Unscrambling Fluorophore Blinking for Comprehensive Cluster Detection via PALM

ABSTRACT 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.