Assemblies of Polymerized Phospholipid Vesicle Structures Chemisorbed on Au Films Visualized in situ by Environmental Sc
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Assemblies of Polymerized Phospholipid Vesicle Structures Chemisorbed on Au Films Visualized in situ by Environmental Scanning Electron Microscopy Ivan Stanish, Richard I. Ray, and Alok Singh Center for Bio/Molecular Science and Engineering Naval Research Laboratory 4555 Overlook Ave. Washington D.C. 20375 ABSTRACT Submicron vesicles immobilized on gold films were visualized in situ using environmental scanning electron microscopy (ESEM). Electron micrographs show that surface immobilized vesicles composed of diacetylenic phospholipids with 1 mole percent disulfide functionality and that encapsulate NaCl are structurally stable for at least three days. Furthermore, energy dispersive spectroscopy (EDS) provides compositional evidence supporting the formation of surface immobilized vesicles. Using ESEM coupled with EDS, a two-layer vesicle structure was imaged and found to contain NaCl and lipid elements sulfur and phosphorous. INTRODUCTION Several methods exist for immobilizing and characterizing (primarily) non-polymerized vesicles onto solid supports. Structural characterization of vesicles on surfaces includes atomic force microscopy (AFM) [1-10] and scanning tunneling microscopy (STM) [11] to provide information about surface topography, quartz crystal microbalance (QCM) [2,12] to measure adsorbate weight, and ellipsometry [13] and surface plasmon resonance (SPR) [3,14] to measure structure height. When considering vesicles immobilized on a non-porous (i.e., non-grid) metal substrate, transmission electron microscopy (TEM) is impractical since electron beams cannot readily penetrate these surfaces. From the prospective of quality control, a rapid and noninvasive method that can provide morphological information of vesicle-layered surfaces is needed. We report for the first time a technique, specifically environmental scanning electron microscopy (ESEM), that rapidly measures immobilized vesicle size, shape, dispersity, number density, and spatial organization on solid surfaces and furthermore provides direct visualization of three dimensional (3-D) vesicle structures under near in situ conditions. Our objective is to synthesize and characterize stable single- and multi-layer chemisorbed vesicle structures. EXPERIMENTAL A dispersion of vesicles in 125 mM NaCl consisting of 1,2-bis(tricosa-10,12-diynoyl)-sn3-glycerophosphocholine (DC8,9PC) at 5 mg/mL doped at one mole percent with 1,2-bis(tricosa10,12-diynoyl)-sn-3-glycerophospho-2-[(2-hydroxyethyl) disulfanyl)] ethanol (DC8,9PDSEtOH) was extruded ten times at 65 oC through a 0.2 µm Nucleopore membrane using a Lipex extruder (Lipex Biomembranes Inc., Vancouver BC), subsequently photopolymerized at 8 oC for 10 minutes by UV-irradiation in a Rayonett Photochemical reactor having sixteen 75 W Hg lamps (So. New England Ultraviolet Co., Hamden, CT), and size-characterized using dynamic light scattering (DLS; 196 nm +/- 25 nm; Coulter Electronic, Inc., Miami, Fl) and TEM (208 nm +/FF6.2.1
52 nm; Zeiss EM-10). Glass substrates coated with 25 nm gold films by thermal eva
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