To see or not to see: lateral organization of biological membranes and fluorescence microscopy.

In the last few years several experimental strategies based on epi-, confocal and two photon excitation fluorescence microscopy techniques have been employed to study the lateral structure of membranes using giant vesicles as model systems. This review article discusses the methodological aspects of the aforementioned experimental approaches, particularly stressing the information obtained by the use of UV excited fluorescent probes using two-photon excitation fluorescence microscopy. Additionally, the advantages of utilizing visual information, to correlate the lateral structure of compositionally simple membranes with complex situations, i.e., biological membranes, will be addressed.

[1]  T. E. Thompson,et al.  Lateral diffusion and percolation in two-phase, two-component lipid bilayers. Topology of the solid-phase domains in-plane and across the lipid bilayer. , 1992, Biochemistry.

[2]  D. Haverstick,et al.  Influence of proteins on the reorganization of phospholipid bilayers into large domains. , 1989, Biophysical journal.

[3]  E. Evans,et al.  Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers. , 1988, Biochemistry.

[4]  M. Edidin The state of lipid rafts: from model membranes to cells. , 2003, Annual review of biophysics and biomolecular structure.

[5]  E Gratton,et al.  Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence. , 1994, Biophysical journal.

[6]  Enrico Gratton,et al.  A Model for the Interaction of 6‐Lauroyl‐2‐(N,N‐dimethylamino)naphthalene with Lipid Environments: Implications for Spectral Properties , 1999, Photochemistry and photobiology.

[7]  J. Ipsen,et al.  Modelling the phase equilibria in two-component membranes of phospholipids with different acyl-chain lengths. , 1988, Biochimica et biophysica acta.

[8]  P. Schwille,et al.  SNAREs Prefer Liquid-disordered over “Raft” (Liquid-ordered) Domains When Reconstituted into Giant Unilamellar Vesicles*[boxs] , 2004, Journal of Biological Chemistry.

[9]  D. Hilgemann Getting ready for the decade of the lipids. , 2003, Annual review of physiology.

[10]  B. Lentz,et al.  Phospholipid lateral organization in synthetic membranes as monitored by pyrene-labeled phospholipids: effects of temperature and prothrombin fragment 1 binding. , 1986, Biochemistry.

[11]  E. Sackmann,et al.  On Domain Structure and Local Curvature in Lipid Bilayers and Biological Membranes , 1977, Zeitschrift fur Naturforschung. Section C, Biosciences.

[12]  E. Sackmann,et al.  Membrane bending energy concept of vesicle‐ and cell‐shapes and shape‐transitions , 1994, FEBS letters.

[13]  H. Mcconnell,et al.  Lateral phase separation in phospholipid membranes. , 1973, Biochemistry.

[14]  Patricia Bassereau,et al.  A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. , 2004, Biophysical journal.

[15]  L. Yang,et al.  Membrane domains containing phosphatidylserine and substrate can be important for the activation of protein kinase C. , 1995, Biochemistry.

[16]  E. Gratton,et al.  Membrane lipid domains and dynamics as detected by Laurdan fluorescence , 1995, Journal of Fluorescence.

[17]  P. W. V. van Dijck,et al.  Miscibility properties of binary phosphatidylcholine mixtures. A calorimetric study. , 1977, Biochimica et biophysica acta.

[18]  L. Bagatolli,et al.  Cholesterol Rules , 2004, Journal of Biological Chemistry.

[19]  T. E. Thompson,et al.  Fluid-phase connectivity and translational diffusion in a eutectic, two-component, two-phase phosphatidylcholine bilayer. , 1991, Biochemistry.

[20]  Watt W. Webb,et al.  Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension , 2003, Nature.

[21]  Jeremy M. Tavaré,et al.  Fluorescent and luminescent probes , 1999 .

[22]  R. New,et al.  Liposomes : a practical approach , 1990 .

[23]  E. Gratton,et al.  Dipolar relaxations in glycerol: a dynamic fluorescence study of 4-2'-(dimethylamino)-6'-naphthoylcyclohexanecarboxylic acid (DANCA) , 1987 .

[24]  P. Schwille,et al.  Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol. , 2003, Biophysical journal.

[25]  E. Gratton,et al.  Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases. , 1998, Biophysical journal.

[26]  E. Gratton,et al.  Detecting membrane lipid microdomains by two-photon fluorescence microscopy , 1999, IEEE Engineering in Medicine and Biology Magazine.

