According to the website of the Chinese Academy of Sciences, the National Key Laboratory of Transient Optics and Photonics of the Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, recently developed a two-photon excitation laser scanning real-time stereo microscope. The stereoscopic microscopy method based on binocular vision and high-resolution two-photon excitation laser scanning fluorescence microscopy combine to achieve high-speed stereo imaging of three-dimensional fluorescence samples. The relevant research results were published in the December 2016 issue of PLOS ONE Magazine. And is authorized to national invention patent (patent number ZL201210384895.4).
Contemporary life science research has placed increasing demands on optical microscopy technology - higher spatial resolution, greater imaging depth, and faster imaging speed. Especially for microscopic imaging of living organisms, the scattering of light by biological tissues greatly increases the noise, which seriously affects the spatial resolution and imaging depth. In order to improve the imaging depth, two-photon excitation laser scanning fluorescence microscopy has been widely used in neuroimaging and other fields since its introduction in the 1990s, but its point-by-point imaging method severely restricts imaging speed. Because the depth of field of a high-resolution optical microscope is very small, it is usually necessary to superimpose and reconstruct a two-dimensional image of dozens or even hundreds of layers for three-dimensional imaging of a sample. Image acquisition and processing generally takes several minutes or even tens of minutes. It is very difficult to acquire and display 3D images in real time in real time.
Under the leadership of researchers Yao Baoli and Ye Wei, the transient room super-resolution imaging team proposed a laser beam stereo scanning device consisting of four galvanometers based on the binocular vision principle and the Bessel beam-generated extended focal field. The three-dimensional control of the lateral position and inclination of the Bessel beam is realized, which breaks through the limitation that the conventional laser scanning with only two degrees of freedom cannot switch the angle of view in real time. Through the optimized design and control of the four-magnet mirror stereo scanning device, the three-degree-of-freedom fast scanning of the Bessel beam can be realized, and the dual-view switching can be performed in the order of milliseconds, thereby solving the double in the laser scanning stereomicroscopic imaging system. For the first time, the stereoscopic microscopic imaging and display system based on dual-view real-time laser scanning is realized. The system can perform stereoscopic dynamic imaging and real-time binocular stereoscopic observation of samples, and its three-dimensional imaging speed is one to two orders of magnitude higher than the traditional point-by-point scanning method. The two-photon stereomicroscopy system provides a new observation tool for 3D real-time imaging and display of living organisms.
“It allows us to observe the dynamic 3D microscopic world in real time like a stereoscopic movie, without the need for light slicing, without the need for time-consuming 3D image reconstruction.†Yang Yanlong summed up the characteristics of the system, he was responsible for the design and completion of the three-dimensional The key parts of the scanning and imaging display. "Binocular vision imaging is a very efficient way to acquire 3D information, but with existing stereo microscopes, spatial resolution and depth of field are mutually constrained. We use the three-degree-of-freedom scanning Bessel beam for nonlinear fluorescence excitation to break through this. limit."
This research has been supported by the "Hundred Talents Program" of the Chinese Academy of Sciences and the National Natural Science Foundation. From basic principle verification, key technology breakthroughs, to the completion of the prototype, it has gone through all aspects from basic research to application integration. At present, the research group is conducting collaborative research on biomedical applications with relevant research institutions at home and abroad, and it is expected that this technology will be applied to the three-dimensional rapid imaging and display of living organisms as soon as possible.
Red and blue stereo images of pollen and fluorescent beads samples (viewable with red and blue glasses)
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