FLUCTUATION CORRELATION SPECTROSCOPY IN CELLS: Determination of molecular aggregation

Fluorescence Correlation Spectroscopy (FCS) was first introduced by Elson, Madge and Webb (1-5) for studying the binding process between ethydium bromide and DNA. When the ethydium dye binds to DNA its fluorescence quantum yield changes by a large factor. It is essentially not fluorescent when free in solution and it becomes strongly fluorescent when bound to double strand DNA. Although the processes are very different in nature, the instrumentation used for the FCS experiment is derived from dynamic light scattering. There are however major differences between dynamic light scattering and FCS. In the FCS experiment the fluorescence fluctuation arises because of the chemical reaction that changes the fluorescence properties of the dye and because the bound ethydium molecules could enter and leave the volume of excitation due to diffusion of the molecule. In dynamic light scattering the fluctuations arise from changes in the index of refraction due to local changes in the concentration of molecules. Therefore FCS is sensitive to all chemico-physical processes that could change the fluorescence intensity in a small volume. For the fluctuation in intensity to be measurable it is crucial that the volume of observation to be small so that only a few molecules are at any instant of time in the volume of observation. The realization of small exaction volume was a major problem hindering the use of this technique and only recently, with the introduction of confocal microscopy and twophoton microscopy FCS the generation of sub-femtoliter volumes of observation has become a routine procedure. At nanomolar concentration a femtoliter volume contains only few molecules. As a consequence of the Poisson distribution of the occupation number,