High-speed atomic force microscopy (HS-AFM) is a powerful technique that provides dynamic movies of biomolecules at work. We successfully used HS-AFM to take movies – and determine dynamic parameters – of membrane trafficking systems such as ESCRT-III and clathrin, transporters and channels.

To break current temporal limitations to characterize molecular dynamics using HS-AFM, we developed HS-AFM height spectroscopy (HS-AFM-HS), a technique whereby we oscillate the HS-AFM tip at a fixed position and detect the motions of the molecules under the tip. This gives sub- nanometer spatial resolution combined with microseconds temporal resolution of molecular fluctuations. HS-AFM-HS can be used in conjunction with HS-AFM imaging modes, thus giving access to a wide dynamic range.

To break current resolution limitations, we developed Localization AFM (LAFM). By applying localization image reconstruction algorithms to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius and reach quasi-atomic resolution on soft protein surfaces in native and dynamic conditions. The LAFM method allows the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, opening new avenues for single molecule structural analysis.