Widefield microscopy for live imaging of lipid domains and membrane dynamics

Within the lateral organisation of plasma membranes of polarized cell types there exist heterogenous microdomains of distinct lipid composition, the small size of which (10–200 nm) makes them difficult to discern with traditional microscopic techniques, but which can be distinguished on the basis of lipid packing. These microdomains or rafts can be concentrated in larger more visible liquid-ordered regions, particularly by cross-linking of their constituents as in the immunological synapse or in features of the polarized cell such as pseudopodia or flagella. One technique, Laurdan fluorescence microscopy, has proven very useful for distinguishing such regions but has hitherto relied on 2-photon confocal microscopy. This has to some extent limited its utility to living systems and its widespread adoption in studying membrane dynamics on the surface of living cells. Here we describe and validate the adaptation of a standard widefield fluorescence microscope for live imaging of Laurdan stained cell membranes.