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Fig. 1. Typical digitized fluorescence images and plots of fluorescence recovery after photobleaching. With (anisomycin) or without (control) anisomycin treatment for 60 minutes, cells were labelled with 5,6-carboxyfluorescein diacetate. Suitable fields of cells were identified using a 40x objective lens. Such fields contained many cells that were in contact with each other but not too confluent. Each field was scanned to generate a digital image of fluorescence (Prebleach). After the initial scan, selected cells were photobleached (0 minute, numbers 1-6). Sequential scans were then carried out at 30 second intervals to detect recovery of fluorescence in the bleached cells (4 minute, numbers 1-6). Images were digitally recorded for analysis. Several unbleached cells were also monitored to provide control data (number 7). Typical plots of fluorescence recovery after photobleaching are shown (proportion of prebleaching against time). A rising slope indicates the recovery of fluorescence. The percentage recovery of fluorescence over time was determined for each selected cell and the data were corrected for the background loss of fluorescence in one area (number 7).
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