Towards nanoscale metrology for biomolecular imaging by atomic force microscopy

Atomic force microscopy (AFM) provides three-dimensional images with resolution at or near the atomic level. Many researchers have addressed the metrological aspects of AFM imaging of physical samples, producing a range of recognized standards for calibrated spatial measurements. However, because of the complex interplay of forces between an AFM tip and biological samples, the dynamic environment in which such imaging takes place, and difficulties in immobilization, no satisfactory equivalents exist for the biological AFM community. Here an exploration of the effects of AFM imaging parameters on apparent biomolecular dimensions in aqueous environments is carried out for a three-component system comprising GroEL protein, plasmid DNA and gold nanoparticles. The biomolecules in this system have been chosen to present differing imaging challenges, while gold nanoparticles serve as an internal reference for tip performance. Concurrent immobilization of these entities requires optimization of sample preparation methodology, described here as it is hoped the system may act as a template for future bio-metrological standards. Investigation of differences in measured heights and lateral dimensions of DNA, GroEL and gold spheres under a range of imaging conditions and the implications of these results for accurate imaging of biological samples and as a potential bio-metrological standard are discussed.