Biomedical Imaging Group

Several recent studies have shown that fluorescent particles can be localized with an accuracy that is well beyond tra-ditional 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 be-tween 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 es-timator, sub-resolution localization at the nanometer scale is demonstrated on experimental data. The results provide op-timal conditions for particle tracking and localization experi-ments