Photobleaching regions of living cells to monitor membrane traffic

Eukaryotic cells are composed of an intricate system of internal membranes that are organized into different compartments-including the endoplasmic reticulum (ER), the nuclear envelope, the Golgi complex (GC), lysosomes, endosomes, caveolae, mitochondria, and peroxisomes-that perform specialized tasks within the cell. The localization and dynamics of intracellular compartments are now being studied in living cells because of the availability of green fluorescent protein (GFP)- fusion proteins and recent advances in fluorescent microscope imaging systems. This protocol outlines two methods for photobleaching living cells to monitor membrane traffic. The first method involves selective photobleaching using a confocal laser-scanning microscope (CLSM) that can bleach discrete selected regions of interest. As outlined in the second method, photobleaching can also be performed with older CLSMs that lack the capacity for selective photobleaching. In this case, photobleaching is accomplished by zooming into a small region of the cell and scanning with full laser power.