Setup of the atomic force microscope (AFM) used in this study (A) for nanomechanical analysis of the endothelial glycocalyx (eGC) and an original tracing of a force indentation curve performed on the endothelial surface of a rat aorta (B) <i>ex vivo.</i>

<p>1) The AFM tip travels vertically towards the endothelial surface. 2) Upon indentation of the endothelial glycocalyx (eGC), the AFM cantilever, which serves as a soft spring, is deflected. The cantilever deflection is measured as a laser beam reflected from the back of the cantilever and is plotted as a function of sample position along the z axis. The resulting curve is transformed into a force-versus-indentation curve using the cantilever’s spring constant and the optical lever sensitivity. The slope of a force indentation curve then directly reflects the stiffness (expressed in pN/nm), which is necessary to indent the eGC for a certain distance. The first slope indicates the stiffness (in this trace 0.38 pN/nm) of the very first layer, the endothelial glycocalyx (eGC). The second slope indicates the stiffness of the plasma membrane and the cortical actin web. Practically, the slope of a force indentation curve is deduced from a manually plotted regression line (similar to the line in the trace) in PUNIAS (Protein Unfolding and Nano-Indentation Analysis Software, Version 1.0, Release 1.8, http:// <a href="http://punias.voila.net/" target="_blank">punias.voila.net/</a>). In our experience, this slope is virtually linear over its entire length, resulting in a single fixed stiffness value. The distance between the starting point of eGC indentation and the starting point of the second slope (projected to the x-axis) corresponds to the thickness of the eGC (in this trace 422.6 nm).