Quantification of protein delivery in live cells using fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a quantitative single-molecule method that measures the concentration and rate of diffusion of fluorophore-tagged molecules, both large and small, in vitro and within live cells, and even within discrete cellular compartments. FCS is exceptionally well-suited to directly quantify the efficiency of intracellular protein delivery—specifically, how well different “cell-penetrating” proteins and peptides guide proteinaceous materials into the cytosol and nuclei of live mammalian cells. This article provides an overview of the procedures necessary to execute robust FCS experiments and evaluate endosomal escape efficiencies: preparation of fluorophore-tagged proteins, incubation with mammalian cells and preparation of FCS samples, setup and execution of an FCS experiment, and a detailed discussion of and custom MATLAB® script for analyzing the resulting autocorrelation curves in the context of appropriate diffusion models.