Experimentalists are increasingly confronted with the demand to single out rare interaction events under a vast excess of non-interacting molecules. We recently presented an approach how to virtually dilute fluorescently labeled membrane constituents by combining photobleaching and single molecule microscopy, termed “Thinning Out Clusters while Conserving the Stoichiometry of Labeling” (TOCCSL; Moertelmaier et al., Appl. Phys. Lett., 2005, 87, 263903). Using this approach, single molecule microscopy can be performed even at arbitrarily high surface densities of fluorescent probe molecules. Here, we extended this method for two color microscopy. We provide a detailed statistical description of false positives and false negatives. In particular, we quantified the increase in sensitivity by tracking the colocalized objects over successive images. Proof of principle experiments were performed by measuring the interaction between Alexa647-labeled Cholera Toxin B (CTX-B-Alexa647) and Bodipy-labeled GM1 (Bodipy-GM1) diffusing in a fluid supported lipid bilayer. We directly observed single Cholera Toxin B molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. Each colocalized spot could be further analyzed with respect to its diffusion constant, yielding a clear anticorrelation between occupancy and mobility. We finally demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified.
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