Single-cell time-lapse imaging of intracellular O₂ in response to metabolic inhibition and mitochondrial cytochrome-c release

The detection of intracellular molecular oxygen (O₂) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6– pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O₂-independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O₂. Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O₂ accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at ‘physiological’ tissue O₂ levels (5% O₂). Multiplexing also allowed us to monitor intracellular O₂ during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O₂ despite a decrease in mitochondrial membrane potential.