Fluorescence phospholipid monolayers

Grazing incidence excitation of a fluorescent probe in a phospholipid monolayer can also be used to indicate order. The collective tilt of the molecules in a domain inferred from such measurements is indicative of long-range orientational order [222]. 

N. L. Thompson, H. M. McConnell, and T. P. Burghardt, Order in supported phospholipid monolayers detected by dichroism of fluorescence excited with polarized evanescent illumination, Biophys. J. 46, 739-747 (1984). 

M. L. Pisarchick and N. L. Thompson, Binding of a monoclonal antibody and its Fab fragment to supported phospholipid monolayers measured by total internal reflection fluorescence microscopy,. Biophys. J. 58, 1235-1239 (1990). 

Peters, R. Beck, K. Translational diffusion in phospholipid monolayers measured by fluorescence microphotolysis. Proc. Natl. Acad. Sci. USA 1983, 80, 7183. 

A very suitable method for measurement of the lateral diffusion of molecules adsorbed at the foam film surfaces is Fluorescence Recovery after Photobleaching (FRAP) ([491-496], see also Chapter 2). Measurements of the lateral diffusion in phospholipid microscopic foam films, including black foam films, are of particular interest as they provide an alternative model system for the study of molecular mobility in biological membranes in addition to phospholipid monolayers at the air/water interface, BLMs, single unilamellar vesicles, and multilamellar vesicles. 

Weis, R. M. (1991). Fluorescence microscopy of phospholipid monolayer phase transitions. Chemistry and Physics of Lipids 57 227-239. 

Using optieal microscopie teehniques (fluorescence microscopy, Brewster angle microseopy and microscopic ellipsometry) the formation, size and shape of domains in the LE-LC coexistence region of phospholipid monolayers have been studied extensively. Furthermore, structures within condensed domains and phases have been visualized, the contrast resulting from the optical anisotropy caused by long-range tilt orientational order. 

One role of the class Y amphipathic helices found in apoA-IV and apoA-I appears to be to serve as low-affinity lipid-associating domains. The snorkel hypothesis predicts that this class of amphipathic helix would not penetrate as deeply into phospholipid surfaces as would class A and thus would have lower lipid affinity. This prediction is supported by experimental evidence (based on Trp fluorescence blue shifts, ease of Trp fluorescence quenching, and liposomal leakage) that apoA-IV sits higher in a phospholipid monolayer than do the other exchangeable apolipoproteins that contain class A amphipathic helices (Weinberg and Jordan, 1990). 

Ke PC, Naumann CA (2001) Hindered diffusion in polymer-tethered phospholipid monolayers at the air-water interface a single molecule fluorescence imaging study. Langmuir 17 5076-5081... 

The phase behaviour of phospholipid monolayers at electrolyte/gas interfaces is studied by fluorescence microscopy. At the LE/LC phase transition, phase separation leads to a WignerH ype lattice structure. The observations are quantified using digital image processing. The results show that the phase transition comprises three different regimes. 

Teissie and coworkers detected rapid lateral movement of protons on a phospholipid monolayer-water interface by a number of measurements fluorescence from a pH indicator dye near the membrane surface, electrical surface conductance, and surface potentiaL These investigators found that the conduction of protons along the surface is considerably faster than proton conduction in the bulk phase (2 to 3 min versus 40 min for a comparable distance in their measurement setup). This novel conduction mechanism is proton-specific, as confirmed by a radioactive electrode measurement as well as by replacement with deuterated water. It is a consequence of cooperativity between neighboring phospholipid molecules the conduction mechanism disappears when phospholipid molecules are not in contact with each other. 

The attachment of pyrene or another fluorescent marker to a phospholipid or its addition to an insoluble monolayer facilitates their study via fluorescence spectroscopy [163]. Pyrene is often chosen due to its high quantum yield and spectroscopic sensitivity to the polarity of the local environment. In addition, one of several amphiphilic quenching molecules allows measurement of the pyrene lateral diffusion in the mono-layer via the change in the fluorescence decay due to the bimolecular quenching reaction [164,165]. 

There has been extensive activity in the study of lipid monolayers as discussed above in Section IV-4E. Coexisting fluid phases have been observed via fluorescence microscopy of mixtures of phospholipid and cholesterol where a critical point occurs near 30 mol% cholesterol [257]. 

Unlike electron and scanning tunneling microscopy, the use of fluorescent dyes in monolayers at the air-water interface allows the use of contrast imaging to view the monolayer in situ during compression and expansion of the film. Under ideal circumstances, one may observe the changes in monolayer phase and the formation of specific aggregate domains as the film is compressed. This technique has been used to visualize phase changes in monolayers of chiral phospholipids (McConnell et al, 1984, 1986 Weis and McConnell, 1984 Keller et al., 1986 McConnell and Moy, 1988) and achiral fatty acids (Moore et al., 1986). 

In order to confirm the large hydration behavior of phospholipid LB films only around their Tc, two-dimensional morphology of DMPE monolayers was observed by a fluorescence microscope. The two-... 

Numerous techniques have been employed to examine the monolayer structure of phospholipids at the air/water interface including surface tension, fluorescence, neutron and X-ray reflection, and IR and Raman spectroscopy. In contrast, very few techniques are suitable to examine monolayers at the oil/water interface. Surface tension and fluorescence microscopy [46-48] have shed some light on these buried monolayers, but most other surface techniques are hampered because of effects from the bulk liquids. Since VSFS is insensitive to the bulk, it is an excellent technique for probing these monolayers. 

There are many cases in which other techniques have been applied to biphasic systems in order to establish the nature of mixing. For example, fluorescence microscopy of DPPC monolayers containing 2% of a fluorescent probe have shown the coexistence of solid and fluid phases of DPPC at intermediate pressures (Weis, 1991). Similar results have been achieved with a variety of other phospholipids using the same technique (Vaz et al., 1989). The recent application of laser light scattering to this area (Street et al., unpublished data) has yet to produce any conclusive evidence, but the future for this particular technique is also promising. It also provides information about the viscoelastic properties of the monolayer and how these are affected by the inclusion of penetration enhancers. 

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