The influence of movement on the localization precision of sub-resolution particles in fluorescence microscopy

Obtaining sub-resolution particle positions in fluorescence microscopy images is essential for single particle tracking and high-resolution localization microscopy. While the localization precision of stationary single molecules or particles is well understood, the influence of particle motion during image acquisition has been largely neglected. Here, we address this issue and provide a theoretical description on how particle motion influences the centroid localization precision, both in case of 2-D and 3-D diffusion. In addition, a novel method is proposed, based on dual-channel imaging, for the experimental determination of the localization precision of moving particles. For typical single particle tracking experiments, we show that the localization precision is approximately two-fold worse than expected from the stationary theory. Strikingly, we find that the most popular localization method, based on the fitting of a Gaussian distribution, breaks down for lateral diffusion. Instead, the centroid localization method is found to perform well under all conditions.