Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

René Platzer, Benedikt K. Rossboth, Magdalena C. Schneider, Eva Sevcsik, Florian Baumgart, Hannes Stockinger, Gerhard J. Schütz, Johannes B. Huppa () and Mario Brameshuber ()
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René Platzer: Medical University of Vienna
Benedikt K. Rossboth: Institute of Applied Physics, TU Wien
Magdalena C. Schneider: Institute of Applied Physics, TU Wien
Eva Sevcsik: Institute of Applied Physics, TU Wien
Florian Baumgart: Institute of Applied Physics, TU Wien
Hannes Stockinger: Medical University of Vienna
Gerhard J. Schütz: Institute of Applied Physics, TU Wien
Johannes B. Huppa: Medical University of Vienna
Mario Brameshuber: Institute of Applied Physics, TU Wien

Nature Communications, 2020, vol. 11, issue 1, 1-12

Abstract: Abstract 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.

Date: 2020
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DOI: 10.1038/s41467-020-18726-9

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