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Published October 3, 2005 | public
Journal Article Open

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 umber 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.

Additional Information

© 2005 Optical Society of America Received 9 August 2005; revised 20 September 2005; accepted 21 September 2005 We thank K. McHale for discussions of statistical estimation and C. Lee for experimental assistance. This work was supported by the Institute for Collaborative Biotechnologies through grant DAAD19-03-D-0004 from the U.S. Army Research Office and by the NSF through grants DBI-0242705 and EIA-0323542.


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