Three-dimensional tracking of fluorescent particles

Single molecule measurements are generally made in conditions that depart from physiological conditions, such as with molecules excised from cells or even immobilized on surfaces. Such departures can easily cause measurements on biomolecules to be inexact. A tracking instrument to follow a single molecule's path in three dimensions inside a living cell would be a major step towards enabling single-molecule observations in physiological conditions. We describe an instrument that will extend the state of the art in single-molecule tracking technology, allowing extended observations of single particles as they diffuse and are transported. Computations show that our approach should be capable of tracking a protein-sized object diffusing at intracellular speeds for average times of over two seconds - long enough to track a typical fluorescent molecule from capture to photobleaching.