Principles of Fluorescence Correlation and Dual-Color Cross-Correlation Spectroscopy

Abstract

Fluorescence Correlation Spectroscopy (FCS) is a non-invasive, highly sensitive technique for measuring the diffusive and photophysical properties of fluorescent species as well as their interactions. All of this information needs to be reliably extracted from the fluctuating fluorescence signal and interpreted in a theoretical framework. In this chapter, we describe the derivation of the basic equations governing FCS correlation curves. By pointing out their limitations and the underlying approximations and assumptions we hope to facilitate applications and the development of more elaborate models for more complex systems. Two detection channels are included to accommodate dual-color Fluorescence Cross-Correlation Spectroscopy. Moreover, we provide a generalized description for the separation of spatial movement and intramolecular change, taking translational diffusion (changes in position), rotational diffusion (changes in orientation) and fluorescence blinking (changes in the quantum mechanical state) into account. Since, experimentally, particles are often labeled with multiple fluorophores, besides multiple dynamics and multiple species of particles, multiple fluorophores per particle are also part of the description.