Monitoring Membrane Hydration with 2-(Dimethylamino)-6-Acylnaphtalenes Fluorescent Probes

  • Luis A. BagatolliEmail author
Part of the Subcellular Biochemistry book series (SCBI, volume 71)


A family of polarity sensitive fluorescent probes (2-(dimethylamino)-6-acylnaphtalenes, i.e. LAURDAN, PRODAN, ACDAN) was introduced by Gregorio Weber in 1979, with the aim to monitor solvent relaxation phenomena on protein matrices. In the following years, however, PRODAN and particularly LAURDAN, were used to study membrane lateral structure and associated dynamics. Once incorporated into membranes, the (nanosecond) fluorescent decay of these probes is strongly affected by changes in the local polarity and relaxation dynamics of restricted water molecules existing at the membrane/water interface. For instance, when glycerophospholipid containing membranes undertake a solid ordered (gel) to liquid disordered phase transition the fluorescence emission maximum of these probes shift ~ 50 nm with a significant change in their fluorescence lifetime. Furthermore, the fluorescence parameters of LAURDAN and PRODAN are exquisitely sensitive to cholesterol effects, allowing interpretations that correlate changes in membrane packing with membrane hydration. Different membrane model systems as well as innate biological membranes have been studied with this family of probes allowing interesting comparative studies. This chapter presents a short historical overview about these fluorescent reporters, discusses on different models proposed to explain their sensitivity to membrane hydration, and includes relevant examples from experiments performed in artificial and biological membranes.


Generalized polarization Fluorescent probes Laurdan 



This work is supported in part by a grant from the Danish Research Council (12-124751).


  1. Almaleck H, Gordillo GJ, Disalvo A (2013) Water defects induced by expansion and electrical fields in DMPC and DMPE monolayers: contribution of hydration and confined water. Colloids Surf B Biointerfaces 102:871–878CrossRefPubMedGoogle Scholar
  2. Antollini SS, Barrantes FJ (1998) Disclosure of discrete sites for phospholipid and sterols at the protein-lipid interface in native acetylcholine receptor-rich membrane. Biochemistry 37:16653–16662CrossRefPubMedGoogle Scholar
  3. Arnulphi C, Levstein PR, Ramia ME, Martin CA, Fidelio GD (1997) Ganglioside hydration study by 2H-NMR: dependence on temperature and water/lipid ratio. J Lipid Res 38:1412–1420PubMedGoogle Scholar
  4. Bagatolli LA (2006) To see or not to see: lateral organization of biological membranes and fluorescence microscopy. Biochim Biophys Acta 1758:1541–1556CrossRefPubMedGoogle Scholar
  5. Bagatolli LA (2013) LAURDAN fluorescence properties in membranes: a journey from the fluorometer to the microscope. In: Mely Y, Duportail G (eds) Fluorescent methods to study biological membranes. Springer, Heidelberg/New York, pp 3–36Google Scholar
  6. Bagatolli LA, Gratton E (1999) Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles. Biophys J 77:2090–2101PubMedCentralCrossRefPubMedGoogle Scholar
  7. Bagatolli LA, Gratton E (2000a) A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: a two-photon fluorescence microscopy study. Biophys J 79:434–447PubMedCentralCrossRefPubMedGoogle Scholar
  8. Bagatolli LA, Gratton E (2000b) Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys J 78:290–305PubMedCentralCrossRefPubMedGoogle Scholar
