1. Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA
2. Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA

Correspondence to: Watt W. Webb¹ Email: www2@cornell.edu

Lipid bilayer membranes—ubiquitous in biological systems and closely associated with cell function—exhibit rich shape-transition behaviour, including bud formation¹ and vesicle fission². Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles^{3, 4}. Detailed theoretical frameworks^{5, 6, 7, 8, 9, 10, 11} link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains.