Membrane Glycolipid Trafficking in Living, Polarized Pancreatic Acinar Cells: Assessment by Confocal Microscopy

This chapter examines the membrane glycolipid trafficking in living, polarized pancreatic acinar cells using confocal microscopy. When the confocal scanning laser microscope (CLSM) is equipped for video imaging through coupling to a digital host computer with image processing capabilities and a shuttering system, it becomes possible to image living cells with increased clarity and increased resolution over that possible with conventional light microscopes. Once images obtained with the CSLM are stored in digitized form either directly on the computer hard disk or more practically on a laser disk, they can be subdivided into picture elements called “pixels.” The chapter uses pixelation to quantify the ceramide fluorescence in pancreatic acinar cells as it moves with time from the Golgi to membranes of the post-Golgi compartments that compose the secretory pathway. The fluorescent analogs of ceramide and the vectorial secretory transport pathway in pancreatic acinar cells are used to (1) define the kinetics of entry of the fluorescent lipid derivatives into elements of the Golgi complex, and (2) examine the kinetics and routes of post-Golgi transport of membrane glycolipids into the distal compartments of the secretory pathway, terminating in secretory granules at the cell apex in rat pancreatic acinar cells.