Orientation of lipophilic dye molecules within a biological
membrane can report on the molecular environment, i.e., the physical and
chemical properties of the surrounding membrane. This fact, however, remains
under-utilized, largely because of our limited quantitative knowledge of
molecular orientational distributions and the fact that robust techniques
allowing experimental observation of molecular orientations of dyes in biological
membranes are only being developed. In order to begin filling this lack of
knowledge and to develop appropriate tools, we have investigated the membrane
orientational distribution of the 3-hydroxyflavone-based membrane dye F2N12S.
Results of our single- and two-photon polarization microscopy observations of
linear dichroism of F2N12S-labeled giant unilamellar vesicles are consistent 
with a Gaussian-like orientational distribution of the
transition dipole moment of the dye, with a mean tilt angle 
of 53.2 +/- 0.1° with respect to the bilayer normal and a standard
deviation of 13.3 +/- 0.6°. Independently, by combining quantum 
chemical calculations and molecular dynamics simulations, we
obtained very similar values; a mean tilt angle of 48 +/- 4° and 
a standard deviation of 13 +/- 2°. The good agreement between the
experimentally and computationally obtained values cross-validates 
both approaches and gives confidence to the results obtained.
The results open a door to robust quantitative determinations of 
orientational distributions of fluorescent molecules (ranging
from simple synthetic dyes to fluorescent proteins attached to 
membrane proteins) associated with lipid membranes. Such
determinations enable rational development of a novel class of 
sensitive fluorescent optical probes, reporting on cellular events
through changes in linear dichroism.