Single-fluorophore imaging with an unmodified epifluorescence microscope and conventional video camera

Single fluorophores in aqueous solution were imaged in real time with a conventional silicon-intensified target video camera connected to an unmodified commercial microscope (IX70, Olympus) with epifluorescence excitation with a high-pressure mercury lamp. Neither a powerful laser nor an extremely sensitive video camera was required. Three experimental systems were used to demonstrate quantitatively that individual, moving or stationary Cy3 fluorophores could be imaged with the microscope: Cy3-gelsolin attached to an actin filament sliding over heavy meromyosin, sliding actin filaments sparsely labelled with Cy3, and heavy meromyosin labelled with one or two Cy3 fluorophores. The results should encourage many laboratories to attempt 'single-molecule physiology' in which the functions and mechanisms of molecular machines are studied at the single-molecule level in an environment where the biological machines are fully active.