Fluorescence imaging of pyrene-labeled lipids in living cells

Microscopic imaging of fluorescent lipid derivatives is a powerful tool to study membrane organization and lipid trafficking but it is complicated by cellular autofluorescence background and photobleaching of the fluorophore as well as by the difficulty to selectively image membranes stacked on top of each other. Here we describe protocols that strongly alleviate such prob lems when pyrene-labeled lipids are being used. First, photobleaching of th ese lipids is virtually eliminated when oxygen is depleted from the medium by using a gentle and simple enzymatic method. Second, an image practically free of cellular autofluorescence contribution can be obtained simply by s ubtracting from the pyrene image the background image obtained at a slightl y different excitation wavelength. This type of background subtraction more properly accounts for the typically uneven distribution of cellular backgr ound fluorescence than other, commonly used methods. Third, it is possible to selectively image the pyrene lipids in the plasma membrane by using plas ma membrane-specific quencher trinitrophenyl lysophosphatidylethanolamine a nd image subtraction. Importantly, either the outer or the inner leaflet ca n be selectively imaged by labeling the cells with pyrene phosphatidylcholi ne or phosphatidylserine, respectively. These protocols should be of consid erable help when studying organization of the plasma membrane or intracellu lar lipid trafficking. (C) 2000 Elsevier Science B.V. All rights reserved.