IMAGING AND NANO-MANIPULATION OF SINGLE BIOMOLECULES

We have developed a new technique for imaging single fluorescent dye m olecules by refining epifluorescence and total internal reflection flu orescence microscopies. In contrast to previously reported single fluo rescent molecule imaging methods, in which specimens were immobilized on an air-dried surface, our method enables video-rate imaging of sing le molecules in aqueous solution, This approach enabled us to directly image the processive movement of individual fluorescently labeled kin esin molecules along a microtubule. This method was also used to visua lize individual ATP turnover reactions of single myosin molecules. The method can be combined with molecular manipulation using an optical t rap. A single kinesin molecule attached to a polystyrene bead was brou ght into contact with a microtubule adsorbed onto the glass surface. T he lifetime of bound Cy3-nucleotide in the absence or presence of the microtubule was 10 s or 0.08 s, respectively, showing that ATPase acti vity of the kinesin is strongly activated by microtubules. As the pres ent system is equipped with a nanometer sensor, elemental steps of a s ingle kinesin molecule can also be measured. By simultaneously measuri ng the individual ATP turnovers and elementary mechanical events of a single kinesin molecule, we will be able to obtain a clear answer to t he fundamental problem of how the mechanical events are coupled to the ATPase reaction. (C) 1997 Elsevier Science B.V.