Information optimization of laser scanning microscopes for real-time feedback-driven single particle tracking

Real-time feedback-driven single particle tracking (RT-FD-SPT) is a class of microscopy techniques that uses measurements of finite excitation/detection volume in a feedback control loop to actuate that volume and track with high spatio-temporal resolution a single particle moving in three dimensions. A variety of methods have been developed, each defined by a set of user-defined choices. Selection of those values is typically done through ad hoc, off-line tuning for the best perceived performance. Here we present a mathematical framework, based on optimization of the Fisher information, to select those parameters such that the best information is acquired for estimating parameters of interest, such as the location of the particle, specifics of the excitation beam such as its dimensions or peak intensity, or the background noise. For concreteness, we focus on tracking of a fluorescently-labeled particle and apply this framework to determine the optimal parameters for three existing fluorescence-based RT-FD-SPT techniques with respect to particle localization.