Combined optical single molecule and atomic force microscopy to elucidate enzyme-induced collagen degradation kinetics

Collagen fibrils are one of the main building blocks of our body’s tissues. Collagen fibrils provide strength and structure to organs such as skin, tendons, lungs, and the heart. Fibrils allow tissues to be resistant against stretching forces — for example, they make tendons strong enough to withstand the pull of muscles when we walk. Collagen fibrils are not static structures. Cells constantly make them, modify and break them down in a natural process called collagen turnover. This process is essential for keeping tissues healthy, by allowing them to adapt to changes and to heal after injury. But, when this process becomes imbalanced, it can lead to diseases such as tissue scarring (fibrosis) or certain heart conditions. While we know collagen turnover is essential for tissue health, we understand very little about how collagen fibrils are broken down at the nanometer scale (1 nm is 100 thousand times smaller than the width of a hair). In this project, we developed experimental methods to study how single collagen fibrils change in shape and stiffness during enzymatic degradation. By combining two powerful imaging techniques — atomic force microscopy (AFM) and a method of super-resolution optical microscopy called STORM (Stochastic Optical Reconstruction Microscopy) — we discovered a way to track these changes with very high precision. Importantly, the combined AFM-STORM approach allowed us to measure the optical properties of collagen fibrils, such as their refractive index. These measurements can hint to molecular changes happening inside collagen fibrils. Because this method can be extended to other biological materials, it opens new possibilities for studying how molecular composition and structure change in health and disease. Our work provides new tools to study how tissues remodel at the level of collagen fibril and may ultimately help scientists better understand and treat diseases where collagen degradation plays a key role, such as in fibrosis or heart conditions.