Fluorescence intensity profiles from spindles after photobleaching and photoactivation. Time-lapse videos of a confocal section through spindles containing Cy5 microtubules and photoactivatable Eg5-paGFP were recorded simultaneously in the Cy5 and GFP channel of a confocal fluorescence microscope. Between the first and the second image of a time series, one or several rectangular stripes in the confocal section were bleached and simultaneously photoactivated. To convert the images of the two time series into fluorescence intensity profiles, the area of the spindle in the Cy5 microtubule image before the photobleach was determined by applying an intensity threshold to the image (I). This area served then as a mask for the entire time series of the Cy5 microtubule images and the Eg5-paGFP (illustrated here only for the second frame of the time series). The intensity values were projected onto the spindle axis. This created fluorescence intensity profiles (II and V). The turnover of microtubules and of Eg5 was extracted from time series of these intensity profiles (II and V; see Materials and methods). To determine the exact position of the maximum amount of bleached microtubules, the Cy5 intensity profiles were subtracted from the prebleach profile, resulting in an inverted intensity difference profile (III). The positions of the maximum of the intensity difference profiles of Cy5 microtubules and of the intensity profiles of photoactivated Eg5-paGFP were determined from a Gaussian fit (blue) to the profiles (IV and VI) and used to calculate the velocity of the movements of the peaks (see Materials and methods).