his chapter describes the fundamentals of an O2 imaging technique based on the quenched-phosphorescence detection of Pt-porphyrin probes. The wide-field, confocal and multi-photon microscopy and methodological aspects of quenched phosphorescence icO2 imaging techniques, theoretical and practical considerations, are briefly described and critically assessed. This is followed by a comprehensive set of practical examples in which the imaging of various biological models, including conventional cell cultures, spheroids (neurospheres), larger organisms such as C. elegans worms and microfluidic devices were analysed. Critical factors that determine the performance of such imaging experiments are also identified and discussed providing a broad prospective on the possible applications of these techniques, particularly in the studies of cell and tissue physiology, the role of O2 in metabolism, hypoxia and other areas of biomedical research.