Robust real-time 3D single-particle tracking using a dynamically moving laser spot.

Real-time three-dimensional (3D) single-particle tracking uses optical feedback to lock on to freely diffusing nanoscale fluorescent particles, permitting precise 3D localization and continuous spectroscopic interrogation. Here we describe a new method of real-time 3D single-particle tracking wherein a diffraction-limited laser spot is dynamically swept through the detection volume in three dimensions using a two-dimensional (2D) electro-optic deflector and a tunable acoustic gradient lens. This optimized method, called 3D dynamic photon localization tracking (3D-DyPLoT), enables high-speed real-time tracking of single silica-coated non-blinking quantum dots (∼30  nm diameter) with diffusive speeds exceeding 10  μm²/s at count rates as low as 10 kHz, as well as YFP-labeled virus-like particles. The large effective detection area (1  μm×1  μm×4  μm) allows the system to easily pick up fast-moving particles, while still demonstrating high localization precision (σ_x=6.6  nm, σ_y=8.7  nm, and σ_z=15.6  nm). Overall, 3D-DyPLoT provides a fast and robust method for real-time 3D tracking of fast and lowly emitting particles, based on a single excitation and detection pathway, paving the way to more widespread application to relevant biological problems.