Proteins are essential for life. Most vital phenomena are governed and coordinated by the activity of proteins and their networks. Proteins are nano-machines. They are tiny molecules but perform sophisticated functions infeasible with man-made machines. To understand how they work is crucial in learning the strategies that life has adopted and eventually creating useful nano-machinery. To this end, various approaches have been developed and used. The detailed structure of numerous proteins has been revealed but limited to static snapshots, while proteins are dynamic in nature. The dynamic behavior of individual protein molecules has been studied by single-molecule fluorescence microscopy but protein molecules themselves are invisible in the observation. This means that we have to infer how proteins operate to function from gleaned data with significant resolution gaps. Directly observing individual protein molecules in action at high spatiotemporal resolution has therefore been a holy grail for biological science. This long-quested dream has now come true by high-speed atomic force microscopy (HS-AFM) that we have developed [1-4].

Unlike single-molecule fluorescence microscopy, various dynamic events of molecules appear in an AFM movie without planned selection. Therefore, we can learn the details of several facts of molecular action in one imaging experiment. Even for biomolecular systems that have already been studied in detail by other approaches, HS-AFM allows us to inquire into more details or questions that have been difficult or impossible to address by other methods. Moreover, the visualized molecular action that appears on video can often be interpreted straightforward without intricate analyses and interpretations, facilitating the attainment of convincing conclusions. These excellent general features of HS-AFM imaging markedly facilitate and accelerate our understanding of the functional mechanism of proteins. This talk will present the fundamentals of this microcopy, recently filmed images of proteins in action, and ongoing and future studies to expand the scope of its application.