Determining local viscoelastic properties of collagen systems using optical tweezers

In this work, I aimed to develop and apply a technique capable of measuring the viscoelastic properties of collagen at different levels of hierarchy. Collagen is the predominant structural protein in vertebrates, and its self-assembly into well-defined structures including fibrils underlies the formation of a wide variety of biological structures with a broad range of functions. Here, in order to understand the correlation between collagen’s structure and its mechanical properties, the viscoelastic properties of different collagen systems were characterized, ranging from solutions of molecules to self-assembled forms of fibrillar gels and gelatin. To determine rheological properties, optical tweezers were used to trap and monitor thermal fluctuations of an embedded micron-sized particle, producing measurements of viscoelastic response of collagen systems at a high bandwidth (> 10^4 Hz). To validate these measurements, I reproduced results on a previously characterized system (polyethylene oxide). The obtained viscoelastic response is affected by the timescales of the interactions between polymers, which play a critical role in conferring elasticity to the system. To provide guidance to the microrheology experiments, the structure of collagen in acidic solution was probed using dynamic light scattering. My microrheology studies of collagen molecules in acidic solution showed that elastic response becomes comparable to viscous response at the highest concentration studied here, 5 mg/ml. Here, the significant elasticity observed at frequencies above ~200 Hz is due to collagens’ intermolecular interactions, which I found were not due to electrostatic interactions. However, elasticity was found to decrease following the removal of collagen’s telopeptides, consistent with their role in facilitating fibril formation. At the fibrillar level, unlike in solutions of collagen, I observed spatial heterogeneity in viscoelastic properties. The elastic modulus varies by an order of magnitude at different locations within fibrillar collagen gels. By making measurements over 100-minute timescales as collagen self-assembled into fibrils, I probed the development of microscale heterogeneity and concluded that heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase.