Figure 2. 3D SR imaging reveals cell-surface protrusions as glycocalyx-covered membrane tubules suitable for quantitation.

(A) 3D reconstruction of Sia on BT-20 cells. Tetraacylated N-azidoacetylmannosamine (ManNAz) incorporated BT-20 cells were labeled with AF647 using Cu-click and subjected to TILT3D microscopy. ManNAz was used rather than periodate to label sialic acids in order to minimize coverslip background. Left and right insets correspond to tubules that extend perpendicular to and within the xy-plane, respectively. (B) Projection of the 3D reconstruction onto the xy-plane. The z-slices had a thickness of ~250 nm. (C) Effect of the orientation of tubules on their projections. Shown is a magnification of the tubules located at the asterisk in Figure 2A. (D) Magnification of the base of a single tubule taken from a z-slice (dagger in Figure 2B). (E) Quantitation of tubule width in 3D. A traverse section of three tubules was taken, and a histogram of single molecule localizations was plotted. (F) Schematic of the tubules as glycocalyx-covered membrane protrusions along with the two key parameters to be determined. The lipid-determined tubule width corresponds to the plasma membrane-to-plasma membrane distance. The GalNAz- and Sia-determined tubule widths correspond to lipid-determined tubule width summed with the glycocalyx height on each side of tubule.