Simultaneous Imaging of Individual Molecules Aligned Both Parallel and Perpendicular to the Optic Axis

Abstract

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 $\left(z\right)$ 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 ${\mathrm{DiIC}}_{18}$ embedded in polymethyl-methacrylate (PMMA), calculated images accurately model the main features of observed emission patterns.