Using Time-Resolved Fluorescence Anisotropy of di-4-ANEPPDHQ and F2N12S to Analyze Lipid Packing Dynamics in Model Systems

  • Harmen B. B. Steele
  • Matthew J. Sydor
  • Donald S. Anderson
  • Andrij Holian
  • J. B. Alexander RossEmail author


The fluorescence probes di-4-ANEPPDHQ and F2N12S have solvochromatic emission spectra and fluorescence lifetimes that are sensitive to order within the environment of lipid membranes. We show in this communication that the time-resolved fluorescence anisotropy of these probes, analyzed either by the wobble-in-a-cone model or by the model-independent order parameter S2, provides complementary information about dynamics and lipid packing in a variety of homogeneous lipid membranes systems.


di-4-ANEPPDHQ F2N12S Wobble-in-a-cone Order parameter Lipid packing Lipid dynamics Fluorescence anisotropy 



The research in this publication was performed in the BioSpectroscopy Core Research Laboratory at the University of Montana, which is supported by National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health CoBRE award P20 GM103546 to the Center for Biomolecular Structure and Dynamics. Additional funding was provided by National Institutes of Health (NIGMS) CoBRE award P30 GM103338 and National Institute of Environmental Health Sciences (NIEHS) award R01 ES023209. DSA received support from the NIEHS of the National Institutes of Health under NSRA award number F32 ES027324. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Supplementary material

10895_2019_2363_MOESM1_ESM.docx (81 kb)
ESM 1 (DOCX 81 kb)


