Fluorescence correlation spectroscopy in vivo

Fluorescence Correlation Spectroscopy (FCS) is an optical technique used to accurately probe the dynamics, local concentration, and photophysics of single molecules both in vitro and in vivo. It is based on the analysis of intensity fluctuations of fluorescently labeled molecules diffusing through a focused laser beam. Fluorescence Cross-correlation Spectroscopy (FCCS) is a powerful extension to FCS which allows quantitative analysis of molecular interactions between differently labeled molecules. This review will focus on the application and potential of FCS and FCCS in vivo. Practical issues and sources of artifacts when performing measurements in living cells are discussed. Finally, several extensions to conventional FCS, such as multiphoton excitation, scanning FCS, Fluorescence Lifetime Correlation Spectroscopy, multiplexing FCS and recent approaches to reach smaller excitation volumes are reviewed.