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 synchronize laser light stimulation with confocal imaging independently. In this study, we used 405 nm laser for light stimulating 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 essential for the autophagy degradation. This experimental system is a good platform for the application of laser scanning confocal SIM technique. This technique ensures that those cellular reactions during light stimulation are visible, making SIM the most powerful tool for fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) and photo-activation research.