title: Two-photon Laurdan studies of the ternary lipid mixture DOPC:SM:cholesterol reveal a single liquid phase at sphingomyelin:cholesterol ratios lower than 1.
abstract: The ternary lipid mixture DOPC:eggSM:cholesterol in excess water has been studied in the form of giant unilamellar vesicles using two-photon fluorescence microscopy. Previous publications based on single-photon fluorescence microscopy had reported heterogeneous phase behavior (phase coexistence) in the region of the triangular phase diagram corresponding to SM:cholesterol molar ratios <1. We have examined this region by two-photon microscopy of Laurdan-labeled mixtures and have found that, under our conditions, only a single liquid phase exists. We have shown that macroscopic phase separation in the above region can be artifactually induced by one-photon excitation of the fluorescent probes and ensuing photooxidation and is prevented using two-photon excitation. The main effect of increasing the concentration of cholesterol in mixtures containing 30 mol % SM was to increase the rigidity of the disordered domains. Increasing the concentration of SM in mixtures containing 20 mol % cholesterol gradually augmented the rigidity of the ordered domains, while the disordered domains reached minimal order at a SM:cholesterol 2.25:1 molar ratio, which then increased again. Moreover, the detailed measurement of Laurdan generalized polarization across the whole phase diagram allowed the representation, for both the single- and two-phase regions, of the gradual variation of membrane lateral packing along the diagram, which we found to be governed largely by SM:cholesterol interactions.
authors: Unidad de Biofísica (CSIC, UPV/EHU), Barrio de Sarriena s/n, 48490 Leioa, Spain.
authors: Pablo
authors: P
authors: Carravilla
authors: José L
authors: JL
authors: Nieva
authors: Félix M
authors: FM
authors: Goñi
authors: Jose
authors: J
authors: Requejo-Isidro
authors: Nerea
authors: N
authors: Huarte
mesh_terms: D002784:Cholesterol / Q000737:chemistry*; D036641:Microscopy, Fluorescence, Multiphoton; D044367:Phase Transition; D010713:Phosphatidylcholines / Q000737:chemistry*; D013109:Sphingomyelins / Q000737:chemistry*
chemical_list: D010713:Phosphatidylcholines; D013109:Sphingomyelins; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine