Atomic Force Microscopy Using Thermal Fluctuation of Cantilever for Observing Soft Biological Macromolecules

In atomic force microscopy (AFM), the detection of interactions between the probe tip and the sample surface is the basis of topography imaging. It is crucial to minimize the tip-sample interaction to prevent deformation or damage to soft biological macromolecules in AFM imaging. Here, a scanning mode of AFM was developed to image soft biological macromolecules with a sub-10 pN load force. In this scanning mode, the reduction in the thermal fluctuation of the cantilever was monitored at subnanometer tip-sample separations, where interaction with the sample begins to influence cantilever behavior. The height of the sample was controlled such that the magnitude of the thermal fluctuation of the cantilever remained constant. The magnitudes of the fluctuations and forces in this scanning mode were formulated within the framework of statistical mechanics. It was estimated that the repulsive force acting between the probe tip and sample surface originated largely from the entropic effect of the reduction in cantilever fluctuation. Two proteins (GroEL and bacteriorhodopsin) were imaged to demonstrate the capability of this scanning mode for acquiring topography with a marginal load force. The fragile double-ring structure of GroEL was preserved after repeated scans. The flexible C-terminal region of bacteriorhodopsin was clearly visualized. Thus, the presented AFM imaging mode is highly noninvasive for soft and fragile biological macromolecules.