Maximizing far-field optical microscopy resolution through selected fluorophore transitions

Abstract

Stimulated emission depletion (STED) microscopy and related nanoscopy techniques, which utilize a saturable optical transition between a bright and a dark state, overcome the diffraction barrier by confining one of the states to an area smaller than the Airy disk. Scanning this area across the specimen yields subdiffraction images by registering inseparable fluorescent markers sequentially in time. Despite the progress made in nanoscopy so far, maximizing the resolution has been hampered by the efficiency of the utilized optical transition and the photostability of the fluorophores. Here, the optical transition responsible for breaking the barrier was studied in order to maximize its efficiency. For a range of fluorophores (dyes, proteins, quantum dots, color centers) the nature of the responsible process could be clarified. It was also investigated whether heat could serve as an imaging contrast to provide an alternative to fluorescence. This work demonstrates a resolving power of down to 6 nm in unprocessed recordings, corresponding to lambda/135, which is to date the highest obtained in far-field optics. These measurements, which show no sign of photobleaching or blinking, were performed with diamond color centers using STED and ground state depletion (GSD) microscopy.