title: Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.
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.affiliation: Dept. of Ophthalmology, Medical University of South Carolina, USA; Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Leonard
authors.affiliation: Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Cameron
authors.affiliation: Dept. of Public Health Sciences, Medical University of South Carolina, USA.
authors.forename: Jaime L
authors.initials: JL
authors.lastname: Speiser
authors.affiliation: Dept. of Public Health Sciences, Medical University of South Carolina, USA.
authors.forename: Bethany J
authors.initials: BJ
authors.lastname: Wolf
authors.affiliation: Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
authors.forename: Yuri K
authors.initials: YK
authors.lastname: Peterson
authors.affiliation: Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
authors.forename: Rick G
authors.initials: RG
authors.lastname: Schnellmann
authors.affiliation: Dept. of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, USA.
authors.forename: Craig C
authors.initials: CC
authors.lastname: Beeson
authors.affiliation: Dept. of Ophthalmology, Medical University of South Carolina, USA; Ralph H. Johnson Memorial Veteran's Administration Hospital, USA.
authors.forename: Bärbel
authors.initials: B
authors.lastname: Rohrer
mesh_terms: D000818:Animals; D001185:Artificial Intelligence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000031:analogs & derivatives / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004579:Electron Transport / Q000187:drug effects; D021621:Imaging, Three-Dimensional / Q000379:methods*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D016877:Oxidants / Q000633:toxicity; D010641:Phenotype; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects; D020122:tert-Butylhydroperoxide / Q000378:metabolism
chemical_list: D016877:Oxidants; C108897:carbonylcyanide 4-trifluoromethoxyphenylhydrazone; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D020122:tert-Butylhydroperoxide; D000254:Sodium-Potassium-Exchanging ATPase
keywords: High content microscopy; Image cytometry; Mitochondrial dynamics; Mitochondrial function; Mitochondrial toxicology; Morphometry