Helmuth Möhwald

Scanning tunneling microscopy of lipid films and embedded biomolecules

Abstract We have observed the ordered molecular structure of fatty acid and phospholipid Langmuir-Blodgett films with a scanning tunneling microscope. Besides confirming earlier studies of cadmium arachidate bilayers on graphite, we have observed bilayers of di-myristoyl-phosphatic acid (DMPA) on cleaved graphite, and studied monolayers of DMPA on oxidized graphite and on gold-coated calcite in air. We present the first images of a bilayer containing the protein concanavalin A. These experiments confirm the possibility of obtaining STM images by electron transport through layers of organic molecules more than 50 A thick. Sample preparation, microscopic technique, and results are discussed.

Electrostatic interactions in phospholipid membranes. I: Influence of monovalent ions

Electrostatic interactions in monolayers and vesicles of acidic phospholipids are studied by thermodynamical and optical techniques in conjunction with numerical calculations. A nonmonotonic ionic strength dependence with an extremum at 0.1 M (NaCl) is observed for the phase transition temperature of vesicles as well as for the surface pressure of monolayers at low molecular density. This finding is in accordance with the calculations predicting the dominance of charge screening by monovalent counterions only for concentrations above 0.1 M. For lower salt content, however, its increase causes an elevation of the degree of dissociation and thus also electrostatic repulsion. This leads to a higher surface pressure, a lower transition temperature and a smaller size of solid domains observed in the liquid/solid coexistence range of monolayers. This supports the previously published idea, that finite size and repulsion of the domains arise from a different surface charge density in fluid and solid lipid phases.

Direct characterization of monolayers at the air-water interface☆

Abstract The microstructure of lipid monolayers is studied by the new techniques of fluorescence microscopy and synchrotron X-ray diffraction. In connection with thermodynamic, surface potential and electron diffraction measurements different ordering processes are illuminated. It is shown tha first-order phase transitions can be followed by means of characteristics textures of coexisting phases. Formation of two-dimensional domains is observed. Because of long-range electrostatic interactions these domainsexhibit uniform sizes and may form superlattices. These lattice can be manipulated via ionic conditions, pressure, temperature, impurity content, external fields and nucleation kinetics. Intermediate phase with lonr-range oriental order and short-range positional order are observed. These may be due to peculiar two-dimensional features or due to the fact that chain alignment and positional ordering may be decoupled. Correlation of molecular structure and microstructure shows that systems can be classified as those where aliphatic chains or where head groups determine the structure. The latter resemble classical solids with regard to diffraction pattern and domain morphology whereas the former resemble phases between fluid and solid. Depending on the system, states from condensed liquid to defect-rich solid may be encountered. This presumably results from the large probability of chain defects in aliphatic systems and calls for further studies in this direction.

Hydrogen-bonded multilayers of self-assembling silanes: structure elucidation by combined Fourier transform infra-red spectroscopy and X-ray scattering techniques

Abstract Hydrogen-bonded multilayer stacks of laterally interconnected long-chain silanes are a new class of synthetic self-assembling thin film organizates endowed with a somewhat unusual combination of structural and dynamic characteristics. In this paper we present experimental results obtained from a combined Fourier transform infra-red (FTIR) spectroscopic and X-ray scattering study, on the basis of which it is possible to derive a rather detailed picture of some of the main features of the microstructure of these novel multilayer films and their monolayer precursors. The films are shown to be composed of discrete monolayers, coupled to each other in a flexible, non-epitaxial manner, via interlayer multiple hydrogen bonds. The hydrocarbon tails assume a perpendicular average orientation on the layer planes and form a ‘rotator phase’-like hexagonal lattice with a lateral packing density of ca. 21 A 2 per molecule and a positional coherence length of ca.70 A. Extensive lateral coupling of the silane head groups appears to be responsible for the high structural robustness and defect self-healing capability of these films, while the interlayer hydrogen bonding accounts for the facile post-assembly intercalation of various polar guest species into their vertically expandable interlayer polar regions. As the SiO bond is too short to permit extensive intralayer polymerization under the steric constraints imposed by the compact packing of the perpendicularly oriented hydrocarbon tails, the observed high interconnectivity of the silane head groups is rationalized in terms of a dynamic equilibrium model involving continuous redistribution of the SiO bonds within a two-dimensional network of oligomeric siloxane and silanol species. This model of dynamic equilibriation of siloxane linkages can also help to explain other intriguing properties of such silane monolayers.

Microscopically observed preparation of Langmuir-Blodgett films

Abstract It is shown that by combining newly developed preparation and characterization techniques Langmuir-Blodgett films with well-defined crystallinity can be prepared. This is achieved by growing two-dimensional crystals on the water surface under fluorescence microscopy control, transferring these crystals onto a solid surface by modified Langmuir-Blodgett techniques and further characterizing the film by ellipsometry, electron microscopy and electron diffraction. Coexisting fluid and solid phases can be observed and controlled over a wide density range. The dimensions of the single crystals of, for example, the phospholipid L-α-dimyristoyl phosphatidic acid (DMPA) amount to 100 μm and hexagonal superlattices of crystallites can be established. Two different phases can be transferred onto a solid support, the difference in thickness being below 0.4 nm. A transition to a less condensed phase at near-zero pressure appears at a molecular density of 1 molecule nm −2 for DMPA and of 1 molecule (0.24nm 2 ) −1 for arachidic acid. Ellipsometry and fluorescence microscopy consistently show that the change in molecular density amounts to less than a factor of 5. This raises doubt as to whether is corresponds to a classical gas-liquid transition.

