Fig. 5. Photobleaching analyses of EYFP-PP1. EYFP-PP1 was transiently expressed in HeLa cells and imaged live on a confocal laser scanning microscope. Panel A shows a FRAP (fluorescence recovery after photobleaching) experiment in which a nucleolar pool of EYFP-PP1 in a cell was photobleached at full laser intensity (region indicated in dashed brackets) for a total of four seconds. The recovery of the signal was monitored by repeated scanning of the entire field of view at low laser power. An unbleached neighboring cell is included for comparison. Panel B shows a FLIP (fluorescence loss in photobleaching) experiment in which repeated photobleaching within a defined region (dashed box) was shown to deplete the fluorescent signal throughout the cell. The entire field of view was scanned at low laser power, and the boxed region then bleached with 2 scans at full laser intensity (4 seconds total). This sequence was repeated 15 times, with the bleached cell gradually becoming dimmer while the unbleached control cell remained bright. Times indicated are total photobleaching times at full laser intensity. Arrows indicate photobleached cells whereas arrowheads indicate nucleoli. The scale bar is 10 µM.