Brownian dynamics simulations of fluorescence fluctuation spectroscopy

We have developed a program for the simulation of the fluorescence fluctuat ions as detected from highly diluted samples of (bio)molecules. The model i s applied to translational diffusion and takes into account the hydrodynami c interactions. The solution concentration is kept constant by assuming per iodic boundary conditions and spans here the range 0.5 < C < 10 nM. We show that the fluorescence correlation functions can be accurately computed on systems of limited size (a few molecules per simulation box) by simulating for a total time similar to 100-300 times the diffusion relaxation time of the fluorescence autocorrelation function. The model is applied also to the simulation of the scanning fluorescence correlation spectroscopy (FCS) and of the photon counting histograms for the confocal collection configuratio n. Scanning FCS simulations of highly diluted samples (C approximate to0.5 nM) show anticorrelation effects in the autocorrelation functions of the fl uorescence signal that are less evident for higher concentrations. We sugge st here that this effect may be due to the non-uniform occupancy of the sca nning area by the fluorophores.