Loss of fluorescence of YFP because of photobleaching. Fluorescence from an YFP-fusion protein expressed in a living cell (a nondiffusible cytoskeletal component) was recorded using 514 nm excitation light in a laser-scanning confocal microscope. The illumination intensity, dwell time per pixel, and photomultiplier gain were adjusted to give an image of acceptable quality using a pinhole diameter equivalent to ∼1 Airy disk. The average fluorescence intensity in a small region was then recorded over the course of repeated scans. The fluorescence decreased by approximately one-half after 100 scans.