title: Fluorescence lifetime imaging of membrane lipid order with a ratiometric fluorescent probe.
abstract: To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N(∗)) and tautomer (T(∗)) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T(∗) form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N(∗) form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis.
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France.
authors.forename: Vasyl
authors.initials: V
authors.lastname: Kilin
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France.
authors.forename: Oleksandr
authors.initials: O
authors.lastname: Glushonkov
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France.
authors.forename: Lucas
authors.initials: L
authors.lastname: Herdly
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France.
authors.forename: Andrey
authors.initials: A
authors.lastname: Klymchenko
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Richert
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France. Electronic address: yves.mely@unistra.fr.
authors.forename: Yves
authors.initials: Y
authors.lastname: Mely
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: D008563:Membrane Lipids; D053835:Unilamellar Liposomes