Measuring 3D orientation properties of single fluorescent emitters including their angle wobbling, as well as their position, is a challenge that would enrich super-resolution techniques with structural molecular information. We present a polarized microscopy technique that provides all 3D orientation parameters unambiguously, using four-polarization splitting of the image plane and intensity filtering in the back focal plane. Using an inverse problem approach we can retrieve 3D orientation, wobbling and 2D position of the fluorophores with high precision. We validated the technique using fluorescent nano-beads and applied it to the structural study of fluorescently labelled F-actin filaments.
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