Axotomy stress depolarizes mitochondria in the vicinity of injured sites. Mature cortical neurons grown on microfluidic chambers were infected with pLenti-GFP. Axons in the terminal chambers were loaded with 25 nM TMRE dye at DIV12, followed by laser axotomy and time-lapse imaging. In brief, for real-time image capture of laser axotomy, both acquisition and bleach setting were reused in separate blocks. The images were first recorded at 5-s intervals for a total of 50 frames, and then the 730-nm laser was powered-up with 70% output and pixel dwell time >20 µs in cropped imaging region for bleaching. The consecutive post-axotomy recording was collected at 5-s intervals for a total of 100 frames. For a successful laser axotomy, the axons in the DIC image were observed with a structural break down or invasion of extracellular dye. Note that GFP-labeled axons are quickly broken up upon axotomy (left) and mitochondria in the vicinity suddenly lose their TMRE staining (right).
