Orientation fluorescence techniques

The properties of membranes commonly studied by fluorescence techniques include motional, structural, and organizational aspects. Motional aspects include the rate of motion of fatty acyl chains, the head-group region of the phospholipids, and other lipid components and membrane proteins. The structural aspects of membranes would cover the orientational aspects of the lipid components. Organizational aspects include the distribution of lipids both laterally, in the plane of the membrane (e.g., phase separations), and across the membrane bilayer (phospholipid asymmetry) and distances from the surface or depth in the bilayer. Finally, there are properties of membranes pertaining to the surface such as the surface charge and dielectric properties. Fluorescence techniques have been widely used in the studies of membranes mainly since the time scale of the fluorescence lifetime coincides with the time scale of interest for lipid motion and since there are a wide number of fluorescence probes available which can be used to yield very specific information on membrane properties. 

Using the principle of ion pair formation between ammonium cations and the phosphate anions of lipids, Matile et al.33 prepared 8, an amphiphilic polyamine dendrimer. Rather than acting as a membrane channel, 8 was expected to form reversible membrane defects in the lipid bilayer. The steroid moiety was expected to act as the hydrophobic anchor for bilayer orientation and steric bulk was expected to prevent the polyamine penetrating the bilayer. Proton transport was assessed in unilamellar vesicles using the pH-fluorescence technique in which the external pH was increased to 7.8 relative to the internal pH at 7.4. The results demonstrated that 8 was almost as active as gramicidin, and maximal flux was achieved in ca. 20 s. 

Lipid A was investigated by FTIR, X-ray diffraction, and fluorescence techniques (Naumann et al., 1987). Analysis of the CH2 scissoring vibrations showed that lamellar structures exist at temperatures below 30 °C, but that no stable closed vesicles are formed. Orientation measurements of lipopolysaccharides prepared from E. coli and Salmonella minnesota by ATR-FTIR also showed a highly ordered gel phase. Besides, the transition from the gel pha.se a to the liquid crystalline phase was also studied (Brandenburg and Seydel, 1988). 

Polarised fluorescence has been used to study the orientation of non-crystalline polymer chains and a fluorescence technique has been developed to quantify the amount of microgel in polyacryl-amide. Time resolved photoluminescence analysis of poly( -phenyl-... 

Fluorescence. The fluorescence technique is based on addition of a molecule with a fluorescing group (chromophore) to a polymer (26-28,69,70,112). It gives much more information on orientation than second-moment procedures such as birefringence and dichroism. This is due to the fact that fluorescence involves two processes absorption of radiation and, after a delay called lifetime of excitation, a sequential emission at a different wavelength. The effect on the absorption and emission steps is to develop a fourth-moment dependence (113). More specifically, the emitted intensity I depends on... 

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

Viovy,J.L. and Monnerie, L. Fluorescence Anisotropy Technique Using Synchroton Radiation as a Powerful Means for Studying the Orientation Correlation Functions of Polymer Chains. Vol. 67, pp. 99—122. 

In this review the definition of orientation and orientation functions or orientation averages will be considered in detail. This will be followed by a comprehensive account of the information which can be obtained by three spectroscopic techniques, infra-red and Raman spectroscopy and broad line nuclear magnetic resonance. The use of polarized fluorescence will not be discussed here, but is the subject of a contemporary review article by the author and J. H. Nobbs 1. The present review will be completed by consideration of the information which has been obtained on the development of molecular orientation in polyethylene terephthalate and poly(tetramethylene terephthalate) where there are also clearly defined changes in the conformation of the molecule. In this paper, particular attention will be given to the characterization of biaxially oriented films. Previous reviews of this subject have been given by the author and his colleagues, but have been concerned with discussion of results for uniaxially oriented systems only2,3). 

Other optical and spectroscopic techniques are also important, particularly with regard to segmental orientation. Some examples are fluorescence polarization, deuterium nuclear magnetic resonance (NMR), and polarized IR spectroscopy [4,246,251]. Also relevant here is some work indicating that microwave techniques can be used to image elastomeric materials, for example, with regard to internal damage [252,253]. 

Figure 7.8 Set-up for field emission and field ionization microscopy. Both techniques produce an image of the concave end of a single crystal tip on the fluorescent screen, as explained in the text. The tip exposes many facets of different crystallographic orientations.

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