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.
journal_name
Nat Communjournal_title
Nature communicationsauthors
Platzer R,Rossboth BK,Schneider MC,Sevcsik E,Baumgart F,Stockinger H,Schütz GJ,Huppa JB,Brameshuber Mdoi
10.1038/s41467-020-18726-9subject
Has Abstractpub_date
2020-10-05 00:00:00pages
4993issue
1issn
2041-1723pii
10.1038/s41467-020-18726-9journal_volume
11pub_type
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