A multi-channel light-sheet microscopic imaging device and its operation method

Abstract

This invention discloses a multi-channel light-slice microscopic imaging device. The image acquisition module includes multiple image detectors. A laser array emits multiple excitation beams of different wavelengths, which are then combined into a single incident beam by an optical path module. This incident beam passes through a scanning galvanometer and an imaging module before illuminating the sample on a stage mounted on a three-dimensional displacement platform. The imaging objective in the imaging module collects fluorescence signals from the sample, which are then split by the beam-splitting module to form multiple fluorescence signals of different wavelengths and projected onto the multiple image detectors. Each image detector forms an image imaging channel. Multiple image imaging channels receive multiple fluorescence signals of different wavelengths and transmit them to the corresponding image detectors. The three-dimensional displacement platform enables three-dimensional displacement movement of the sample slice. By employing a strategy of continuous motion imaging of the sample slice and simultaneous detection and acquisition of fluorescence signals by multiple image detectors, the time interval between sample preparation and imaging, as well as the pauses during imaging, can be reduced.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, specifically to a multi-channel light-sheet microscopic imaging device and its operating method. Background Technology

[0002] In modern microscopy, three-dimensional microscopy is rapidly developing. A key objective of three-dimensional microscopy is to rapidly and continuously image fluorescently labeled samples without sacrificing resolution.

[0003] Generally, 3D digital imaging detects (excites) a sample by generating a contrast response (such as fluorescence), records this contrast by photoelectric conversion and digitization, and uses the digitized contrast of each volumetric unit within the test area as a volume pixel as the final output. Therefore, the number of volume pixels—that is, the volume of the test area divided by the volume corresponding to the volume pixel (determined by the required resolution)—and the digitization speed determine an upper limit to the speed of 3D digital imaging. A strategy of simultaneous imaging with multiple image detectors can significantly shorten the imaging time for multicolor-labeled samples, thereby further increasing the upper limit of 3D digital imaging speed.

[0004] Taking existing light-sheet microscopy as an example, the existing paper, Wang, Hao, et al. "Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution." National Science Review 6.5 (2019):982-992, discloses that imaging the whole mouse brain using high-throughput three-dimensional fluorescence imaging (VISoR) with a single channel takes nearly 1.5 hours, with a resolution of 0.5μm × 0.5μm × 3.5μm. Compared to other imaging techniques, VISoR imaging technology has firmly established itself in the top tier in terms of imaging speed and resolution. Although this technology has achieved uninterrupted continuous moving imaging during microscopy, in order to study the distribution of different cells or genes in the brain, different markers are needed to specifically express each cell or gene, thus requiring multiple imaging sessions using a single channel, which takes a long time.

[0005] The aforementioned microscopic imaging technique involves numerous interruptions in the imaging process. After each sample imaging is completed, the filter and laser source must be changed before imaging can continue, resulting in prolonged imaging interruptions. When performing multiple rounds of laser irradiation and microscopic imaging at different wavelengths, the above operations and imaging must be repeated. This is extremely time-consuming for imaging multi-fluorescent labeled sample sections. Furthermore, when the sample volume is large, high resolution is required, and there are many labels, the imaging time will be even longer, severely impacting imaging efficiency. Summary of the Invention

[0006] The technical problem to be solved by this invention is: how to solve the problems of low imaging efficiency and long imaging time caused by single-channel imaging for microscopic imaging of multi-labeled samples.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] A multi-channel light-sheet microscopic imaging device includes a laser array, an optical path module, a scanning galvanometer, an imaging module, a beam splitting module, a three-dimensional displacement platform, and an image acquisition module. The image acquisition module includes multiple image detectors. The laser array emits multiple excitation beams of different wavelengths, which are then combined into a single incident beam by the optical path module. This incident beam passes through the scanning galvanometer and the imaging module and illuminates the sample on the stage, which is mounted on the three-dimensional displacement platform. The imaging objective in the imaging module collects fluorescence signals from the sample, which are then split by the beam splitting module to form multiple fluorescence signals of different wavelengths, which are projected onto the multiple image detectors.

[0009] This invention employs multiple image detectors, each forming an image imaging channel. These multiple imaging channels receive fluorescence signals of different wavelengths and transmit them to their respective detectors. This significantly improves imaging efficiency compared to existing technologies (where N is the number of image detectors). Simultaneously, a three-dimensional displacement platform stores sample slices and enables their three-dimensional displacement. Therefore, by using a strategy of continuous motion imaging of sample slices and simultaneous detection and acquisition of fluorescence signals by multiple image detectors, the time interval between sample preparation and imaging can be reduced, minimizing imaging pauses in three-dimensional microscopy. Compared to existing single-channel imaging techniques, this invention allows for simultaneous imaging of samples using multiple channels, increasing three-dimensional imaging throughput, improving imaging quality, increasing imaging speed, shortening imaging time, and enhancing imaging accuracy. It can be applied to three-dimensional imaging of multi-fluorescent labeled samples.

