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Experimental Parameters Leading to Optimal Bilayers for Total Internal Reflection Fluorescence Microscopy Visualization

Published online by Cambridge University Press:  23 February 2017

Elisabeth Mantil ,
Trinda Crippin ,
Anatoli Ianoul  and
Tyler J. Avis
Elisabeth Mantil
Affiliation:
Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Trinda Crippin
Affiliation:
Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Anatoli Ianoul
Affiliation:
Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Tyler J. Avis*
Affiliation:
Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
*
*Corresponding author. tyler.avis@carleton.ca
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Abstract

Supported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.

Keywords

total internal reflection fluorescence microscopysmall unilamellar vesiclesextrusionlipid phase separationsupported lipid bilayers
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 23 , Issue 1 , February 2017 , pp. 97 - 112
Copyright
© Microscopy Society of America 2017 

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References

Almeida, P.F.F. (2009). Thermodynamics of lipid interactions in complex bilayers. Biochim Biophys Acta 1788(1), 72–85.Google Scholar
Attwood, S.J., Choi, Y. & Leonenko, Z. (2013). Preparation of DOPC and DPPC supported planar lipid bilayers for atomic force microscopy and atomic force spectroscopy. Int J Mol Sci 14(2), 3514–3539.CrossRefGoogle ScholarPubMed
Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89(1), 141–145.Google Scholar
Bagatolli, L.A. (2006). To see or not to see: Lateral organization of biological membranes and fluorescence microscopy. Biochim Biophys Acta 1758(10), 1541–1556.CrossRefGoogle ScholarPubMed
Baumgart, T., Hunt, G., Farkas, E.R., Webb, W.W. & Feigenson, G.W. (2007). Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. Biochim Biophys Acta 1768(9), 2182–2194.Google Scholar
Bouvrais, H., Pott, T., Bagatolli, L.A., Ipsen, J.H. & Méléard, P. (2010). Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers. Biochim Biophys Acta 1798(7), 1333–1337.CrossRefGoogle ScholarPubMed
Brian, A.A. & McConnell, H.M. (1984). Allogeneic stimulation of cytotoxic T cells by supported planar membranes. Proc Natl Acad Sci 81(19), 6159–6163.CrossRefGoogle ScholarPubMed
Castellana, E.T. & Cremer, P.S. (2006). Solid supported lipid bilayers: From biophysical studies to sensor design. Surf Sci Rep 61(10), 429–444.Google Scholar
Cha, T., Guo, A. & Zhu, X.Y. (2006). Formation of supported phospholipid bilayers on molecular surfaces: Role of surface charge density and electrostatic interaction. Biophys J 90(4), 1270–1274.CrossRefGoogle ScholarPubMed
Chan, Y.-H.M. & Boxer, S.G. (2007). Model membrane systems and their applications. Curr Opin Chem Biol 11(6), 581–587.Google Scholar
Chen, D. & Santore, M.M. (2014). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-rich domain formation in binary phospholipid vesicle membranes: Two-dimensional nucleation and growth. Langmuir 30(31), 9484–9493.CrossRefGoogle Scholar
Choucair, A., Chakrapani, M., Chakravarthy, B., Katsaras, J. & Johnston, L.J. (2007). Preferential accumulation of Aβ(1–42) on gel phase domains of lipid bilayers: An AFM and fluorescence study. Biochim Biophys Acta 1768(1), 146–154.Google Scholar
Cornell, B.A., Fletcher, G.C., Middlehurst, J. & Separovic, F. (1982). The lower limit to the size of small sonicated phospholipid vesicles. Biochim Biophys Acta 690(1), 15–19.CrossRefGoogle Scholar
Cremer, P.S. & Boxer, S.G. (1999). Formation and spreading of lipid bilayers on planar glass supports. J Phys Chem B 103(13), 2554–2559.CrossRefGoogle Scholar
Cremer, P.S., Groves, J.T., Kung, L.A. & Boxer, S.G. (1999). Writing and erasing barriers to lateral mobility into fluid phospholipid bilayers. Langmuir 15(11), 3893–3896.CrossRefGoogle Scholar
Daniel, S., Diaz, A.J., Martinez, K.M., Bench, B.J., Albertorio, F. & Cremer, P.S. (2007). Separation of membrane-bound compounds by solid-supported bilayer electrophoresis. J Am Chem Soc 129(26), 8072–8073.Google Scholar
De Vries, J.J. & Berendsen, H.J.C. (1969). Nuclear magnetic resonance measurements on a macroscopically ordered smectic liquid crystalline phase. Nature 221(5186), 1139–1140.Google Scholar
