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

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