Abstract
The determination of molecular interactions in cells has become a field of great interest in the post-genomic era. Ideally, we will like to know which protein is interacting with which, the migration and the localization of the proteins in the cell. During the past years very powerful optical methods have been developed that have the sensitivity to detect single molecules and molecular interactions. Although most of these methods have been demonstrated in solutions or on molecules immobilized on a surface, we will like to extend this methodology to the cell environment. Fluctuation correlation spectroscopy has been proposed as a method to detect single molecules, determine some of the chemico-physical parameters such as local diffusion and molecular brightness. Finally, advances in the determination of spatio-temporal correlations in a cell will be discussed.
Highlights
Fluorescence Correlation Spectroscopy (FCS) was first introduced by Elsoii, Madge and Webb [1,2,3,4,5] for studying the binding process between ethydium bromide and DNA
The processes are very different in nature, the instrumentation used for the FCS experiment is derived from dynamic light scattering
We have shown that the statistical analysis of fluctuations of the fluorescence signal is a powerful tool for the study of chemical reactions both in solutions and in the interior of cells
Summary
Fluorescence Correlation Spectroscopy (FCS) was first introduced by Elsoii, Madge and Webb [1,2,3,4,5] for studying the binding process between ethydium bromide and DNA. This molecular species is different form the monomers because in carries twice the number of fluorescent moieties When this aggregate enters the volume of excitation, it will cause a larger fluctuation of the intensity than a single monomer. The analytical expression for the PCH distribution for a single molecular species of a given brightness has been derived for the 3D-Gaussian illumination profile [11] and is reported below In this expression, Vo is the volume of illumination, s is the brightness of the molecule and k is the number of photons in a give time interval. The heterogeneity of the diffusion could be due to interactions of the protein with other cellular components which results in slowing the motion of the protein
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