Single Molecule Localization and Orientation Mapping Using a Joint Inversion Technique

For rotationally-constrained molecules, single molecule (SM) localization based super-resolution imaging can incur losses in accuracy when not accounting for the effect of SM orientation on the point spread function. It is important to determine molecular orientation both to maximize localization accuracy and also because orientation provides information about the local molecular environment, which determines features such as chemical reactivity, biomolecular interactions, local polymer mobility, etc. To map the 3D orientation of SMs, we collect the intensity projected onto four different polarization orientations simultaneously and use the intensities in the different channels to reconstruct each dipole’s 3D orientation. These data provide 1) information on the SM’s local environment, and 2) constraints for image point spread function-fitting and localization. SM images acquired from a second objective positioned on the opposite side of the sample versus the four-channel collection system are utilized for a joint inversion technique for position. We present techniques of registration, localization, and polarization analysis, along with an uncertainty analysis and assessment of the developed methods.