Relatively simple modifications of an ordinary epifluorescence microscope have greatly reduced its background luminescence, allowing continuous and real time imaging of single fluorophores in an aqueous medium. Main modifications were changing the excitation light path and setting an aperture stop so that stray light does not scatter inside the microscope. A simple and accurate method using actin filaments is presented to establish the singularity of the observed fluorophores. It was possible, at the video rate of 30 frames/s, to image individual tetramethylrhodamine fluorophores bound to actin filaments sliding over heavy meromyosin. The successful imaging of moving fluorophores demonstrates that conventional microscopes may become a routine tool for studying dynamic interactions among individual biomolecules in physiological environments.
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