Combination of polarized TIRF and ATR spectroscopies for determination of the second and fourth order parameters of molecular orientation in thin films and construction of an orientation distribution based on the maximum entropy method.

This article describes two mathematical formalisms for the determination of the second and fourth order parameters of molecular films using optical spectroscopy. Method A uses polarized total internal reflection fluorescence (TIRF) to calculate the second and fourth order parameters, {P2(cos theta)} and {P4(cos theta)}, using an independently determined value for the angle between the absorption and emission dipoles, gamma. Method B uses {P2(cos theta)} obtained from attenuated total reflectance (ATR) data, along with polarized TIRF measurements to calculate {P4(cos theta)} and {cos2 gamma}. The choice of a specific method should rely on experimental considerations. We also present a method to separate the contributions of substrate surface roughness and dipole orientation with respect to the molecular axis from the spectroscopically determined second and fourth order parameters. Finally, a maximum entropy approach for construction of an orientation distribution from order parameters is compared with the commonly used delta and Gaussian distributions.

Order parameters and orientation distributions of solution adsorbed and microcontact printed cytochrome c protein films on glass and ITO.

The structure of solution adsorbed and microcontact printed (muCP) cytochrome c (cyt c) films on glass and indium tin oxide (ITO) was investigated using attenuated total reflectance (ATR) and total internal reflectance fluorescence (TIRF) spectroscopies to determine the orientation of the heme groups in the films. The second and fourth order parameters of the heme as well as information on the angle between the absorption and emission dipoles of the heme, gamma, were experimentally determined. The order parameters of the heme are related to the order parameters of the protein molecule using the known angle between the heme plane and the electrostatic dipole moment of the cyt c protein. The effect of the surface roughness of the substrates (glass and ITO) was also taken into account quantitatively using AFM data. Physically possible order parameters were obtained for the heme group in both solution adsorbed and muCP films, but not for the electrostatic dipole moment of the protein. In addition, the experimental values of {cos2 gamma} for immobilized zinc-substituted cyt c are greater than the values of {cos2 gamma} determined in viscous solutions, which could be an indication that the environment of the heme groups changes upon adsorption. The electron transfer behavior of solution adsorbed and muCP films on ITO, determined using electrochemical methods, is compared to their orientation distribution and surface coverage as determined by spectroscopic methods.

Determination of anisotropic optical constants and surface coverage of molecular films using polarized visible ATR spectroscopy. Application to adsorbed cytochrome c films.

This article describes a method to determine the anisotropic optical constants and surface coverage of molecular films using polarized attenuated total reflectance (ATR) absorbance measurements. We have extended the transfer-matrix formalism to describe birefringent and dichroic films in ATR geometries and have combined it with an iterative numerical procedure to determine the anisotropic values of both the real (n) and imaginary (k) parts of the complex refractive index of the film under investigation. Anisotropic values of the imaginary part of the refractive index (k) allow for the determination of the surface coverage and one order parameter of the film. To illustrate this approach, we have used cytochrome c (cyt c) protein films adsorbed to glass and indium tin oxide (ITO) surfaces. Experimental results show that cyt c films on these surfaces, which were formed under identical conditions, have significant differences in their surface coverages (11.2 +/- 0.4 pmol/cm(2) on glass and 21.7 +/- 0.9 pmol/cm(2) on ITO); however, their order parameters are similar (0.30 +/- 0.02 on glass and 0.36 +/- 0.04 on ITO).