Studies of flow-metabolism coupling often presume that microvessel architecture is a surrogate for blood flow. To test this assumption, we introduce an in vivo Dynamic Contrast Optical Coherence Tomography (DyC-OCT) method to quantify layer-resolved microvascular blood flow and volume across the full depth of the mouse neocortex, where the angioarchitecture has been previously described. First, we cross-validate average DyC-OCT cortical flow against conventional Doppler OCT flow. Next, with laminar DyC-OCT, we discover that layer 4 consistently exhibits the highest microvascular blood flow, approximately two-fold higher than the outer cortical layers. While flow differences between layers are well-explained by microvascular volume and density, flow differences between subjects are better explained by transit time. Finally, from layer-resolved tracer enhancement, we also infer that microvascular hematocrit increases in deep cortical layers, consistent with predictions of plasma skimming. Altogether, our results show that while the cortical blood supply derives mainly from the pial surface, laminar hemodynamics ensure that the energetic needs of individual cortical layers are met. The laminar trends reported here provide data that links predictions based on the cortical angioarchitecture to cerebrovascular physiology in vivo
