Effects of laser excitation saturation in measurements of biomolecule binding by FCS.

POSTER

Abstract

Fluorescence Correlation Spectroscopy (FCS) is a technique developed by physicists in the 1970's that is now widely used for determination of the nanomolar concentrations of fluorescently-labeled biomolecules within microliter-sized droplets of solutions. The conventional theory, which dates from the early papers, states that the amplitude of the autocorrelation function of fluctuations in the fluorescence signal is inversely proportional to the mean number of fluorescent molecules in the focused laser beam. However, we have recently shown that excitation saturation causes dependence of the autocorrelation function on laser power even in the limit of infinitesimal laser power, and thereby a bias in the determined concentration. Here we use computer simulations to investigate the effects of such bias on two-species concentration determinations for application to measurements of molecular binding in pharmaceutical drug discovery.

Authors

  • You Li

    The University of Tennessee Space Institute

  • Rebecca Scott

    UCSB, LBNL, NCSU, University of North Carolina at Chapel Hill, Wright State University, Department of Physics, The University of Memphis, TN 38152, North Carolina State University, Department of Chemical and Biomolecular Engineering, Raleigh, NC, Clemson University, Fisk University, University of North Dakota (Grand Forks), Southern Illinois University, The University of Tennessee Space Institute, SC Governor's School for Science and Mathematics, Francis Marion University, University of North Alabama, Lousiana State University, Department of Physics, Dhaka University, Dhaka, Bangladesh, Head of Government Relations, American Physical Society, Vanderbilt University, Mississippi State University, Emory University, College of William and Mary, University of Tennessee Space Institute, Morehead State University

  • Rebecca Scott

    UCSB, LBNL, NCSU, University of North Carolina at Chapel Hill, Wright State University, Department of Physics, The University of Memphis, TN 38152, North Carolina State University, Department of Chemical and Biomolecular Engineering, Raleigh, NC, Clemson University, Fisk University, University of North Dakota (Grand Forks), Southern Illinois University, The University of Tennessee Space Institute, SC Governor's School for Science and Mathematics, Francis Marion University, University of North Alabama, Lousiana State University, Department of Physics, Dhaka University, Dhaka, Bangladesh, Head of Government Relations, American Physical Society, Vanderbilt University, Mississippi State University, Emory University, College of William and Mary, University of Tennessee Space Institute, Morehead State University