title: Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM.
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.
authors.affiliation: Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK. g.chennell13@imperial.ac.uk.|Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. g.chennell13@imperial.ac.uk.|Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. g.chennell13@imperial.ac.uk.
authors.forename: George
authors.initials: G
authors.lastname: Chennell
authors.affiliation: Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK. robin.willows08@imperial.ac.uk.
authors.forename: Robin J W
authors.initials: RJ
authors.lastname: Willows
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research and St Vincent's Clinical School, University of New South Wales, Darlinghurst, NSW 2010, Australia. s.warren@garvan.org.au.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Warren
authors.affiliation: Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK. david.carling@imperial.ac.uk.|Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. david.carling@imperial.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Carling
authors.affiliation: Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. paul.french@imperial.ac.uk.
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
authors.affiliation: Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. christopher.dunsby@imperial.ac.uk.|Centre for Pathology, Department of Medicine, Imperial College London, London W12 0NN, UK. christopher.dunsby@imperial.ac.uk.
authors.forename: Chris
authors.initials: C
authors.lastname: Dunsby
authors.affiliation: Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. a.sardini@imperial.ac.uk.|Whole Animal Physiology and Imaging, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK. a.sardini@imperial.ac.uk.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sardini
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D015374:Biosensing Techniques / Q000379:methods*; D018929:Cell Culture Techniques; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D057054:Molecular Imaging / Q000379:methods*; D061848:Optical Imaging / Q000379:methods; D011494:Protein Kinases / Q000302:isolation & purification*; D018874:Spheroids, Cellular / Q000166:cytology
chemical_list: C527701:enhanced cyan fluorescent protein; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; D000091162:AMP-Activated Protein Kinase Kinases
keywords: 2-photon; 3D culture; AMPK; FLIM; FRET; TCSPC; biosensor; spheroid