Superresolution Microscopy on the Basis of Engineered Dark States

Abstract

New concepts for superresolution fluorescence microscopy by subsequent localization of single molecules using photoswitchable or photoactivatable fluorophores are rapidly emerging and provide new ways to resolve structures beyond the diffraction limit. Here, we demonstrate that superresolution imaging can be carried out with practically every single-molecule compatible, synthetic fluorophore by controlling their emission properties. We prepare dark states by removing oxygen that extends the triplet state lifetime to several milliseconds. We further increase the duration of the off-states using electron transfer reactions to create radical ion states of severalfold longer lifetimes. Imaging single molecules, actin filaments, and microtubules in fixed cells as well as simulations demonstrate that the thus created dark states are sufficiently long for resolution of approximately 50 nm.