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Simultaneous quantitative measurement and automated analysis of mitochondrial morphology, mass, potential, and motility in living human skin fibroblasts
Author(s) -
Koopman Werner J. H.,
Visch HenkJan,
Smeitink Jan A. M.,
Willems Peter H. G. M.
Publication year - 2005
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20198
Subject(s) - mitochondrion , motility , rhodamine 123 , microbiology and biotechnology , confocal , biology , confocal microscopy , live cell imaging , biophysics , biochemistry , cell , physics , multiple drug resistance , optics , antibiotics
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. © 2005 Wiley‐Liss, Inc.