5, characterized in that: The light field imaging optical path and the light sheet imaging optical path work in their respective working time periods, that is, when the light field imaging mode works, only the illumination light field enters the rear pupil plane of the imaging objective lens; when the light sheet imaging mode works, only the illumination light sheet enters the rear pupil plane of the imaging objective lens; When the light field imaging optical path is working, a microlens array is arranged between the camera and the imaging lens, and the photosensitive surface of the camera is arranged at the focal plane of the microlens array; when the light sheet imaging optical path is working, the excited fluorescence is projected onto the photosensitive surface of the camera through the imaging lens to form an image, and the photosensitive surface of the camera is arranged at the focal plane of the imaging lens.

7. The light field light sheet dual-mode region of interest three-dimensional fluorescence imaging system according to claim 4, characterized in that: The imaging optical paths of the light field imaging mode and the light sheet imaging mode are spatially decoupled, and the fluorescence collected by the imaging optical paths of the light field imaging mode and the light sheet imaging mode are respectively collected and imaged by cameras; A flip mirror is provided on the rear pupil surface of the objective lens to select the fluorescence to enter the light field imaging optical path or the light sheet imaging optical path; The light field illumination optical path and the light sheet illumination optical path have a selector mechanically or signal-related to the flip mirror, which is used to select the illumination light field generated by the light field illumination optical path or the illumination light sheet generated by the light sheet illumination optical path to enter the rear pupil plane of the objective lens.

8. A method for three-dimensional fluorescence imaging of a region of interest based on a light field light sheet dual-mode, characterized in that: Imaging is performed using the light field light sheet dual-mode region of interest three-dimensional fluorescence imaging system as described in any one of claims 1 to 7.

9. The method for three-dimensional fluorescence imaging of a region of interest based on a light field light sheet dual mode as claimed in claim 8, characterized in that: The following steps are involved: Imaging the sample in a light field imaging mode to obtain a three-dimensional light field image of the sample; dividing a region of interest in the three-dimensional light field image to determine a coordinate range of the region of interest within a field of view in the light field mode and a depth range within a depth of field; In the light sheet imaging mode, the Rayleigh range of the illumination light sheet is scanned to cover the corresponding field of view coordinate range and depth range of the light field mode, and the sample is imaged and reconstructed into a three-dimensional image of the region of interest.

10. The method for three-dimensional fluorescence imaging of a region of interest based on a light field light sheet dual-mode as claimed in claim 8, characterized in that: The division of the region of interest in the three-dimensional light field image is specifically as follows: The three-dimensional image is input into a trained and converged region of interest recognition model to automatically recognize the region of interest.

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