  9. Bagatolli LA, Gratton E (2001) Direct observation of lipid domains in free-standing bilayers using two-photon excitation fluorescence microscopy. J Fluoresc 11:141–160CrossRefGoogle Scholar
  10. Bagatolli LA, Maggio B, Aguilar F, Sotomayor CP, Fidelio GD (1997) Laurdan properties in glycosphingolipid-phospholipid mixtures: a comparative fluorescence and calorimetric study. Biochim Biophys Acta 1325:80–90CrossRefPubMedGoogle Scholar
  11. Bagatolli LA, Gratton E, Fidelio GD (1998) Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence. Biophys J 75:331–341PubMedCentralCrossRefPubMedGoogle Scholar
  12. Bagatolli LA, Parasassi T, Fidelio GD, Gratton E (1999) A model for the interaction of 6-lauroyl-2-(N, N-dimethylamino)naphthalene with lipid environments: implications for spectral properties. Photochem Photobiol 70:557–564CrossRefPubMedGoogle Scholar
  13. Bernardino De La Serna J, Oradd G, Bagatolli LA, Simonsen AC, Marsh D, Lindblom G, Perez-Gil J (2009) Segregated phases in pulmonary surfactant membranes do not show coexistence of lipid populations with differentiated dynamic properties. Biophys J 97:1381–1389CrossRefPubMedGoogle Scholar
  14. Bernardino De La Serna J, Hansen S, Berzina Z, Simonsen AC, Hannibal-Bach HK, Knudsen J, Ejsing CS, Bagatolli LA (2013) Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice. Biochim Biophys Acta 1828:2450–2459CrossRefPubMedGoogle Scholar
  15. Bernchou U, Brewer J, Midtiby HS, Ipsen JH, Bagatolli LA, Simonsen AC (2009) Texture of lipid bilayer domains. J Am Chem Soc 131:14130–14131CrossRefPubMedGoogle Scholar
  16. Bloksgaard M, Bek S, Marcher AB, Neess D, Brewer J, Hannibal-Bach HK, Helledie T, Fenger C, Due M, Berzina Z, Neubert R, Chemnitz J, Finsen B, Clemmensen A, Wilbertz J, Saxtorph H, Knudsen J, Bagatolli L, Mandrup S (2012a) The acyl-CoA binding protein is required for normal epidermal barrier function in mice. J Lipid Res 53:2162–2174PubMedCentralCrossRefPubMedGoogle Scholar
  17. Bloksgaard M, Svane-Knudsen V, Sorensen JA, Bagatolli L, Brewer J (2012b) Structural characterization and lipid composition of acquired cholesteatoma: a comparative study with normal skin. Otol Neurotol 33:177–183CrossRefPubMedGoogle Scholar
  18. Bloksgaard M, Brewer J, Pashkovski E, Ananthapadmanabhan KP, Ahm Sørensen J, Bagatolli LA (2014) Effect of detergents on the physico-chemical properties of skin stratum corneum: a two-photon excitation fluorescence microscopy study. Int J Cosmet Sci 36(1):39–45CrossRefPubMedGoogle Scholar
  19. Brewer J, Bernardino De La Serna J, Wagner K, Bagatolli LA (2010) Multiphoton excitation fluorescence microscopy in planar membrane systems. Biochim Biophys Acta 1798:1301–1308CrossRefPubMedGoogle Scholar
  20. Carrer DC, Vermehren C, Bagatolli LA (2008) Pig skin structure and transdermal delivery of liposomes: a two photon microscopy study. J Control Release 132:12–20CrossRefPubMedGoogle Scholar
  21. Celli A, Gratton E (2010) Dynamics of lipid domain formation: fluctuation analysis. Biochim Biophys Acta 1798:1368–1376PubMedCentralCrossRefPubMedGoogle Scholar
  22. Chong PL (1988) Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers and cellular membranes. Biochemistry 27:399–404CrossRefPubMedGoogle Scholar
  23. Chong PL-G (1990) Interactions of LAURDAN and PRODAN with membranes at high pressure. High Pressure Res 5:761–763CrossRefGoogle Scholar
  24. Dietrich C, Bagatolli LA, Volovyk ZN, Thompson NL, Levi M, Jacobson K, Gratton E (2001) Lipid rafts reconstituted in model membranes. Biophys J 80:1417–1428PubMedCentralCrossRefPubMedGoogle Scholar
