Influence of the excitation polarization on single molecule 3D orientation imaging

Abstract

Single Molecule Orientation and Localization Microscopy (SMOLM) is gaining an increasing interest in the community of localization microscopy, due to the capability to monitor orientational information in addition to spatial reconstruction. In many cases, molecule’s orientations are not random and their linker to a protein of interest is sufficiently rigid to be able to report orientation information from this protein. While several strategies exist to report single molecule orientation based on polarization splitting and point spread function engineering, the effect of the incident polarization has been often neglected by supposing its effect embedded in the retrieved angular parameters, or by supposing the excitation to be isotropic or the rotational diffusion to be very fast. In this work we quantify the amount of possible bias brought in SMOLM readout due to the incident polarization effect, using analytical derivations. We illustrate this effect experimentally on single molecules attached to a surface in presence of wobbling.