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We devise a methodology to fixate and image dynamic fluid domain patterns of giant unilamellar vesicles (GUVs) at sub-optical length scales. Individual GUVs are rapidly transferred to a solid support forming planar bilayer patches. These are taken to represent a fixated state of the free standing membrane, where lateral domain structures are kinetically trapped. High-resolution images of domain patterns in the liquid-ordered (lo) and liquid-disordered (ld) co-existence region in the phase-diagram of ternary lipid mixtures are revealed by atomic force microscopy (AFM) scans of the patches. Macroscopic phase separation as known from fluorescence images is found, but with superimposed fluctuations in the form of nanoscale domains of the lo and ld phases. The size of the fluctuating domains increases as the composition approaches the critical point, but with the enhanced spatial resolution, such fluctuations are detected even deep in the coexistence region. Agreement between the area-fraction of domains in GUVs and the patches respectively, supports the assumption that the thermodynamic state of the membrane remains stable. The approach is not limited to specific lipid compositions, but could potentially help uncover lateral structures in highly complex membranes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We investigate the relationship between stripe domains and the ripple phase in membranes. These have previously been observed separately without being linked explicitly. Past results have demonstrated that solid and ripple phases exhibit rich textural patterns related to the orientational order of tilted lipids and the orientation of ripple corrugations. Here we reveal a highly complex network pattern of ripple and solid domains in DLPC, DPPC bilayers with structures covering length scales from 10 nm to 100 μm. Using spincoated double supported membranes we investigate domains by correlated AFM and fluorescence microscopy. Cooling experiments demonstrate the mode of nucleation and growth of stripe domains enriched in the fluorescent probe. Concurrent AFM imaging reveals that these stripe domains have a one-to-one correspondence with a rippled morphology running parallel to the stripe direction. Both thin and thick stripe domains are observed having ripple periods of 13.5±0.2 nm and 27.4±0.6 nm respectively. These are equivalent to previously observed asymmetric/equilibrium and symmetric/metastable ripple phases, respectively. Thin stripes grow from small solid domains and grow predominantly in length with a speed of ~3 times that of the thick stripes. Thick stripes grow by templating on the sides of thinner stripes or can emerge directly from the fluid phase. Bending and branching angles of stripes are in accordance with an underlying six fold lattice. We discuss mechanisms for the nucleation and growth of ripples and discuss a generic phase diagram that may partly rationalize the coexistence of metastable and stable phases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.

A number of morphological and statistical aspects of domain formation in singly and doubly supported ternary membranes have been investigated. Such ternary membranes produce macroscopic phase separation in two fluid phases and are widely used as raft models. We find that membrane interactions with the support surface can have a critical influence on the domain shapes if measures are not taken to screen these interactions. Combined AFM and fluorescence microscopy demonstrate small (500 nm) irregular domains and incomplete formation of much larger (5 microm) round domains. These kinetically trapped structures are the result of interactions between the membrane and the support surface, and they can be effectively removed by employing doubly supported membranes under physiological salt concentrations. These decoupled supported membranes display macroscopic round domains that are easily perturbed by fluid shear flow. The system allows a quantitative characterization of domain coarsening upon being cooled into the coexistence region. We determine the domain growth exponent alpha = 0.31, which is in close agreement with the theoretical value of 1/3. Analysis of the spatial domain pattern in terms of Voronoi polygons demonstrates a close similarity to equilibrated cellular structures with a maximized configurational entropy.

An atomic force and fluorescence microscopy investigation of the structure of spin-coated lipid films is presented. In the surface of the dry film, lipids are found to orient in a conformation where acyl chains are pointing outward while laterally the individual layers of the multilamellar film exhibit a dewetting pattern similar to what is found in polymer thin films. Hydration of the film in liquid water promotes detachment of bilayers from the surface while a single membrane remains on the mica substrate. This supported membrane is highly uniform and defect-free as compared to supported membranes prepared by conventional methods. It is further demonstrated that supported membranes of binary lipid mixtures prepared by this method exhibit gel-fluid domain coexistence in accordance with expectations from the phase diagrams.