Using fluorescence recovery after photobleaching to measure lipid diffusion in membranes

The lateral diffusion of lipids is crucial to the biogenesis and function of biological membranes. In this chapter, approaches for observing the lateral diffusion of lipids using fluorescence recovery after photobleaching are described. The procedures described can be carried out with a standard laser-scanning confocal microscope. The membrane of interest is stained with a lipophilic fluorophore or fluorescent lipid analog. The confocal laser spot is then used to photobleach fluorescence in a small region of the sample. Subsequent spread and recovery of the bleach, reports on the diffusion of the fluorophore. The results provide a measure of membrane fluidity, and the extent to which lipid diffusion in the membrane might be constrained. Fluorescence recovery after photobleaching measurements may be carried out in vivo, in systems ranging from bacteria to mammalian cells, or in vitro in isolated membrane fragments. Procedures for preparing the biological sample, performing the measurement, and quantitative data analysis are explained.