[27]  L. Bagatolli,et al.  Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol. , 2006, Biophysical journal.

[28]  T. E. Thompson,et al.  Topology of gel-phase domains and lipid mixing properties in phase-separated two-component phosphatidylcholine bilayers. , 1996, Biophysical journal.

[29]  L. Bagatolli,et al.  Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[30]  M. Longo,et al.  Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions. , 2006, Biophysical journal.

[31]  R N Zare,et al.  Rapid preparation of giant unilamellar vesicles. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Enrico Gratton,et al.  Laurdan and Prodan as Polarity-Sensitive Fluorescent Membrane Probes , 1998, Journal of Fluorescence.

[33]  Sarah L Veatch,et al.  Organization in lipid membranes containing cholesterol. , 2002, Physical review letters.

[34]  G. Fidelio,et al.  Molecular interactions and thermotropic behavior of glycosphingolipids in model membrane systems. , 1986, Chemistry and physics of lipids.

[35]  C. Barrow,et al.  Surface behavior and lipid interaction of Alzheimer beta-amyloid peptide 1-42: a membrane-disrupting peptide. , 2005, Biophysical journal.

[36]  E. Gratton,et al.  Giant vesicles, Laurdan, and two-photon fluorescence microscopy: evidence of lipid lateral separation in bilayers. , 2003, Methods in enzymology.

[37]  E. Gratton,et al.  Direct Observation of Lipid Domains in Free-Standing Bilayers Using Two-Photon Excitation Fluorescence Microscopy , 2001, Journal of Fluorescence.

[38]  E Gratton,et al.  A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study. , 2000, Biophysical journal.

[39]  D. Hammer,et al.  Interaction of the influenza hemagglutinin fusion peptide with lipid bilayers: area expansion and permeation. , 1997, Biophysical journal.

[40]  P. Schwille,et al.  Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS. , 2006, Biophysical journal.

[41]  Petra Schwille,et al.  Probing Lipid Mobility of Raft-exhibiting Model Membranes by Fluorescence Correlation Spectroscopy* , 2003, Journal of Biological Chemistry.

[42]  T. E. Thompson,et al.  Translational diffusion and fluid domain connectivity in a two-component, two-phase phospholipid bilayer. , 1989, Biophysical journal.

[43]  Brian Herman,et al.  Fluorescence imaging spectroscopy and microscopy , 1996 .

[44]  E. Gratton,et al.  Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types. , 1993, Biochimica et biophysica acta.

[45]  O. G. Mouritsen,et al.  Temperature-controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers. , 2003, Biophysical journal.

[46]  K. Gousset,et al.  Evidence for a physiological role for membrane rafts in human platelets , 2002, Journal of cellular physiology.

[47]  Sarah L Veatch,et al.  Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. , 2003, Biophysical journal.

[48]  P. Kinnunen,et al.  Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. , 2000, Biophysical journal.

[49]  Enrico Gratton,et al.  Time-resolved fluorescence microscopy using two-photon excitation , 1995 .

[50]  Ole G Mouritsen,et al.  Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy. , 2002, Biophysical journal.

[51]  P. Schwille,et al.  Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles. , 2005, Biochemistry.

[52]  Kai Simons,et al.  Model systems, lipid rafts, and cell membranes. , 2004, Annual review of biophysics and biomolecular structure.

[53]  E. Gratton,et al.  Two-photon fluorescence microscopy studies of bipolar tetraether giant liposomes from thermoacidophilic archaebacteria Sulfolobus acidocaldarius. , 2000, Biophysical journal.

[54]  de Mendoza J,et al.  Model systems , 1998, Current opinion in chemical biology.

[55]  E. Gratton,et al.  Time-resolved fluorescence emission spectra of Laurdan in phospholipid vesicles by multifrequency phase and modulation fluorometry. , 1986, Cellular and molecular biology.

[56]  D. S. Dimitrov,et al.  A mechanism of liposome electroformation , 1988 .

[57]  T. E. Thompson,et al.  Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 1. Single component phosphatidylcholine liposomes. , 1976, Biochemistry.

[58]  T. E. Thompson,et al.  Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 2 Two-component phosphatidylcholine liposomes. , 1976, Biochemistry.

[59]  M. Lillo,et al.  Cholesterol effect on the physical state of lipid multibilayers from the platelet plasma membrane by time-resolved fluorescence. , 1995, Biochimica et biophysica acta.

