Unscrambling Fluorophore Blinking for Comprehensive Cluster Detection via PALM
Benedikt K. Rossboth, Rene Platzer, Eva Sevcsik, Florian Baumgart, Hannes Stockinger, Gerhard J. Schütz, Johannes B. Huppa and Mario Brameshuber
Received Date: 23rd February 19
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. Employing a custom-designed platform we accurately determined complex blinking behavior of single photoswitchable CFP2 molecules with blinking cycles on time scales of several seconds. We demonstrate the necessity to identify and incorporate outlier fluorophore blinking behavior in the evaluation of individually recorded localization maps and present a readily applicable simulation-based methodology to do so for any PALM-compatible fluorophore.
Read in full at bioRxiv.
This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.