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

Lookup NU author(s): Emeritus Professor Anthony Harriman, Dr Joshua Karlsson, Dr Alex LaudeORCiD, Dr Michael HallORCiD



This is the authors' accepted manuscript of an article that has been published in its final definitive form by Wiley-VCH Verlag , 2019.

For re-use rights please refer to the publisher's terms and conditions.


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.

Publication metadata

Author(s): Harriman A, Karlsson JKG, Laude A, Hall MJ

Publication type: Article

Publication status: Published

Journal: Chemistry - A European Journal

Year: 2019

Volume: 25

Issue: 65

Pages: 14983-14998

Print publication date: 22/11/2019

Online publication date: 13/09/2019

Acceptance date: 11/09/2019

Date deposited: 14/09/2019

ISSN (print): 0947-6539

ISSN (electronic): 1521-3765

Publisher: Wiley-VCH Verlag


DOI: 10.1002/chem.201904117


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Funder referenceFunder name
BBSRC (BB/K013971/1,
BBSRC BB/M001563/1)
Newcastle University for financial support, including a research scholarship for JKGK