[60]  E. Gratton,et al.  Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-1,3,5-hexatriene fluorescence. , 1984, The Journal of biological chemistry.

[61]  Sarah L Veatch,et al.  Miscibility phase diagrams of giant vesicles containing sphingomyelin. , 2005, Physical review letters.

[62]  M. Dogterom,et al.  Membrane tube formation from giant vesicles by dynamic association of motor proteins , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[63]  F. Goñi,et al.  Detergent-resistant, ceramide-enriched domains in sphingomyelin/ceramide bilayers. , 2006, Biophysical journal.

[64]  F. Menger,et al.  Chemistry and physics of giant vesicles as biomembrane models. , 1998, Current opinion in chemical biology.

[65]  E Gratton,et al.  Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. , 1991, Biophysical journal.

[66]  E. Gratton,et al.  Modulation of concentration fluctuations in phase-separated lipid membranes by polypeptide insertion. , 2002, Biophysical journal.

[67]  D. Haverstick,et al.  Visualization of Ca2+-induced phospholipid domains. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[68]  M. Angelova,et al.  Interactions of DNA with giant liposomes. , 1999, Chemistry and physics of lipids.

[69]  E. Gratton,et al.  Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation. , 2004, Journal of the American Chemical Society.

[70]  D. Haverstick,et al.  Visualization of domain formation in the inner and outer leaflets of a phospholipid bilayer , 1988, The Journal of cell biology.

[71]  E Gratton,et al.  Giant phospholipid vesicles: comparison among the whole lipid sample characteristics using different preparation methods: a two photon fluorescence microscopy study. , 2000, Chemistry and physics of lipids.

[72]  Enrico Gratton,et al.  A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles. , 2002, Biophysical journal.

[73]  A. Lee,et al.  Fluorescence studies of chlorophyll a incorporated into lipid mixtures, and the interpretation of "phase" diagrams. , 1975, Biochimica et biophysica acta.

[74]  R. Dowben,et al.  Formation and properties of thin‐walled phospholipid vesicles , 1969, Journal of cellular physiology.

[75]  K. Arnold,et al.  31p-NMR investigations of phase separation in phosphatidylcholine/phosphatidylethanolamine mixtures. , 1981, Biochimica et biophysica acta.

[76]  E. Ikonen,et al.  Functional rafts in cell membranes , 1997, Nature.

[77]  M. Angelova,et al.  Preparation of giant vesicles by external AC electric fields. Kinetics and applications , 1992 .

[78]  Chen-Yuan Dong,et al.  Multiphoton polarization imaging of the stratum corneum and the dermis in ex-vivo human skin. , 2003, Optics express.

[79]  S. Singer,et al.  The fluid mosaic model of the structure of cell membranes. , 1972, Science.

[80]  J. Korlach,et al.  Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[81]  E. Gratton,et al.  Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption. , 2006, Journal of the American Chemical Society.

[82]  E. Gratton,et al.  Visualizing lipid structure and raft domains in living cells with two-photon microscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[83]  P. Schwille,et al.  Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol. , 2005, Chemistry and physics of lipids.

[84]  G. Feigenson,et al.  Ternary phase diagram of dipalmitoyl-PC/dilauroyl-PC/cholesterol: nanoscopic domain formation driven by cholesterol. , 2001, Biophysical journal.

[85]  Manuel Prieto,et al.  Lipid rafts have different sizes depending on membrane composition: a time-resolved fluorescence resonance energy transfer study. , 2005, Journal of molecular biology.

[86]  E Gratton,et al.  Lipid rafts reconstituted in model membranes. , 2001, Biophysical journal.

[87]  E Gratton,et al.  Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach. , 1996, Biophysical journal.

[88]  L. Bagatolli Direct observation of lipid domains in free standing bilayers: from simple to complex lipid mixtures. , 2003, Chemistry and physics of lipids.

[89]  Christer S. Ejsing,et al.  Polyene-lipids: A new tool to image lipids , 2005, Nature Methods.

[90]  K. Fujita [Two-photon laser scanning fluorescence microscopy]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[91]  E. Evans,et al.  Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 degrees C below to 10 degrees C above the liquid crystal-crystalline phase transition at 24 degrees C. , 1988, Biochemistry.

[92]  H. Itoh,et al.  Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. , 1996, Biophysical journal.

[93]  J. Silvius Partitioning of membrane molecules between raft and non-raft domains: insights from model-membrane studies. , 2005, Biochimica et biophysica acta.

