Simultaneous imaging of individual molecules aligned both parallel and perpendicular to the optic axis

We report the first room temperature observation of an emission pattern from a single fluorescent molecule signifying that the emission dipole is aligned along the optic (z) axis of a microscopic imaging system. This technique takes advantage of the x, y, and z-polarized evanescent fields generated in total internal reflection and the usually nettlesome aberrations commonly encountered when imaging biological samples with high numerical aperture oil-immersion objectives. For both z-oriented and transverse-oriented individual molecules of the carbocyanine dye DiIC(18) embedded in polymethyl-methacrylate (PMMA), calculated images accurately model the main features of observed emission patterns.