Time lapse studies were performed using a live cell imaging Zeiss Axiovert Fluorescence microscope equipped with a Hamamatsu Orca-ER camera and a self-contained incubation chamber providing 37°C and 5% CO2. Frames were taken every 2 seconds for a total period of 3 minutes for mitochondrial trafficking studies as well as for imaging mitochondrial fusion and fission events using a 63× oil objective (Plan-Apochromat, 1.4 NA). For visualization of mitochondria, cells were transduced with CellLight® Mito-GFP, BacMam 2.0 (Invitrogen) 16 hours prior to imaging. The staining media was replaced with fresh neuronal media before imaging. Time-lapse imaging of neurons for neuronal outgrowth studies was carried out using a 20× phase-contrast objective. Frames were taken every 2 minutes for a period of 25 minutes.