  1. 1.
    Jin L, Millard AC, Wuskell JP, Clark HA, Loew LM (2005) Cholesterol-enriched lipid domains can be visualized by di-4-ANEPPDHQ with linear and nonlinear optics. Biophys J 89:L04–L06. CrossRefGoogle Scholar
  2. 2.
    Owen DM, Lanigan PMP, Dunsby C, Munro I, Grant D, Neil MAA, French PMW, Magee AI (2006) Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells. Biophys J 90:L80–L82. CrossRefGoogle Scholar
  3. 3.
    Shynkar VV, Klymchenko AS, Kunzelmann C, Duportail G, Muller CD, Demchenko AP, Freyssinet JM, Mely Y (2007) Fluorescent biomembrane probe for ratiometric detection of apoptosis. J Am Chem Soc 129:2187–2193. CrossRefGoogle Scholar
  4. 4.
    Obaid a L, Loew LM, Wuskell JP, Salzberg BM (2004) Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis. J Neurosci Methods 134:179–190. CrossRefGoogle Scholar
  5. 5.
    Kilin V, Glushonkov O, Herdly L, Klymchenko A, Richert L, Mely Y (2015) Fluorescence lifetime imaging of membrane lipid order with a Ratiometric fluorescent probe. Biophys J 108:2521–2531. CrossRefGoogle Scholar
  6. 6.
    Dinic J, Biverståhl H, Mäler L, Parmryd I (2011) Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing. Biochim Biophys Acta Biomembr 1808:298–306. CrossRefGoogle Scholar
  7. 7.
    Jin L, Millard AC, Wuskell JP, Dong X, Wu D, Clark HA, Loew LM (2006) Characterization and application of a new optical probe for membrane lipid domains. Biophys J 90:2563–2575. CrossRefGoogle Scholar
  8. 8.
    Klymchenko AS, Oncul S, Didier P, Schaub E, Bagatolli L, Duportail G, Mély Y (2009) Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone. Biochim Biophys Acta Biomembr 1788:495–499. CrossRefGoogle Scholar
  9. 9.
    Das R, Klymchenko AS, Duportail G, Mély Y (2008) Excited state proton transfer and solvent relaxation of a 3-Hydroxyflavone probe in lipid bilayers. J Phys Chem B 112:11929–11935. CrossRefGoogle Scholar
  10. 10.
    Klymchenko AS, Mély Y, Demchenko AP, Duportail G (2004) Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes. Biochim Biophys Acta Biomembr 1665:6–19. CrossRefGoogle Scholar
  11. 11.
    Timr Š (2013) Simulation of processes in cellular membranes. Charles University in PragueGoogle Scholar
  12. 12.
    Timr Š, Bondar A, Cwiklik L, Štefl M, Hof M, Vazdar M, Lazar J, Jungwirth P (2014) Accurate determination of the orientational distribution of a fluorescent molecule in a phospholipid membrane. J Phys Chem B 118:855–863. CrossRefGoogle Scholar
  13. 13.
    Darwich Z, Kucherak OA, Kreder R, Richert L, Vauchelles R, Mély Y, Klymchenko AS (2013) Rational design of fluorescent membrane probes for apoptosis based on 3-hydroxyflavone. Methods Appl Fluoresc 1:025002. CrossRefGoogle Scholar
  14. 14.
    Klymchenko AS, Kreder R (2014) Fluorescent probes for lipid rafts: from model membranes to living cells. Chem Biol 21:97–113. CrossRefGoogle Scholar
  15. 15.
    Le Marois A, Owen DM, Suhling K (2015) Investigating cell membrane structure and dynamics with TCSPC-FLIM. In: Periasamy A, So PTC, König K (eds) SPIE proceedings. p 932938Google Scholar
  16. 16.
    Kinosita K, Ikegami A, Kawato S (1982) On the wobbling-in-cone analysis of fluorescence anisotropy decay. Biophys J 37:461–464. CrossRefGoogle Scholar
  17. 17.
    Kinosita K, Kawato S, Ikegami A (1977) A theory of fluorescence polarization decay in membranes. Biophys J 20:289–305. CrossRefGoogle Scholar
  18. 18.
    Kawato S, Kinosita K, Ikegami A (1977) Dynamic structure of lipid bilayers studied by nanosecond fluorescence techniques. Biochemistry 16:2319–2324. CrossRefGoogle Scholar
  19. 19.
    Heyn MP (1979) Determination of lipid order parameters and rotational correlation times from fluorescence depolarization experiments. FEBS Lett 108:359–364. CrossRefGoogle Scholar
  20. 20.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York, NYCrossRefGoogle Scholar
  21. 21.
    Chen LA, Dale RE, Roth S, Brands L (1977) Nanosecond time-dependent fluorescence depolarization of diphenylhexatriene in Dimyristoyllecithin vesicles and the. J Biol Chem 252:2163–2169Google Scholar
  22. 22.
    Dale RE, Chen LA, Brand L (1977) Rotational relaxation of the “microviscovity” probe diphenylhexatriene in paraffin oil and egg lecithin vesicles. J Biol Chem 252:7500–7510Google Scholar
  23. 23.
    Bonneau L, Gerbeau-Pissot P, Thomas D, der C, Lherminier J, Bourque S, Roche Y, Simon-Plas F (2010) Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells. Biochim Biophys Acta Biomembr 1798:2150–2159. CrossRefGoogle Scholar
  24. 24.
    Oncul S, Klymchenko AS, Kucherak OA, Demchenko AP, Martin S, Dontenwill M, Arntz Y, Didier P, Duportail G, Mély Y (2010) Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: effects of cholesterol depletion and apoptosis. Biochim Biophys Acta Biomembr 1798:1436–1443. CrossRefGoogle Scholar
  25. 25.
    Minazzo AS, Darlington RC, Ross JBA (2009) Loop dynamics of the extracellular domain of human tissue factor and activation of factor VIIa. Biophys J 96:681–692. CrossRefGoogle Scholar
  26. 26.
    YashRoy RC (1990) Determination of membrane lipid phase transition temperature from 13C-NMR intensities. J Biochem Biophys Methods 20:353–356. CrossRefGoogle Scholar
  27. 27.
    Silvius JR (1982) Thermotropic phase transitions of pure lipids in model membranes and their modifications by membrane proteins. John Wiley & Sons, Inc, New York, NYGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry & BiochemistryUniversity of MontanaMissoulaUSA
  2. 2.Center for Biomolecular Structure & DynamicsUniversity of MontanaMissoulaUSA
  3. 3.Department of Biomedical and Pharmaceutical Sciences, Center for Environmental Health SciencesUniversity of MontanaMissoulaUSA

Personalised recommendations