The development and application of a methodology for measurement of oxygen within single mammalian cells is presented which employs novel macromolecular NIR (near infrared) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, low cyto- and photo-toxicity, are loaded into cells by simple transfection procedures and subsequently analysed by high resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptor mediated Ca²⁺ influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas such as cardiovascular and neurological research, metabolic diseases, and cancer.