Dewa, T., Vigmond, S.J. & Regen, S.L. (1996). Lateral heterogeneity in fluid bilayers composed of saturated and unsaturated phospholipids. J Am Chem Soc 118(14), 3435–3440.CrossRefGoogle Scholar
Dimitrievski, K. (2010). Deformation of adsorbed lipid vesicles as a function of vesicle size. Langmuir 26(5), 3008–3011.Google Scholar
Dulin, D., Le Gall, A., Perronet, K., Soler, N., Fourmy, D., Yoshizawa, S., Bouyer, P. & Westbrook, N. (2010). Reduced photobleaching of BODIPY-FL. Phys Proc 3(4), 1563–1567.CrossRefGoogle Scholar
Epand, R.M. & Epand, R.F. (2009). Lipid domains in bacterial membranes and the action of antimicrobial agents. Biochim Biophys Acta 1788(1), 289–294.Google Scholar
Galush, W.J., Nye, J.A. & Groves, J.T. (2008). Quantitative fluorescence microscopy using supported lipid bilayer standards. Biophys J 95(5), 2512–2519.CrossRefGoogle ScholarPubMed
Green, D.E. & Tzagoloff, A. (1966). Role of lipids in the structure and function of biological membranes. J Lipid Res 7(5), 587–602.CrossRefGoogle ScholarPubMed
Gromelski, S., Saraiva, A.M., Krastev, R. & Brezesinski, G. (2009). The formation of lipid bilayers on surfaces. Colloids Surf B 74(2), 477–483.CrossRefGoogle ScholarPubMed
Hageneder, S., Bauch, M. & Dostalek, J. (2016). Plasmonically amplified bioassay—total internal reflection fluorescence versus epifluorescence geometry. Talanta 156–157, 225–231.CrossRefGoogle Scholar
Hamai, C., Yang, T., Kataoka, S., Cremer, P.S. & Musser, S.M. (2006). Effect of average phospholipid curvature on supported bilayer formation on glass by vesicle fusion. Biophys J 90(4), 1241–1248.CrossRefGoogle ScholarPubMed
Hardy, G.J., Nayak, R. & Zauscher, S. (2013). Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion. Curr Opin Colloid Interface Sci 18(5), 448–458.Google Scholar
Holden, M.A., Jung, S.-Y., Yang, T., Castellana, E.T. & Cremer, P.S. (2004). Creating fluid and air-stable solid supported lipid bilayers. J Am Chem Soc 126(24), 6512–6513.CrossRefGoogle ScholarPubMed
Honerkamp-Smith, A.R., Veatch, S. & Keller, S.L. (2009). An introduction to critical points for biophysicists; observations of compositional heterogeneity in lipid membranes. Biochim Biophys Acta 1788(1), 53–63.CrossRefGoogle ScholarPubMed
Ira, & Johnston, L.J. (2008). Sphingomyelinase generation of ceramide promotes clustering of nanoscale domains in supported bilayer membranes. Biochim Biophys Acta 1778(1), 185–197.CrossRefGoogle ScholarPubMed
Ira, , Zou, S., Ramirez, D., Vanderslip, S., Ogilvie, W., Jakubek, Z. & Johnston, L. (2009). Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers. J Struct Biol 168(1), 78–89.Google Scholar
Johnston, L.J. (2007). Nanoscale imaging of domains in supported lipid membranes. Langmuir 23(11), 5886–5895.Google Scholar
Juhasz, J., Davis, J. & Sharom, F. (2010). Fluorescent probe partitioning in giant unilamellar vesicles of ‘lipid raft’ mixtures. Biochem J 430(3), 415–423.CrossRefGoogle ScholarPubMed
Kaasgaard, T., Leidy, C., Crowe, J.H., Mouritsen, O.G. & Jørgensen, K. (2003). Temperature-controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers. Biophys J 85(1), 350–360.CrossRefGoogle ScholarPubMed
Kalb, E., Frey, S. & Tamm, L.K. (1992). Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers. Biochim Biophys Acta 1103(2), 307–316.CrossRefGoogle ScholarPubMed
Kashimura, Y., Durao, J., Furukawa, K. & Keiichi, T. (2008). Self-spreading behavior of supported lipid bilayer through single sub-100-nm gap. Jpn J Appl Phys 47(4S), 3248–3252.CrossRefGoogle Scholar
Kashimura, Y., Furukawa, K. & Keiichi, T. (2010). Self-spreading supported lipid bilayer passing through single nanogap structure: Effect of position of dyes in lipid molecules. Jpn J Appl Phys 49(4S), 04DL15.Google Scholar
Lasic, D.D. (1988). The mechanism of vesicle formation. Biochem J 256(1), 1–11.CrossRefGoogle ScholarPubMed
Loura, L.M.S. & Ramalho, J.P.P. (2011). Recent developments in molecular dynamics simulations of fluorescent membrane probes. Molecules 16(7), 5437–5452.Google Scholar
Mattheyses, A.L., Simon, S.M. & Rappoport, J.Z. (2010). Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123(21), 3621–3628.CrossRefGoogle ScholarPubMed
McConnell, H.M., Watts, T.H., Weis, R.M. & Brian, A.A. (1986). Supported planar membranes in studies of cell-cell recognition in the immune system. Biochim Biophys Acta 864(1), 95–106.Google Scholar
