Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps. Determining molecular clustering in Photoactivated Localization Microscopy (PALM) experiments requires knowledge of the blinking properties of the fluorophore to prevent overcounting artefacts. Here the authors develop an experimental and analytical framework to determine the blinking parameters of fluorophores and incorporate this information into cluster analysis.