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Chemistry. 2019 Sep 13. doi: 10.1002/chem.201904117. [Epub ahead of print]

Photoisomerization of the Cyanine Dye Alexa-Fluor® 647 (AF-647) in the Context of dSTORM Super-Resolution Microscopy.

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University of Newcastle, Department of Chemistry, Bedson Building, NE1 7RU, Newcastle upon Tyne, UNITED KINGDOM.
Newcastle University, UNITED KINGDOM.


Cyanine dyes, as used in super-resolution fluorescence microscopy, undergo light-induced "blinking", enabling localization of fluorophores with spatial resolution beyond the optical diffraction limit. Despite a plethora of studies, the molecular origins of this blinking are not well understood. Here, we examine the photophysical properties of a bio-conjugate cyanine dye (AF-647), used extensively in dSTORM imaging. In the absence of a potent sacrificial reductant, light-induced electron transfer and intermediates formed via the meta-stable, triplet-excited state are considered unlikely to play a significant role in the blinking events. Instead, it is found that, under conditions appropriate to dSTORM microscopy, AF-647 undergoes reversible photo-induced isomerization to at least two long-lived dark species. These photo-isomers are characterized spectroscopically and their interconversion probed by computational means. The first-formed isomer is light sensitive and transforms to a longer-lived species in modest yield that could be involved in dSTORM related blinking. Permanent photo-bleaching of AF-647 occurs with very low quantum yield and is partially suppressed by the anaerobic redox buffer.


cyanine dyes; electron transfer; fluorescence; photochemistry; super-resolution microscopy


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