Electro-formation and fluorescence microscopy of giant vesicles with coexisting liquid phases

Giant unilamellar vesicles (GUVs) are routinely used to study coexisting liquid phases in bilayer membranes. Liquid domains are observed in a wide variety of ternary GUV membranes containing phospholipids and cholesterol, and are thought to model raft domains in cell membranes. GUVs are attractive model systems because vesicles are easily prepared using standard and inexpensive laboratory equipment, and phase-separated vesicles can be visualized using optical microscopy. In this chapter, a detailed method is presented to form and view 10-100-microm diameter single-walled vesicles of charged or uncharged lipid mixtures. GUVs can be visualized by fluorescence microscopy and methods are presented to measure miscibility transition temperatures and to distinguish solid (gel) and liquid domains. Numerous experimental artifacts associated with GUV preparation and viewing are discussed, including the effects of nonideal growth conditions and perturbations of fluorescent probes and other impurities.