title: Simultaneous quantitative measurement and automated analysis of mitochondrial morphology, mass, potential, and motility in living human skin fibroblasts.
abstract: 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.
authors.affiliation: Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: Visch
authors.forename: Jan A M
authors.initials: JA
authors.lastname: Smeitink
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
mesh_terms: 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*
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D012402:Rotenone; D009243:NAD; D042967:Electron Transport Complex I