Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle.

Laser illumination used in both conventional widefield epi-fluorescence as well as in total internal reflection fluorescence (TIRF) microscopy is subject to nonuniformities in intensity that obscure true image details. These intensity variations are interference fringes arising from coherent light scattering and diffraction at every surface in the laser light's optical path, including the lenses, mirrors, and coverslip. We present an inexpensive technique for effectively eliminating these interference fringes based upon introduction of the excitation laser beam by oblique through-the-objective incidence coupled with rapid azimuthal rotation of the plane of incidence. Although this rotation can be accomplished in several ways, a particularly simple method applicable to a free laser beam is to use an optical wedge, spun on a motor, which diverts the beam into a hollow cone of fixed angle. A system of lenses converts this collimated beam cone into a focused spot that traces a circle at the objective's back focal plane. Consequently, a collimated beam with fixed polar angle and spinning azimuthal angle illuminates the sample. If the wedge is spun rapidly, then the different interference patterns at every particular azimuthal incidence angle average out over a single camera exposure to produce an effectively uniform field of illumination.