[94]  Sarah Kefayati,et al.  Confocal and two-photon microscopy : image enhancement / , 2008 .

[95]  E. Gratton,et al.  Surface properties of cholesterol-containing membranes detected by Prodan fluorescence. , 2001, Biochimica et biophysica acta.

[96]  E. Gratton,et al.  Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. , 1997, Biophysical journal.

[97]  D. Jameson,et al.  Spectral properties of environmentally sensitive probes associated with horseradish peroxidase. , 1996, Biochemistry.

[98]  R. Griffin,et al.  Phase equilibria, molecular conformation, and dynamics in phosphatidylcholine/phosphatidylethanolamine bilayers. , 1982, Biochemistry.

[99]  C. Mateo,et al.  Lateral heterogeneity in human platelet plasma membrane and lipids from the time-resolved fluorescence of trans-parinaric acid , 1991, European Biophysics Journal.

[100]  L. Bagatolli,et al.  Laurdan properties in glycosphingolipid-phospholipid mixtures: a comparative fluorescence and calorimetric study. , 1997, Biochimica et biophysica acta.

[101]  E Gratton,et al.  Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles. , 1999, Biophysical journal.

[102]  G. Weber,et al.  Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene. , 1979, Biochemistry.

[103]  Sarah L Veatch,et al.  Seeing spots: complex phase behavior in simple membranes. , 2005, Biochimica et biophysica acta.

[104]  P. Wolber,et al.  Fluorescence lifetime and time-resolved polarization anisotropy studies of acyl chain order and dynamics in lipid bilayers. , 1981, Biochemistry.

[105]  Robert B. Macgregor,et al.  Estimation of the polarity of the protein interior by optical spectroscopy , 1986, Nature.

[106]  A. Diaspro Confocal and two-photon microscopy : foundations, applications, and advances , 2001 .

[107]  T. E. Thompson,et al.  Fluid phase connectivity in an isomorphous, two-component, two-phase phosphatidylcholine bilayer. , 1990, Biophysical journal.

[108]  H. Garda,et al.  Possible compensation of structural and viscotropic properties in hepatic microsomes and erythrocyte membranes of rats with essential fatty acid deficiency. , 1994, Journal of lipid research.

[109]  E. Gratton,et al.  Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS). , 2004, Biophysical journal.

[110]  D. Wiersma,et al.  Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion. , 2001, Biophysical journal.

[111]  L. Bagatolli,et al.  Direct visualization of membrane leakage induced by the antibiotic peptides: maculatin, citropin, and aurein. , 2005, Biophysical journal.

[112]  L. Bagatolli,et al.  Segregation of saturated chain lipids in pulmonary surfactant films and bilayers. , 2002, Biophysical journal.

[113]  M. Angelova,et al.  DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles , 1999, European Biophysics Journal.

[114]  F. Amat-Guerri,et al.  New Transmembrane Polyene Bolaamphiphiles as Fluorescent Probes in Lipid Bilayers. , 2001, Angewandte Chemie.

[115]  M. Caffrey,et al.  A temperature gradient method for lipid phase diagram construction using time-resolved x-ray diffraction. , 1987, Biophysical journal.

[116]  L. Bagatolli,et al.  Activation of Dynamin II by POPC in Giant Unilamellar Vesicles: A Two-Photon Fluorescence Microscopy Study , 2002, Journal of protein chemistry.

[117]  I. V. Polozov,et al.  Liquid domains in vesicles investigated by NMR and fluorescence microscopy. , 2004, Biophysical journal.

[118]  O. G. Mouritsen,et al.  Phase separation dynamics and lateral organization of two-component lipid membranes. , 1995, Biophysical journal.

[119]  J M Sturtevant,et al.  Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[120]  B. Lentz,et al.  Use of fluorescent probes to monitor molecular order and motions within liposome bilayers. , 1993, Chemistry and physics of lipids.

[121]  D. Brown,et al.  Structure and Origin of Ordered Lipid Domains in Biological Membranes , 1998, The Journal of Membrane Biology.

[122]  P. Luisi,et al.  Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations. , 1996, Chemistry & biology.

[123]  J. Korvink,et al.  Phase equilibria. , 1993, Science.

[124]  M. Prieto,et al.  Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts. , 2003, Biophysical journal.

[125]  E Gratton,et al.  Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. , 2000, Biophysical journal.