4] Fluorescence correlation spectroscopy

Abstract

Publisher Summary This chapter describes fluorescence correlation spectroscopy (FCS). FCS is an attractive technique for intracellular applications. First, FCS determines kinetic processes from equilibrium fluctuations. Thus, no external perturbation is required to obtain kinetic information. Second, FCS provides excellent spatial resolution. FCS characterizes any kinetic process that leads to changes in the fluorescence, because the spontaneous fluctuations at thermodynamic equilibrium are governed by the same laws that describe the kinetic relaxation of a system to equilibrium. Thus, FCS offers a very convenient method for determining kinetic properties at equilibrium without requiring a physical perturbation of the sample. This is especially important for systems in which the use of perturbation techniques is extremely difficult and challenging, such as measurements in living cells. The concept of measuring signal fluctuations has direct consequences for the experimental realization of FCS. When performing FCS measurements inside the nucleus, it can be found that the fluorescence intensity is very stable. In the cytoplasm, however, the fluorescence intensity depends on the spatial location, and sometimes strong fluctuations, which persist for a few seconds, are observed, whereas at other times the intensity is as stable as inside the nucleus.