Calculation of Vascular Density, Red Blood Cell Filling, and perfused boundary region from recordings of the sublingual circulation.

(A) The Vascular Density is estimated by the total length of the vessels identified in the recorded area. Automatically identified vessels are marked by transversal markers placed at intervals of 10 μm longitudinally along the course of the vessel; each marker therefore contributes 10 μm to the total length. (B) The Red Blood Cell Filling is the proportion of 21 markers (the above main marker and 20 micromarkers placed at 0.5 μm intervals, 10 at either side of the main marker) occupied by Red Blood Cells across all frames of the video recording. (C) The Perfused Boundary Region is the width of the superficial component of the endothelial glycocalyx permeable to Red Blood Cells, estimated by collecting the measured widths of the flowing Red Blood Cell column at each main marker over all recording frames, linearly extrapolating the width of the overall RBC permeable lumen from the 50^th (median RBC column width) and the 75^th percentiles of the distribution of measured widths, and subtracting the median RBC column width from the width of the overall RBC permeable lumen.