Time-dependent rotational rates of excited fluorophores. A linkage between fluorescence depolarization and solvent relaxation

It is generally assumed that the rotational diffusion coefficients of fluorophores are independent of time subsequent to excitation, and that the rotational diffusion coefficients of the ground and the excited states are the same. We now describe a linkage between the extent of solvent relaxation and the rate of fluorescence depolarization. Specifically, if a fluorophore displays time-dependent solvent relaxation it may also show a time-dependent decrease in its rotational rate. A decreased rate of rotation could result from the increased interaction with polar solvent molecules which occurs as a result of solvent relaxation. The decays of anisotropy predicted from our model closely mimic those often observed for fluorophores which are bound to macromolecules. For example, the decays are more complex than a single exponential, and the time-resolved anisotropy can display a limiting value which does not decay to zero. The effect of solvent relaxation upon the rates of rotational diffusion is expected to be most dramatic for solvent-sensitive fluorophores in a viscous environment. These conditions are frequently encountered for fluorophore-macromolecule complexes. Consideration of the linkage between solvent relaxation and rotational diffusion leads to two unusual predictions. First even spherical fluorophores in an isotropic environment could display multi- or nonexponential decays of fluorescence anisotropy. Secondly, for the special case in which the fluorophore dipole moment decreases upon excitation, the theory predicts that the anisotropy decay rate may increase with time subsequent to pulsed excitation. The predictions of this theory are consistent with published data on the effects of red-edge excitation upon the apparent rotational rates of fluorophores in polar solvents.