title: Characterization and application of a new optical probe for membrane lipid domains.
abstract: In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut 06030-1507, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Jin
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Millard
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Wuskell
authors.forename: Xuemei
authors.initials: X
authors.lastname: Dong
authors.forename: Dianqing
authors.initials: D
authors.lastname: Wu
authors.forename: Heather A
authors.initials: HA
authors.lastname: Clark
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Loew
mesh_terms: D000818:Animals; D001703:Biophysics / Q000379:methods*; D001854:Bone Marrow Cells / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology*; D008051:Lipid Bilayers / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry; D008564:Membrane Potentials; D008566:Membranes / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013053:Spectrophotometry; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008055:Lipids; D002784:Cholesterol