Microscopic distribution membranes

In addition to fluorescence methods, another study [27] developed a method to permit electron microscopic localization of Ras anchor domains on cytoplasmic membrane surfaces by immunogold labeling. The particle neighbor distances can be analyzed to obtain information about possible domain structure. Expressing H-Ras and K-Ras in baby hamster kidney cells, a nonrandom particle distribution was obtained from which the estimated mean raft size was 7.5-22 nm and about 35% of the membrane area consists of rafts. The same technique applied to cells that had been incubated with [3-cydodextrin to reduce cholesterol produced completely random distributions of H-Ras. This cholesterol dependence suggests some type of coupling of rafts across the inner and outer membrane leaflets. 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

Ceynowa performed electron microscopic studies of 60—80 nm thick microtomed Nafion 125 membranes that were converted, for the purpose of affecting electron density contrast, to the Pb2+ form, and all of the excess cations and co-ions were removed. It is the heavy metal that provides electron density contrast between the phase in which it resides and the surrounding phase. These membranes were then exposed to ethanol and 1,2-epoxypropane, although these solvents would not have remained in the samples under the vacuum in the microscope column. The micrographs consisted of uniformly distributed points that were presumed to be ion clusters that were 3—6 nm in diameter. 

Weber and Newman do the averaging by using a capillary framework. They assume that the two transport modes (diffusive for a vapor-equilibrated membrane and hydraulic for a liquid-equilibrated one) are assumed to occur in parallel and are switched between in a continuous fashion using the fraction of channels that are expanded by the liquid water. Their model is macroscopic but takes into account microscopic effects such as the channel-size distribution and the surface energy of the pores. Furthermore, they showed excellent agreement with experimental data from various sources and different operating conditions for values of the net water flux per proton flux through the membrane. 

Eikerling et al. ° used a similar approach except that they focus mainly on convective transport. As mentioned above, they use a pore-size distribution for Nafion and percolation phenomena to describe water flow through two different pore types in the membrane. Their model is also more microscopic and statistically rigorous than that of Weber and Newman. Overall, only through combination models can a physically based description of transport in membranes be accomplished that takes into account all of the experimental findings. 

Another microscopic technique is to freeze the specimen and then fracture it with a knife. A knife cutting through the frozen specimen splits the membrane down the middle, exposing the inside of the bilayer (fig. 17.13a). If the Davson-Danielli model for membrane structure were correct, the two exposed surfaces would be featureless. However, electron micrographs of metallic casts of such samples reveal surfaces studded with particles of various sizes (fig. 17.13(f)- Additional studies indicate that these particles are proteins that are deeply embedded in the membrane. The particles seen on the inner and outer leaflets of the bilayer usually differ in size and distribution because of an asymmetrical disposition of the proteins across the bilayer. 

Most important macroscopic transport properties (i.e., permeabilities, solubilities, constants of diffusion) of polymer-based membranes have their foundation in microscopic features (e.g., free-volume distribution, segmental dynamics, distribution of polar groups, etc.) which are not sufficiently accessible to experimental characterization. Here, the simulation of reasonably equilibrated and validated atomistic models provides great opportunities to gain a deeper insight into these microscopic features that in turn will help to develop more knowledge-based approaches in membrane development. 

The distribution of proteins in membranes can be revealed by electron microscopy using the freeze-fracture technique (Fig. 3b). In this technique, a membrane specimen is rapidly frozen to the temperature of liquid nitrogen (-196°C) and then fractured by a sharp blow. The bilayer often splits into mono-layers, revealing the interior. The exposed surface is then coated with a film of carbon and shadowed with platinum in order for the surface to be viewed in the electron microscope (see Topic A3). The fractured surface of the membrane is revealed to have numerous randomly distributed protuberances that correspond to integral membrane proteins. 

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