title: Laurdan fluorescence lifetime discriminates cholesterol content from changes in fluidity in living cell membranes.
abstract: Detection of the fluorescent properties of Laurdan has been proven to be an efficient tool to investigate membrane packing and ordered lipid phases in model membranes and living cells. Traditionally the spectral shift of Laurdan's emission from blue in the ordered lipid phase of the membrane (more rigid) toward green in the disordered lipid phase (more fluid) is quantified by the generalized polarization function. Here, we investigate the fluorescence lifetime of Laurdan at two different emission wavelengths and find that when the dipolar relaxation of Laurdan's emission is spectrally isolated, analysis of the fluorescence decay can distinguish changes in membrane fluidity from changes in cholesterol content. Using the phasor representation to analyze changes in Laurdan's fluorescence lifetime we obtain two different phasor trajectories for changes in polarity versus changes in cholesterol content. This gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content and thus perform a more comprehensive analysis of cell membrane heterogeneity. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in the membrane water content during cell migration.
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, USA.
authors.forename: Ottavia
authors.initials: O
authors.lastname: Golfetto
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hinde
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival; D002784:Cholesterol / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D007848:Laurates / Q000737:chemistry*; D008560:Membrane Fluidity / Q000187:drug effects; D051379:Mice; D041681:NIH 3T3 Cells; D010713:Phosphatidylcholines / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D007848:Laurates; D010713:Phosphatidylcholines; D012996:Solutions; D004815:Epidermal Growth Factor; D002784:Cholesterol; D015081:2-Naphthylamine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; C065580:laurdan