Partitioning of acyl chain-labeled lipid analogues in coexisting fluid phases in GPMVs. Equatorial (A–C) or tangential (D) sections of GPMVs, colabeled after formation, with Rh-DOPE and 12-NBD-SM, 12-NBD-PC, 12-NBD-Ceramide, or 6-NBD-SM and imaged at 20°C. (A–B) Acyl-chain labeled 12-NBD-SM and 12-NBD-PC exhibit differential partitioning in GPMVs: NBD-SM partitions equally between Lo-like and Ld-like phases (A), whereas, NBD-PC shows similar partitioning as Rh-DOPE (Ld-phase) (B). (C) Acyl chain labeled 12-NBD-Ceramide exhibits equal partitioning between Lo-like and Ld-like phases. (D) Acyl chain labeled 6-NBD-SM prefers the Lo-like phase and exhibits partitioning complementary to Rh-DOPE. (Scale bars, 5 μm.)