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 problems when pyrene-labeled lipids are being used. First, photobleaching of these 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 subtracting from the pyrene image the background image obtained at a slightly different excitation wavelength. This type of background subtraction more properly accounts for the typically uneven distribution of cellular background fluorescence than other, commonly used methods. Third, it is possible to selectively image the pyrene lipids in the plasma membrane by using plasma membrane-specific quencher trinitrophenyl lysophosphatidylethanolamine and image subtraction. Importantly, either the outer or the inner leaflet can be selectively imaged by labeling the cells with pyrene phosphatidylcholine or phosphatidylserine, respectively. These protocols should be of considerable help when studying organization of the plasma membrane or intracellular lipid trafficking.