Three dimensional single-molecule tracking with confocal-feedback microscope

Three dimensional single-molecule tracking (3D-SMT) holds great promises of providing insights into molecular transport and localization dynamics in live cells. Sub-diffraction localization precision, sub-millisecond temporal resolution and tens-of-seconds observation period are the benchmarks of recent SMT techniques. Among various techniques, the optical fiber-based confocal-feedback tracking microscope has advantages in large axial tracking range, high signal-to-noise ratio, and time-resolved fluorescence analysis. Here we report the optimization of a confocal-feedback tracking system through Monte Carlo simulation, and show it´s possible to combine molecular tracking with fluorescence resonance energy transfer (FRET) measurements.