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In vivo imaging of cerebral tissue oxygenation is important in defining healthy physiology and pathological departures associated with cerebral disease. We used a recently developed two-photon microscopy method, based on a novel phosphorescent nanoprobe, to image tissue oxygenation in the rat primary sensory cortex in response to sensory stimulation. Our measurements showed that a stimulus-evoked increase in tissue pO₂ depended on the baseline pO₂ level. In particular, during sustained stimulation, the steady-state pO₂ at low-baseline locations remained at the baseline, despite large pO₂ increases elsewhere. In contrast to the steady state, where pO₂ never decreased below the baseline, transient decreases occurred during the “initial dip” and “poststimulus undershoot.” These results suggest that the increase in blood oxygenation during the hemodynamic response, which has been perceived as a …