[0010] As a further aspect of the present invention: the laser group includes four lasers, namely laser one, laser two, laser three and laser four, which emit lasers of different wavelengths, and the four lasers are horizontally distributed at equal intervals on the working stage of the imaging device.

[0011] As a further aspect of the present invention: the optical path module is installed on the worktable of the imaging device and located on one side of the laser group; the optical path module includes a reflector one, a reflector two, a reflector three and a reflector four, wherein the reflector one, the reflector two, the reflector three and the reflector four correspond to the laser one, the laser two, the laser three and the laser four respectively.

[0012] As a further embodiment of the present invention: the optical path module further includes reflector five, reflector six, reflector seven, reflector eight, reflector ten, reflector eleven, reflector twelve, lens one, and lens two, wherein reflector five, reflector six, reflector seven, and reflector eight correspond to reflector one, reflector two, reflector three, and reflector four, respectively.

[0013] The optical path module also includes reflector nine, beam combiner one, beam combiner two, and beam combiner three, wherein reflector nine, beam combiner one, beam combiner two, and beam combiner three correspond to reflector five, reflector six, reflector seven, and reflector eight, respectively.

[0014] As a further embodiment of the present invention: the four reflected beams from the reflector nine, beam combiner one, beam combiner two, and beam combiner three are combined into one incident beam, which then passes sequentially through the reflector ten, reflector eleven, reflector twelfth, scanning galvanometer, lens one, and lens two before entering the equilateral triangular prism. The equilateral triangular prism reflects the received laser light onto the pentaprism one, and the pentaprism one reflects the received excitation light into the illumination objective lens. The equilateral triangular prism and the pentaprism one are mounted on the working stage of the imaging device.

[0015] As a further aspect of the present invention: the imaging module includes an illumination objective and an imaging objective disposed on the worktable of the imaging device, wherein the focal line of the illumination objective is perpendicular to the focal line of the imaging objective, and they are respectively placed at a 45° angle to the tabletop of the stage.

[0016] The illumination objective and the imaging objective are located directly above the stage. The illumination objective illuminates the sample in the stage with the received laser light, and the imaging objective reflects the collected fluorescence signal to the beam splitter through a pentaprism II, which is located at the end of the imaging objective.

[0017] As a further embodiment of the present invention: a precision translation stage is provided on the rear side of the illumination objective lens, wherein the precision translation stage is fixed to the end of the imaging device stage, and the drive shaft of the precision translation stage is detachably connected to the illumination objective lens through a connector.

[0018] As a further aspect of the present invention: the beam splitting module includes dichroic mirrors of different types, namely, dichroic mirror one, dichroic mirror two, and dichroic mirror three, and also includes filter one, filter two, filter three, and filter four, wherein the effective wavelength range of the dichroic mirrors and filters corresponds to the wavelength of the fluorescence signal generated by the sample being irradiated by the incident light beam.

[0019] As a further aspect of the present invention: the plurality of image detectors include image detector one, image detector two, image detector three and image detector four, wherein filter one, filter two, filter three and filter four are respectively located at the front end of the detector heads of image detector one, image detector two, image detector three and image detector four.

[0020] As a further aspect of the present invention: the first dichroic mirror divides the received fluorescence signal into two fluorescence signals that are perpendicular in direction and do not overlap in wavelength; the two divided fluorescence signals are then passed through the second and third dichroic mirrors to form two more fluorescence signals that are perpendicular in direction and do not overlap in wavelength; the fluorescence signals in the four directions are filtered by four filters and then acquired by the corresponding image detectors.

[0021] As a further aspect of the present invention, it also includes a control module, which synchronously controls the emission of multiple laser sources from the laser array, the movement of the scanning galvanometer, and the exposure of the image acquisition module;

[0022] The control module includes a motion control card, which outputs analog signals to control the amplitude and polarization of the scanning galvanometer vibration, and outputs digital signals to control the triggering of the multi-channel laser source of the laser array and the image acquisition module.

[0023] The present invention also provides an operating method for a multi-channel light-film microscopic imaging device, characterized by comprising the following steps:

[0024] S1. First, set the parameters of each component, including the laser group, optical path module, scanning galvanometer, imaging module, beam splitting module, and image acquisition module.

