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Sensors 2016, 16(8), 1312; doi:10.3390/s16081312

Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

1
Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK
2
Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK
3
Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK
4
Centre for Pathology, Department of Medicine, Imperial College London, London W12 0NN, UK
5
Whole Animal Physiology and Imaging, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK
6
The Kinghorn Cancer Centre, Garvan Institute of Medical Research and St Vincent’s Clinical School, University of New South Wales, Darlinghurst, NSW 2010, Australia
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editors: Niko Hildebrandt, Igor Medintz and Russ Algar
Received: 20 June 2016 / Revised: 10 August 2016 / Accepted: 12 August 2016 / Published: 19 August 2016
(This article belongs to the Special Issue FRET Biosensors)
View Full-Text   |   Download PDF [6143 KB, uploaded 19 August 2016]   |  

Abstract

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. View Full-Text
Keywords: FRET; FLIM; AMPK; spheroid; 2-photon; biosensor; TCSPC; 3D culture FRET; FLIM; AMPK; spheroid; 2-photon; biosensor; TCSPC; 3D culture
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MDPI and ACS Style

Chennell, G.; Willows, R.J.W.; Warren, S.C.; Carling, D.; French, P.M.W.; Dunsby, C.; Sardini, A. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM. Sensors 2016, 16, 1312.

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