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Two-photon phosphorescence lifetime microscopy (2PLM) has been used recently for depth measurements of oxygen partial pressure (PO(2)) in the rodent brain. In capillaries of olfactory bulb glomeruli, 2PLM has also allowed simultaneous measurements of PO(2) and blood flow and revealed the presence of erythrocyte-associated transients (EATs), which are PO(2) gradients that are associated with individual erythrocytes. We investigated the extent to which EAT properties in capillaries report local neuronal activity. We find that at rest, PO(2) at EAT peaks overestimates the mean PO(2) by 35 mm Hg. PO(2) between two EAT peaks is at equilibrium with, and thus reports, PO(2) in the neuropil. During odor stimulation, there is a small PO(2) decrease before functional hyperemia, showing that the initial dip in PO(2) is present at the level of capillaries. We conclude that imaging oxygen dynamics in capillaries provides a unique and noninvasive approach to map neuronal activity.

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.