Linear dichroism measurements on oriented purple membranes between parallel polarizers: Contribution of linear birefringence and applications to chromophore isomerization

Absorption measurements with linearly polarized light were performed with oriented purple membranes in the wavelength range from 350 to 750 nm. The membranes were oriented in a magnetic field and subsequently immobilized in a gel. Alternatively orientation was achieved by anisotropic swelling of a dehydrated gel. This new method of orientation, which is similar to the gel-squeezing technique, yields a negative second-order parameter S-2 (e.g., the membranes orient preferentially with their planes parallel to the direction of expansion). In contrast to standard linear dichroism measurements, we used a setup where the oriented sample is inbetween two parallel polarizers. The dependence of the transmitted intensity on the angle between the orientation axis and the polarizer direction shows that the phase difference due to linear birefringence contributes substantially to the signal. Experimental data of high accuracy were obtained by taking measurements at 14 angles from -20 degrees to 110 degrees in steps of 10 degrees. Fitting of the polarization data at each wavelength yields the absorption spectrum, the anisotropy and the linear birefringence. Analogous measurements were carried out on chromophore-free samples to correct for light scattering and to extract-the chromophore contribution to the linear birefringence. The chromophore part of the linear birefringence is in good agreement with numerical calculations from absorption and anisotropy via the Kramers-Kronig transform. The method was applied to investigate possible changes in transition dipole moment orientation associated with the isomerization of the chromophore in light-dark adaptation (all-trans to 13-cis) and the blue-pink transition (all-trans to 9-cis). Analysis of the anisotropy indicates that there is no change in the orientation of the transition dipole moment with respect to the orientation axis between the light and dark adapted states. Only a small increase (approximate to 2.5 degrees) of the chromophore angle occurs between the blue state and the pink state. Comparison of the measured birefringence changes and the calculations from absorption and anisotropy changes confirm the validity of the approach.