DOI: 10.1002/cphc.201300874 Increasing the Brightness of Cyanine Fluorophores for Single-Molecule and Superresolution Imaging Kathrin Klehs, [a] Christoph Spahn, [a] Ulrike Endesfelder, [a] Steven F. Lee, [b] Alexandre Fürstenberg,* [c] and Mike Heilemann* [a] In spite of their relatively low fluorescence quantum yield, cya- nine dyes such as Cy3, Cy5, or Cy7 are widely used in single- molecule fluorescence applications due to their high extinction coefficients and excellent photon yields. We show that the fluorescence quantum yield and lifetime of red-emitting cya- nine dyes can be substantially increased in heavy water (D 2 O) compared with water (H 2 O). We find that the magnitude of the quantum yield increase in D 2 O scales with the emission wave- length, reaching a particularly high value of 2.6-fold for the most red-emitting dye investigated, Cy7. We further demon- strate a higher photon yield in single-molecule superresolution experiments in D 2 O compared to H 2 O, which leads to an im- proved localization precision and hence better spatial resolu- tion. This finding is especially beneficial for biological applica- tions of fluorescence microscopy, which are typically carried out in aqueous media and which greatly profit from the red spectral range due to reduced cellular auto-fluorescence. Cyanine dyes, in particular indocarbocyanines, are widely used in fluorescence microscopy and spectroscopy, including in single-molecule fluorescence techniques such as fluorescence energy transfer or superresolution imaging. [1–4] They owe their popularity to their unusually high extinction coefficients and to their availability as different derivatives that cover most of the visible spectrum and that can selectively be attached to biomolecules. [5] The photophysics of cyanines such as Cy3, Cy5, Cy7, and other analogues has therefore been intensively studied over many years. [1, 6–11] A new field of applications for cyanines was opened after the discovery of their light-induced photoswitching capability. With Cy5, in the presence of reducing agents, it was found that a transition into a non-fluorescent and stable off-state could be stimulated by irradiation in the S 1 absorption band, and that this transition could be reverted to the fluorescent on-state by irradiation with light at shorter wavelengths. [12] The photoswitching efficiency was shown to be higher when an ac- tivator fluorophore (not necessarily a cyanine dye) was placed in close vicinity (1–2 nm) of the dye, the activator being thought to act as an antenna. [13] This phenomenon turned cya- nine dyes into excellent fluorophores for a novel kind of mi- croscopy emerging at that time, which enables one to bypass the limit of resolution imposed by diffraction, [14, 15] namely single-molecule localization microscopy (SMLM). In SMLM, the fluorescence emission of the ensemble of fluorophores is con- fined in time by controlled photoswitching of a subset of fluo- rophores, before their spatial position is determined with nanometer accuracy and an image is reconstructed after re- peating the process over many subsets. [16] The mechanism of photoswitching is associated with reversible reduction of the dye [8, 9, 17–19] and multicolor superresolution imaging has been achieved with combinations of multiple spectrally distinct cya- nine dyes. [20] In contrast to their high extinction coefficients, the fluores- cence quantum yields of indocarbocyanines are usually less than 30 % in aqueous media. This has generally been ascribed to non-radiative deactivation via cis–trans isomerization in the excited-state or efficient intersystem crossing to triplet states. [7, 11] Somewhat higher fluorescence quantum yields have nonetheless been observed in organic solvents. [5, 21] Despite their relatively low quantum yield in water, cyanine dyes have been broadly used in biophysical applications owing to their high radiative rates leading to large photon yields when com- pared with other dyes [1, 22] and due to a lack of suitable alterna- tives. Nonetheless, single-molecule fluorescence applications in general demand as many photons as possible, in particular single-molecule-based superresolution techniques in which the achievable spatial resolution scales inversely with the square root of the number of photons emitted per single fluoro- phore. [23] Therefore, even for cyanines any increase in the fluo- rescence quantum yield would be beneficial, especially in the near-infrared where the yields are usually lower due to more efficient internal conversion as the energy gap between the ground and the excited electronic states decreases and Franck–Condon factors increase (energy gap law). [24] Whereas others have tried to improve the photon yield of cyanines by chemically modifying the dye molecules, [25] we in- vestigate in the present work how the fluorescence quantum [a] K. Klehs, C. Spahn, Dr. U. Endesfelder, Prof. Dr. M. Heilemann Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Max-von-Laue-Str. 7 60438 Frankfurt (Germany) E-mail : heilemann@chemie.uni-frankfurt.de [b] Dr. S. F. Lee Department of Chemistry University of Cambridge Lensfield Road, Cambridge, CB2 1EW (United Kingdom) [c] Dr. A. Fürstenberg Department of Human Protein Sciences University of Geneva CMU, Rue Michel-Servet 1 1211 Genve 4 (Switzerland) E-mail : alexandre.fuerstenberg@unige.ch Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201300874.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2014, 15, 637 – 641 637 CHEMPHYSCHEM COMMUNICATIONS