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.