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

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Title: Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM
Authors: Chennell, G
Willows, RJW
Warren, SC
Carling, D
French, PMW
Dunsby, C
Sardini, A
Item Type: Journal Article
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.
Issue Date: 19-Aug-2016
Date of Acceptance: 12-Aug-2016
ISSN: 1424-8239
Publisher: MDPI
Journal / Book Title: Sensors
Volume: 16
Issue: 8
Copyright Statement: This is an open access article distributed under the Creative Commons Attribution License (CC BY 4.0).
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (BBSRC)
Funder's Grant Number: BB/M006786/1
Keywords: Analytical Chemistry
Electrical And Electronic Engineering
Publication Status: Published
Article Number: 1312
Appears in Collections:Clinical Sciences
Molecular Sciences
Faculty of Medicine

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