Fluorescence Anisotropy Measurements of Fluoroscein Mixed with Ficoll Solutions

Abstract

We have conducted fluorescence anisotropy measurements of fluorescein fluorophores embedded in Ficoll solutions. Ficoll, a highly branched, hydrophilic polysaccharide, is used as a model crowding agent for studies of the effects of crowding on the behavior of nanoparticles. Fluorescein is a bright fluorophore with well-known fluorescence spectrum. We have collected the measurements with an ISS K2 frequency-domain fluorometer (470-nm excitation wavelength and 1-100 MHz modulation frequency), and determined the fluorescence lifetime, the fluorescence anisotropy, and the rotational correlation coefficient of the fluorescein as a function of ficoll concentration (up to 1200 mg/ml.) As the ficoll concentration is increased, the lifetime of the fluorophores show little change from 4.08 in water to 4.23 in 1200 mg/ml ficoll. However, the rotational correlation coefficient measurements show significant changes. At low ficoll concentrations no measureable interactions was observed. As the ficoll concentration is increased, two rotational modes emerge for better fitting of the data, which can be associated with free fluorophores and fluorophore-ficoll interactions. Above 1000 mg/ml ficoll solutions the data can be fit with a single rotational mode due to slowing down of the rotation of fluorophores. We compare the results from the fluorescence anisotropy and lifetime measurements with those from viscosity measurements and fluorescence correlation spectroscopy measurements, the latter being related to the translation diffusion.