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

摘要

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.