Coordinate-targeted and coordinate-stochastic super-resolution microscopy with the reversibly switchable fluorescent protein dreiklang Journal Article


Author(s): Jensen, Nickels A; Danzl, Johannes G; Willig, Katrin I; Lavoie-Cardinal, Flavie; Brakemann, Tanja; Hell, Stefan W; Jakobs, Stefan
Article Title: Coordinate-targeted and coordinate-stochastic super-resolution microscopy with the reversibly switchable fluorescent protein dreiklang
Affiliation
Abstract: Diffraction-unlimited far-field super-resolution fluorescence (nanoscopy) methods typically rely on transiently transferring fluorophores between two states, whereby this transfer is usually laid out as a switch. However, depending on whether this is induced in a spatially controlled manner using a pattern of light (coordinate-targeted) or stochastically on a single-molecule basis, specific requirements on the fluorophores are imposed. Therefore, the fluorophores are usually utilized just for one class of methods only. In this study we demonstrate that the reversibly switchable fluorescent protein Dreiklang enables live-cell recordings in both spatially controlled and stochastic modes. We show that the Dreiklang chromophore entails three different light-induced switching mechanisms, namely a reversible photochemical one, off-switching by stimulated emission, and a reversible transfer to a long-lived dark state from the S1 state, all of which can be utilized to overcome the diffraction barrier. We also find that for the single-molecule- based stochastic GSDIM approach (ground-state depletion followed by individual molecule return), Dreiklang provides a larger number of on-off localization events as compared to its progenitor Citrine. Altogether, Dreiklang is a versatile probe for essentially all popular forms of live-cell fluorescence nanoscopy.
Keywords: dreiklang; fluorescent proteins; nanoscopy; photoswitching; resolution
Journal Title: ChemPhysChem
Volume: 15
Issue 4
ISSN: 1439-7641
Publisher: Wiley-Blackwell  
Date Published: 2014-03-17
Start Page: 756
End Page: 762
DOI: 10.1002/cphc.201301034
Open access: no