3-Dimensional Tracking of Non-blinking

While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or 'blinking,' which causes ambiguity in particle trajectory analysis and limits tracking duration. Here, we exploit non-blinking 'giant' quantum dots (gQDs) to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3-dimensional (3D) SPT microscope. We find a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to commercially available QDs. We further implement a time-gated photon-pair correlation analysis to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which we quantify using a time-dependent diffusion coefficient and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, especially in cases were changes in cellular dynamics are occurring on the time scale of several minutes.