Measuring Single Collagen Fibril Fluctuations and Relaxation Times with Activity Microscopy

Collagen is the primary component of the extra-cellular matrix (ECM), a network composed of individual filaments that gives tissues structure and helps mediate mechanical communication between cells. Because cells in the ECM primarily exert forces on the collagen fibrils in their immediate environment, the mechanical properties of individual fibrils and their integration in the network are of particular interest. A fibril’s thermally driven shape fluctuations offer a valuable probe of properties such as its bending stiffness, length, spatial modes, and the forces the fibril is under. However, the stiffness of collagen fibrils results in nanometer scale thermal fluctuations that occur on a sub millisecond time scale, making them challenging to measure. We introduce activity microscopy as a method capable of resolving the thermal fluctuations of a single fibril within a network, without the need for external mechanical forces or fluorescent labels. In the research presented here, activity microscopy is applied to collagen I fibrils within in vitro networks. Fibril axes are located with nanometer precision and their transverse thermal fluctuations are measured with 100 kHz bandwidth. Power spectral density (PSD) analysis of the fibril fluctuations is used to characterize their dynamics and extract characteristic relaxation times. The shape of a fibril’s power spectra show a strong dependence on the network’s properties and the individual fibril’s attachment to surrounds structures.