S-polarized light-sheets improve resolution and light-efficiency in oblique plane microscopy

Abstract

Abstract Oblique plane microscopy (OPM) offers 3D optically sectioned imaging with high spatial- and temporal-resolution while enabling conventional sample mounting. The technique uses a concatenation of three microscopes, two for remote focusing and a tilted tertiary microscope, often including an immersion objective, to image an oblique sample plane. This design induces Fresnel reflections and a reduced effective aperture, thus impacting the resolution and light efficiency of the system. Using vectorial diffraction simulations, the system performance was characterized based on illumination angle and polarization, signal to noise ratio, and refractive index of the tertiary objective immersion. We show that for samples with high fluorescent anisotropy, s-polarized light-sheets yield higher average resolution for all system configurations, as well as higher light-efficiency. We also provide a tool for performance characterization of arbitrary light-sheet imaging systems.