Fig. 3. Simultaneous imaging (co-localization) of mitochondrial flavoproteins (A) and calcium (B) in permeabilized myocardial fibers loaded with Rhod-2. (A) As in the case of isolated cells, autofluorescence of mitochondrial flavoproteins in a fully oxidized state in the absence of mitochondrial substrates shows a regular arrangement of mitochondria in saponin-permeabilized fibers. (B) Fluorescence of Rhod-2 trapped in mitochondria allows the localization and quantification of mitochondrial free calcium. As in cardiomyocytes (Fig. 1), clear co-localization of flavoproteins (green fluorescence) and mitochondrial calcium (red fluorescence) can be seen.