Photoconversion of Far‐Red Organic Dyes: Implications for Multicolor Super‐Resolution Imaging. Issue 5 (13th March 2018)

• Record Type: Journal Article
• Authors: Dirix, Lieve; Kennes, Koen; Fron, Eduard; Debyser, Zeger; van der Auweraer, Mark; Hofkens, Johan; Rocha, Susana
• Is Part Of: ChemPhotoChem. Volume 2:Issue 5(2018)
• Page Start: 433
• Page End: 441
• Publication Date: 2018-03-13
• Subjects: far-red organic dyes -- fluorescence spectroscopy -- fluorescent probes -- photochromism -- super-resolution fluorescence microscopy; Photochemistry -- Periodicals; Periodicals; Electronic journals; 541.35
• DOI: 10.1002/cptc.201700216
• Languages: English
• ISSNs: 2367-0932

Abstract

Abstract: 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. Abstract : One direction only : Upon intense 561 nm irradiation, various far‐red organic dyes, including Alexa Fluor 647, irreversibly photoconvert to a blue‐shifted emissive derivate. This has severe implications for multicolor super‐resolution microscopy, since this derivative overlaps extensively with the spectral range of photoconverting fluorescent proteins like mEos3.2, typically used as the second color.