Comprehensive Fluorophore Blinking Analysis Platform as a Prerequisite for Cluster Detection via Photoavticated Localization Microscopy

Determining nanoscale protein distribution via Photoactivation Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore's photophysics. If not accounted for, blinking of dyes on time-scales of typical PALM experiments will invariably cause overcounting artifacts, which become even more pronounced in fixed cells with predominantly immobile proteins. Here, we developed a lipid bilayer-based imaging platform as a means to determine the blinking behavior of two fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE of two photoswitchable 635). For all investigated fluorophores we revealed blinking cycles on time scales of several seconds. Our strategy is amenable to determining blinking signatures for any fluorophore of choice to support robust conclusions related to PALM. Furthermore, the gained blinking statistics can be utilized to discriminate clustered from randomly distributed membrane proteins.