Ordered/disordered phase separation occurring in bio-membranes has piqued researchers’ interest because these ordered domains, called lipid rafts, regulate important biological functions. The structure of the ordered domain has been examined with artificial membranes, which undergo macroscopic ordered/disordered phase separation. However, owing to technical difficulties, the local structure inside ordered domains remains unknown. In this study, we employed electron diffraction to examine the packing structure of the lipid carbon chains in the ordered domain. First, we prepared dehydrated monolayer samples using a rapid-freezing and sublimation protocol, which attenuates the shrinkage of the chain-packing lattice in the dehydration process. Then, we optimised the electron flux to minimise beam damage to the monolayer sample. Finally, we developed low-flux scanning electron diffraction and assessed the chain packing structure inside the ordered domain formed in a distearoylphosphatidylcholine/dioleoylphosphatidylcholine binary monolayer. Consequently, we discovered that the ordered domain contains multiple subdomains with different crystallographic axes. Moreover, the size of the subdomain is larger in the domain centre than that near the phase boundary. To our knowledge, this is the first study to reveal the chain packing structures inside an ordered domain.
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