Microscopy of lipids in living cells is currently hampered by a lack of adequate fluorescent tags. The most frequently used tags, NBD and BODIPY, strongly influence the properties of lipids, yielding analogs with quite different characteristics. Here, we introduce polyene-lipids containing five conjugated double bonds as a new type of lipid tag. Polyene-lipids exhibit a unique structural similarity to natural lipids, which results in minimal effects on the lipid properties. Analyzing membrane phase partitioning, an important biophysical and biological property of lipids, we demonstrated the superiority of polyene-lipids to both NBD- and BODIPY-tagged lipids. Cells readily take up various polyene-lipid precursors and generate the expected end products with no apparent disturbance by the tag. Applying two-photon excitation microscopy, we imaged the distribution of polyene-lipids in living mammalian cells. For the first time, ether lipids, important for the function of the brain, were successfully visualized.