Performance of TMRM and Mitotrackers in mitochondrial morphofunctional analysis of primary human skin fibroblasts.

Mitochondrial membrane potential (Δψ) and morphology are considered key readouts of mitochondrial functional state. This morphofunction can be studied using fluorescent dyes ("probes") like tetramethylrhodamine methyl ester (TMRM) and Mitotrackers (MTs). Although these dyes are broadly used, information comparing their performance in mitochondrial morphology quantification and Δψ-sensitivity in the same cell model is still scarce. Here we applied epifluorescence microscopy of primary human skin fibroblasts to evaluate TMRM, Mitotracker Red CMXros (CMXros), Mitotracker Red CMHXros (CMH2Xros), Mitotracker Green FM (MG) and Mitotracker Deep Red FM (MDR). All probes were suited for automated quantification of mitochondrial morphology parameters when Δψ was normal, although they did not deliver quantitatively identical results. The mitochondrial localization of TMRM and MTs was differentially sensitive to carbonyl cyanide-4-phenylhydrazone (FCCP)-induced Δψ depolarization, decreasing in the order: TMRM ≫ CHM2Xros = CMXros = MDR > MG. To study the effect of reversible Δψ changes, the impact of photo-induced Δψ "flickering" was studied in cells co-stained with TMRM and MG. During a flickering event, individual mitochondria displayed subsequent TMRM release and uptake, whereas this phenomenon was not observed for MG. Spatiotemporal and computational analysis of the flickering event provided evidence that TMRM redistributes between adjacent mitochondria by a mechanism dependent on Δψ and TMRM concentration. In summary, this study demonstrates that: (1) TMRM and MTs are suited for automated mitochondrial morphology quantification, (2) numerical data obtained with different probes is not identical, and (3) all probes are sensitive to FCCP-induced Δψ depolarization, with TMRM and MG displaying the highest and lowest sensitivity, respectively. We conclude that TMRM is better suited for integrated analysis of Δψ and mitochondrial morphology than the tested MTs under conditions that Δψ is not substantially depolarized.