Domain Growth, Shapes, and Topology in Cationic Lipid Bilayers on Mica by Fluorescence and Atomic Force Microscopy

Domain formation in mica-supported cationic bilayers of dipalmitoyltrimethylammoniumpropane (DPTAP) and dimyristoyltrimethylammoniumpropane (DMTAP), fluorescently doped with an NBD (((7-nitro-2–1, 3-benzoxadiazol-4-yl)amino)caproyl) phospholipid, was investigated with fluorescence microscopy and atomic force microscopy. Heating above the acyl chain melting temperature and cooling to room temperature resulted in nucleation and growth of domains with distinguishable patterns. Fractal patterns were found for DPTAP, whereas DMTAP domains were elongated and triangular with feathery edges. Reducing the cooling rate or probe concentration for DPTAP bilayers resulted in larger, filled-in domains with more rounded edges. However, for DMTAP, cooling rates mainly affected size and only slightly modified domain morphology. In a saline environment, the domains were dark, and the surrounding continuous region was bright and thus contained the fluorescent probe. However, as the salt concentration was decreased, the dark regions percolated (connected), resulting in bright domains. Atomic force microscopy scans along domain edges revealed that the dark regions in fluorescence images were approximately 1.4 nm thicker than the light regions. Additionally, the dark regions were of bilayer thickness, approximately 4 nm. Comparison of these results in bilayers to well-documented behavior in Langmuir monolayers has revealed many similarities (and some differences) and is therefore useful for understanding our observations and identifying possible growth mechanisms that may occur in domain formation in cell membranes or supported membrane systems.