Remarkably, the bR-bR interaction in the transiently formed assembly elicits both positive and negative cooperative effects on the decay kinetics as the initial bR recovers. By the bipolar nature of the cooperativity, however, the turnover rate of the phtocycle is maintained constant on average, irrespective of the light intensity.Thus, the direct and high-resolution visualization of dynamically acting molecules is a powerful new approach to gaining insight into elaborate bimolecular processes. 2 The biological function of proteins is closely associated with their ability to undergo structural changes. In many cases, these structural changes are triggered by external stimuli including pH, temperature, ligand binding, mechanical stress, and light.Although their direct real-space and real-time visualization is a straightforward approach to understanding the dynamic molecular processes, the lack of suitable techniques has precluded it. Atomic force microscopy (AFM) is a versatile technique to image proteins in liquids at sub-molecular resolution, but its poor temporal resolution has meant an availability of only static or slow time-lapse images of proteins [1][2][3][4][5] . In the last decade, various efforts have been carried out to increase the scan speed of AFM 6-9 .As a result, single protein molecules exhibiting Brownian motion are captured on video at a highest temporal resolution of ~30 ms 10 . However, dynamic visualization of physiologically relevant conformational changes in proteins has been difficult because tip-sample interaction tends to interfere with the physiological functions. To solve this problem, a new method has recently been developed which allows fast and precise control of the tip-sample distance with a minimum load to the sample 7 . This report presents the first ever exemplification of dynamic imaging of a functioning biological sample.Bacteriorhodopsin (bR) is a well-known example of the association between stimulus-triggered structural dynamics and biological function 11,12 , and its direct visualization has long been a goal. bR contains seven transmembrane α-helices (named A-G) enclosing the chromophore retinal 13,14 . In the photocycle, a series of spectral intermediates, designated J, K, L, M, N, and O, occur in that order 12 . The light-induced conformational changes in bR have been investigated by various methods 15-25 , leading to a consensus that the proton channel at the cytoplasmic surface is opened by the tilting of helix F away from the protein center 21,23,24 . Sass et al. reported helix F displacement of ~0.1 nm in the late M state, based on X-ray diffraction of the three-dimensional crystal of wild type (WT) 21 . However, a larger structural change in bR was reported in 3 the electron crystallography study of the D96G, F171C, F219L triple mutant of bR: displacement of helix F by ~0.35 nm away from the center of the protein 23 . The electron crystallography study of the F219L mutant further reported that helices E and F tilt away from the center of the protein, which is ...