Abstract Oxygen plays an important role in aerobic energy metabolism and signal transduction. The detection of intracellular or subcellular molecular oxygen (O2) levels is important for the understanding of cell physiology and mitochondrial energy metabolism, and the alterations correlated with cancer. Detection of O2 has recently been improved significantly with the development of live cell time lapse microscopy compatible oxygen sensitive probes. We here utilized nanoparticle probe MitoImageTM-MM2 with embedded poly(9,9-dioctylfluorene) (PFO) as an O2-independent component and Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) to perform time lapse ratiometric measurements of intra-cellular oxygen levels. Loading of HeLa cervical cancer cells with MM2 enables us to image kinetics of intracellular O2 concentration in response to the mitochondrial respiratory chain inhibitors Antimycin A (complex III) or sodium azide (complex IV). These changes were detected in respiration-supporting medium equilibrated with 5% (53 μM) and 20% (225 μM)O2. Also exposing cells to varying extracellular oxygen concentrations (21 - 225 μM) showed strong dependence of the intracellular oxygen levels. The mitochondrial membrane potential was monitored by simultaneous imaging of Tetra-Methyl-Rhodamine-Methyl ester (TMRM) in the same sample. Multiplexing with GFP finally allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilisation (MOMP) in HeLa expressing cytochrome-c-eGFP and stained with TMRM. In conclusion, the MitoImageTM-MM2 nanoparticle probe enables single-cell, time-lapse imaging of alterations in intracellular oxygen levels in cancer cells, and can be employed in multiplex assays to investigate alterations in intracellular oxygen levels relative to protein dynamics and mitochondrial function. Keywords: Intracellular oxygen; phosphorescent oxygen-sensitive nanoparticles; HeLa cells; MitoImage ratiometric probes; confocal microscopy. Citation Format: Heiko Düssmann, Sergio Perez, Ujval Anil Kumar, Dmitri B. Papkovsky, Jochen HM Prehn. Multiplexing single cell time lapse imaging of intra-cellular oxygen in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4224.