Investigating two-photon photophysics with fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a sensitive research tool for studying molecular dynamics at the single molecule level. Photophysical dynamics often dramatically influence FCS measurements, as we show here in characterizing the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the influence of excitation saturation on the two-photon fluorescence observation volume, and derive an analytical expression for the correlation function that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.