Imaging of single molecule diffusion.

In recent years observations at the level of individual atoms and molecules became possible by microscopy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholipids carrying one rhodamine dye molecule. For this methodology, fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bioscience--for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoichiometries of individual components.