基于激光共聚焦同步双扫描技术的LC3脱乙酰化修饰调控自噬的机理研究

A Study on the Mechanism of LC3 Deacetylation in Regulating Autophagy Based on Laser Scanning Confocal Microscopy with Simultaneous Scanner

激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。

The simultaneous （ SIM ） scanner technique is based on an additional compact scanning unit used to syn-chronize laser light stimulation with confocal imaging independently. In this study, we used 405 nm laser for light stimu-lating and 488 nm laser for imaging. The formation of the LC3 complex and optical dynamic of LC3 before and after acetylation was real-time monitored by real-time live cell imaging, revealing that LC3 deacetylation modification is essen-tial for the autophagy degradation. This experimental system is a good platform for the application of laser scanning confo-cal SIM technique. This technique ensures that those cellular reactions during light stimulation are visible, making SIMthe most powerful tool for fluorescence recovery after photobleaching （ FRAP）,fluorescence loss in photobleaching （FLIP） and photo-activation research.