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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.

Type Journal
ISBN 1424-8220 (Electronic) 1424-8220 (Linking)
Authors Chennell, G.; Willows, R. J.; Warren, S. C.; Carling, D.; French, P. M.; Dunsby, C.; Sardini, A.;
Publisher Name SENSORS
Published Date 2016-01-01
Published Volume 16
Published Issue 8
Status Published in-print
URL link to publisher's version http://www.ncbi.nlm.nih.gov/pubmed/27548185
OpenAccess link to author's accepted manuscript version https://publications.gimr.garvan.org.au/open-access/13769