Statistical analysis of single-molecule colocalization assays

In chemical assays, specific molecular recognition events result in close p hysical proximity of two molecular species, e.g., Ligands and receptors, Mi croscopy techniques that are able to image individual molecules allow for a chieving a positional accuracy far beyond the resolution limit. Therefore, independent position determination, e.g,, by dual-color microscopy, becomes possible, permitting determination of intermolecular distances beyond the resolution limit. Nonzero measured distances occur due to experimental inac curacies in case of a recognition event or due to accidental close proximit y between ligand-receptor pairs. Using general statistical considerations, finite measured distances between single ligand-receptor pairs are directly translated into probabilities for true molecular recognition or mere accid ental proximity, This enables a quantitative statistical analysis of single recognition events. It is demonstrated that in a general assay, even in th e presence of strong unspecific background, the probability for a certain d iagnosis and a measure for its reliability can be extracted from the observ ation of a few binding events. The power of the method is demonstrated at t he example of a single-molecule DNA hybridization assay. Our findings are o f major importance for future assay miniaturization and assaying with minut e amounts of analyte.