TY - JOUR T1 - Unscrambling Fluorophore Blinking for Comprehensive Cluster Detection via Photoactivated Localization Microscopy JF - bioRxiv DO - 10.1101/545152 SP - 545152 AU - Benedikt K. Rossboth AU - Rene Platzer AU - Eva Sevcsik AU - Florian Baumgart AU - Hannes Stockinger AU - Gerhard J. Schütz AU - Johannes B. Huppa AU - Mario Brameshuber Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/06/20/545152.abstract N2 - Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts distorting results. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore for maximal spatial resolution. Using a custom-designed platform we revealed complex blinking of single photoswitchable CFP2 (PS-CFP2) molecules with blinking cycles on time scales of several seconds, which we incorporated in our simulation-based analysis package to robustly evaluate individually recorded PS-CFP2 localization maps for molecular clustering. GRAPHICAL ABSTRACT ER -