A prism combination for near isotropic fluorescence excitation by total internal reflection.

Abstract

Total internal reflection fluorescence (TIRF) microscopy is finding increasing application for selectively detecting molecules at or near a glass-water surface. As with all fluorescence methods, the efficiency of excitation of a fluorophore is potentially sensitive to the polarization state of the source. In TIRF, s-polarized excitation produces an evanescent field that is perpendicular to the incident plane (y direction), whereas p-polarized light generates a more complex pattern but one dominated by a field that is vertical to the surface (z direction). Thus, fluorophores whose absorption dipoles are fixed in the x direction are not favourably aligned for excitation. Here we describe a beam-splitting prism arrangement that allows excitation by two orthogonal beams, thus giving isotropic excitation in the x-y plane with s-polarized light. With linearly polarized light at the magic angle, near isotropic excitation in three dimensions should be achieved. This prism design should find application in polarized fluorescence microscopy to investigate the rotational motions of macromolecules or to minimize flickering of fluorescence emission arising from molecular rotations in single molecule studies.