Tracking-FCS: Fluorescence correlation spectroscopy of individual particles

We exploit recent advances in single-particle tracking to perform fluorescence correlation spectroscopy on individual fluorescent particles, in contrast to traditional methods that build up statistics over a sequence of many measurements. By rapidly scanning the focus of an excitation laser in a circular pattern, demodulating the measured fluorescence, and feeding these results back to a piezoelectric translation stage, we track the Brownian motion of fluorescent polymer microspheres in aqueous solution in the plane transverse to the laser axis. We discuss the estimation of particle diffusion statistics from closed-loop position measurements, and we present a generalized theory of fluorescence correlation spectroscopy for the case that the motion of a single fluorescent particle is actively tracked by a time-dependent laser intensity. We model the motion of a tracked particle using Ornstein-Uhlenbeck statistics, using a general theory that contains a number of existing results as specific cases. We find good agreement between our theory and experimental results, and discuss possible future applications of these techniques to passive, single-shot, single-molecule fluorescence measurements with many orders of magnitude in time resolution. (c) 2005 Optical Society of America.