Manipulation and characterization of photo-induced transient states of Merocyanine 540 by fluorescence correlation spectroscopy

In this study, fluorescence correlation spectroscopy (FCS) is used to inves tigate the photo-induced transient states of Merocyanine 540 (MC540), a flu orescent agent for photodynamic therapy. Two relaxation processes are obser ved in the FCS measurements, which can be attributed to trans-cis isomeriza tion and triplet state formation. Under the photostationary conditions pres ent in the detection volume of the FCS measurements, the steady state popul ations of the photo-isomer and the triplet state, as well as their relaxati on rates, can be determined. The population of the triplet states was notic eably reduced by light-induced deactivation at 515 nm excitation, and by si multaneous excitation at 647 nm the triplet state build-up could be almost eliminated with a concomitant increase in fluorescence intensity. By applyi ng a simplified kinetic model for the measured fluorescence fluctuations it is possible to determine the rates for intersystem crossing, triplet state decay, as well as photo-induced isomerization and back-isomerization. In r elation to other present techniques, FCS offers a relatively simple way to monitor photo-induced trans-cis isomerization, and cis-trans back-isomeriza tion. For MC540, it is desirable to increase the triplet state formation at the expense of trans-cis isomerization in order to optimize the photodynam ic action. FCS is well suited to monitor these processes on a microscopic s cale, and thus to follow the local potency of MC540 as a photodynamic agent , at its site of action in target cells.