Spectroscopic and photophysical investigations on the nature of localization of rhodamine-123 and its dibromo derivative in different cell lines

Steady-state and time-resolved spectroscopic properties of rhodamine-123 (rh123) and 4,5-dibromorhodamine methyl ester (dbr123) bound to different cell lines are evaluated. Studies are also performed on the dye bound to extracted mitochondria. Results are compared with those obtained in homogeneous and microheterogeneous media. Results suggest that these dyes can specifically bind only with cell mitochondria. As a result of binding, excitation and emission spectra are red shifted by 10 to 12 nm. The fluorescence decay of these dyes bound to mitochondria shows two lifetimes. Values are about 4.0 and 2.0 ns forrh123 and about 1.9 and 0.5 ns fordbr123. Detailed global analysis of emission wavelength and dye concentration dependences of the fluorescence decay is performed. Results indicate that these dyes are bound to two different binding sites at mitochondria. The decay-associated fluorescence spectrum for the species corresponding to each binding site is recovered. Species1, corresponding to the longer lifetime, is found to be more red shifted compared to species2. The fluorescence of species2 is heavily quenched. The origin of this quenching is explained in terms of resonance energy transfer between donor species2 and acceptor species1. The possible nature of the two binding sites is also discussed.