Note: Time-gated 3D single quantum dot tracking with simultaneous spinning disk imaging

• Published in: Review of scientific instruments Vol. 86; no. 12; p. 126102
• Main Authors: DeVore, M S, Stich, D G, Keller, A M, Cleyrat, C, Phipps, M E, Hollingsworth, J A, Lidke, D S, Wilson, B S, Goodwin, P M, Werner, J H
• Format: Journal Article
• Language: English
• Published: United States 01.12.2015
• Subjects: Cell Tracking - instrumentation; Equipment Design; Equipment Failure Analysis; Image Enhancement - instrumentation; Imaging, Three-Dimensional - instrumentation; Microscopy, Fluorescence - instrumentation; Molecular Imaging - instrumentation; Quantum Dots; Reproducibility of Results; Sensitivity and Specificity; Signal Processing, Computer-Assisted - instrumentation
• ISSN: 1089-7623
• DOI: 10.1063/1.4937477

We describe recent upgrades to a 3D tracking microscope to include simultaneous Nipkow spinning disk imaging and time-gated single-particle tracking (SPT). Simultaneous 3D molecular tracking and spinning disk imaging enable the visualization of cellular structures and proteins around a given fluorescently labeled target molecule. The addition of photon time-gating to the SPT hardware improves signal to noise by discriminating against Raman scattering and short-lived fluorescence. In contrast to camera-based SPT, single-photon arrival times are recorded, enabling time-resolved spectroscopy (e.g., measurement of fluorescence lifetimes and photon correlations) to be performed during single molecule/particle tracking experiments.