FIG. 2.

Photobleaching properties and fluorescence intensities of fluorescently labelled kinesin. uhK560-Cy3 was adsorbed onto a fused-silica slide, and the intensities of individual fluorescent spots plotted against time. Typical examples of one-step (a) or two-step (b) photobleaching events are shown, reflecting one or two Cy3 dye molecules attached to kinesin, respectively. The distribution of the fluorescence intensities of isolated uhK560-Cy3 spots adsorbed onto the slide (c) or moving along the axoneme (d) are comparable, which suggests that moving fluorescent spots are single molecules and not higher-order aggregates. Intensities were measured using a rolling 8-frame average to reduce fluctuations, summing intensities from a 7 × 7 pixel window, and subtracting the background intensity from an adjacent region on the slide. The intensities of two dye molecules (b) and three dye molecules (not shown) are in the linear range of the camera, as photobleaching caused approximately equal decreases in intensity.