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.

Date Published: 10 July 2003
PDF: 12 pages
Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.485597

Published in SPIE Proceedings Vol. 4963:
Multiphoton Microscopy in the Biomedical Sciences III
Ammasi Periasamy; Peter T. C. So, Editor(s)