Mitochondrial flashes are detected with mitoHyper and mitoSypHer. Experiments were performed on 5–8-wk-old male OF1 mice (Charles River). In vivo transfection of mitoHyper or mitoSypHer within the flexor digitorum brevis of the animals, single fiber isolation, TMRM, and MitoSOX loading were performed as described in Pouvreau (2010). Bi-wavelength imaging of mitoHyper/mitoSypHer and TMRM or MitoSOX was performed by line-interleaving excitation at 488 and 543 nm, and emission was collected at 505–545 and >560 nm, respectively. Regions of interest (ROI) were detected as described previously (Pouvreau, 2010) and correspond to areas of flashing mitochondria. All experiments and procedures were performed in accordance with the guidelines of the French Ministry of Agriculture (87/848), the European Community (86/609/EEC). (A) Time course of changes in normalized fluorescence of mitoHyper (top) and TMRM (bottom) within one ROI. (B and D) Time course of changes in normalized fluorescence of mitoHyper/mitoSypHer (top) and MitoSOX (bottom) within three ROI. (C and E) Fluorescence intensity of mitoHyper (C) or mitoSypHer (E) in a cell incubated with 1.5 or 2 mM of H2O2, respectively.