[0025] S2. Take a sample slice, place the sample slice on the stage, and determine the position information of the sample slice within the stage;

[0026] S3. Subsequently, the three-dimensional displacement platform carries the sample slice and starts moving from the starting position along the x-axis of the displacement platform. The control module generates a synchronization signal at a given frequency. Within one exposure time, multiple lasers are turned on at the set power level, and the scanning galvanometer begins to complete one scan at the given amplitude and bias.

[0027] S4. A staining mark in one plane of the sample slice is used to excite fluorescence. The excited fluorescence is split into four beams of different wavelengths after passing through three sets of dichroic mirrors.

[0028] S5. The four beams are captured by the image acquisition module after passing through the filter. The image acquisition module completes the exposure and stores the image data of this plane until the sample slice moves to the end position. The above operation is repeated to complete the multi-channel continuous imaging of the sample slice.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] I. This invention utilizes multiple image detectors, each forming an image imaging channel. These multiple imaging channels receive fluorescence signals from multiple different wavelengths and transmit them to the corresponding image detectors. During the imaging process, its imaging efficiency is increased by N times compared to existing technologies (where N is the number of image detectors). Simultaneously, a three-dimensional displacement platform stores the sample slices and enables their three-dimensional displacement. Therefore, by employing a strategy of continuous motion imaging of the sample slices and simultaneous detection and acquisition of fluorescence signals by multiple image detectors, the time interval between sample preparation and imaging can be reduced, minimizing imaging pauses in three-dimensional microscopy. Compared to existing single-channel imaging techniques, this invention can simultaneously image samples using multiple channels, increasing three-dimensional imaging throughput, improving imaging quality, increasing imaging speed, shortening imaging time, and improving imaging accuracy. It can be applied to three-dimensional imaging of multi-fluorescent labeled samples.

[0031] Second, by setting up multiple lasers, each with a different excitation wavelength, the present invention can collect excitation light of multiple wavelengths simultaneously. In contrast, traditional imaging devices need to interrupt and adjust to the next wavelength after collecting excitation light of one wavelength, and then repeat the above movement and imaging. The present invention does not need to repeat the above movement, and can collect excitation light of multiple wavelengths in one go, thereby improving imaging efficiency.

[0032] Third, this invention sets up multiple image detectors, which simultaneously acquire images under the reflection of multiple dichroic mirrors, resulting in a very high data transmission rate.

[0033] Fourth, by setting up multi-channel imaging, the number of times the sample is continuously moved is far less than that of existing single-channel imaging technology. This greatly reduces the displacement error generated by the three-dimensional displacement platform during continuous movement and avoids the problem of deviation at the same position in multiple single-channel imaging. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the structure of a multi-channel light sheet imaging micro-imaging device according to an embodiment of the present invention;

[0035] Figure 2 This is an optical path diagram of the laser array, optical path module, and scanning galvanometer according to an embodiment of the present invention;

[0036] Figure 3 This is a schematic diagram of the imaging module according to an embodiment of the present invention;

[0037] Figure 4 This is an embodiment of the present invention. Figure 1 Top view;

[0038] Figure 5This is a top view of another embodiment of the multi-channel light sheet microscopic imaging device of the present invention;

[0039] Figure 6 This is an embodiment of the present invention. Figure 5 Optical path diagram;

[0040] Figure 7 This is an embodiment of the present invention. Figure 4 Optical path diagram;

[0041] Figure 8 This is a schematic diagram illustrating the three-dimensional imaging principle of an embodiment of the present invention;

[0042] Figure 9 This is a schematic diagram illustrating the three-dimensional imaging principle during movement in an embodiment of the present invention.

[0043] Figure 10 This is a schematic diagram illustrating the principle of three-dimensional imaging during continuous movement according to an embodiment of the present invention.

