Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy.

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 Commun

journal_title

Nature communications

authors

Platzer R,Rossboth BK,Schneider MC,Sevcsik E,Baumgart F,Stockinger H,Schütz GJ,Huppa JB,Brameshuber M

doi

10.1038/s41467-020-18726-9

subject

Has Abstract

pub_date

2020-10-05 00:00:00

pages

4993

issue

1

issn

2041-1723

pii

10.1038/s41467-020-18726-9

journal_volume

11

pub_type

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