High-speed AFM and nano-visualization of biomolecular processes

Abstract

Conventional atomic force microscopes (AFMs) take at least 30-60 s to capture an image, while dynamic biomolecular processes occur on a millisecond timescale or less. To narrow this large difference in timescale, various studies have been carried out in the past decade. These efforts have led to a maximum imaging rate of 30-60 ms/ frame for a scan range of~250 nm, with a weak tip-sample interaction force being maintained. Recent imaging studies using high-speed AFM with this capacity have shown that this new microscope can provide straightforward and prompt answers to how and what structural changes progress while individual biomolecules are at work. This article first compares high-speed AFM with its competitor (single-molecule fluorescence microscopy) on various aspects and then describes high-speed AFM instrumentation and imaging studies on biomolecular processes. The article concludes by discussing the future prospects of this cutting-edge microscopy.

AAA+ Chaperone ClpX Regulates Dynamics of Prokaryotic Cytoskeletal Protein FtsZ

“…The cantilever (Olympus) has a resonant frequency of ϳ1 MHz in water and a spring constant of 0.1-0.2 newton/m. An amorphous carbon tip was grown on the original cantilever tip by electron beam deposition (26). For high speed AFM observation, FtsZ (6 M) was incubated in buffer H at 30°C for 5 min.…”

Structural Changes in Bacteriorhodopsin in Response to Alternate Illumination Observed by High‐Speed Atomic Force Microscopy

“…[13][14][15] Recent advances in fast scanning techniques of AFM have demonstrated that it is possible to observe dynamic behavior of single protein molecules in action. [16] Using high-speed atomic force microscopy (HS-AFM), we recently succeeded in the real-space and real-time observation of light-induced conformational changes of bR under physiological conditions. [17] The photocycle of the wild type at neutral pH proceeds very fast (~10 ms), and hence, the conformational changes cannot be clearly imaged even by our HS-AFM.…”

Structure determination of channel and transport proteins by high-resolution microscopy techniques

“…Such time resolutions only allow slow dynamics and conformational changes of proteins to be captured. Recently, high-speed AFM has been introduced making the visualization of dynamic biomolecular processes possible at millisecond and nanometer resolution (Humphris et al, 2005;Ando et al, 2008).…”