The precision of estimating a parameter is bounded by the Quantum Cramér-Rao Lower Bound (QCRLB), which provides a bound on parameter estimates for any measurement allowed by quantum mechanics (i.e., physics). We show that the common method of directly imaging single molecules onto a camera and estimating their position is not optimal for freely rotating dipoles imaged with high NA objectives. We further demonstrate that by separating the emission into two orthogonal polarization states, we can improve the localization precision. Using horizontal and vertical polarization, we see that the point spread function (PSF) shrinks along one axis relative to the polarization direction, providing added information about emitter position. Simulations confirm that these polarization-specific PSF features enhance localization precision compared to conventional single-channel methods. We implemented this technique experimentally using DNA-PAINT nanorulers and cellular samples. The dual-channel polarization strategy consistently improved localization accuracy and revealed richer structural detail in reconstructed images. These findings establish polarization-resolved SMLM as a powerful method to extract more information from fluorescence emission, offering a practical path toward higher-resolution and more informative super-resolution microscopy.
Shotorban et al. (Sun,) studied this question.