Optical measurement of microvascular oxygenation and blood flow responses in awake mouse cortex during functional activation

The cerebral cortex has a number of conserved morphological and functional characteristics across brain regions and species. Among them, the laminar differences in microvascular density and mitochondrial cytochrome c oxidase staining suggest potential laminar variability in the baseline O₂ metabolism and/or laminar variability in both O₂ demand and hemodynamic response. Here, we investigate the laminar profile of stimulus-induced intravascular partial pressure of O₂ (pO2) transients to stimulus-induced neuronal activation in fully awake mice using two-photon phosphorescence lifetime microscopy. Our results demonstrate that stimulus-induced changes in intravascular pO₂ are conserved across cortical layers I–IV, suggesting a tightly controlled neurovascular response to provide adequate O₂ supply across cortical depth. In addition, we observed a larger change in venular O₂ saturation (ΔsO₂) compared to arterioles, a gradual increase in venular ΔsO₂ response towards the cortical surface, and absence of the intravascular “initial dip” previously reported under anesthesia. This study paves the way for quantification of layer-specific cerebral O₂ metabolic responses, facilitating investigation of brain energetics in health and disease and informed interpretation of laminar blood oxygen level dependent functional magnetic resonance imaging signals.