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[ Instrument R&D of Instrument Network ] At 23 o'clock on August 10th, Nature Biotechnology published an online publication by the Chinese Academy of Sciences Brain Science and Intelligent Technology Excellence Innovation Center (Neuroscience Institute), Shanghai Brain Science and Brain-like Research Center, and National Key Laboratory of Neuroscience Researcher Wang Kai's research group completed a research paper entitled "Fast Body Imaging of Mouse and Zebrafish Brain by Confocal Light Field Microscope". The research developed a new type of volume imaging technology: Confocal light field microscopy, which can quickly and large-scale volume imaging of the nerve and blood vessel network in the deep brain tissue of living animals.
How large-scale neurons across brain regions integrate information and affect behavior is a core problem in neuroscience. Answering this question requires tools to capture the dynamic changes of a large number of neuronal activities at higher spatial and temporal resolution. Traditional tools used in in vivo brain imaging, such as confocal microscopy and two-photon microscopy, are based on point scanning, which has low time resolution and is difficult to study the rapid changes of neurons in a large area of ​​the brain. Therefore, in recent years, researchers have been committed to developing faster imaging methods. Among a variety of new technologies, light field microscopy has potential and has attracted wide attention. Its characteristic is that it can record signals from different depths of objects at the moment of a single exposure of the camera, and reconstruct the entire three-dimensional volume through the deconvolution algorithm. Rapid body imaging has been initially applied to small model animals such as nematodes and zebrafish juveniles.
Traditional light field microscopes have two difficult problems, which limit its application in biological imaging. First of all, the reconstructed result will be distorted. In 2017, the eXtended field-of-view Light Field Microscopy (XLFM) developed by Wang Kai’s research group solved this problem and applied it to the whole brain neurons of free-behaving zebrafish juveniles In functional imaging, the first three-dimensional record of zebrafish juveniles' changes in brain neuron activity during complete predation behavior. Second, the existing light field microscopy imaging technology lacks optical sectioning capabilities, and cannot image thick tissues, such as the brain of mice. Let the light field microscope have the same optical sectioning capability as the confocal microscope, and filter out the background signal except the focal layer in the large sample to improve the signal-to-noise ratio, which is the key to improving the image quality and being widely used.
However, the traditional confocal microscope uses laser point-by-point scanning and conjugate point pinhole detection to reduce the noise outside the focal plane, which is not suitable for three-dimensional light field microscopes. Facing this challenge, the research team innovatively proposed the concept of generalized confocal detection, which can be combined with the three-dimensional imaging strategy of light field microscopes to effectively filter out background noise without sacrificing volume imaging speed, and improve sensitivity and resolution. rate. This new type of light field microscopy imaging technology is called confocal light field microscope.
The research team tested the imaging capabilities of the confocal light field microscope on different animal samples. The team members performed whole brain calcium imaging on the embedded live zebrafish juveniles, and compared the imaging results of confocal and traditional light field microscopes, and found that with the addition of optical sectioning capabilities, the image resolution and signal-to-noise ratio improved, and more can be detected Weak calcium activity. Furthermore, the confocal light field microscope and high-speed three-dimensional tracking system are combined to perform whole brain calcium imaging of free-behaving zebrafish juveniles, achieving a spatial resolution of 2 x 2 x 2.5 μm3 in a volume of ø 800 μm x 200 μm Rate and 6Hz time resolution. Benefiting from higher resolution and sensitivity, it is possible to identify changes in the calcium ion activity of individual neurons in the process of preying on paramecium by zebrafish juveniles.
The team members verified the imaging effect of the confocal light field microscope on the brain of mice. Calcium imaging of the visual cortex of awake mice can simultaneously record the activity of nearly a thousand neurons in a volume of ø 800 μm x 150 μm. Up to about 400 μm, and stable recording of more than 100,000 frames for more than 5 hours without obvious photobleaching. The team members further tried to use a confocal light-field microscope to image blood cells in the mouse brain with a depth of up to 600 μm and a shooting speed of 70 Hz. At the same time, they recorded the flow of blood cells in thousands of blood vessel branches and calculated the speed of blood cells. Compared with the previous traditional imaging method, the throughput has increased by more than a hundred times.
The research team proved that confocal light field microscopes have higher resolution and sensitivity on free-behaving zebrafish juveniles and mouse brains, providing new tools for studying the functions of large-scale neural networks and vascular networks. At the same time, this technology is not only suitable for brain tissue imaging, but also can flexibly adjust the resolution, imaging range and speed according to the type of sample to be imaged, and can be applied to fast dynamic imaging of other thick tissues.
Under the guidance of Wang Kai, the research was mainly done by doctoral students Zhang Zhenkun, Bai Lu, and assistant researcher Conglin. Wang Kai's research group Yu Peng and Zhang Tianlei, University of Science and Technology of China Shi Wanzhuo, Du Jiulin's research group Li Funing contributed, and researcher Du Jiulin participated in the cooperation and gave guidance. The research was supported by the experimental animal platform of the Center for Excellence in Brain Intelligence, Chinese Academy of Sciences. The research work was funded by the Ministry of Science and Technology, the Chinese Academy of Sciences, the National Natural Science Foundation of China and Shanghai.