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.
Koopman
Werner J H
WJ
Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Visch
Henk-Jan
HJ
Smeitink
Jan A M
JA
Willems
Peter H G M
PH
D002478:Cells, Cultured; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors; D005347:Fibroblasts / Q000648:ultrastructure*; D005456:Fluorescent Dyes; D006801:Humans; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology*; D008933:Mitochondrial Swelling / Q000187:drug effects / Q000502:physiology; D009243:NAD / Q000378:metabolism; D012235:Rhodamines; D012402:Rotenone / Q000494:pharmacology; D012867:Skin / Q000166:cytology*
D005456:Fluorescent Dyes; D012235:Rhodamines; D012402:Rotenone; D009243:NAD; D042967:Electron Transport Complex I