Phosphorescence lifetime imaging (PLIM), combined with specific indicator dyes allows for quantitative measurements of physiologically relevant O2 gradients. In order to achieve reliable and specific measurement of O2, phosphorescent indicator dye must be protected from self-quenching and environmental interferences, and have sufficient brightness and photostability. Here, we report the two approaches for PLIM-based measurement of O2 in cultured adherent cells, 3D spheroids and ex vivo tissue models: (i) cell-penetrating small molecule and nanoparticle probes and (ii) O2-sensitive solid-state phosphorescent 3D porous scaffold material. Using click-modification of Pt-porphyrin (PtTFPP) with thio-glucose we produced conjugate Pt-Glc, which displayed microsecond decay times and sufficient brightness for one-photon PLIM. Pt-Glc efficiently stained the cytoplasm of cultured mammalian cells, neurospheres and ex vivo cultures of brain and intestine. We also developed a panel of bright “multi-modal” O2 polymeric nanoparticles compatible with both one- and two-photon excitation modes and harboring different surface charges. Depending on the cell model, various types of nanoparticles (cationic, anionic, zwitter-ionic) displayed efficient intracellular staining, comparable with small molecules. Using all these O2-sensitive materials and confocal PLIM, we showed that multi-cellular aggregates of PC12 and HCT116 cancer cells experience profound and dynamic character of deoxygenation, which is affected by their size and metabolic status. They showed responses to stimulation with pharmacological agents targeting mitochondria and selectively killing hypoxic cells. The dynamic O2 gradients monitored in situ affected cell death and differentiation. Collectively, O2 PLIM imaging provides a powerful tool for the quantitative assessment of hypoxia in live cells.Supported by Science Foundation Ireland grants 13/SIRG/2144.