Electron microscopy and diffraction study of phospholipid monolayers transferred from water to solid substrates

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Abstract
Monomolecular films of phospholipids (phosphatidylcholines and phosphatidicacids) and arachidonic-acid were transferred from air/water interface onto electron transparent hydrophilic substrates such as 1) carbon films, 2) crystalline graphite-oxide layers, 3) SiO2-covered Formvar foils and were studied by conventional transmission electron microscopy (TEM), by scanning transmission STEM in the darkfield mode and by electron diffraction. Positive staining with uranylacetate and platinum shadowing, using a low angle of incidence, was applied. The latter allowed measurements of the thickness of the transferred lipid layers to an accuracy of 10 %. Imaging of monolayers in the transmission mode at magnifications of x 2 000-50 000 was achieved for the first time. Electron diffraction patterns were taken from layers which in all cases have been characterized simultaneously by conventional EM. The structure of monolayers transferred 1) from the completely condensed solid state, 2) from the intermediate state, 3) from the expanded (fluid) state and 4) from the region of the main phase transition are compared. Evidence is provided that the state of the monolayer on the air-water interface is at least partially preserved upon transfer. In the completely condensed state macroscopically continuous monolayers are formed by fatty acids and phosphatidic acid. The chains form a triangular lattice (lattice constant d100 = 4.2 A; area per molecule 40 Å 2) and are oriented normal to the surface. The transferred phosphatidylcholines exhibit a spider's web of elongated 100 Å wide cracks. This lipid is tilted with respect to the monolayer normal by about 20 degrees and forms and orthorhombic lattice both at the air/water interface and on substrate. A comparison of the radial widths of the electron diffractions of the monolayer with that of graphite oxide substrates provides evidence that the high pressure phase of all lipids do not exhibit a true long-range order of the chains. In the intermediate pressure region — also called fluid condensed region — the film consists of an accumulation of platelets of about 1 μm diameter which according to electron diffraction — are certainly crystalline on the substrate. The large size of the platelets shows that the bulk of the monolayer is also crystalline on the water surface, while a small fraction may still be in the fluid state. The fluid-like viscosity of the intermediate phase is attributed to the low shearing strength of the accumulation of platelets. The 2nd order like transition into the completely condensed crystalline phase is attributed to the merging of the platelets. Evidence is provided that in the region of main phase transition the monolayer consists of fluid and crystalline domains of some 100 A diameter. The finite slope of the isotherm at the main transition is thus not an artefact but is due to the finite size of the cooperative unit undergoing a simultaneous transition. This confirms our previous interpretation and a recent Monte-Carlo study of the nature of the phase transition by Georgallas and Pink (Can. J. Phys. 60 (1982) 1678)