Tracking single fluorescent particles in three dimensions via extremum seeking.

The ability to track single fluorescent particles in three-dimensions with sub-diffraction limit precision as well as sub-millisecond temporal resolution has enabled the understanding of many biophysical phenomena at the nanometer scale. While there are several techniques for achieving this, most require complicated experimental setups that are expensive to implement. These methods can offer superb performance but their complexity may be overwhelming to the end-user whose aim is only to understand the feature being imaged. In this work, we describe a method for tracking a single fluorescent particle using a standard confocal or multi-photon microscope configuration. It relies only on the assumption that the relative position of the measurement point and the particle can be actuated and that the point spread function has a global maximum that coincides with the particle's position. The method uses intensity feedback to calculate real-time position commands that "seek" the extremum of the point spread function as the particle moves through its environment. We demonstrate the method by tracking a diffusing quantum dot in a hydrogel on a standard epifluorescent confocal microscope.