Determining chemical rate coefficients using time-gated fluorescence correlation spectroscopy

Abstract

In recent years, fluorescence correlation spectroscopy (FCS) has become an important technique for studying dynamic processes of molecules in thermodynamic equilibrium. Fluorescent organic molecules are excited by laser light, and the emitted light quanta from a small number of molecules in a volume of ∼1 fl are collected using a high numerical aperture microscope objective and photon counting detection. Translational and rotational diffusion, chemical reactions (including photochemistry) and conformational changes of the molecules give rise to temporal correlations in the fluorescence intensity fluctuations that can be revealed by autocorrelation analysis. A method is presented to improve the sensitivity of FCS measurements on samples containing multiple fluorescent species. Using pulsed laser excitation in conjunction with electronic gating in the detection channel, we preferentially suppress the emission from the short lifetime components by fluorescence lifetime separation. We demonstrate the usefulness of this technique by applying it to the binding reaction of the organic dye 1-anilino-8-naphthalenesulfonic acid in the interior of the small globular protein apomyoglobin. When studying this chemical reaction with FCS, a relaxation component appears in the autocorrelation function which can be enhanced by the time gating technique. Furthermore, the analysis is considerably simplified and both kinetic and equilibrium coefficients of the reaction can be determined. Copyright © 2000 John Wiley & Sons, Ltd.