High-Speed Nanomechanical Mapping of the Early Stages of Collagen Growth by Bimodal Force Microscopy

High-speed atomic force microscopy (AFM) enabled the imaging of protein interactions with millisecond time resolutions (10 fps). However, the acquisition of nanomechanical maps of proteins is about 100 times slower. Here, we developed a high-speed bimodal AFM that provided high-spatial resolution maps of the elastic modulus, the loss tangent, and the topography at imaging rates of 5 fps. The microscope was applied to identify the initial stages of the self-assembly of the collagen structures. By following the changes in the physical properties, we identified four stages, nucleation and growth of collagen precursors, formation of tropocollagen molecules, assembly of tropocollagens into microfibrils, and alignment of microfibrils to generate microribbons. Some emerging collagen structures never matured, and after an existence of several seconds, they disappeared into the solution. The elastic modulus of a microfibril (similar to 4 MPa) implied very small stiffness (similar to 3 X 10(-6) N/m). Those values amplified the amplitude of the collagen thermal fluctuations on the mica plane, which facilitated microribbon build-up.

Automated summary

High-speed bimodal AFM was developed to provide high-spatial resolution maps of protein nanomechanics, allowing rapid imaging of the initial stages of collagen self-assembly and identification of four stages of change.