Near-field optical and shear-force microscopy of single fluorophores and DNA molecules

Abstract

Photodynamics of individual fluorescence molecules has been studied using an aperture-type near-field scanning optical microscope with two channel fluorescence polarisation detection and tuning fork shear-force feedback. The position of maximum fluorescence from individual molecules could be localised with an accuracy of 1 nm. Dynamic processes such as translational and rotational diffusion were observed for molecules adsorbed to a glass surface or embedded in a polymer host. The in-plane molecular dipole orientation could be determined by monitoring the relative contribution of the fluorescence signal in the two perpendicular polarised directions. Rotational dynamics was investigated on 10 ms–1000 s timescale. Shear-force phase feedback was used to obtain topographic imaging of DNA fragments, with a lateral and vertical resolution comparable to scanning force microscopy. A DNA height of 1.4 nm has been measured, an indication of the non-disturbing character of the shear force mechanism.