Abstract

Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. We introduce a general approach for multicolor super-resolution fluorescence imaging based on photoswitching of standard small organic fluorophores. Photoswitching of organic rhodamine and oxazine fluorophores, i.e. the reversible transition from a fluorescent to a non-fluorescent state in aqueous buffers exploits the formation of long-lived radical anions through reaction with thiol compounds (e.g. glutathione or mercaptoethylamine) and repopulation of the singlet ground state by reaction with molecular oxygen. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. We unravel the underlying switching mechanism and demonstrate super-resolution imaging with different commercially available organic fluorophores with ~ 20 nm optical resolution in fixed and living cells. Combining the genetic labeling approach with small, bright and photostable organic fluorophores represents the method of choice for future super-resolution imaging and precision colocalization experiments.