[0044] Figure 11 This is a flowchart illustrating the operation of the microscopic imaging device according to an embodiment of the present invention;

[0045] Explanation of reference numerals in the attached diagram: 1. Image Detector 1; 2. Image Detector 2; 3. Image Detector 3; 4. Image Detector 4; 5. Dichroic Mirror 1; 6. Dichroic Mirror 2; 7. Dichroic Mirror 3; 8. Laser Group; 81. Laser 1; 82. Laser 2; 83. Laser 3; 84. Laser 4; 9. Optical Path Module; 91. Reflector 1; 92. Reflector 2; 93. Reflector 3; 94. Reflector 4; 95. Reflector 5; 96. Reflector 6; 97. Reflector 7; 98. Reflector 8; 99. Mirror 9; 910, Beam combiner 1; 911, Beam combiner 2; 912, Beam combiner 3; 913, Mirror 10; 914, Mirror 11; 915, Mirror 12; 916, Lens 1; 917, Lens 2; 10, Scanning galvanometer; 11, Three-dimensional displacement platform; 12, Stage; 13, Right triangular prism; 14, Pentagon prism 1; 15, Illumination objective; 16, Imaging objective; 17, Pentagon prism 2; 18, Precision translation stage; 19, Filter 1; 20, Filter 2; 21, Filter 3; 22, Filter 4. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] A multi-channel light-slide microscopy imaging device is disclosed, relating to the field of microscopy imaging technology, and more specifically to a device for multi-channel light-slide microscopy imaging of fluorescently labeled samples with biological and medical applications. This device is used in a four-channel three-dimensional microscopy imaging system. It should be noted that this invention can be extended to light-slide microscopy imaging with more channels. This invention only provides one implementation of a four-channel light-slide microscopy imaging system; the implementation can be adapted according to the actual situation.

[0048] See Figure 1 As shown, the four-channel microscopic imaging device includes image detector 1, image detector 2, image detector 3, image detector 4, dichroic mirror 1, dichroic mirror 2, dichroic mirror 3, laser group 8, optical path module 9, scanning galvanometer 10, three-dimensional displacement platform 11, stage 12, prism 13, pentaprism 14, pentaprism 2 17, illumination objective 15, imaging objective 16, precision translation stage 18, filter 19, filter 20, filter 3 21, filter 4 22, control module, and processing host. The control module synchronously controls the emission of multiple laser sources from the laser group 8, the movement of the scanning galvanometer 10, and the exposure of the image acquisition module. The core of this invention is multi-channel simultaneous imaging, specifically four-channel simultaneous imaging, to reduce unnecessary imaging interruptions. This involves a combination of technologies, including continuous imaging while the sample is moving continuously, reducing the time interval between sample preparation and imaging, minimizing the need to interrupt imaging for field-of-view transitions, and using four image detectors to simultaneously acquire fluorescent excitation light of different wavelengths.

[0049] It should be noted that the control module includes a motion control card. The motion control card outputs analog signals to control the amplitude and polarization of the scanning galvanometer 10. The motion control card outputs digital signals to control the triggering of the multi-channel laser source of the laser group 8 and the image acquisition module. The processing host controls the on / off state and laser power of different wavelength lasers in the multi-channel laser source, the movement speed and movement distance of the three-dimensional displacement platform 11 in each direction, the imaging area of ​​the image detector, the triggering mode and exposure time, and the analog and digital output signals of the control module. The image detector 1, image detector 2, image detector 3, and image detector 4 of this invention are used to receive fluorescence signals of different wavelengths to obtain image data and transmit the image data to the host for storage.

[0050] It should be noted that the image detector can use a Hamamatsu ORCA-Fusion digital CMOS camera, with a maximum acquisition range of 2304×2304 pixels, a pixel size of 6.5×6.5 micrometers, and a full-frame frame rate of 89Hz, or 11.2 milliseconds per frame.

[0051] Reference Figure 2The laser array 8 includes four lasers—laser 1 81, laser 2 82, laser 3 83, and laser 4 84—that emit lasers of different wavelengths. These four lasers are horizontally and equidistantly distributed on the imaging device's worktable. The optical path module 9 is also mounted on the imaging device's worktable and is located on one side of the laser array 8.

