Sub-Resolution Maximum-Likelihood Based Localization of Fluorescent Nanoparticles in Three Dimensions
Several recent studies have shown that fluorescent particles can be localized with an accuracy that is well beyond traditional resolution limits. Using a theoretical model of the image formation process that accounts for possible sources of noise, Cramér-Rao bounds have been used to define the theoretical limits. A crucial influence on these bounds is the mismatch of refractive indices that is usually present between immersion medium and specimen. This results in an axially shift-variant point spread function, meaning that the bounds change as a function of the particle's position in the z-direction. We investigate the theoretical bounds for this shift-variant model, and propose a maximum-likelihood estimator for the particle position in 3D (XYZ position). Using this estimator, sub-resolution localization at the nanometer scale is demonstrated on experimental data. The results provide optimal conditions for particle tracking and localization experiments.