Determining chemical rate coefficients using time-gated fluorescence correlation spectroscopy

In recent years, fluorescence correlation spectroscopy (FCS) has become an important technique for studying dynamic processes of molecules in thermody namic equilibrium. Fluorescent organic molecules are excited by laser light , and the emitted light quanta from a small number of molecules in a volume of similar to1 fl are collected using a high numerical aperture microscope objective and photon counting detection. Translational and rotational diff usion, chemical reactions (including photochemistry) and conformational cha nges of the molecules give rise to temporal correlations in the fluorescenc e intensity fluctuations that can be revealed by autocorrelation analysis. A method is presented to improve the sensitivity of FCS measurements on sam ples containing multiple fluorescent species. Using pulsed laser excitation in conjunction with electronic gating in the detection channel, we prefere ntially suppress the emission from the short lifetime components by fluores cence lifetime separation. We demonstrate the usefulness of this technique by applying it to the binding reaction of the organic dye 1-anilino-8-napht halenesulfonic acid in the interior of the small globular protein apomyoglo bin. When studying this chemical reaction with FCS, a relaxation component appears in the autocorrelation function which can be enhanced by the time g ating technique. Furthermore, the analysis is considerably simplified and b oth kinetic and equilibrium coefficients of the reaction can be determined. Copyright (C) 2000 John Wiley & Sons, Ltd.