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Int. J. Mol. Sci. 2015, 16(7), 14695-14716; doi:10.3390/ijms160714695

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

1
Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK
2
Structural and Molecular Biology, University College London, London WC1E 6BT, UK
*
Author to whom correspondence should be addressed.
Academic Editor: Herbert Schneckenburger
Received: 20 May 2015 / Revised: 15 June 2015 / Accepted: 17 June 2015 / Published: 30 June 2015
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Abstract

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. View Full-Text
Keywords: time resolved fluorescence anisotropy imaging (TR-FAIM); FRET; multiplexed imaging; homo-FRET time resolved fluorescence anisotropy imaging (TR-FAIM); FRET; multiplexed imaging; homo-FRET
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Warren, S.C.; Margineanu, A.; Katan, M.; Dunsby, C.; French, P.M.W. Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells. Int. J. Mol. Sci. 2015, 16, 14695-14716.

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