Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy

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.