The biophysical properties and organization of cell membranes regulate many membrane-based processes, including electromotility in outer hair cells (OHCs) of the cochlea. Studies of the membrane environment can be carried out by measuring the orientation of membrane-bound fluorophores using fluorescence polarization microscopy (FPM). Due to the cylindrical shape of OHCs, existing FPM theory developed for spherical cells is not applicable. We develop a new method for analyzing FPM data suitable for the quasi-cylindrical OHC. We present the theory for this model, as well as a study of the orientation of the fluorescent probe pyridinium, 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl) (di-8-ANEPPS) in the OHC membrane. Our results indicate that the absorption transition dipole moment of di-8-ANEPPS orients symmetrically about the membrane normal at 27 deg with respect to the plane of the membrane. The observed agreement between theoretical predictions and experimental measurements establishes the applicability of FPM to study OHC plasma membrane properties.
Many biological processes are accompanied by changes in the orientation of membrane-embedded molecules. Fluorescence polarization microscopy (FPM) is an optical technique that can be used to determine how the transition dipole of a fluorophore is oriented with respect to the membrane and therefore may detect these changes. The FPM experimental setup is essentially an anisotropy measurement executed on a microscope. A linear polarizer is placed in both the excitation and emission light paths of an inverted microscope, and images are taken with the polarizers parallel or orthogonal. Intensity measurements are recorded at regions on the sample and compared with theoretical predictions to validate a model of dye orientation. We have applied FPM to determine the orientation of 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (PE-rhodamine) in pure 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC) giant unilamellar vesicles (GUVs). Vesicles are widely used as model membranes, and GUVs are particularly useful as cellular models because of their large diameter size (~10-40 μm). A model for membrane dye orientation follows theory developed previously and indicates that PE-rhodamine molecules orient at ~31° with respect to the membrane. A computational routine was developed to minimize deviations between predicted and experimental results. We have also applied FPM to study the orientation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in GUVs. Preliminary results indicate that DiI is oriented at ~23°.