title: Correlation-matrix analysis of two-color coincidence events in single-molecule fluorescence experiments.
abstract: We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.
authors.affiliation: Applied Laser Physics and Laser Spectroscopy, Bielefeld University, Bielefeld, Germany.
authors.forename: Idir
authors.initials: I
authors.lastname: Yahiatène
authors.forename: Sören
authors.initials: S
authors.lastname: Doose
authors.forename: Thomas
authors.initials: T
authors.lastname: Huser
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
mesh_terms: D001483:Base Sequence; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000235:genetics; D004058:Diffusion; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D008956:Models, Chemical; D015233:Models, Statistical; D008969:Molecular Sequence Data; D011008:Pneumococcal Infections / Q000175:diagnosis / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes