Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.

Journal: Biochimica et biophysica acta
Published Date: Nov 13, 2014

Abstract

Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.

Authors

  • Anthony P Leonard
    Dept. of Ophthalmology, Medical University of South Carolina, USA; Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
  • Robert B Cameron
    Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
  • Jaime L Speiser
    Dept. of Public Health Sciences, Medical University of South Carolina, USA.
  • Bethany J Wolf
    Dept. of Public Health Sciences, Medical University of South Carolina, USA.
  • Yuri K Peterson
    Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
  • Rick G Schnellmann
    Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
  • Craig C Beeson
    Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
  • Bärbel Rohrer
    Dept. of Ophthalmology, Medical University of South Carolina, USA; Ralph H. Johnson Memorial Veteran's Administration Hospital, USA.

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