Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy

Abstract

Cy5 is one of a few commercially available dyes in the near-infrared wavelength range. In this study, the fluorescence fluctuations of Cy5 have been investigated under steady-state excitation conditions by fluorescence correlation spectroscopy (FCS). The fluctuations in fluorescence are compatible with and can be used to characterize the photoinduced isomerization and back-isomerization, as well as the transitions between the singlet and triplet states of the dye. By employing a simple kinetic model, the rate constants of these processes can be determined. The model was used over a broad range of experimental conditions, where the influence on the isomerization properties of solvent viscosity, polarity, and temperature, excitation intensity and wavelength, and the presence of different side groups was investigated. We propose FCS as a useful and simple complementary approach to study isomerization processes of cyanine dyes yielding information about the rates of both the photoinduced isomerization and the back-isomerization, as well as of the kinetic properties of the triplet states. Our data show that for most excitation conditions relevant for ultrasensitive fluorescence spectroscopy a photostationary equilibrium is established between the isomeric forms, where approximately 50% of the Cy5 dye molecules can be expected to be in their weakly fluorescent cis states. The fluorophores therefore lose about half of their fluorescence capacity. This is of relevance for the performance of the dye in all applications of fluorescence spectroscopy where a high sensitivity or a fast readout is required, such as in single-molecule detection experiments and in many applications of confocal laser scanning microscopy.

Trans–Cis Isomerization of Lipophilic Dyes Probing Membrane Microviscosity in Biological Membranes and in Live Cells

“…When attached to a lipid membrane, MC540 is likely to display slower isomerization rates, due to the presumably larger viscous drag experienced. This is well in line with previous FCS solution studies, where for the dye Cy5 was found to increase in proportion to the viscosity of the solution 21 . In the FCS curves recorded from the liposome samples one extra exponential relaxation process in the time range of 100 ns could be noticed, reasonably well in agreement with the theoretically expected rotational correlation times ( = 100 ns, from the Debye expression) of the vesicles used (100 nm diameter).…”

“…These findings are well in line with the excitation irradiance dependence of the isomerization parameters and , as recorded by FCS in this work ( Figures 1C,D) and in previous studies. 21,22 Taken together, this allowed us to associate the dark state observed in the TRAST measurements with the isomerization of MC540. The recorded TRAST were found to be well described by a single exponential term according to Equation 10 and 11, indicating the presence of only a single dark state.…”

Mg 2+ -dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

“…t2 is much slower than the relaxation time of Cy5 in the presence of oxygen, which was measured to be on the order of Ϸ1 s (20). However, the triplet-state lifetime of Cy5 might be dramatically lengthened in the absence of oxygen, a very effective triplet-state quencher.…”

“…2A). These relaxations may be tentatively identified as a combination of a trans-cis isomerization and triplet-state relaxation (20). To mimic a situation of direct excitation of Cy5, S15 protein (3-h off rate) was attached to the junction so that the molecule is in the folded configuration, giving high FRET efficiency (17).…”

Studying Ion Exchange in Solution and at Biological Membranes by FCS

“…This changes the fluorescence emission per molecule, and the fluorescence brightness coefficient Q of the studied molecules. This strategy, has been exploited to monitor inter-or intra-molecular dynamics, influenced by fluorescence quenching (Bonnet et al, 1998), (Chattopadhyay et al, 2002), a range of photophysical processes inherent to the fluorophore marker molecules, including triplet state formation (Widengren et al, 1995), photo-isomerisation (Widengren and Schwille, 2000), and photo-induced charge transfer (Widengren et al, 2007), but also other processes influencing fluorescence blinking, such as ion exchange involving ion-sensitive fluorophores, as discussed in this review.…”