Unscrambling Fluorophore Blinking for Comprehensive Cluster Detection via Photoactivated Localization Microscopy
作者： Eva Sevcsik , Johannes B Huppa , Hannes Stockinger , Rene Platzer , Florian Baumgart
DOI: 10.1101/545152
关键词:
摘要: Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore9s blinking properties to counteract overcounting artifacts that distort resulting biomolecular distributions. Here, we present a readily applicable methodology determine, optimize and quantitatively account for behavior any PALM-compatible fluorophore. Using custom-designed platform revealed complex two photoswitchable fluorescence proteins (PS-CFP2 mEOS3.2) photoactivatable organic fluorophores (PA Janelia Fluor 549 Abberior CAGE 635) with cycles on time scales several seconds. Incorporating such detailed information in our simulation-based analysis package allowed robust evaluation molecular clustering based individually recorded single molecule localization maps.
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