Local Stoichiometries Determined by Counting Individual Molecules

The capability of optical single-molecule microscopy for quantitative determination of local stoichiometries is demonstrated. Biotin−streptavidin embedded in a phospholipid membrane was chosen as a model system. At a local resolution of 30 nm and a time resolution of 35 ms, it was possible to determine local stoichiometries between one and four occurring in this system. The “stoichiometric resolution” of the method is analyzed by statistical means. From this a quantitative criterion is derived for the reliability in assigning a defined number of fluorophores to each individual fluorescence peak observed. It is shown that only a few ( n ≈ 10) observations are sufficient for reliable assignments.