The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450–650 kHz) and small spring constants (150–280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 × 100 pixel ² image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm ).

High-speed atomic force microscopy can be used to record the structure and dynamics of biomolecules simultaneously. These authors use this method to directly observe the dynamics of the motor protein myosin V moving along actin filaments, with unprecedented time resolution. The high-resolution movies provide evidence supporting the 'swinging lever-arm' model of myosin motility, and provide important insights into the mechanism of motor movement.

Directly observing individual protein molecules in action at high spatiotemporal resolution has long been a holy grail for biological science. This is because we long have had to infer how proteins function from the static snapshots of their structures and dynamic behavior of optical makers attached to the molecules. This limitation has recently been removed to a large extent by the materialization of high-speed atomic force microscopy (HS-AFM). HS-AFM allows us to directly visualize the structure dynamics and dynamic processes of biological molecules in physiological solutions, at subsecond to sub-100-ms temporal resolution, without disturbing their function. In fact, dynamically acting molecules such as myosin V walking on an actin filament and bacteriorhodopsin in response to light are successfully visualized. In this review, we first describe theoretical considerations for the highest possible imaging rate of this new microscope, and then highlight recent imaging studies. Finally, the current limitation and future challenges to explore are described.

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9–RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.