平鋪在基板(如:雲母片)上的脂質雙層 (supported lipid bilayer)，常被當成研究細胞膜的模型，且已被廣泛使用在生物物理相關領域。但雲母片 (mica) 對脂質雙層的影響少有研究。我們利用原子力顯微鏡探測在雲母片和在POPC塗佈雲母片 (POPC-coated mica) 上的1:1 DPPC/DOPC表面形貌，本研究中大部分的量測是在液體環境中進行。為了在液態環境中鋪平脂質雙層，我們使用不同方法製作脂質雙層，相較旋轉塗佈方法 (spin coating)，脂球融合方法 (vesicle fusion) 所製備的樣品在液態環境中有更好的品質。1:1 DPPC/DOPC 脂質雙層平鋪在POPC塗佈雲母片上的AFM影像，顯示出兩種脂質雙層厚度分布：較厚的脂質雙層推測對應到“富含DPPC的膠態區塊”(DPPC-rich gel phase domain) ; 較薄的脂質雙層推測對應到“富含DOPC的液態區塊”(DOPC-rich liquid crystalline phase domain)。且POPC塗佈雲母片上的脂質雙層和懸浮的多層脂球 (multilamellar vesicle) 有著相似的相變溫度 (gel-to-liquid crystalline transition temperature)，顯示在POPC塗佈雲母片上，脂質雙層可近似懸浮樣品。另一方面，在雲母片上的1:1 DPPC/DOPC有較高的相變溫度，此結果指出雲母片會迫使脂質雙層排列更緊密有序，而藉由預先塗佈一層POPC脂質雙層，夾在1:1 DPPC/DOPC 脂質雙層和雲母片之間，可減少此有序效應。此外，我們推測雲母片-脂質單層相互影響 (mica- leaflet interaction)和脂質雙層間相互影響 (inter-leaflet coupling) 可能是決定脂質雙層對稱性質 (bilayer symmetry) 的因素，這方面的研究還必須做更進一步的實驗驗證。

Supported planar lipid bilayers are versatile models of the biological membrane, which have been widely used in biophysical studies. However, limited consideration has been given to the effect of mica on the lipid bilayer. In this thesis, we study the morphology of the 1:1 DPPC/DOPC bilayer on mica and on POPC-coated mica using atomic force microscopy (AFM). Most of AFM images were acquired in buffer. To obtain a good-quality sample, bilayers prepared from different methods were compared. We found that the bilayer prepared from vesicle fusion yields better quality for imaging in buffer and shorter preparation time than that from spin coating. The images of 1:1 DPPC/DOPC bilayer on POPC-coated mica display two different bilayer thicknesses. The thicker and thinner bilayers, presumably correspond to the DPPC-rich gel phase and DOPC-rich liquid crystalline phase, respectively. We found that this bilayer on POPC-coated mica can be regarded as a free-standing bilayer effectively as it has a gel-to-liquid crystalline transition temperature close to that of the bilayers in multilamellar vesicle suspension. In contrast, the 1:1 DPPC/DOPC bilayer on mica exhibits a higher gel-to-liquid crystalline transition temperature, suggesting that mica induces an ordering effect on the lipid bilayer. This effect can be reduced by inserting a POPC bilayer between the 1:1 DPPC/DOPC bilayer and mica. Furthermore, there is a slight indication that the mica-leaflet interaction and and inter-leaflet coupling may dominate in bilayers on mica and on POPC-coated mica, respectively, which may result in different bilayer symmetry. Further studies will be needed to examine this in detail.

[1] Drennan, P. M., M. T. Smith, D. Goldsworthy, and J. Van Staden. The occurrence of trehalose in the leaves of the desiccation-tolerant angiosperm Myrothamnus flabellifolius Welw. Plant Physiol. 142:493–496 (1993).
[2] T. Kaasgaard, C. Leidy, John H. Crowe, Ole G. Mourtisen and Kent Jorgensen. Temperature-Controlled Structure and Kinetics of Ripple Phases in One- and Two-Component Supported Lipid Bilayers. Biophysical Journal, 85, 350-360 (2003).
[3] C. Leidy, T. Kaasgaard, John H. Crowe,and Ole G. Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy. Biological Journal, 83, 2625 (2002).
[4] M. Rappolt, G. Pabst, G. Rapp, M. Kriechaum, H. Amenitsch, C. Krenn, S. Bernstorff,and P. Laggner. New evidence for gel-liquid crystalline phase coexistence in the ripple phase of phosphatidylcholines European Biophysical Journal, 29, 125 (2000).
[5] Lewis, R. N. A. H. Mak, and R. N. McElhaney. A differential scanning calorimetric study of the thermotropic phase behavior of model membranes composed of phosphatidylcholines containing linear saturated fatty acyl chains. Biochemistry, 26, 6118 (1987).
[6] Deborah R. Fattal, David Andelman,and Avinoam Ben-Shaul. The Vesicle- Micelle Transition in Mixed Lipid-Surfactant Systems: A Molecular Model. Langmuir, 11, 1154 (1995)
[7] J. M. Seddon,and R. H. Templer. Polymorphism of Lipid-Water Systems, from the Handbook of Biological Physics, Vol. 1, ed. R. Lipowsky, and E. Sackmann. (c) 1995.
[8] Gadd, G. M., K. Chalmers, and R. H. Reed. The role of trehalose in dehydration resistance of Saccharomyces cerevisiae. FEMS Microb. Letts. 48:249–254 (1987).
