Photoactivatable Fluorescent Protein mEos2 Displays Repeated Photoactivation after a Long-Lived Dark State in the Red Photoconverted Form

Illumination with 405 nm light can recover the emission for single green fluorescent protein (GFP) mutants that have gone into a long-lived dark state. The reported behavior is the standard for the reverse photoswitchable protein Dronpa and its mutants. However, conventional knowledge regarding the mEos2 photoactivatable fluorescent protein (PA-FP) is that, once bleached, this fluorophore is hardly reactivated, aside from a minority population that might display this behavior. Here we show that, in a typical experiment approximately 50% of the investigated single molecule time traces display multiple reactivations, making this a seemingly inherent feature of the mEos2 PA-FP. These results hint to some similarities between mEos2 and other reversibly photoactivatable probes such as Dronpa. We investigate the consequences of this phenomenon in the framework of photoactivated localization microscopy (PALM) experiments.