The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging

Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. 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.