Single molecule confocal fluorescence lifetime correlation spectroscopy for accurate nanoparticle size determinationElectronic supplementary information (ESI) available: Advantage of the FLCS measurement, laser excitation power-dependent fluorescence lifetimes of Alexa 488 dyes, and SEM images of the beads. See DOI: 10.1039/c4cp01197j

We report on an experimental procedure in confocal single molecule fluorescence lifetime correlation spectroscopy (FLCS) to determine the range of excitation power and molecular or particulate concentration in solution under which the application of an unmodified model autocorrelation function is justified. This procedure enables fitting of the autocorrelation to an accurate model to measure diffusion length ( r ) and diffusion time ( τ D ) of single molecules in solution. We also report on the pinhole size dependency of r and τ D in a confocal FLCS platform. This procedure determines a set of experimental parameters with which the Stokes-Einstein (S-E) equation accurately measures the hydrodynamic radii of spherical nanoparticles, enabling the determination of the particle size range for which the hydrodynamic radius by the S-E equation measures the real particle radius. Experimental procedure in confocal single molecule fluorescence lifetime correlation spectroscopy to determine the excitation power and molecular or particulate concentration under which the application of an unmodified model autocorrelation function is justified.