Fast, robust and precise 3D localization for arbitrary point spread functions

We present a fitter for 3D single-molecule localization of arbitrary, experimental point spread functions (PSFs) that reaches minimum uncertainty for EMCCD and sCMOS cameras, and achieves more than 10 5 fits/s. We provide tools to robustly model experimental PSFs and correct for depth induced aberrations, which allowed us to achieve an unprecedented 3D resolution with engineered astigmatic PSFs, and acquire high quality 3D superresolution images even on standard microscopes without 3D optics.