Collagen is the major structural protein in bones, tendons, ligaments, and connective tissues. Its most fundamental unit is the collagen fibril, whose structure, mechanical and optical properties have been the focus of extensive research, as collagen plays not only a central role in biomechanics, but also in wound healing and tissue remodelling, mediated by matrix metalloproteinases (MMPs). Therefore, investigating collagen fibrils remains important for the understanding of physiology, as well as pathological processes.This work presents a comprehensive characterization of the morphology of collagen fibrils in a near-native environment, by deploying a multimodal approach using Single Molecule Localization Microscopy (SMLM) and Atomic Force Microscopy (AFM). The results show the flexible and diverse shapes of collagen fibrils and their attachment behaviour on glass coverslips. Furthermore, a novel, universal method was developed as part of this work, to measure the refractive index in biological samples that are diffraction limited. This method utilizes the point spread function in SMLM measurements and was applied to retrieve the refractive index of single collagen fibrils. The results uncovered significant variability both across different fibrils and within single fibrils. Additionally, an efficient and cost-effective protocol was established for expressing and fluorescently labeling MMP1, providing a framework for future studies on collagen degradation dynamics. The activity of the expressed MMP1 was validated through enzymatic assays on a test substrate and through live digestion experiments on collagen fibrils.
Simon Jaritz (Wed,) studied this question.