McKiernan, A.E., Ratto, T.V. & Longo, M.L. (2000). Domain growth, shapes, and topology in cationic lipid bilayers on mica by fluorescence and atomic force microscopy. Biophys J 79(5), 2605–2615.Google Scholar
Neville, F., Cahuzac, M., Konovalov, O., Ishitsuka, Y., Lee, K.Y.C., Kuzmenko, I., Kale, G.M. & Gidalevitz, D. (2006). Lipid headgroup discrimination by antimicrobial peptide LL-37: Insight into mechanism of action. Biophys J 90(4), 1275–1287.Google Scholar
Oreopoulos, J. & Yip, C.M. (2009). Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM. J Struct Biol 168(1), 21–36.CrossRefGoogle Scholar
Polyakova, S.M., Belov, V.N., Yan, S.F., Eggeling, C., Ringemann, C., Schwarzmann, G., de Meijere, A. & Hell, S.W. (2009). New GM1 ganglioside derivatives for selective single and double labelling of the natural glycosphingolipid skeleton. Eur J Org Chem 2009(30), 5162–5177.CrossRefGoogle Scholar
Ramirez, D.M.C., Ogilvie, W.W. & Johnston, L.J. (2010). NBD-cholesterol probes to track cholesterol distribution in model membranes. Biochim Biophys Acta 1798(3), 558–568.Google Scholar
Ravier, M.A., Tsuboi, T. & Rutter, G.A. (2008). Imaging a target of Ca2+ signalling: Dense core granule exocytosis viewed by total internal reflection fluorescence microscopy. Methods 46(3), 233–238.CrossRefGoogle ScholarPubMed
Schönherr, H., Johnson, J.M., Lenz, P., Frank, C.W. & Boxer, S.G. (2004). Vesicle adsorption and lipid bilayer formation on glass studied by atomic force microscopy. Langmuir 20(26), 11600–11606.CrossRefGoogle ScholarPubMed
Seelig, J. & Macdonald, P.M. (1987). Phospholipids and proteins in biological membranes. Deuterium NMR as a method to study structure, dynamics, and interactions. Acc Chem Res 20(6), 221–228.Google Scholar
Seifert, U. & Lipowsky, R. (1990). Adhesion of vesicles. Phys Rev A 42(8), 4768–4771.CrossRefGoogle ScholarPubMed
Shaw, J.E., Slade, A. & Yip, C.M. (2003). Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers. J Am Chem Soc 125(39), 11838–11839.CrossRefGoogle ScholarPubMed
Singer, S.J. & Nicolson, G.L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175(4023), 720–731.CrossRefGoogle ScholarPubMed
Skaug, M.J., Longo, M.L. & Faller, R. (2011). The impact of Texas Red on lipid bilayer properties. J Phys Chem B 115(26), 8500–8505.Google Scholar
Somerharju, P., Virtanen, J.A., Cheng, K.H. & Hermansson, M. (2009). The superlattice model of lateral organization of membranes and its implications on membrane lipid homeostasis. Biochim Biophys Acta 1788(1), 12–23.Google Scholar
Thompson, N.L., Pearce, K.H. & Hsieh, H.V. (1993). Total internal reflection fluorescence microscopy: Application to substrate-supported planar membranes. Eur Biophys J 22(5), 367–378.CrossRefGoogle ScholarPubMed
Thompson, N.L., Wang, X. & Navaratnarajah, P. (2009). Total internal reflection with fluorescence correlation spectroscopy: Applications to substrate-supported planar membranes. J Struct Biol 168(1), 95–106.CrossRefGoogle ScholarPubMed
Travkova, O.G. & Brezesinski, G. (2013). Adsorption of the antimicrobial peptide arenicin and its linear derivative to model membranes—a maximum insertion pressure study. Chem Phys Lipids 167–168, 43–50.CrossRefGoogle ScholarPubMed
Uster, P.S. & Pagano, R.E. (1986). Resonance energy transfer microscopy: Observations of membrane-bound fluorescent probes in model membranes and in living cells. J Cell Biol 103(4), 1221–1234.CrossRefGoogle ScholarPubMed
Vanegas, J.M., Contreras, M.F., Faller, R. & Longo, M.L. (2012). Role of unsaturated lipid and ergosterol in ethanol tolerance of model yeast biomembranes. Biophys J 102(3), 507–516.Google Scholar
Veatch, S.L. & Keller, S.L. (2003). Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys J 85(5), 3074–3083.Google Scholar
Veatch, S.L. & Keller, S.L. (2005). Seeing spots: Complex phase behavior in simple membranes. Biochim Biophys Acta 1746(3), 172–185.CrossRefGoogle ScholarPubMed
Yuan, J., Hao, C., Chen, M., Berini, P. & Zou, S. (2013). Lipid reassembly in asymmetric langmuir–blodgett/langmuir–schaeffer bilayers. Langmuir 29(1), 221–227.CrossRefGoogle ScholarPubMed
Zhao, J., Wu, J., Shao, H., Kong, F., Jain, N., Hunt, G. & Feigenson, G. (2007). Phase studies of model biomembranes: Macroscopic coexistence of Lα+Lβ, with light-induced coexistence of Lα+Lo phases. Biochim Biophys Acta 1768(11), 2777–2786.Google Scholar