Polarization of excitation light influences molecule counting in single-molecule localization microscopy

Abstract

Single-molecule localization microscopy has been widely applied to count the number of biological molecules within a certain structure. The percentage of molecules that are detected significantly affects the interpretation of data. Among many factors that affect this percentage, the polarization state of the excitation light is often neglected or at least unstated in publications. We demonstrate by simulation and experiment that the number of molecules detected can be different from -40 up to 100 % when using circularly or linearly polarized excitation light. This is determined mainly by the number of photons emitted by single fluorescent molecule, namely the choice of fluorescence proteins,..