  25. Dodes Traian MM, González Flecha FL, Levi V (2012) Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope. J Lipid Res 53(3):609–616PubMedCentralCrossRefPubMedGoogle Scholar
  26. Fidorra M, Duelund L, Leidy C, Simonsen AC, Bagatolli LA (2006) Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol. Biophys J 90:4437–4451PubMedCentralCrossRefPubMedGoogle Scholar
  27. Fidorra M, Heimburg T, Bagatolli LA (2009) Direct visualization of the lateral structure of porcine brain cerebrosides/POPC mixtures in presence and absence of cholesterol. Biophys J 97:142–154PubMedCentralCrossRefPubMedGoogle Scholar
  28. Gaus K, Gratton E, Kable EP, Jones AS, Gelissen I, Kritharides L, Jessup W (2003) Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc Natl Acad Sci U S A 100:15554–15559PubMedCentralCrossRefPubMedGoogle Scholar
  29. Golfetto O, Hinde E, Gratton E (2013) Laurdan fluorescence lifetime discriminates cholesterol content from changes in fluidity in living cell membranes. Biophys J 104:1238–1247PubMedCentralCrossRefPubMedGoogle Scholar
  30. Henshaw JB, Olsen CA, Farnbach AR, Nielson KH, Bell JD (1998) Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2. Biochemistry 37:10709–10721CrossRefPubMedGoogle Scholar
  31. Hutterer R, Schneider FW, Sprinz H, Hof M (1996) Binding and relaxation behaviour of prodan and patman in phospholipid vesicles: a fluorescence and 1H NMR study. Biophys Chem 61:151–160CrossRefPubMedGoogle Scholar
  32. Jameson DM, Croney JC, Moens PD (2003) Fluorescence: basic concepts, practical aspects, and some anecdotes. Methods Enzymol 360:1–43CrossRefPubMedGoogle Scholar
  33. Juhasz J, Davis JH, Sharom FJ (2010) Fluorescent probe partitioning in giant unilamellar vesicles of ‘lipid raft’ mixtures. Biochem J 430:415–423CrossRefPubMedGoogle Scholar
  34. Jurkiewicz P, Sykora J, Olzynska A, Humpolickova J, Hof M (2005) Solvent relaxation in phospholipid bilayers: principles and recent applications. J Fluoresc 15:883–894CrossRefPubMedGoogle Scholar
  35. Jurkiewicz P, Olzynska A, Langner M, Hof M (2006) Headgroup hydration and mobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study. Langmuir 22:8741–8749CrossRefPubMedGoogle Scholar
  36. Jurkiewicz P, Cwiklik L, Jungwirth P, Hof M (2012) Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations. Biochimie 94:26–32CrossRefPubMedGoogle Scholar
  37. Kim HM, Choo HJ, Jung SY, Ko YG, Park WH, Jeon SJ, Kim CH, Joo T, Cho BR (2007) A two-photon fluorescent probe for lipid raft imaging: C-laurdan. Chembiochem 8:553–559CrossRefPubMedGoogle Scholar
  38. Korlach J, Schwille P, Webb WW, Feigenson GW (1999) Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci U S A 96:8461–8466PubMedCentralCrossRefPubMedGoogle Scholar
  39. Krasnowska EK, Gratton E, Parasassi T (1998) Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases. Biophys J 74:1984–1993PubMedCentralCrossRefPubMedGoogle Scholar
  40. Krasnowska EK, Bagatolli LA, Gratton E, Parasassi T (2001) Surface properties of cholesterol-containing membranes detected by Prodan fluorescence. Biochim Biophys Acta 1511:330–340CrossRefPubMedGoogle Scholar
  41. Kubiak J, Brewer J, Hansen S, Bagatolli LA (2011) Lipid lateral organization on giant unilamellar vesicles containing lipopolysaccharides. Biophys J 100:978–986PubMedCentralCrossRefPubMedGoogle Scholar
  42. Lakowicz JR, Sheppard JR (1981) Fluorescence spectroscopic studies of Huntington fibroblast membranes. Am J Hum Genet 33:155–165PubMedCentralPubMedGoogle Scholar
  43. Lakowicz JR, Weber G (1973) Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale. Biochemistry 12:4171–4179CrossRefPubMedGoogle Scholar
