Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO2) 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 PO2 can be accurately measured in the brain at depths up to 300 mm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO2 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 PO2 in capillaries and by a biphasic PO2 response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO2 opens new avenues for studies of brain metabolism and blood flow regulation.