Imaging single fluorophores at very high resolution by near-field optical microscopy

Abstract

A new probe for scanning near-field optical microscopy (SNOM) that exploits field concentration at a metal tip allows to image single dye molecules at a resolution far beyond the diffraction limit. This probe combines the high resolution of the apertureless SNOM with the single molecule sensitivity of the aperture SNOM. An image of single Cy-3 fluorophores covalently attached to the termini of DNA. In fluorescence, the dyes appear as characteristic patterns with details of 10 nm in width. The patterns indicate field concentration at the metal tip and simultaneously fluorescence quenching. Modeling of the data allows determining the position of the dyes at sub-nm accuracy and, at the same time, the 3-D orientation of the dyes. As an important feature of our probe, the metal tip simultaneously provides high-resolution topographic imaging. The evaluation of two independent co-localized signals (fluorescence and topography) allows a much clearer interpretation of the sample than possible with one signal alone. With this unique combination of qualities the new probe promises exciting applications in various fields of nanoscience, in particular in molecular and cellular biology