A statistical analysis of fluorescence correlation data

Fluorescence correlation spectroscopy (FCS) is a relatively recent techniqu e in which the diffusion coefficient of fluorescently labeled molecules can be determined. The change in diffusion behavior when these molecules inter act with others can also be used to study interactions in solution. A new s tatistical method is proposed to analyze FCS measurements that cannot be ev aluated with a classical autocorrelation function, which is normally used t o analyze FCS data. It applies to binding studies where one of the interact ing particles has a much brighter fluorescence intensity with respect to th e other, which causes high fluorescence bursts whenever these molecules are detected, This biases the autocorrelation function, making it in most case s impossible to use this function as a fitting equation. Here, a statistica l approach is used to quantify the amount of fluorescence found in bursts, thereby enabling to perform binding studies in cases where the fluorescence per molecule of both interacting species differs greatly. The method is de monstrated on a system of known composition, making it a promising tool for future FCS measurements.