Gold-nanorod-enhanced FCS of fluorophores with high quantum yield in lipid bilayers

Abstract

Plasmonic fluorescence enhancement is used to study fluorescence correlation spectroscopy (FCS) at higher concentrations than in regular diffraction-limited FCS experiments. Previous studies suffered from sticking to the substrate and were performed mainly with poorly emitting dyes. A lipid bilayer forms a passivating surface preventing sticking of the dye or the protein and allows for specific anchoring of probe molecules. For dyes with high quantum yields, the fluorescence background of unenhanced molecules is high, and the fluorescence enhancement is weak, less than about 10. Nonetheless, we show that FCS is possible at micromolar concentrations of the probe molecule. Enhanced FCS is recorded by selecting signals on the basis of their shortened lifetime. This selection enhances the contrast of the correlation by more than an order of magnitude. The lipid bilayer can be used to anchor biomolecules and perform enhanced FCS, as we show for a dye-labeled protein.