[0052] Reference Figure 2 The optical path module 9 includes reflectors 91, 92, 93, 94, 95, 96, 97, 98, and 99; beam combiners 910, 911, and 912; reflectors 911, 912, 913, 914, and 915; lens 916; and lens 917. Reflectors 91, 92, 93, and 94 are respectively connected to lasers 81, 82, 83, and 84. 83 corresponds to laser 4 and 84; that is, the excitation light emitted by laser 1, laser 2, laser 3, and laser 4 is received by the corresponding reflector 1 91, reflector 2 92, reflector 3 93, and reflector 4 94; reflector 5 95, reflector 6 96, reflector 7 97, and reflector 8 98 correspond to reflector 1 91, reflector 2 92, reflector 3 93, and reflector 4 94 respectively, that is, the excitation light emitted by reflector 1 91, reflector 2 92, reflector 3 93, and reflector 4 94 is received by the corresponding reflector 5 95. Reflectors 6 (96), 7 (97), and 8 (98) receive the light; reflectors 9 (99), 1 (910), 2 (911), and 3 (912) correspond to reflectors 5 (95), 6 (96), 7 (97), and 8 (98), respectively. That is, the excitation light emitted by reflectors 5 (95), 6 (96), 7 (97), and 8 (98) is received by the corresponding reflectors 9 (99), 1 (910), 2 (911), and 3 (912). Subsequently, reflectors 9 (99), 1 (910), 2 (911), and 3 (912) receive the light. The four reflected beams from mirror 3 912 are combined into one incident beam, which then passes sequentially through mirror 10 913, mirror 11 914, mirror 12 915, scanning galvanometer 10, lens 1 916, and lens 2 917 before entering prism 13. Prism 13 reflects the received laser light onto prism 14, which in turn reflects the received excitation light into the illuminator 15. Prism 13 and prism 14 are also located on the worktable of the imaging device, and scanning galvanometer 10, lens 1 916, and lens 2 917 are on the same horizontal line.

[0053] Reference Figure 1The scanning galvanometer 10 of this invention receives continuous triangular wave signals for synchronous scanning, ensuring one in-phase full-field scan within each imaging time. The output signal of the motion control card synchronously controls the exposure of the image detector and the triggering of the scanning galvanometer. Furthermore, the scanning galvanometer 10 of this invention uses the ST210 from Han's Sensor Technology, with a maximum scanning angle of 40°.

[0054] Reference Figure 1 The three-dimensional displacement platform 11 of this invention consists of a high-precision single-axis displacement platform manufactured by Aerotech. The X-axis model is ABL2000, and the Y and Z axes models are PRO165SL. It should be noted that the stage 12 of this invention is mounted on the three-dimensional displacement platform 11, and the stage 12 can be driven by the three-dimensional displacement platform 11, that is, the displacement adjustment of the stage 12 in three directions of the X-axis, Y-axis and Z-axis can be realized, thereby realizing the all-round position adjustment of the sample on the stage and ensuring the integrity of subsequent imaging.

[0055] Reference Figure 3 The focal line of the illumination objective 15 is perpendicular to the focal line of the imaging objective 16, and both are placed at a 45° angle to the tabletop of the stage 12. The illumination objective 15 and the imaging objective 16 are located directly above the stage 12. The illumination objective 15 illuminates the sample in the stage 12 with the received laser light, and the imaging objective 16 reflects the collected fluorescence signal to the beam splitter via a pentaprism 17, which is located at the end of the imaging objective 16. It should be noted that the illumination objective 15 is a TL20X-MPL from THROLABS, with a numerical aperture of 0.8 and a working distance of 3 mm; while the imaging objective 16 is an N16XLWD-PF from THROLABS, with a numerical aperture of 0.6 and a working distance of 5.5 mm.

[0056] Furthermore, refer to Figure 3 A precision translation stage 18 is also provided on the rear side of the illumination objective lens 15. The precision translation stage 18 is fixed to the end of the imaging device's worktable. The drive shaft of the precision translation stage 18 is detachably connected to the illumination objective lens 15 through a connector. The precision translation stage 18 is used to adjust the position of the illumination objective lens 15 so that the beam waist of the laser after being focused by the illumination objective lens 15 is in the middle of the image detector's field of view, thereby obtaining the thinnest light sheet. The precision translation stage 18 adopts the GCM-T13 precision translation stage from Daheng Optoelectronics, with a working stroke of ±6.5mm.

[0057] Reference Figure 5 and Figure 6 It also provides structural diagrams and optical path diagrams of a novel four-channel light sheet imaging microscopy device, including the imaging method, imaging apparatus, and... Figure 1 Similarly, the ray path diagram and Figure 7 resemblance.

[0058] Figure 8 , Figure 9 and Figure 10 The three-dimensional imaging principle of the four-channel imaging device is demonstrated. The optical path of the device (including the illumination optical path and the imaging optical path) and the movement of the sample are independent of each other. During imaging, the sample and the imaging system maintain continuous and uniform motion. The direction of motion is not in the imaging direction, nor is it perpendicular to the imaging direction. In this way, the scale of the sample movement range and direction is not limited by the imaging device. Image acquisition can be continuous for a long time and it can be applied to the imaging of large-sized samples. Figure 8 Examples are shown where the direction of motion forms a 45° angle with the imaging direction; such as Figure 9 and Figure 10 As shown, the sample is continuously imaged at new positions until a three-dimensional image of the sample is completed. It is evident that the sample movement direction is not perpendicular to the imaging direction, thus ensuring that adjacent images do not correspond to coplanar regions within the sample, and there is no overlap between adjacent images.

