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

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Abstract

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 (30nm diameter) with diffusive speeds exceeding 10μm2/s at count rates as low as 10 kHz, as well as YFP-labeled virus-like particles. The large effective detection area (1μm×1μm×4μm) allows the system to easily pick up fast-moving particles, while still demonstrating high localization precision (σx=6.6nm, σy=8.7nm, and σz=15.6nm). 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.

© 2017 Optical Society of America

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Supplementary Material (5)

NameDescription
Visualization 1: MP4 (15045 KB)      Real-time movie of a 100 nm fluorescent bead being tracked by the 3D-DyPLoT system. The 3D plot is the movement of the piezoelectric stage to hold the diffusing nanoparticle in the objective focal volume. The inset is the sCMOS readout.
Visualization 2: MP4 (1723 KB)      Real-time movie of a gQD@SiO2 particle being tracked by 3D-DyPLoT. The 3D plot is the movement of the piezoelectric stage to hold the diffusing gQD@SiO2 in the objective focal volume. The inset is the sCMOS readout (3x3 Gaussian filter, false-color).
Visualization 3: MP4 (1434 KB)      Real-time movie of a VSV-G-YFP virion being tracked by 3D-DyPLoT. The 3D plot is the movement of the piezoelectric stage to hold the diffusing virion in the objective focal volume. The inset is the sCMOS readout (3x3 Gaussian filter, false-color).
Visualization 4: MP4 (19277 KB)      Movie of the 3D-DyPLoT system picking up sequential 100 nm beads, showing both the robustness of the tracking and the ease with which the system can pick up diffusing particles. The inset is the sCMOS readout. The movie is played at 5x real time.
Visualization 5: MP4 (1811 KB)      Real-time movie of the 3D-DyPLoT system picking up sequential gQD@SiO2 particles showing the ability to easily catch particles even at high diffusive speeds and lower count rates. The inset is the sCMOS readout (3x3 Gaussian filter, false-color).

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Figures (6)

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Equations (4)

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