Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background Environments

We demonstrate a custom confocal fluorescence-microscope that is capable of tracking individual quantum dots undergoing three-dimensional Brownian motion (diffusion coefficient ∼ 0.5 μm2/s) in environments with a signal-to-background ratio as low as 2:1, significantly worse than observed in a typical cellular environment. By utilizing a pulsed excitation source and time-correlated single photon counting, the time-resolved photon stream can be used to determine changes in the emission lifetime as a function of position and positively identify single quantum dots via photon-pair correlations. These results indicate that this microscope will be capable of following protein and RNA transport throughout the full three-dimensional volume of a live cell for durations up to 15 s.