  44. Lakowicz JR, Bevan DR, Maliwal BP, Cherek H, Balter A (1983) Synthesis and characterization of a fluorescence probe of the phase transition and dynamic properties of membranes. Biochemistry 22:5714–5722CrossRefGoogle Scholar
  45. Lippert E (1957) Spektroskopische Bestimmung des Dipolmomentes aromatischer Verbindungen im ersten angeregten Singulettzustand. Z Elektrochem 61:962–975Google Scholar
  46. Macgregor RB, Weber G (1986) Estimation of the polarity of the protein interior by optical spectroscopy. Nature 319:70–73CrossRefPubMedGoogle Scholar
  47. Montes LR, Alonso A, Goni FM, Bagatolli LA (2007) Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions. Biophys J 93:3548–3554PubMedCentralCrossRefPubMedGoogle Scholar
  48. Nag K, Pao JS, Harbottle RR, Possmayer F, Petersen NO, Bagatolli LA (2002) Segregation of saturated chain lipids in pulmonary surfactant films and bilayers. Biophys J 82:2041–2051PubMedCentralCrossRefPubMedGoogle Scholar
  49. Nielsen SB, Otzen DE (2010) Impact of the antimicrobial peptide Novicidin on membrane structure and integrity. J Colloid Interface Sci 345:248–256CrossRefPubMedGoogle Scholar
  50. Norlen L, Plasencia I, Bagatolli L (2008) Stratum corneum lipid organization as observed by atomic force, confocal and two-photon excitation fluorescence microscopy. Int J Cosmet Sci 30:391–411CrossRefPubMedGoogle Scholar
  51. Olzynska A, Zan A, Jurkiewicz P, Sykora J, Grobner G, Langner M, Hof M (2007) Molecular interpretation of fluorescence solvent relaxation of Patman and 2H NMR experiments in phosphatidylcholine bilayers. Chem Phys Lipids 147:69–77CrossRefPubMedGoogle Scholar
  52. Parasassi T, Gratton E (1992) Packing of phospholipid vesicles studied by oxygen quenching of Laurdan fluorescence. J Fluoresc 2:167–174CrossRefPubMedGoogle Scholar
  53. Parasassi T, Gratton E (1995) Membrane lipid domains and dynamics as detected by LAURDAN fluorescence. J Fluoresc 5:59–69CrossRefPubMedGoogle Scholar
  54. Parasassi T, Conti F, Gratton E (1986a) Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry. Cell Mol Biol 32:99–102PubMedGoogle Scholar
  55. Parasassi T, Conti F, Gratton E (1986b) Time-resolved fluorescence emission spectra of Laurdan in phospholipid vesicles by multifrequency phase and modulation fluorometry. Cell Mol Biol 32:103–108PubMedGoogle Scholar
  56. Parasassi T, De Stasio G, D’ubaldo A, Gratton E (1990) Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys J 57:1179–1186PubMedCentralCrossRefPubMedGoogle Scholar
  57. Parasassi T, De Stasio G, Ravagnan G, Rusch RM, Gratton E (1991) Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. Biophys J 60:179–189PubMedCentralCrossRefPubMedGoogle Scholar
  58. Parasassi T, Loiero M, Raimondi M, Ravagnan G, Gratton E (1993a) Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types. Biochim Biophys Acta 1153:143–154CrossRefPubMedGoogle Scholar
  59. Parasassi T, Ravagnan G, Rusch RM, Gratton E (1993b) Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence. Photochem Photobiol 57:403–410CrossRefPubMedGoogle Scholar
  60. Parasassi T, Di Stefano M, Loiero M, Ravagnan G, Gratton E (1994a) Cholesterol modifies water concentration and dynamics in phospholipid bilayers: a fluorescence study using Laurdan probe. Biophys J 66:763–768PubMedCentralCrossRefPubMedGoogle Scholar
  61. Parasassi T, Di Stefano M, Loiero M, Ravagnan G, Gratton E (1994b) Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence. Biophys J 66:120–132PubMedCentralCrossRefPubMedGoogle Scholar
