Spectral characterization of fluorescent 5-iodoacetamidotetramethylrhodamine and its N-acetylcysteine derivative

This study describes a spectroscopic characterization of the fluorescent pr obe 5-iodoacetamidotetramethylrhodamine and its N-acetylcysteine derivative in various solvents. The monomeric character and the purity of the two com pounds was etablished in ethanol, ethanol-water, water, dimethylformamide, dimethyl sulfoxide, glycerol, polyvinylalcohol and nitrocellulose films, us ing a combination of absorption and fluorescence spectroscopy and mass spec trometry. In all the polar solvents studied here, the probes were present i n the open form only and thus, from the spectral parameters, the natural li fetime and Forster distance in those solvents could be calculated. Fluoresc ence lifetimes were determined and the homogeneity of probe distribution in both stretched and unstretched polymer films was checked. For this, a comb ination of confocal microscopy and polarized fluorescence experiments was u sed. Finally, applying angle-resolved fluorescence depolarization for immob ilized molecules in uniaxially stretched polymer films, the orientation of both rhodamines in stretched polyvinylalcohol films was examined and the or ientation of the transition dipole moments within the molecular frame was d etermined. There were no differences between the 5-isomer and its N-acetylc ysteine derivative, but a strong environmental dependence of most optical p roperties was observed. This dependence was especially intriguing for the d ifference angle between the absorption and emission dipole moment. The impl ications of our results and suggestions for future research are discussed.