Thickness and temperature dependent structure of Cd arachidate Langmuir-Blodgett films

Abstract The structure of monolayers of Cd arachidate on water and on solid support, and the thickness dependent changes when building up a multilayer via the LB technique are studied by means of grazing incidence diffraction of X-ray. In monolayers the perpendicularly oriented amphiphilic molecules are arranged in a hexagonal lattice, whereas for thicker layers (even for three layers) they crystallize in an orthorhombic unit cell with a reduced molecular are ( A = 18.2 A 2 ) compared to that of the monolayer ( A = 19.7 A 2 ). In-plane diffraction measurements with wave vector transfer perpendicular to the surface (rod scans) could prove for multilayers a maximum tilt angle of 2°. The data prove orientational as well as positional correlation between adjacent monolayers. For multilayers, a thermally induced phase transition accompanied by an expansion of the lattice from an orthorhombic ( A = 18.2 A 2 ) to a hexagonal ( A = 19.7 A 2 ) lattice is found. This structural change is reversible below 110 °C.

Impurity controlled phase transitions of phospholipid monolayers

The phase diagram of monolayers of l-α-dimyristoyl phosphatidic acid has been studied by fluorescence microscopy. For pressures corresponding to the nearly horizontal slope in the pressure area diagram the growth of crystalline platelets can be observed. They are of dendritic nature; their sizes can be controlled via pressure, compression speed, temperature and pH, and increased up to 100 μm. Due to repulsive interaction a hexagonal arrangement of crystalline platelets can be established.It is shown that the textures do not depend on the dye probe for concentrations below 3 mol%. On the other hand via incorporation of impurities in concentrations of about 1 mol% the coexistence of lipid and solid phases can be controlled. Since, for a constant surface pressure, this coexistence can be maintained, these monolayers are suitable model systems to study the interactions of proteins and vesicles with coexisting fluid and solid membrane areas.

Formation of Langmuir-Blodgett films via electrostatic control of the lipid/water interface

Abstract Monolayers of diacylphosphatidic acids have been studied, utilizing film balance and fluorencence microscopic experiments in combination with numerical calculations. Special attention was devoted to the fluid/gel phase transition and to shifts in this transition caused by the presence of mono- and divalent ions in the subphase. It has been shown that the pressure corresponding to this transition increases with increasing monovalent ion concentration and decreases with increasing divalent ion concentration. The effects are ascribed to an increase in surface charge density in the former case and to screening of coulombic forces in the latter. They can be partly described by Gouy-Chapman-Stern theory and partly ascribed to structural changes brought about by ion binding. The size and shape of the crystalline domains on the water surface can be varied with the ionic conditions. Dendritic and regular structures thus formed can be transferred onto solid supports allowing uniform multilayer formation.

An X-ray scattering study of lipid monolayers at the air-water interface and on solid supports

Abstract Monolayers of the lipid arachidic acid (C20) and of the phospholid dimyristolyphosphatidic acid (PMDA) have been studied by X-ray reflection and diffraction technique, using a purpose-built Langmuir trough installed at the sample stage of our high-resolution X-ray diffractometer at the DORIS synchroton X-ray source in Hamburg. For comparison we also report data for monolayers of C20 on a solid support using a 10 kW rotating anode X-ray source. By the X-ray reflection method, the density profile across the interface is probe, while in-plane diffraction measurements gauge the two-dimensional crystalline properties of the monolayers. Flourescence microscopy experiments of DMPA monolayers idicates coexistence of a “fluid” and the denser “gel” phase between surface pressures πc and πc where the π-A isotherm changes slope. X-ray data show that the positional correlation length increase from less than 10 to about 100 lattice spacing on increasing the pressure beyond πs.X-ray reflectivity data indicate 40% increase in monolayer thickness when the surface pressure is increased from below πc to above πs. For C20 monolayers, the reflectivity measurements indicates similar monolyer thickness on water surfaces and on solid supports. On water, the correlation length was 22 lattice spacing while, on thermally oxidized silicon ξ≈ 8 lattice spacings.

Development of equilibrium domain shapes in phospholipid monolayers

Fluorescence micrographs of monolayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC) at the air water interface are analyzed. Ordered phase domains in coexistence with the fluid lipid phase change their shapes with time and with lateral pressure. With domains fixed under an electrode and well separated from their neighbours a peculiar instability of the boundary lines is observed. It is ascribed to the onset of an electrostatically induced shape transition. Elaborating the boundary regions where domain fusion and fission, respectively, are observed leads to a model on in-plane dipole orientation.