Diffusion and molecular binding in crowded vesicle solutions measured by fluorescence correlation spectroscopy

Abstract

Fluorescence Correlation Spectroscopy (FCS) in concentrated complex media such as vesicle or colloidal suspensions is obscured by light scattering and hydrodynamic crowding effects. Nevertheless, quantitative measurements are desirable for intracellular live cell measurements and studies on pharmaceutical or food products. Here we study the diffusion of green fluorescent protein (GFP) and fluorescently labeled antibodies (IGG) in suspensions of PEGylated liposomes. As a function of vesicle concentration the apparent particle number in the focal volume, as well as the structure parameter increase. This finding is consistent with a distortion of the focal volume due to scattering. We also find that the diffusion times of the proteins increase in agreement with theory of diffusion in concentrated solutions of hard spheres. Taking both effects into account we are able to demonstrate using Annexin V binding to PS vesicles that FCS binding experiments yield the same binding constant in buffer and in concentrated liposome solutions.