Super-Resolution Imaging Conditions for enhanced Yellow Fluorescent Protein (eYFP) Demonstrated on DNA Origami Nanorulers

Photostability is one of the crucial properties of a fluorophore which strongly influences the quality of single molecule-based super-resolution imaging. Enhanced yellow fluorescent protein (eYFP) is one of the most widely used versions of fluorescent proteins in modern cell biology exhibiting fast intrinsic blinking and reversible photoactivation by UV light. Here, we developed an assay for studying photostabilization of single eYFP molecules with respect to fast blinking and demonstrated a 6-fold enhanced photostability of single eYFP molecules with a beneficial influence on the blinking kinetics under oxygen removal and addition of aliphatic thiols (dSTORM-buffer). Conjugation to single stranded DNA and immobilization via DNA hybridization on a DNA origami 12 helix bundle in aqueous solution allowed photophyiscal studies of eYFP at the single-molecule level and at close to physiological conditions. The benefit of improved photophysical properties for localization-based super-resolution microscopy is demonstrated and quantitatively characterized by imaging 12 helix bundle DNA origami nanorulers with binding sites at designed distances of 160 and 100 nm and by imaging microtubules in fixed mammalian Vero cells.