A triplet state mechanism of primed conversion enables rational engineering of photoconvertible fluorescent proteins for dual color fluorescence nanoscopy
 

A triplet state mechanism of primed conversion enables rational engineering of photoconvertible fluorescent proteins for dual color fluorescence nanoscopy


Photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs. Among these, pr-mEos2 exhibits excellent properties for primed PALM, allowing a new dual color PALM modality that does not occupy additional spectral space.

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