Membrane lipids fluorescence

Ambrosini, A., Bertoli, E. and Zolese, G. (1996). Effect of organotin compounds on membrane lipids fluorescence spectroscopy studies, Appl. Organomet. Chem., 10, 53-59. 

Hartel S, Tykhonova S, Haas M, Diehl HA (2002) The susceptibility of non-UV fluorescent membrane dyes to dynamical properties of lipid membranes. J Fluoresc 12(3) 465 179... 

The polarization properties of light in combination with fluorescence can be used as a powerful tool for determining motional properties of membranes. This is possible due to the fact that the time scale of interest for membrane lipids falls within the time frame of the fluorescence decay phenomena (0-100+ ns). This, coupled with high sensitivity, low perturbing properties of fluorescent probes, and the large number of available probes, makes the fluorescence approach the method of choice for membrane motional studies. 

The first decision to be made in designing an experiment to measure the motional properties of membrane lipids concerns the type of probe molecule. Too often, this choice is made from the point of view of convenience or tradition rather than suitability, although there is now a considerable range of suitable fluorophores from which to choose. The second consideration is the type of measurement to be made. The most detailed and complete motional information is obtained from a time-resolved fluorescence anisotropy measurement which is able to separate the structural or orientational aspects from the dynamic aspects of fluorophore motion. Steady-state anisotropy measurements, which are much easier to perform, provide a more limited physical parameter relating to both of these aspects. 

The rx term is the anisotropy at times long compared to the fluorescence lifetime, whereas in Eq. (5.9) 2 will be long. If there is no rM, then Eq. (5.8) reduces to the familiar Perrin equation for an isotropic rotator. Earlier, some confusion existed in this field since it was not recognized that an rro term was required for the case of membrane lipid bilayers. For the most part, time-resolved anisotropy measurements have a short rotational correlation time and an term. However, it has been recognized that a more adequate description may be to use two rotational correlation times, where the second may be quite long but not infinite as the rm implies/35 36 ... 

Table 5.3. Alteration of Membrane Lipid Properties and the Resultant Approximate Directions of Changes of Fluorescence Parameters of a Typical Membrane Fluorophore Probe of the Fatty Acyl Chain Region0...

Over 50 different pyridazin-3-ones were evaluated for biological activity in a wheat (Triticum aestivum L.) test system described previously (1). Briefly, seeds were germinated in 9-cm petri dishes on three layers of filter paper. Pyridazinones were dissolved in acetone and the filter papers were impregnated with 1 ml of acetone solution. After the soluent evaporated, 10 ml of distilled water were added to form an inhibitor concentration of 100 yM. Seeds were planted directly on the moist papers and germinated for 4 days in a controlled environment chamber on a 16-hr photoperiod with 27+lC day temperature and 21+lC night temperature. Light intensity from both fluorescent and incandescent bulbs was 28 klux at dish level. Lipids were extracted and recovered from 1 g of lyophilized shoot tissue, separated into membrane and non-membrane lipids, and analyzed by gas chromatography as described (1). 

Looks at the role of caveolins in the formation of membrane caveolae Covers the investigation of hop diffusion of membrane lipids using FRAP (fluorescence recovery after photobleaching)... 

Turunen, T.M., et al. 1994. Effect of some penetration enhancers on epithelial membrane lipid domains Evidence from fluorescence spectroscopy studies. Pharm Res 11 288. 

Carvedilol (11), which contains a CBZ moiety, is a multiple-action antihypertensive drug that has been shown to protect cell membranes from lipid perox-idative damages. Cheng et al. have studied [131] carvedilol, CBZ and 4-hydroxy-carbazole in a 9 1 DMPC DMPG membrane. Fluorescence anisotropy measurement showed that carvedilol is relatively mobile and does not have a rigidly defined molecular orientation in the membrane. The fluorescence spectra... 

Langner and S. W. Huie. Iodide penetration into lipid bilayers as a probe of membrane lipid organization. Chem. and Phys. of Lipids 60 127-132 (1991). Chalpin and A. M. Kleinfeld. Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes. Biochim. et Biophys. Acta 73I 465-A14 (1983). 

The method of choice to determine, under meaningful conditions, the location of synthetic ion channels and pores in bilayer membranes is fluorescence depth quenching (FDQ) [4, 11]. In this well developed but costly analysis, the position of a quencher in the lipid bilayer is varied systematically. Analysis of the dependence of the efficiency to quench a fluorescent synthetic ion channel or pore on the position of the quencher reveals transmembrane, central or interfacial location (Fig. 11.13b-d). 

A third technique for studying foam films is the fluorescence recovery after photobleaching (FRAP). This techniques was applied by Clarke et al. [36] for lateral diffusion in foam films, and involves irreversible photobleaching by intense laser light of fluorophore molecules in the sample. The time of redistribution of probe molecules (which are assumed to be randomly distributed within the constitutive membrane lipids in the film) is monitored. The lateral diffusion coefficient, D, is calculated from the rate of recovery of fluorescence in the bleaching region due to the entry of unbleaching fluoroprobes of adjacent parts of the membranes. 

Murcia MJ, Garg S, Naumann CA (2007) Single molecule fluorescence microscopy to determine phospholipid lateral diffusion. In Dopico A (ed) Methods in membrane lipids. Humana, Totowa, pp 277-294... 

Merocyanine 540 is a red-emitting fluorescent dye that appears to be a sensitive measure of apoptosis-induced alterations in cytoplasmic membrane lipid structure (68). In apoptotic cells, the fluorescence increases in some models increased fluorescence correlates well with increased annexin V binding (68,71). Merocyanine 540 staining has been successfully used together with Hoechst 33342 to identify cell-cycle specific apoptotic events (72). 

C20 5 >3 C22 6 >3) fatty acyl residues were preferentially hydrogenated compared with oleic and linoleic acids. The physical state of the membrane lipid matrix, as judged by the motion of fluorescent probes, became more viscous as a consequence of catalytic hydrogenation. 

A EXPERIMENTAL FIGURE 5-6 Fluorescence recovery after photobleaching (FRAP) experiments can quantify the lateral movement of proteins and lipids within the plasma membrane, (a) Experimental protocol. Step H Cells are first labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein. Step B A laser light is then focused on a small area of the surface, irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area. Step B In time, the fluorescence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is... 

A EXPERIMENTAL FIGURE 5-10 Some membrane lipids and proteins colocalize in lipid rafts. The results of biochemical studies suggested that GM1, a glycosphingolipid, and placental alkaline phosphatase (PLAP), a lipid-anchored membrane protein, aggregate together into lipid rafts, whereas the transferrin receptor (TfR), which traverses the entire membrane, does not. To locate these components in the intact plasma membrane, cells were treated with fluorescence-labeled cholera toxin (green), which cross-links closely spaced GM1 molecules, and with fluorescence-labeled antibodies (red) specific for either PLAP or TfR. Each antibody can cross-link closely spaced... 

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