Comparison between fluorescence correlation spectroscopy and time-resolvedfluorescence anisotropy as illustrated with a fluorescent dextran conjugate

The motional properties of rhodamine green alone and conjugated to 10-kDa d extran have been studied by fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA). With FCS the translational d iffusion times of the fluorescent particles can be determined, which are di rectly proportional to the shear viscosity as shown in aqueous solutions of different sucrose concentrations. With TRFA the rotational correlation tim es of the fluorescent particles can be determined. TRFA experiments in the case of fluorescent dextran reveal a distinct restricted internal motion of the fluorescent probe independent of the slower overall rotation of the po lysaccharide. The fast depolarization is most likely due to internal motion and not to energy transfer between different rhodamine green molecules in the same dextran, since a higher viscosity of the solvent increases the cor relation time for internal motion proportionally. FCS and TRFA yield comple mentary information in the sense that the correlation time for overall dext ran rotation can be accurately determined from the translational diffusion coefficient.