激光共聚焦同步双扫描技术在斑马鱼前脑神经发育研究中的应用  

Application of confocal simultaneous scanner unit in the study of forebrain neurodevelopment in zebrafish

作　　者：石莹[1] 高景霞[1] 彭刚[1] 姜民[1] 

机构地区：[1]复旦大学脑科学研究院、医学神经生物学国家重点实验室,上海200032

出　　处：《生理学报》2013年第1期77-82,共6页Acta Physiologica Sinica

基　　金：supported by the National 985 Program from the Ministry of Education of China

摘　　要：随着标记蛋白光转换等实验技术在生物科研领域的开展,对激光共聚焦取图技术也提出了越来越高的要求。本文旨在以斑马鱼前脑神经发育为研究对象,建立应用激光共聚焦同步双扫描系统的实验模式。本实验将36～48h的转基因Tg(lhx5:kaede)斑马鱼胚胎麻醉固定后,以光诱导变色荧光蛋白kaede标记的前脑神经元作为实验观察目标,使用激光共聚焦同步双扫描系统的405nm紫外激光刺激扫描感兴趣区域的同时,进行488nm和559nm激光刺激取图,实时观察kaede蛋白光学信号的转换状态,并在光转换后记录紫外激光刺激区域神经元的投射形态图像。结果显示,激光刺激引起kaede蛋白从绿色转变为红色,其在感兴趣区域的分布变化指示出目标神经元的胞体位置及其轴突投射路径。由上述结果可见,激光共聚焦同步双扫描技术的应用满足了kaede蛋白光转换实验的刺激和扫描同步的要求,该技术在斑马鱼胚胎特定神经元形态发育研究中的应用,可作为神经发育研究的一种实验模式在很多实验设计中进行延伸,也为激光共聚焦双扫描技术的推广提供了一个很好的平台。With the application of the photoconversion technology of genetically expressed fluorescent proteins in biologic field, more powerful confocal imaging ability was demanded. The aim of the present study was to establish an experimental model employing confocal simultaneous scanner unit for simultaneous laser stimulation and imaging, taking study of forebrain neurodevelopment in ze- brafish as an example. In the present study, 36-M8-hour-old Tg（lhx5：kaede） zebrafish embryos were mounted with 1.2% low melting temperature agarose. The forebrain neurons marked with kaede were observed using the simultaneous scanner unit of confocal mi- croscopy. The 405 nm laser was used to stimulate the region of interest （ROI）, while 488 and 559 nm lasers were used to acquire im- ages at the same time. The photoconversion state of kaede protein was then reviewed, and the projecting pattern of neurons stimulated by the ultraviolet laser was examined. The results showed that, the fluorescence of stimulated kaede turned from green to red, and the photoconversion of kaede demonstrated anterior dorsal telencephalon （ADt） neurons projected axons ventrally into the anterior com- missure （AC） and supraoptic tract （SOT）. These results suggest the confocal simultaneous scanner unit meets the demand of the pho- toconversion experiment. The application of confocal simultaneous scanning technology in examining Tg（lhx5：kaede） zebrafish em- bryos affords an ideal experimental model in neurodevelopment study.

关 键 词：激光共聚焦同步双扫描技术 斑马鱼 光转换 kaede 神经发育 

分 类 号：Q42[生物学—神经生物学]