Simultaneous quantitative measurement and automated analysis of mitochondrial morphology, mass, potential, and motility in living human skin fibroblasts.

Background: Understanding the interdependence of mitochondrial and cellular functioning in health and disease requires detailed knowledge about the coupling between mitochondrial structure, motility, and function. Currently, no rapid approach is available for simultaneous quantification of these parameters in single living cells.

Methods: Human skin fibroblasts were pulse-loaded with the mitochondria-selective fluorescent cation rhodamine 123. Next, mitochondria were visualized using video-rate (30 Hz) confocal microscopy and real-time image averaging. To highlight the mitochondria, the acquired images were binarized using a novel image processing strategy.

Results: Our approach enabled rapid and simultaneous quantification of mitochondrial morphology, mass, potential, and motility. It was found that acute inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) by means of rotenone transiently reduced mitochondrial branching, area, and potential. In contrast, mitochondrial motility was permanently reduced.

Conclusions: We present and validate a novel approach for rapid, unbiased, and simultaneous quantification of multiple mitochondrial parameters in living cells. Because this method is automated, large numbers of cells can be analyzed in a short period of time.