[0059] The specific operating principle of this invention is as follows:

[0060] Before operation, the parameters of each device are set, including the on / off state and power of the laser source; the amplitude and bias of the scanning galvanometer 10; the synchronization signal of the motion control card; and the image size information, image storage location, and exposure time of the four image detectors. Then, a sample slice is taken and placed on the stage 12, and its position within the stage 12 is determined. After determining the position of the sample slice within the stage, this parameter is imported, which represents the start and end positions of the displacement platform 11 during continuous movement. The movement speed of the displacement platform 11 is set, and then the three-dimensional displacement platform 11, carrying the sample slice, begins to move from the starting position along the x-axis of the displacement platform. The control module generates a synchronization signal at a given frequency. Within one exposure time, multiple sets of... The laser is turned on at the set power level, and the scanning galvanometer 10 begins to complete one scan with a given amplitude and bias. The incident laser is reflected by the scanning galvanometer 10 and focused by the lens and the illumination objective 15 before illuminating the sample slice. The staining mark in one plane of the sample slice is excited by the incident light to generate a fluorescence signal. The fluorescence signal is split into four beams of different wavelengths after passing through three sets of dichroic mirrors. The four beams are then captured by multiple image detectors after passing through specific filters. The image detectors complete the exposure and store the image data of this plane until the sample slice moves to the end position. After the X-axis movement is completed, the Y-axis and Z-axis movements continue, and the above operation is repeated to complete the multi-channel continuous imaging of the sample slice.

[0061] This invention can be applied to samples up to 300 micrometers thick, with resolution requirements at the micrometer or even sub-micrometer level. The specific sample thickness can be no greater than 300 micrometers. The fluorescence of the sample markers upon which imaging depends is provided by fluorescent microspheres excited by 405nm, 488nm, 561nm, and 640nm lasers. The stage 12 contains a refractive index matching solution, and the front ends of the illumination objective 15, the imaging objective 16, and the sample slice are all placed in pure water. The excitation light from the four lasers is selected from continuous lasers at 640nm, 561nm, 488nm, and 405nm, generated by continuous lasers 81, 82, 83, and 84, respectively.

[0062] It is important to note that samples can be selected from mammalian tissue sections (such as mice, monkeys, etc.). Cell nuclei are labeled with Hoechst 33342 staining agent (Hoechst 33342 can bind to nucleic acids) and can be excited by light at a wavelength of 405 nm. Neuronal cell bodies are labeled with NeuroTrace Nissl 500 / 525 green fluorescent staining agent and can be excited by light at a wavelength of 488 nm. Vascular endothelial cell immunostaining involves labeling with primary and secondary antibodies, respectively; the secondary antibody can be excited by light at a wavelength of 561 nm. Neutrophil immunostaining involves labeling with primary and secondary antibodies, respectively; the secondary antibody can be excited by light at a wavelength of 640 nm.

[0063] A 640nm continuous laser beam is transmitted through reflectors 1-91, 5-95, and 9-99 to reflector 10-913.

[0064] The 561nm continuous laser is transmitted through reflector 2 92 and reflector 6 96 to beam combiner 1 910 (LM01-613-25, Semrock), and beam combiner 1 910 reflects the 561nm laser to reflector 10 913.

[0065] The 488nm continuous laser is transmitted through reflector 3 93 and reflector 7 97 to beam combiner 2 911 (LM01-503-25, Semrock), and beam combiner 2 911 reflects the 488nm laser to reflector 10 913.

[0066] A 405nm continuous laser is transmitted through reflectors 494 and 898 to beam combiner 3912 (LM01-427-25, Semrock), and beam combiner 3912 reflects the 405nm laser to reflector 10913.

[0067] Continuous laser beams of 405nm, 488nm, 561nm, and 647nm are all incident on the same position on mirror 913. Mirror 913 reflects the laser beam to scanning mirror 10, which periodically vibrates to generate linear excitation light that is transmitted to lens 916. Lenses 916 and 917 are cemented doublet achromatic lenses, making the back focal plane of scanning mirror 10 conjugate with that of illumination objective 15. The linear laser beam is continuously reflected by prism 13 and pentaprism 14 and incident on the back focal plane of illumination objective 15. Illumination objective 15 focuses the incident laser beam onto the sample in stage 12, generating linear excitation light that scans in a plane perpendicular to the principal axis of imaging objective 16. The excitation beam is focused within the sample to excite fluorescence. The principal axes of the optical paths of illumination objective 15 and imaging objective 16 are perpendicular and form a 45° angle with the tabletop of stage 12. The stage 12 is horizontally fixed on the three-dimensional displacement platform 11, parallel to the horizontal plane. It is used to load sample slices and move the sample slices to the imaging position. During imaging, it drives the sample to move horizontally at a uniform speed and continuously rather than stepwise, thereby improving the sample imaging speed.

