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.pdfVIP

生理学报Acta Physiologica Sinica , February 25, 2013, 65(1): 77–82 77 实验技术 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 * 石 莹，高景霞，彭刚，姜民 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 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36~48 h 的转基因 Tg(lhx5:kaede) kaede 斑马鱼胚胎麻醉固定后，以光诱导变色荧光蛋白 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405 nm紫外激光刺激扫描感兴趣区域的同时，进行488 nm和559 nm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。 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 中图分类号：Q7 Application of confocal simultaneous scanner unit in the study of forebrain neurodevelopment in zebrafish SHI Ying, GAO Jing-Xia, PENG Gang, JIANG Min* Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China Abstract: 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, 36–48-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 w