The cerebral metabolic rate of oxygen () is an essential parameter for evaluating brain function and pathophysiology. However, the currently available approaches for quantifying rely on complex multimodal imaging and mathematical modeling. Here, we introduce a method that allows estimation of based on a single measurement modality—two-photon imaging of the partial pressure of oxygen () in cortical tissue. We employed two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen-sensitive nanoprobe PtP-C343 to map the tissue distribution around cortical penetrating arterioles. is subsequently estimated by fitting the changes of tissue around arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline in anesthetized rats and modulated tissue levels by manipulating the depth of anesthesia. This method provides measurements localized within and it may provide oxygen consumption measurements in individual cortical layers or within confined cortical regions, such as in ischemic penumbra and the foci of functional activation.