Data shown for 100% dual-labeled nano-bead reference.a Mismatching confocal volumes represented by red and blue ellipsoids. Three classes of trajectories of diffusing molecules are depicted as black lines: borderline (A), peripheral (B), and central (C) with respect to centroid of red ellipsoid. Note, that the nano-bead is not on scale. b Segments of inter-photon lag (IPL) time trace. Dips of the IPLs below the burst thresholds reflect single-molecule transits that correspond to trajectories (A), (B), and (C) illustrated in a. c Histogram of number of photons per burst for red channel (Atto 647N). Borderline trajectories (A) with a low number of photons per burst are much more abundant than central trajectories (C) with a high number of photons per bursts. d Fraction of coincident bursts as a function of normalized brightness threshold nbr. Bursts with a low brightness threshold include all trajectories (A), central molecule trajectories (C) reach the expected value of 100% coincidence. Burst and analysis statistics are given in Supplementary Note 1.