Life cell quantification of mitochondrial membrane potential at the single organelle level

Mitochondrial membrane potential (Delta Psi) is key to mitochondrial function and cellular survival. Here, we aimed to develop an automated protocol allowing sensitive quantification of Delta Psi in living cells at the level of individual mitochondria. Human skin fibroblasts were stained with the fluorescent cation tetramethyl rhodamine methyl ester (TMRM), which is sequestered by mitochondria according to their Delta Psi. Cells were visualized by videomicroscopy and the acquired images were processed to generate a mitochondria-specific mask. The latter was superimposed on the original image to allow quantification of TMRM fluorescence. Following validation, our approach revealed that mitochondria with different Delta Psi coexisted within the same cell. Furthermore, our method allowed reproducible detection of small (<10%) reductions in TMRM intensity induced by the complex Ill inhibitor antimycin A. Mitochondrial uncoupling by p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) greatly reduced mitochondrial TMRM fluorescence. Under these conditions faithful mask calculation and TMRM intensity analysis were still possible using a mitochondria-targeted green fluorescence protein (mitoAcGFP1), expressed in the cells using baculoviral transfection. (c) 2007 International Society for Analytical Cytology.