Statistical Analysis of Single-Molecule Colocalization Assays

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Abstract
In chemical assays, specific molecular recognition events result in close physical proximity of two molecular species, e.g., ligands and receptors. Microscopy techniques that are able to image individual molecules allow for achieving 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 inaccuracies in case of a recognition event or due to accidental close proximity 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 accidental proximity. This enables a quantitative statistical analysis of single recognition events. It is demonstrated that in a general assay, even in the presence of strong unspecific background, the probability for a certain diagnosis and a measure for its reliability can be extracted from the observation of a few binding events. The power of the method is demonstrated at the example of a single-molecule DNA hybridization assay. Our findings are of major importance for future assay miniaturization and assaying with minute amounts of analyte.