Detection of Rare Interaction Events Via Combined Photobleaching and Single Molecule Microscopy

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Gathering information on the interplay between different membrane components and the characteristics of protein interactions in live cells requires the design of sensitive methods that allow for the simultaneous study of different probe molecules. Especially the aim to single out rare interaction events under a vast excess of non-interacting molecules remains challenging. The two-color single molecule imaging technique presented here is the advancement of a recently presented approach to virtually dilute fluorescently labeled membrane constituents by photobleaching [1]. Using this technique, single molecule microscopy can be performed at almost arbitrarily high surface densities of fluorescent probe molecules. The method yields information on the fraction of colocalized particles and their position within 40nm accuracy. Supplemental data on the mobility and stoichiometry of the labeled molecule species can also be deduced. The sensitivity of our two-color single molecule imaging technique is significantly increased by tracking colocalized spots over consecutive images. We present a detailed statistical description of false positives and false negatives and quantify the sensitivity of our method. 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 molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. We demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified by tracking colocalized spots. We also present preliminary results on the application to live cells investigating the interaction between Bodipy-GM1 and Ab-Alexa647 labeled CD59. [1] Moertelmair et al., Appl. Phys Lett. 87, 263903, 2005.