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-field 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.