Monolayer fluorescence microscopy

The ability to image lateral heterogeneity in Langmuir monolayers dates back to Zocher and Stiebel s 1930 study with divergent light illumination [166]. More recently the focus shifted toward the use of fluorescence microscopy of mono-layers containing a small amount of fluorescent dye [167]. Even in single-corn-... 

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]. 

SPMs, fluorescence microscopy, and SIMS of HC-FC phase-separated mixed monolayers... 

Fluorescence microscopy of phase separated mixed monolayer by a convnetional fluorescence microscope and SNOAM... 

The sizes of the phase separated HC islands in the mixed monolayers of HC and FC anionic amphiphiles polyion complexed with cationic polymers were increased by addition of a HC cationic amphiphile. The HC islands sat on the FC sea in a structure of on-top in a two story monolayer in the same way as two-component amphiphile systems. By SSPM and conventional and SNOAM fluorescence microscopies, the cationic HC amphiphile was found to dissolve preferentially into the HC islands, although it also dissolved partially in the FC sea phase. 

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). 

During the next lew years many techniV ues-oricnlcd scientists will he attracted to wink on LB Hints because they provide interesting novel structures whose molecular architecture can be systematically controlled, The quality of (he floating monolayer ts also important and needs to he characterized as does the interlace between the first deposited monolayer and ihe substrate. Fluorescence microscopy and Brillouin and Fourier transform infrared spectroscopies are currently being used to address these problems. 

In fluorescence microscopy (FM) a small amount of a fluorescent dye is added to the mono-layer. To be incorporated into the monolayer the dye must be amphiphilic. The film is illuminated and the lateral distribution of the fluorescent molecules is observed with an optical microscope [589], Depending on the phase condition of the monolayer, the fluorescent molecules distribute unevenly or have a different quantum yield. Usually the dyes are expelled from condensed liquid and solid phases. With this technique the coexistence of different phases in monolayers on water was demonstrated for the first time [590,591],... 

Figure 9.8 Bilayers and a mixed monolayer of biotinylated and fluorescently labeled Cowpea mosaic virus (CPMV) particles on gold slides imaged via fluorescence microscopy The bilayers are shown on the left and the mixed monolayer on the right. A schematic representation of the CPMV layer...

Schwartz, D.K. Rnobler, M. Direct observation of transitions between condensed Langmuir monolayer phases by polarized fluorescence microscopy. J. Phys. Chem. 1993, 97, 8849. 

Knobler, CM. Seeing phenomena m flatland studies of monolayers by fluorescence microscopy. Science 1990, 249,870. 

Brennan et al. used a method to detect the reaction of acetylcholineesterase with acetylcholine [46]. The method was based on the use of a monolayer, consisting of fatty acids having Ci6 chain lengths, which were covalently attached to quartz wafers and which contained a small amount of nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE) (partitioned from water into the membrane). The enzyme substrate reaction produced a decrease in fluorescence intensity from the monolayer, and the detection system was sensitive to the changes in bulk concentration of as small as 0.1 pM, with a limit of detection of 2 pM of acetylcholine. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric methods and fluorescence microscopy. 

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. 

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