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Proceedings Paper

Going beyond 2D: following membrane diffusion and topography in the IgE-Fc[epsilon]RI system using 3-dimensional tracking microscopy
Author(s): Nathan P. Wells; Guillaume A. Lessard; Mary E. Phipps; Peter M. Goodwin; Diane S. Lidke; Bridget S. Wilson; James H. Werner
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Paper Abstract

The ability to follow and observe single molecules as they function in live cells represents a major milestone for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in three dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot labeled IgE antibodies bound to FcεRI membrane receptors in live RBL-2H3 cells. The results are consistent with prior studies of two dimensional membrane diffusion (Andrews et al., Nat. Cell Biol., 10, 955, 2008). In addition, the microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative to the "hills and valleys" of the dynamically changing membrane landscape. This approach is uniquely capable of following single molecule dynamics on live cells with three dimensional spatial resolution.

Paper Details

Date Published: 24 February 2009
PDF: 13 pages
Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 71850Z (24 February 2009);
Show Author Affiliations
Nathan P. Wells, Los Alamos National Lab. (United States)
Guillaume A. Lessard, Los Alamos National Lab. (United States)
Mary E. Phipps, Los Alamos National Lab. (United States)
Peter M. Goodwin, Los Alamos National Lab. (United States)
Diane S. Lidke, Univ. of New Mexico (United States)
Bridget S. Wilson, Univ. of New Mexico (United States)
James H. Werner, Los Alamos National Lab. (United States)

Published in SPIE Proceedings Vol. 7185:
Single Molecule Spectroscopy and Imaging II
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)

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