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High-speed atomic force microscopy (HS-AFM) enables direct nanometer resolution visualisation of single molecules and molecular assemblies in real-time and under physiological conditions, providing unique insights into how biomolecules move and interact. Recent developments in methods and complementarity with other techniques have expanded its applicability to increasingly complex and physiologically relevant systems, bridging gaps between static structural methods and functional dynamics. Here, I present developments in HS-AFM methods and their application to understanding organisation, conformational dynamics, and interactions. By capturing transient events inaccessible to ensemble approaches, HS-AFM is establishing itself as a vital component of the biophysics toolkit for studying the structural dynamics that allow biomolecules to function.