[9] S. J. Singer and Garth L. Nicolson. Cell membranes are viewed as two-dimensional solutions of oriented globular proteins and lipids. Science, 175, 720-731.
[10] H. C. Berg, "Random Walks in Biology". Extended Paperback Ed. ed. (1993).
[11] K. Simons, and E. Ikonen. Functional rafts in cell membranes. Nature, 387, 569-572 (1997).
[12] D.A. Brown, and E. London. Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes? Biochemical And Biophysical Research Communications, 240, 1-7 (1997).
[13] S. Mukherjee, and F.R. Maxfield. Role of membrane organization and membrane domains in endocytic lipid trafficking. Traffic, 1, 203-211 (2000).
[14] Alberts B, Johnson A, Lewis J, et al (2002). Molecular Biology of the Cell (4th ed.).
[15] Pieter R. Cullis, Michael J. Hope, and Colin P.S. Tilcock. Lipid polymorphism and the roles of lipids in membranes. Chemistry and Physics of lipids. 40: 127-144 (1986).
[16] Bazzi MD, Youakim MA, and Nelsestuen GL. Importance of phosphate- dylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes. Biochemistry. 31:1125-34 (1992).
[17] Eric Laflamme, Antonella Badia, Michel Lafleur, Jean-Louis Schwartz, and Raynald Laprade. Atomic Force Microscopy Imaging of Bacillus thuringiensis Cry1 Toxins Interacting with Insect Midgut Apical Membranes. J Membrane Biol 222:127–139 (2008).
[18] G. Binnig, C. F. Quate, and C. H. Gerber. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986).
[19] D. Rugar and P. K. Hansma. Atomic force microscopy. Phys. Today 43, 23–30 (1990).
[20] H. K. Wickramasinghe. Scanned-probe microscopes. Sci. Am. 260, 98–105 (1989).
[21] S. Kasas, N. H. Thomson, B. L. Smith, P. K. Hansma, J. Miklossy, and H. G. Hansma. Biological Applications of the AFM: From Single Molecules to Organs. Vol. 8, 151–161 (1997).
[22] Danielle Keller, Niels B. Larsen, Ian M. Møller, and Ole G. Mouritsen. Decoupled Phase Transitions and Grain-Boundary Melting in Supported Phospholipid Bilayers. PRL 94, 025701 (2005).
[23] Yang, J., and J. Appleyard. The main phase transition of mica-supported phosphatidylcholine membranes. J. Phys. Chem. B. 104:8097–8100 (2000).
[24] A. F. Xie, R. Yamada, A. A. Gewirth, and S. Granick. Materials Science of the Gel to Fluid Phase Transition in a Supported Phospholipid Bilayer. Phys. Rev. Lett. 89, 246103 (2002).
[25] Fuyuki Tokumasu, Albert J. Jin, and James A. Dvorak. Lipid membrane phase behaviour elucidated in real time by controlled environment atomic force microscopy. J. Electron Microsc. 51, 1 (2002).
[26] Leonenko ZV, Finot E, Ma H, Dahms TE,and Cramb DT. Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy. Biophys. J. 86, 3783 (2004).
[27] Giocondi M.C., Pacheco, L., Milhiet, P.E. and Le Grimellec, C. emperature dependence of the topology of supported dimirystoyl-distearoyl phosphate- dylcholine bilayers. Ultramicroscopy, 86: 151–157 (2001).
[28] Bustamante, C. and Keller, D. Scanning force microscopy in biology. Phys. Today 48, 32–38 (1995).
[29] Adapted from Jain, M., and Wagner, R. C., 1980. Introduction to Biological Membranes. New York : John Wiley and Sons; Martonosi , A., ed., 1982. Membranes and Transport, Vol. 1. New York.
[30] Ricker, J. V., N. M. Tsvetkova, W. F. Wolkers, C. Leidy, F. Tablin, M. Longo, and J. H. Crowe. Trehalose maintains phase separation in an air-dried binary lipid mixture. Biophys. J. 84:3045–3051 (2003).
[31] C. A. J. Putman, K. O. Vanderwerf, B. G. Degrooth, N. F. Vanhulst, F. B. Segerink, and J. Greve. Atomic force microscope with inte-grated optical microscope for biological applications Rev. Sci. Instrum. 63, 1914–1917 (1992).
[32] James H. Davis, Jesse James Clair, and Janos Juhasz. Phase Equilibria in DOPC/DPPC-d62/Cholesterol Mixtures. Biophysical Journal. 96, 521–539 (2009).
[33] Anne Feng Xie, Ryo Yamada, Andrew A. Gewirth, and Steve Granick. Materials Science of the Gel to Fluid Phase Transition in a Supported Phospholipid Bilayer. Phys. Rev. Lett. 89, 246103 (2002).
[34] Fuyki Tokumasu, Albert J. Jin, and James A. Dvorak. Lipid membrane phase behavior elucidated in real time by controlled environment atomic force microscopy. Journal of Electron Microscopy 51(1): 1-9 (2002).
[35] Wan-Chen Lin, Craig D. Blanchette, Timothy V. Ratto, zand Marjorie L. Longo. Lipid Asymmetry in DLPC/DSPC-Supported Lipid Bilayers: A Combined AFM and Fluorescence Microscopy Study. Biophysical Journal. 90, 228–237 (2006).
[36] McIntyre JC and Sleight RG. Fluorescence assay for phospholipid membrane asymmetry. Biochemistry. 30(51):11819-27 (1991).