The most straightforward approach to understanding how proteins function is to observe the individual molecules directly while they are performing their function. To achieve this objective, I spearheaded the development of high-speed (HS) AFM, which was initiated in 1993 and completed in 2008 [1, 2].

HS-AFM does not disrupt the structure and function of fragile molecules when the imaging rate is around 10 frames per second (fps). Since 2008, my group has been observing the dynamic processes of various proteins [3]. Alongside the commercial production and dissemination of HS-AFM systems by RIBM and Asylum, HS-AFM imaging studies on biological molecules have increased rapidly worldwide [4, 5].

The presentation will focus on findings derived from HS-AFM imaging of protein molecules, with particular emphasis on the energy-transducing myosin V motor [6, 7], early endosome antigen 1 (EEA1) interacting with Rab5-GTP (Fig.1A) and membranes, axleless F1-ATPase hydrolyzing ATP (Fig. 1B) [8], and intrinsically disordered proteins [9].

Following this, the potential of HS-AFM research in the context of emerging time-resolved cryo-electron microscopy [10,11] and cryo-electron tomography [12] will be discussed. Finally, recent endeavors to increase the imaging rate of HS-AFM to 100–200 fps [13] will be presented.

Fig. 1 High-speed AFM images showing conformational changes of EEA1 upon binding to Rab5-GTP (A) and the α3β3 sub-complex of F1-ATPase hydrolyzing ATP (B). The red circles in (B) indicate the highest pixel positions in respective images.

References
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2. Ando, T. et al. Prog. Surf. Sci. 83, 337-437 (2008). DOI: 10.1016/j.progsurf.2008.09.001
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