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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.

Stimulated emission depletion (STED) nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging. Here, we introduce dual-modulation difference STED microscopy (dmdSTED) to suppress the background noise in traditional STED imaging. By applying respective time-domain modulations to the two continuous-wave lasers, signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies. The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution. We used nanoparticle, fixed cell, and perovskite coating experiments, as well as theoretical demonstration, to confirm the effectiveness of this method. We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality, which substantially enlarges the versatility of STED nanoscopy.