Micron-scale, coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases are straightforward to observe in giant unilamellar vesicles (GUVs) composed of ternary lipid mixtures. Experimentally, uniform membranes undergo demixing when temperature is decreased: domains subsequently nucleate, diffuse, collide, and coalesce until only one domain of each phase remains. The sizes of these two domains are limited only by the size of the system. Under different conditions, vesicles exhibit smaller-scale domains of fixed sizes, leading to the question of what sets the length scale. In membranes with excess area, small domains are expected when coarsening is hindered or when a microemulsion or modulated phase arises. Here, we test predictions of how the size, morphology, and fluorescence levels of small domains vary with the membrane's temperature, tension, and composition. Using GUVs and cell-derived giant plasma membrane vesicles, we find that 1) the characteristic size of domains decreases when temperature is increased or membrane tension is decreased, 2) stripes are favored over circular domains for lipid compositions with low energy per unit interface, 3) fluorescence levels are consistent with domain registration across both monolayer leaflets of the bilayer, and 4) small domains form in GUVs composed of lipids both with and without ester-linked lipids. Our experimental results are consistent with several elements of current theories for microemulsions and modulated phases and inconsistent with others, suggesting a motivation to modify or enhance current theories.
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