Theory of fluorescence correlation spectroscopy on single molecules

A theoretical framework is presented that connects "classic" fluorescence c orrelation spectroscopy (FCS) treating averages of many freely diffusing mo lecules and FCS single-molecule analysis. By assuming a general two-state e mission dynamics of the single molecule, expressions describing the autocor relation of the total fluorescence fluctuations are derived. By studying an idealized experimental situation, the relation between signal-to-noise and signal-to-background ratios are discussed. Under appropriate conditions it is possible to make statistically feasible measurements of single molecule dynamics despite low signal-to-background. The quantum yield ratio of the background molecules to the single molecule as well as the position of the single molecule inside the detection volume are crucial in obtaining good s ignal-to-noise ratios in single-molecule experiments.