Fluorescence correlation spectroscopy with saturated excitation

Abstract

In this paper,we study the errors caused by an intense excitation beam in fluorescence correlation spectroscopy (FCS). Saturated excitation induced by an intense excitation beam will alter the spatial distribution function of the laser-induced fluorescence; therefore it changes the results of FCS measurements. Using the theory for saturated excitation,we derived a general expression for the effective detection volume as a function of the excitation laser intensity. Quantitative relations between the particle number and diffusion time,both obtained from FCS measurements,and the excitation laser intensity are established and used to remove the systematic error due to saturated excitation. The success of this technique is demonstrated in both Monte-Carlo simulations and experiments.