Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

We have developed an engineered Point Spread Function (PSF) to enable the simultaneous estimation of dipole orientation, 3D position and degree of rotational constraint of single-molecule emitters from a single 2D focal plane. Besides giving access to orientation information, the Vortex PSF along with the vectorial PSF fitter avoids localization bias common in localization microscopy for fixed dipole emitters. We demonstrate this technique on reorienting single-molecules and using binding-activated localization microscopy on DNA intercalators, corroborating perpendicular azimuthal angles to the DNA axis for in-plane emitters but find a non-uniform distribution as a function of the polar angle. The Vortex PSF is realized by an affordable glass phase mask and has a compact footprint that can easily be combined with localization microscopy techniques on rotationally constrained emitters.