[0068] Reference Figure 6 and Figure 7 The excitation light generated by the sample is redirected by pentaprism 17 and then illuminates dichroic mirror 5 (specifically model T560lpxr, CHROMA). Fluorescence signals with wavelengths greater than 560nm pass through dichroic mirror 5 and illuminate dichroic mirror 6 (specifically model T647lpxr, CHROMA). Fluorescence signals with wavelengths greater than 647nm pass through and are then filtered by filter 20 (specifically model ET655lp, CHROMA) to extract the useful signal, which is then captured by image detector 2, obtaining the fluorescence information generated by the sample under 647nm laser irradiation. Fluorescence signals with wavelengths less than 647nm are reflected and then filtered by filter 19 (specifically model ET600 / 50m, CHROMA) to extract the useful signal, which is then captured by image detector 1, obtaining the fluorescence information generated by the sample under 561nm laser irradiation.

[0069] Fluorescence signals with wavelengths less than 560 nm are reflected by dichroic mirror 5 and illuminated by dichroic mirror 7 (specifically model T470lpxr, CHROMA). Fluorescence signals with wavelengths greater than 470 nm pass through and are then filtered by filter 21 (specifically model ET520 / 40m, CHROMA) to extract the useful signal, which is then captured by image detector 3 to obtain the fluorescence information generated by the sample under 488 nm laser irradiation. Fluorescence signals with wavelengths less than 470 nm are reflected and then filtered by filter 22 (specifically model ET455 / 50m, CHROMA) to extract the useful signal, which is then captured by image detector 4 to obtain the fluorescence information generated by the sample under 405 nm laser irradiation.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multi-channel light-film microscopic imaging device, characterized in that, It includes a laser array (8), an optical path module (9), a scanning galvanometer (10), an imaging module, a beam splitting module, a three-dimensional displacement platform (11), and an image acquisition module, wherein the image acquisition module includes multiple image detectors; The laser array (8) emits multiple excitation beams of different wavelengths, which are then combined into a single incident beam by the optical path module (9). After passing through the scanning galvanometer (10) and the imaging module, the incident beam illuminates the sample in the stage (12), which is mounted on a three-dimensional displacement platform (11). The imaging objective (16) in the imaging module collects the fluorescence signal in the sample, and then the fluorescence signal in multiple different bands is generated by the beam splitting module and projected into multiple image detectors; The staining mark in one plane of the sample slice is excited by incident light to generate a fluorescence signal. The fluorescence signal is split into four beams of different wavelengths after passing through three sets of dichroic mirrors. The four beams are then collected by multiple image detectors after passing through filter plates. The image detectors complete the exposure and store the image data of this plane until the sample slice moves to the endpoint position. The imaging module includes an illumination objective (15) and an imaging objective (16) mounted on the worktable of the imaging device. The focal line of the illumination objective (15) is perpendicular to the focal line of the imaging objective (16), and they are placed at a 45° angle to the table of the stage (12). The illumination objective (15) and the imaging objective (16) are located directly above the stage (12), and the illumination objective (15) illuminates the sample in the stage (12) with the received laser light, and the imaging objective (16) reflects the collected fluorescence signal to the beam splitter through the pentaprism (17), wherein the pentaprism (17) is located at the end of the imaging objective (16).

2. The multi-channel light-sheet microscopic imaging device according to claim 1, characterized in that: The laser group (8) includes four lasers, namely laser one (81), laser two (82), laser three (83) and laser four (84), which emit lasers of different wavelengths, and the four lasers are horizontally distributed at equal intervals on the worktable of the imaging device.

3. The multi-channel light-sheet microscopic imaging device according to claim 2, characterized in that: The optical path module (9) is installed on the worktable of the imaging device and is located on one side of the laser group (8). The optical path module (9) includes a first reflector (91), a second reflector (92), a third reflector (93) and a fourth reflector (94), wherein the first reflector (91), the second reflector (92), the third reflector (93) and the fourth reflector (94) correspond to the first laser (81), the second laser (82), the third laser (83) and the fourth laser (84) respectively.

