Tracking Nanometer-Scale Fluorescent Particles in Two Dimensions With a Confocal Microscope

A system for tracking multiple nanometer-scale fluorescent particles in a confocal microscope and an experimental validation is described. Position estimates of an individual fluorescent particle are generated from fluorescence intensity measurements taken at a small number of discrete locations. Tracking is achieved by combining the estimation procedure with a linear quadratic Gaussian (LQG) regulator. Multiple particles are tracked by combining the models for individual particles into a single system, applying the same LQG framework, and then cycling the control through each subsystem in turn. Experimental results are presented for single and multiple particles. For validation purposes, during each experiment images from a charge-coupled device camera were captured and analyzed offline using a standard Gaussian fit method. The estimated trajectories were in good agreement with those produced by the LQG algorithm, thereby verifying the tracking scheme.