Single molecules in fluorescence fluctuation spectroscopy: effective volume and photon counting histogram

This paper presents a stochastic theory for the interpretation of photon counting histograms in fluorescence fluctuation spectroscopy (FFS). New concepts of an effective volume and a single molecule probability distribution are introduced to characterize a molecular species. Whereas the effective volume corresponds to the visibility of a molecular species in a given confocal setup, the single molecule probability distribution gives the signal measured for a single visible molecule. Specific properties of the effective volume and the single molecule probability distribution are discussed. Advantages arise for the high precision measurements of concentrations, mixtures, and binding constants especially for complex molecular environment, e.g. in flow systems and cell compartments.