4. The multi-channel light-sheet microscopic imaging device according to claim 3, characterized in that: The optical path module (9) also includes reflector five (95), reflector six (96), reflector seven (97), reflector eight (98), reflector ten (913), reflector eleven (914), reflector twelfth (915), lens one (916) and lens two (917), wherein reflector five (95), reflector six (96), reflector seven (97) and reflector eight (98) correspond to reflector one (91), reflector two (92), reflector three (93) and reflector four (94) respectively; The optical path module (9) also includes a reflector nine (99), a beam combiner one (910), a beam combiner two (911), and a beam combiner three (912), wherein the reflector nine (99), the beam combiner one (910), the beam combiner two (911), and the beam combiner three (912) correspond to the reflector five (95), the reflector six (96), the reflector seven (97), and the reflector eight (98), respectively.

5. The multi-channel light-sheet microscopic imaging device according to claim 4, characterized in that: The four reflected beams from the reflector nine (99), beam combiner one (910), beam combiner two (911), and beam combiner three (912) are combined into one incident beam, which then passes through the reflector ten (913), reflector eleven (914), reflector twelfth (915), scanning galvanometer (10), lens one (916), and lens two (917) in sequence before entering the prism (13). The prism (13) reflects the received excitation light onto the pentaprism one (14). The prism (13) and the pentaprism one (14) are located on the working table of the imaging device.

6. The multi-channel light-sheet microscopic imaging device according to claim 1, characterized in that: The rear side of the illumination objective (15) is also provided with a precision translation stage (18), wherein the precision translation stage (18) is fixed at the end of the imaging device stage, and the drive shaft of the precision translation stage (18) is detachably connected to the illumination objective (15) through a connector.

7. The multi-channel light-sheet microscopic imaging device according to claim 1, characterized in that: The beam splitting module includes dichroic mirrors of different models, namely, one (5), two (6), and three (7), as well as filters one (19), two (20), three (21), and four (22). The effective wavelength range of the dichroic mirrors and filters corresponds to the wavelength of the fluorescence signal generated by the sample being irradiated by the incident light beam.

8. The multi-channel light-sheet microscopic imaging device according to claim 7, characterized in that: The plurality of image detectors include image detector one (1), image detector two (2), image detector three (3) and image detector four (4), and filter one (19), filter two (20), filter three (21) and filter four (22) are respectively located at the front end of the detector heads of image detector one (1), image detector two (2), image detector three (3) and image detector four (4).

9. A multi-channel light-sheet microscopic imaging device according to claim 8, characterized in that: The first dichroic mirror (5) divides the received fluorescence signal into two fluorescence signals that are perpendicular in direction and do not overlap in wavelength. The two fluorescence signals after division are respectively passed through the second dichroic mirror (6) and the third dichroic mirror (7) to form two fluorescence signals that are perpendicular in direction and do not overlap in wavelength. The fluorescence signals in the four directions are filtered by the four filters and then collected by the corresponding image detectors.

10. A multi-channel light-sheet microscopic imaging device according to claim 1, characterized in that: It also includes a control module, which synchronously controls the emission of the multi-channel laser source of the laser group (8), the movement of the scanning galvanometer (10), and the exposure of the image acquisition module; The control module includes a motion control card, which outputs analog signals to control the amplitude and polarization of the scanning galvanometer (10) vibration, and outputs digital signals to control the triggering of the multi-channel laser source and image acquisition module of the laser group (8).

11. An operating method for a multi-channel light-sheet microscopic imaging device according to any one of claims 1-10, characterized in that, Includes the following steps: S1. First, set the parameters of each component, including the laser group, optical path module, scanning galvanometer, imaging module, beam splitting module, and image acquisition module. S2. Take a sample slice, place the sample slice on the stage, and determine the position information of the sample slice within the stage; S3. Subsequently, the three-dimensional displacement platform carries the sample slice and starts moving from the starting position along the x-axis of the displacement platform. The control module generates a synchronization signal at a given frequency. Within one exposure time, multiple lasers are turned on at the set power level, and the scanning galvanometer begins to complete one scan at the given amplitude and bias. S4. A staining mark in one plane of the sample slice is used to excite fluorescence. The excited fluorescence is split into four beams of different wavelengths after passing through three sets of dichroic mirrors. S5. The four beams are captured by multiple image detectors after passing through the filter. The image detectors complete the exposure and store the image data of this plane until the sample slice moves to the end position. After the X-axis movement ends, the Y-axis and Z-axis movement continues, and the above operation is repeated to complete the multi-channel continuous imaging of the sample slice.

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