Atomic Force Microscopy Investigations of Collagen: From Single Molecules to Fibrils

Collagen is a key component of the extracellular matrix, providing structural support and influencing cellular behavior through its physical properties. Understanding its mechanical properties is essential for elucidating its role in tissue integrity and pathology. Collagen forms complex hierarchical structures, ranging from individual proteins to higher-order fibrils, fibers, and networks, each contributing to the mechanical stability of tissues. In this methods chapter, we outline the use of atomic force microscopy (AFM) to investigate collagen's structural properties across multiple hierarchical levels, from single molecules to fibrils. We describe the preparation of collagen samples for AFM analysis of collagen in three forms: proteins, reconstituted fibrils, and tendon-derived fibrils from rat tails. Additionally, we describe a MATLAB-based image analysis method for quantitatively assessing mechanical properties of individual collagen proteins.