Two-photon microscopy of brain endothelial glycocalyx uncovers spatial heterogeneity, vesicular transport, and lectin-binding kinetics in the living brain

The endothelial glycocalyx is a key regulator of cerebrovascular function and remains one of the most difficult structures to study in vivo. Here we uncover new structural and dynamical features of the brain endothelial glycocalyx using in vivo two-photon microscopy. We identified glycocalyx enrichment at endothelial junctions and arteriolar branch points, visualized its Vesicular transport in real-time, and found evidence for its compositional Variations along the arteriovenous axis. Fluorescence recovery after photobleaching revealed two distinct kinetics of wheat germ agglutinin binding, including a previously undescribed one. Finally, super-localization of the glycocalyx estimated glycocalyx thickness as 775{+/-}17 nm and 622{+/-}34 nm before and after enzymatic shedding, reconciling discrepancies between past optical and electron microscopy estimates. Together these findings establish the first miltiscale framework of glycocalyx distribution and heterogeneity, transport, and molecular interaction kinetics in the living brain.