Fluorescence microscopy, phospholipid monolayers

The revelation that monolayers exhibited a rich and varied microstructure of coexisting domains in the microns-to-millimeter size range was first revealed by the application of fluorescence microscopy to monolayers of phospholipids and subsequent application to monolayers of fatty acids and their esters." The fluorescence microscopy technique requires the introduction of a small percentage of a fluorophore-labeled lipid or amphiphile to provide contrast between coexisting phases. The orientation of the fluorescent probe has also been exploited successfully in many cases to image regions of varying amphiphile tilt orientation relative to the interface. The method of fluorescence microscopy has limitations with respect to monolayer studies of supermolecules for example, it is unlikely that... 

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). 

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). 

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. 

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. 

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. 

Are any of these structures typical of those that would be observed in a pure amphiphile The role played by the probe, which is essential to the fluorescence method, is not completely clear. It has been argued that the formation of dendritic structures in phospholipids is the result of constitutional supercooling, a mechanism that depends on the differential solubility of an impurity between two phases. This may not be the case similar patterns have been observed in LB films by surface-plasmon microscopy, for which no probe is added. The foam structures at the LE-G transition have also been attributed by some to the presence of the probe, but foams have also been observed in monolayers composed solely of a labeled amphiphile. 

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