Premium
Photoconversion of Far‐Red Organic Dyes: Implications for Multicolor Super‐Resolution Imaging
Author(s) -
Dirix Lieve,
Kennes Koen,
Fron Eduard,
Debyser Zeger,
van der Auweraer Mark,
Hofkens Johan,
Rocha Susana
Publication year - 2018
Publication title -
chemphotochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.13
H-Index - 18
ISSN - 2367-0932
DOI - 10.1002/cptc.201700216
Subject(s) - alexa fluor , fluorescence , far red , resolution (logic) , intramolecular force , microscopy , photochemistry , laser , irradiation , chemistry , materials science , optics , red light , physics , stereochemistry , botany , artificial intelligence , computer science , nuclear physics , biology
Localization‐based super‐resolution microscopy has become an indispensable tool in biology to study features smaller than the diffraction limit of light. In a multicolor approach, adequate spectral separation of the different photoswitchable probes is required. A far‐red emitting dye is often one of the labels of choice. However, irradiation with high laser intensity can induce photo‐conversion of some of the most frequently used fluorophores. Herein we show that upon intense irradiation with a 561 nm laser line, far‐red organic dyes photoconvert to blue‐shifted emissive species. In the case of Alexa Fluor 647, the most commonly used fluorescent label in super‐resolution microscopy, this derivative is created over time in an intramolecular, irreversible photoinduced chemical reaction. The dynamics of this reaction are altered by the presence of reducing agents. Importantly, the blue‐shifted derivatives emit in the spectral range of the red fluorescent proteins (e. g. PAmCherry and converted mEos3.2), severely implicating multicolor super‐resolution imaging.