  62. Parasassi T, Giusti AM, Raimondi M, Gratton E (1995) Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations. Biophys J 68:1895–1902PubMedCentralCrossRefPubMedGoogle Scholar
  63. Parasassi T, Gratton E, Yu WM, Wilson P, Levi M (1997) Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. Biophys J 72:2413–2429PubMedCentralCrossRefPubMedGoogle Scholar
  64. Parasassi T, Krasnowska EK, Bagatolli LA, Gratton E (1998) LAURDAN and Prodan as polarity sensitive fluorescent membrane probes. J Fluoresc 8:365–373CrossRefGoogle Scholar
  65. Plasencia I, Norlen L, Bagatolli LA (2007) Direct visualization of lipid domains in human skin stratum corneum’s lipid membranes: effect of pH and temperature. Biophys J 93:3142–3155PubMedCentralCrossRefPubMedGoogle Scholar
  66. Sanchez SA, Bagatolli LA, Gratton E, Hazlett TL (2002) A two-photon view of an enzyme at work: crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles. Biophys J 82:2232–2243PubMedCentralCrossRefPubMedGoogle Scholar
  67. Sanchez SA, Tricerri MA, Gratton E (2012) Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo. Proc Natl Acad Sci U S A 109:7314–7319PubMedCentralCrossRefPubMedGoogle Scholar
  68. Sot J, Bagatolli LA, Goni FM, Alonso A (2006) Detergent-resistant, ceramide-enriched domains in sphingomyelin/ceramide bilayers. Biophys J 90:903–914PubMedCentralCrossRefPubMedGoogle Scholar
  69. Stock RP, Brewer J, Wagner K, Ramos-Cerrillo B, Duelund L, Jernshoj KD, Olsen LF, Bagatolli LA (2012) Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate. PLoS One 7:e36003PubMedCentralCrossRefPubMedGoogle Scholar
  70. Sumbilla C, Lakowicz JR (1982) Fluorescence studies of red blood cell membranes from individuals with Huntington’s disease. J Neurochem 38:1699–1708CrossRefPubMedGoogle Scholar
  71. Sun Y, Lo W, Lin SJ, Jee SH, Dong CY (2004) Multiphoton polarization and generalized polarization microscopy reveal oleic-acid-induced structural changes in intercellular lipid layers of the skin. Opt Lett 29:2013–2015CrossRefPubMedGoogle Scholar
  72. Vanounou S, Pines D, Pines E, Parola AH, Fishov I (2002) Coexistence of domains with distinct order and polarity in fluid bacterial membranes. Photochem Photobiol 76:1–11CrossRefPubMedGoogle Scholar
  73. Viard M, Gallay J, Vincent M, Meyer O, Robert B, Paternostre M (1997) Laurdan solvatochromism: solvent dielectric relaxation and intramolecular excited-state reaction. Biophys J 73:2221–2234PubMedCentralCrossRefPubMedGoogle Scholar
  74. Vincent M, De Foresta B, Gallay J (2005) Nanosecond dynamics of a mimicked membrane-water interface observed by time-resolved stokes shift of LAURDAN. Biophys J 88:4337–4350PubMedCentralCrossRefPubMedGoogle Scholar
  75. Weber G, Farris FJ (1979) Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene. Biochemistry 18:3075–3078CrossRefPubMedGoogle Scholar
  76. Yu W, So PT, French T, Gratton E (1996) Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach. Biophys J 70:626–636PubMedCentralCrossRefPubMedGoogle Scholar
  77. Zeng J, Chong PL (1995) Effect of ethanol-induced lipid interdigitation on the membrane solubility of Prodan, Acdan, and Laurdan. Biophys J 68:567–573PubMedCentralCrossRefPubMedGoogle Scholar

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© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Membrane Biophysics and Biophotonics Group/MEMPHYS-Center for Biomembrane Physics, Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdense MDenmark

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