Document Detail


Time-resolved three-dimensional molecular tracking in live cells.
MedLine Citation:
PMID:  20957984     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms to follow three-dimensional molecular motion. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, we follow individual QD-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell.
Authors:
Nathan P Wells; Guillaume A Lessard; Peter M Goodwin; Mary E Phipps; Patrick J Cutler; Diane S Lidke; Bridget S Wilson; James H Werner
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Nano letters     Volume:  10     ISSN:  1530-6992     ISO Abbreviation:  Nano Lett.     Publication Date:  2010 Nov 
Date Detail:
Created Date:  2011-04-18     Completed Date:  2011-06-24     Revised Date:  2013-07-03    
Medline Journal Info:
Nlm Unique ID:  101088070     Medline TA:  Nano Lett     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4732-7     Citation Subset:  IM    
Affiliation:
Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Tracking / instrumentation*
Cells, Cultured
Equipment Design
Equipment Failure Analysis
Imaging, Three-Dimensional / instrumentation*
Mast Cells / cytology*
Microscopy, Fluorescence / instrumentation*
Quantum Dots*
Rats
Grant Support
ID/Acronym/Agency:
AI051575/AI/NIAID NIH HHS; P50GM065794/GM/NIGMS NIH HHS; R21 AI077072-02/AI/NIAID NIH HHS; R21AI07707/AI/NIAID NIH HHS
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