TY - JOUR
T1 - Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning
AU - Leonard, Anthony P.
AU - Cameron, Robert B.
AU - Speiser, Jaime L.
AU - Wolf, Bethany J.
AU - Peterson, Yuri K.
AU - Schnellmann, Rick G.
AU - Beeson, Craig C.
AU - Rohrer, Bärbel
N1 - Funding Information:
The authors thank Robert Graves at GE for the suggestion to implement the decision tree classifier as inclusion criteria within individual target sets (to quantify mitochondrial morphological subtypes separately). The authors also thank Luanna Bartholomew for excellent help in editing the final revision of this manuscript. This work was funded in part by the South Carolina Clinical & Translational Research (SCTR) Institute , with an academic home at the Medical University of South Carolina, through NIH/NCRR TL1 RR029881 and TL1 TR000061 (A.P.L., B.J.W., J.L.S.), by National Eye Institute Grant NIH/NEI 1R01EY019320-01A2 (B.R.), by Department of Education Graduate Assistantship in Areas of National Need Grant P200A040143 (A.P.L.), by the Foundation Fighting Blindness WG-TRAP Award TA-NP-0446-MUSC-WG (B.R., C.C.B., A.P.L.), by NIH Medical Scientist Training Program Grant T32 GM08716 (A.P.L., R.B.C.), by NIH/NIGMS Grant 084147 (R.G.S.), and by the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs Merit grant BX000851 (R.G.S.), Veterans Affairs Grant I01 RX000444 (B.R.), an unrestricted grant to MUSC from Research to Prevent Blindness (Dept. support for A.L., B.R.), and by the Arnold and Mabel Beckman Foundation (B.R.), with building construction support via NIH/NCRR C06 RR-015455 (All Authors).
Publisher Copyright:
© 2014 Elsevier B.V.
PY - 2015/2/1
Y1 - 2015/2/1
N2 - 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.
AB - 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.
KW - High content microscopy
KW - Image cytometry
KW - Mitochondrial dynamics
KW - Mitochondrial function
KW - Mitochondrial toxicology
KW - Morphometry
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U2 - 10.1016/j.bbamcr.2014.11.002
DO - 10.1016/j.bbamcr.2014.11.002
M3 - Article
C2 - 25447550
AN - SCOPUS:84913580346
SN - 0167-4889
VL - 1853
SP - 348
EP - 360
JO - Biochimica et Biophysica Acta - Molecular Cell Research
JF - Biochimica et Biophysica Acta - Molecular Cell Research
IS - 2
ER -