Characterizing Fluorescently Labeled Viral-Like Particles by Fluorescence Imaging

Abstract

Previous studies with fluorescence fluctuation spectroscopy (FFS) on viral-like particles (VLP) formed from fluorescently labeled Gag proteins demonstrated that the VLP samples contain more than a single brightness species. This observation holds both for human immunodeficiency virus type 1 (HIV-1) VLPs and for human T-cell leukemia virus type 1 (HTLV-1) VLPs. Thus, the FFS results imply that the Gag stoichiometry of VLPs is heterogeneous. While FFS analysis identified two brightness species for the VLP samples, the technique cannot differentiate between discrete populations and a broad distribution of Gag stoichiometries. In an effort to characterize the underlying brightness distribution, we combine FFS studies with fluorescence imaging of a VLP population. We first perform a brightness characterization of the VLPs by FFS. Next, the particles from the same sample are immobilized on the surface of a glass coverslip and fluorescence images of the immobile VLPs are collected. The intensity of individual particles is determined by image analysis. We found that the intensity of particles is heterogeneous, which confirms the existence of a distribution of Gag stoichiometries. We characterize the histogram of intensity values and compare it with the FFS results. In addition, we explore the use of particle tracking to determine the diffusion coefficient and the size of the particles by imaging mobile VLPs in a thin sample layer with the goal of relating brightness and size of particles. Combining FFS and fluorescence imaging offers a promising tool for the characterization of protein stoichiometry in viral particles and the heterogeneity. This work was supported by a grant from the National Institutes of Health (R01 GM64589).