The atomic force microscope (AFM) has a unique capability of allowing the high-resolution imaging of biological samples on substratum surfaces in physiological solutions. Recent technological progress of AFM in biological research has resulted in remarkable improvements in both the imaging rate and the tip force acting on the sample. These improvements have enabled the direct visualization of dynamic structural changes and dynamic interactions occurring in individual biological macromolecules, which is currently not possible with other techniques. Therefore, high-speed AFM is expected to have a revolutionary impact on biological sciences. In addition, the recently achieved atomic-resolution in liquids will further expand the usefulness of AFM in biological research. In this article, we first describe the various capabilities required of AFM in biological sciences, which is followed by a detailed description of various devices and techniques developed for high-speed AFM and atomic-resolution in-liquid AFM. We then describe various imaging studies performed using our cutting-edge microscopes and their current capabilities as well as their limitations, and conclude by discussing the future prospects of AFM as an imaging tool in biological research. © 2008 Elsevier Ltd. All rights reserved

A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

F₁ is an adenosine triphosphate (ATP)–driven motor in which three torque-generating β subunits in the α₃β₃ stator ring sequentially undergo conformational changes upon ATP hydrolysis to rotate the central shaft γ unidirectionally. Although extensive experimental and theoretical work has been done, the structural basis of cooperative torque generation to realize the unidirectional rotation remains elusive. We used high-speed atomic force microscopy to show that the rotorless F₁ still “rotates”; in the isolated α₃β₃ stator ring, the three β subunits cyclically propagate conformational states in the counterclockwise direction, similar to the rotary shaft rotation in F₁. The structural basis of unidirectionality is programmed in the stator ring. These findings have implications for cooperative interplay between subunits in other hexameric ATPases.

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.