Kinesin moving through the spotlight: single-motor fluorescence microscopy with submillisecond time resolution.

Kinesin-1 is one of the motor proteins that drive intracellular transport in eukaryotes. This motor makes hundreds of 8-nm steps along a microtubule before releasing. Kinesin-1 can move at velocities of up to approximately 800 nm/s, which means that one turnover on average takes 10 ms. Important details, however, concerning the coordination between the two motor domains have not been determined due to limitations of the techniques used. In this study, we present an approach that allows the observation of fluorescence intensity changes on individual kinesins with a time resolution far better than the duration of a single step. In our approach, the laser focus of a confocal fluorescence microscope is pointed at a microtubule and the photons emitted by fluorescently labeled kinesin motors walking through the spot are detected with submicrosecond accuracy. We show that the autocorrelation of a fluorescence time trace of an individual kinesin motor contains information at time lags down to 0.1 ms. The quality and time resolution of the autocorrelation is primarily determined by the amount of signal photons used. By adding the autocorrelations of several tens of kinesins, fluorescence intensity changes can be observed at a timescale below 100 micros.