Fundamental limits on measuring the rotational constraint of single molecules using fluorescence microscopy

Optical fluorescence imaging is capable of measuring both the translational and rotational dynamics of single molecules. However, unavoidable measurement noise will result in inaccurate estimates of rotational dynamics, causing a molecule to appear to be more rotationally constrained than it actually is. We report a mathematical framework to compute the fundamental limit of accuracy in measuring the rotational mobility of dipolelike emitters. By applying our framework to both in-plane and three-dimensional methods, we provide a means to choose the optimal orientation-measurement technique based on experimental conditions.