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Plos One : N-way Fret Microscopy of Multiple Protein-protein Interactions in Live Cells, Volume 8

By Brody, James, P.

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Book Id: WPLBN0003964098
Format Type: PDF eBook :
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Reproduction Date: 2015

Title: Plos One : N-way Fret Microscopy of Multiple Protein-protein Interactions in Live Cells, Volume 8  
Author: Brody, James, P.
Volume: Volume 8
Language: English
Subject: Journals, Science, Medical Science
Collections: Periodicals: Journal and Magazine Collection
Publication Date:
Publisher: Plos


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Brody, J. P. (n.d.). Plos One : N-way Fret Microscopy of Multiple Protein-protein Interactions in Live Cells, Volume 8. Retrieved from

Description : Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale proteinprotein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.


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