Three-dimensional tracking of a single fluorescent nanoparticle using four-focus excitation in a confocal microscope

We report high sensitivity detection and tracking of a single fluorescent nanoparticle in solution by use of four alternately pulsed laser diodes for fluorescence excitation in a confocal microscope. Slight offsets between the centers of the overlapping laser foci together with time-resolved photon counting enable sub-micron precision position measurements. Real-time correction for diffusional motion with a xyz-piezo stage then enables tracking of a nanoparticle with diffusivity up to ~12 μm(2) s(-1). Fluorescence correlation spectroscopy and calibration measurements indicate a net fluorescence photon detection efficiency of ~6-9%, comparable to that of an optimized single-molecule microscope.