A technique is proposed for determination of the three-dimensional orientat
ion of the transition dipoles of single molecules by use of polarization-se
nsitive detection of fluorescence through a high-N.A. objective. Molecular
orientation can be determined uniquely and rapidly based on the counts from
three detectors that are sensitive to different polarizations of light. (C
) 2001 Optical Society of America OCIS codes: 180.2520, 260.5430.