Use of fluorescence microscopy to probe intracellular lipolysis

Intracellular lipolysis is an important cellular process in key metabolic tissues, and while much is known about the enzymatic basis of lipolysis, our understanding of how these processes are organized and regulated within cells is incomplete. Lipolysis takes place on the surface of intracellular lipid droplets, which are now recognized as bona fide organelles, and a large number of proteins have been found to change their associations with lipid droplets in response to lipolytic stimulation. Intracellular lipolysis has critical spatial and temporal domains that can be investigated using high-resolution imaging of fixed and live cells. Here, we describe techniques for high-resolution imaging of native lipid droplet proteins, of dynamic trafficking and interaction of these proteins in model systems, and of intracellular fatty acid production using fluorescent reporters in live adipocytes.