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Abstract

Currently, very few strategies of sample preparation for single molecule analysis exist. Open volume techniques have the disadvantage that molecules can only be detected for a short time, while immobilization techniques, such as tethering the molecules to a surface or embedding them in gels, may perturb the function of molecules. Here we propose a novel technique of capturing single molecules that combines the major advantages of the currently available approaches. Through use of lipid vesicles the size of a confocal volume element, we can confine the movement of molecules to the spatial dimensions of a focal spot without the need for chemical linkers. Thus, the observable kinetics is mainly determined by the photophysics of the chromophore. Used in conjunction with a laser trap, standard measurements, such as TCSPC, can be performed with the added benefit that the microenvironment surrounding the molecules can be controlled and individually varied. This technique can be extended for use in measuring enzyme reactions kinetics where the confined volume may provide advantageous diffusional constraints to promote reactions not favored in dilute solution.