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 wavele ngth range. In this study, the fluorescence fluctuations of Cy5 have been i nvestigated under steady-state excitation conditions by fluorescence correl ation spectroscopy (FCS). The fluctuations in fluorescence are compatible w ith 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 prope rties 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 iso merization processes of cyanine dyes yielding information about the rates o f both the photoinduced isomerization and the back-isomerization, as well a s of the kinetic properties of the triplet states. Our data show that for m ost excitation conditions relevant for ultrasensitive fluorescence spectros copy a photostationary equilibrium is established between the isomeric form s, where approximately 50% of the Cy5 dye molecules can be expected to be i n their weakly fluorescent cis states. The fluorophores therefore lose abou t half of their fluorescence capacity. This is of relevance for the perform ance of the dye in all applications of fluorescence spectroscopy where a hi gh sensitivity or a fast readout is required, such as in single-molecule de tection experiments and in many applications of confocal laser scanning mic roscopy.