Imaging intracellular membrane traffic and organelle dynamics using confocal microscopy

Abstract

Movements of exocytic and endocytic vesicles within cells are controlled in part by active transport along microtubules. The forces generated by microtubule-based transport are also known to strongly influence the distribution and shapes of membranous organelles, such as mitochondria and the endoplasmic reticulum. The ability to observe such dynamic interactions between cytoskeletal elements and fluorescently-labelled intracellular membranes in living cells has been improved recently by the advent of scanning laser confocal microscopy. Here we describe the use of this technique to study activities of the Golgi apparatus in living cells.Vesicular traffic, diffusive transport and morphological dynamics within the Golgi apparatus of cultured rat hippocampal astrocytes were imaged with a Bio-Rad MRC-500 scanning laser confocal microscope. Golgi elements were labelled with NBD-ceramide, a molecule which serves as a vital stain for the trans-most cisternae of Golgi stacks. Single scans of the specimen, obtained with the laser microscope's slow scan rate (4 sec/image), were stored sequentially on an optical memory disk recorder.