mSOFs are coincident with depolarization of the mitochondrial membrane potential. (A and B) Representative mt-cpYFP (A) or TMRE (B) confocal images of an adult skeletal muscle fiber obtained from a muscle-specific, mt-cpYFP–expressing transgenic mouse co-labeled with TMRE, a mitochondrial membrane potential indicator; boxed region indicates an area containing an mSOF. (C) A standard deviation (STD) map generated from a stack of time-lapsed images from the same fiber shown in A and B (1.24 s/frame, 100 frames), using a custom-developed program (Flash Collector) for automated mSOF detection and analysis. Detected mSOF events are outlined in yellow and numbered in red (event no. 25 corresponds to the boxed flash shown in A and D). (D) Time course of mt-cpYFP and TMRE fluorescence within the boxed region shown in A (mt-cpYFP) and B (TMRE). (Top) Series of pseudo color time-lapse mt-cpYFP (top) and TMRE (bottom) images within the boxed regions. (Bottom) Time course of simultaneously recorded mt-cpYFP (green) and TMRE (red) fluorescence for the individual flash event shown in A and B. Numbers and arrows (1–4) indicate times where the corresponding pseudo color images were taken.