A) Concentration profiles (ΔN) of AF532-HSA vs. distance (μm) above glass-cell interface. Curve shows ΔN (concentration of albumin over cell layer minus concentration of albumin in aqueous solution without cells). ΔN increase nearly 5-fold between 1.5 and 2.5 μm above the glass coverslip. B) Characteristic diffusion times, τD, of AF532-HSA vs. distance above a control cell (control cell) and in aqueous solution over a coverslip without cells (solution). Increases in τD, indicative of slowing of AF532-HSA, can be seen from approximately 1.5 to 2.5 μm above the glass coverslip containing a confluent endothelial monolayer. The combined observation of increases in ΔN and τD(z), occurring at the same distance above the cell surface, identifies the location of a structure that sieves albumin, consistent with the function of the glycocalyx.