Simultaneous super-resolution estimation of single-molecule position and orientation with minimal photon fluxes

The orientation of a single molecule provides valuable information on fundamental biological processes. We report a technique for the simultaneous estimation of single-molecule 2D position and 2D orientation with ultra-high localization precision (∼2-nm precision with ∼500 photons under a typical 100-nm diameter of excitation beam pattern), which is also compatible with tracking in living cells. In the proposed method, the theoretical precision limits are calculated, and the localization and orientation performance along with potential applications are explored using numerical simulations. Compared to other camera-based orientation measurement methods, it is confirmed that the proposed method can obtain reasonable estimates even under very weak signals (∼15 photons). Moreover, the maximum likelihood estimator (MLE) is found to converge to the theoretical limit when the total number of photons is less than 100.