Adhesion force microscopy is sensitive to the charge distribution at the surface of single collagen fibrils

Collagen fibrils are a key component of the extracellular matrix of mammalian tissues where they serve as structural elements and as a ligand for receptor-mediated signaling. As collagen molecules assemble into fibrils, in vitro or in vivo, they acquire a modulation of their molecular and electron densities called the D-band, with a 67 nm spacing, that can be visualized by cryo-electron microscopy. The D-band is composed of a gap region missing one-fifth of the molecules in the cross-section compared to the overlap region. This leads to the gap region having a positive potential and the overlap region a negative potential with respect to an n-doped silicon probe as observed by Kelvin Probe Force Microscopy. In this study, we use the adhesion force between an n-doped silicon probe and a collagen substrate to demonstrate the sensitivity of adhesion force towards charge distribution on the surface of collagen fibrils. We also map the charge distribution at the surface of single in vivo and in vitro assembled collagen fibrils and characterize the three-dimensional location and strength of three sub D-band regions that have been observed previously by cryo-electron microscopy. Our approach provides an adhesion fingerprint unique to each fibril type we analyzed and points to local charge variations at the sub D-band level even along a single fibril. It opens the road for a detailed analysis of collagen fibrils surface modifications due to ligand binding or the accumulation of advanced glycation end products at sub D-band resolution on a fibril by fibril basis.