Imaging of single fluorescent molecules using video-rate confocal microscopy

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nip-kow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled w ith fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecompositi on lifetimes of the fluorophores were similar to 10 s for TMR and similar t o2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluore scence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecu les along microtubules on glass surface and 3D Brownian motion of individua l kinesin molecules in viscous solution could be observed using this micros copy. These results indicated that this method could be applicable to the s tudy of single molecular events in living cells at real time. (C) 2001 Acad emic Press.