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The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the "Holy Grail" of cellular physiology. In an effort to obtain a minimally invasive approach to the mapping of intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were compared in the current study and gave similar results. These were two-photon confocal laser scanning microscopy with pinhole shifting, and picosecond time-resolved epi-phosphorescence microscopy using a single 0.5 μm focused spot. Both methods utilized Ru coordination complex embedded nanoparticles (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of a titanium–sapphire Tsunami lasers (710–1050 nm).