Molecular Orientation Affects Localization Accuracy in Superresolution Far-Field Fluorescence Microscopy

We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-held superresolution microscopy based on stochastic single molecule switching, If tilt angle and defocus are unknown, the localization contains systematic errors up to about +/-125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about +/-32 nm, restricting the attainable resolution accordingly.