Fluorescent observation of ATP binding in Kinesin driven microtubule gliding using nano-slits

In this paper, we report a simultaneous observation of labelled ATP and microtubule gliding motility driven by kinesin. To increase the concentration of fluorescent dye available for single molecule detection, nano-slit devices were used as linear-shaped zero mode waveguides (ZMWs). Proposed experimental setup enabled observation of individual ATP binding and unbinding to and from kinesin at the concentration of 500 nM. Maximum labelled ATP concentration is around ten times higher than the concentration available in conventional total internal reflection fluorescence microscopy (TIRFM). In addition, binding duration measured indicated the existence of short binding duration of ATP to kinesin. We thus demonstrated experimental method for the single molecule fluorescent observation of kinesin motor proteins, which can be applicable to other liner motor proteins such as dynein and myosin.