FUNCTIONAL IMAGING OF MITOCHONDRIA IN SAPONIN-PERMEABILIZED MICE MUSCLE-FIBERS

Confocal laser-scanning and digital fluorescence imaging microscopy we re used to quantify the mitochondrial autofluorescence changes of NAD( P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue, Addition of mitochondrial substrates, A DP, or cyanide led to redox state changes of the mitochondrial NAD sys tem, These changes were detected by ratio imaging of the autofluoresce nce intensities of fluorescent flavoproteins and NAD(P)H, showing inve rse fluorescence behavior. The flavoprotein signal was colocalized wit h the potentiometric mitochondria-specific dye dimethylaminostyryl pyr idyl methyl iodide (DASPMI), or with MitoTracker(TM) Green FM, a const itutive marker for mitochondria, Within individual myofibers we detect ed topological mitochondrial subsets with distinct flavoprotein autofl uorescence levels, equally responding to induced rate changes of the o xidative phosphorylation, The flavoprotein autofluorescence levels of these subsets differed by a factor of four, This heterogeneity was sub stantiated by flow-cytometric analysis of flavoprotein and DASPMI fluo rescence changes of individual mitochondria isolated from mice skeleta l muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluoresce nt marker of the mitochondrial NAD-redox system. Under the present exp erimental conditions these subsets show similar functional responses.