Several methods based on single- and two-photon fluorescence detected linear dichroism have recently been 
used to determine the orientational distributions of fluorescent dyes in lipid membranes. However, these 
determinations relied on simplified descriptions of non-linear anisotropic properties of the dye molecules, 
using a transition dipole moment-like vector instead of an absorptivity tensor. To investigate the validity 
of the vector approximation, we have now carried out a combination of computer simulations and polarization 
microscopy experiments on two representative fluorescent dyes (DiI and F2N12S) embedded in aqueous 
phosphatidylcholine bilayers. Our results indicate that a simplified vector-like treatment of the two-photon 
transition tensor is applicable for molecular geometries sampled in the membrane at ambient conditions. 
Furthermore, our results allow evaluation of several distinct polarization microscopy techniques. In 
combination, our results point to a robust and accurate experimental and computational treatment of 
orientational distributions of DiI, F2N12S and related dyes (including Cy3, Cy5, and others), with 
implications to monitoring physiologically relevant processes in cellular membranes in a novel way.