高通量神经元功能活动在体光场成像技术

With the development of advanced optical imaging methods and fluorescent indicators that are sensitive to neural activities, it is now possible to do in vivo functional monitoring of the exact same group of neurons over extended period of time at subcellular resolution, and with molecular specificity. The currently representative functional optical imaging methods, such as two photon excitation microscope, is based on serial point scanning mechanism and therefore limited in imaging speed, which makes it hardly applicable on studies of large scale neural network dynamics. Light Field Microscopy (LFM) is a special type of imaging method that does not rely on any scanning mechanism and can acquire 3D volume images with one camera exposure. It can potentially increase the speed of functional imaging significantly. Particularly, we recently developed a new type of light field microscope, termed eXtended field-of-view Light Field Microscopy (XLFM), which overcame many drawbacks in conventional LFM and demonstrated high throughput functional imaging of neuronal activities in the whole brain of larval zebrafish. This technology, however, can not be readily applied to in vivo functional imaging in mouse brain due to its vulnerability to optical scattering in deep tissue. Therefore, we will develop new methods to extend the imaging depth of XLFM and achieve high throughput functional imaging deep in scattering tissue. This new technology will help to gain more information for studies on neural population coding and neural network dynamics at both large scale and high speed, and possibly open new windows for neuroscience researches in general.

通过结合对神经元活动敏感的荧光探针技术，光学成像方法可以对具有细胞特异性的群体神经元进行在体、亚细胞分辨率、长时程的功能活动记录。目前，以双光子荧光激发成像技术为代表的光学成像技术使用了串行扫描机制，其速度和通量受限，不能满足一些大规模神经网络功能活动研究的需求。光场成像技术是一种无需扫描，通过一次相机曝光就能够记录整个三维体信息的新技术。而且,我们新近发展了新型扩增视场光场成像技术，克服了传统光场成像分辨率低，视场受限等缺点，可应用于高通量记录斑马鱼全脑的神经元活动。目前，该技术比较适用于组织透明度高的样品。为将该技术进一步发展和推广，我们将探索可以抵抗光散射效应的新型光场成像技术，并应用于小鼠等模式动物中，实现在较大穿透深度下的高通量在体神经元功能活动成像。本课题将为理解群体神经元编码，大规模神经网络的动态功能研究提供更丰富的神经元活动信息，为神经科学研究打开新的窗口。

生命是一个三维的动态过程。研究这一复杂系统的理想工具需要能够同时记录整个三维体中不同细胞群体间的相互作用。以共聚焦扫描荧光显微镜为代表的传统成像技术基于点扫描来串行获取图像信息，无法快速覆盖较大的成像体积。为了应对这一挑战，实现并行化同时获取整个三维体积内荧光信号的新成像技术——三维光场成像技术获得越来越多的关注。传统光场成像技术存在分辨率低，存在重构伪影等技术瓶颈，为此，我们发展了几种新型三维光场成像技术，并拓展其在大规模神经元功能成像中的应用。例如，新型光场显微镜彻底解决了重构伪影问题，并有效缓解了分辨率和成像覆盖范围之间的矛盾，首次实现在自由行为的斑马鱼幼鱼中以70 Hz三维体成像速度记录全脑神经元在捕食草履虫过程中的钙离子活动。此外，我们还进一步发展了具有滤除背景噪声能力的共聚焦光场显微成像技术，能够在小鼠脑中大规模记录神经元的钙离子活动，以及三维血管网络中血细胞流动的快速动态过程。