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Proceedings Paper

3D single-molecule tracking using one- and two-photon excitation microscopy
Author(s): Cong Liu; Evan P. Perillo; Quincy Zhuang; Khang T. Huynh; Andrew K. Dunn; Hsin-Chih Yeh
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Paper Abstract

Three dimensional single-molecule tracking (3D-SMT) has revolutionized the way we study fundamental cellular processes. By analyzing the spatial trajectories of individual molecules (e.g. a receptor or a signaling molecule) in 3D space, one can discern the internalization or transport dynamics of these molecules, study the heterogeneity of subcellular structures, and elucidate the complex spatiotemporal regulation mechanisms. Sub-diffraction localization precision, sub-millisecond temporal resolution and tens-of-seconds observation period are the benchmarks of current 3D-SMT techniques. We have recently built two molecular tracking systems in our labs. The first system is a previously reported confocal tracking system, which we denote as the 1P-1E-4D (one-photon excitation, one excitation beam, and four fiber-coupled detectors) system. The second system is a whole new design that is based on two-photon excitation, which we denote as the 2P-4E-1D (two-photon excitation, four excitation beams, and only one detector) system. Here we compare these two systems based on Monte Carlo simulation of tracking a diffusing fluorescent molecule. Through our simulation, we have characterized the limitation of individual systems and optimized the system parameters such as magnification, z-plane separation, and feedback gains.

Paper Details

Date Published: 4 March 2014
PDF: 9 pages
Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 89501C (4 March 2014); doi: 10.1117/12.2039565
Show Author Affiliations
Cong Liu, The Univ. of Texas at Austin (United States)
Evan P. Perillo, The Univ. of Texas at Austin (United States)
Quincy Zhuang, The Univ. of Texas at Austin (United States)
Khang T. Huynh, The Univ. of Texas at Austin (United States)
Andrew K. Dunn, The Univ. of Texas at Austin (United States)
Hsin-Chih Yeh, The Univ. of Texas at Austin (United States)


Published in SPIE Proceedings Vol. 8950:
Single Molecule Spectroscopy and Superresolution Imaging VII
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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