Amphiphilic hemicyanine dyes are fluorescent probes for voltage transients in nerve cells. Their sensitivity is assumed to be related with an intramolecular charge shift along the oriented chromophore that interacts with the electrical field across the cell membrane. Here we report on a measurement of the molecular orientation of the hemicyanine dye Di8ANEPPS in a lecithin membrane. We took advantage of the features of dipole radiation in front of a mirror. The fluorescence intensity of a stained membrane on oxidized silicon was measured as a function of the thickness of silicon dioxide up to 1000 nm and fitted with an electromagnetic theory accounting for the interference of the exciting light, for the interference of the emitted light and for the change of fluorescence lifetime. We found an angle of 37.8 ± 1.6° between the transition dipole moment and the membrane normal for an uniaxial cone model of the angular distribution function, with an order parameter <P_2>=0.44 similar to the hydrocarbon chains of the lipid matrix.
Fluorescence intensity of Di8ANEPPS in lipid bilayer on silicon. (a) Experimental data in arbitrary units versus the thickness of silicon dioxide. The data are fitted by a Sommerfeld-type theory with a uniaxial cone model of the angular distribution function of the transition dipole moment with an angle of theta = 36° to the membrane normal. (b) Residuals of the fit.
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