Molecular aggregation characterized by high order autocorrelation in fluorescence correlation spectroscopy

The use of high order autocorrelation in fluorescence correlation spectroscopy for investigating aggregation in a sample that contains fluorescent molecules is described. Theoretical expressions for the fluorescence fluctuation autocorrelation functions defined by gm,n(tau) = [(delta fm(t + tau)delta fm(t] - (delta Fm(t] (delta Fn(t]]/(F)m+n, where delta F(t) is the fluorescence fluctuation at time t, (F) is the average fluorescence, and m and n are integers less than or equal to 3, are derived. Methods for determining the number densities and relative fluorescence yields of aggregates of different sizes from a series of Gm,n(0) values are outlined. The method is applied to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate suspended in solutions of water and ethyl alcohol. The technique presented may prove useful in detecting and characterizing aggregates of fluorescent-labeled biological molecules such as cell surface receptors.