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The Single Particle Orientation and Rotational Tracking (SPORT) technique, which utilizes anisotropic plasmonic gold nanorods and differential interference contrast (DIC) microscopy, has shown potential as an effective alternative to fluorescence-based techniques to decipher rotational motions on the cellular and molecular levels. However, localizing gold nanorods from their DIC images with high accuracy and precision is more challenging than the procedures applied in fluorescence or scattering microscopy techniques due to the asymmetric DIC point spread function with bright and dark parts superimposed over a grey background. In this paper, localization accuracy and inherited uncertainties from unique DIC image patterns are elucidated with the assistance of computer simulation. These discussions provide guidance for researchers to properly evaluate their data and avoid making claims beyond the technical limits. The understanding of the intrinsic localization errors and the principle of DIC microscopy leads us to propose a new localization strategy that utilizes the experimentally-measured shear distance of the DIC microscope to improve the localization accuracy.

Localization accuracy of gold nanoparticles in single particle orientation and rotational tracking