An evanescent approach for mitochondrial function assay of living cells

Mitochondria are now known to function physiologically not only in the production of ATP as the major cellular energy source, but also in the regulation of intracellular signaling, in, for example, stress-induced apoptosis and buffering of cytosolic calcium. It should be noted, when interpreting mitochondrial studies in situ, that mitochondria within cells show heterogeneity in both function and location. We applied both conventional epifluorescence microscopy (EPIFM) and total-internal-reflection fluorescence microscopy (TIRFM) in this study. Image data taken from TIRFM are excellent and markedly different from those taken from EPIFM. We further investigated the physiological variations of mitochondrial functions using an EPIFM/TIRFM dual-imaging system. This system permits further analysis of functions of mitochondria and other organelles with more precision than is possible using a traditional platform.