{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T12:37:02Z","timestamp":1777725422737,"version":"3.51.4"},"reference-count":28,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2016,8,19]],"date-time":"2016-08-19T00:00:00Z","timestamp":1471564800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000268","name":"Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["BB\/M006786\/1"],"award-info":[{"award-number":["BB\/M006786\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/s16081312","type":"journal-article","created":{"date-parts":[[2016,8,19]],"date-time":"2016-08-19T09:58:27Z","timestamp":1471600707000},"page":"1312","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5859-361X","authenticated-orcid":false,"given":"George","family":"Chennell","sequence":"first","affiliation":[{"name":"Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK"},{"name":"Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK"},{"name":"Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK"}]},{"given":"Robin","family":"Willows","sequence":"additional","affiliation":[{"name":"Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5253-7147","authenticated-orcid":false,"given":"Sean","family":"Warren","sequence":"additional","affiliation":[{"name":"The Kinghorn Cancer Centre, Garvan Institute of Medical Research and St Vincent\u2019s Clinical School, University of New South Wales, Darlinghurst, NSW 2010, Australia"}]},{"given":"David","family":"Carling","sequence":"additional","affiliation":[{"name":"Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK"},{"name":"Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK"}]},{"given":"Paul","family":"French","sequence":"additional","affiliation":[{"name":"Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK"}]},{"given":"Chris","family":"Dunsby","sequence":"additional","affiliation":[{"name":"Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK"},{"name":"Centre for Pathology, Department of Medicine, Imperial College London, London W12 0NN, UK"}]},{"given":"Alessandro","family":"Sardini","sequence":"additional","affiliation":[{"name":"Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK"},{"name":"Whole Animal Physiology and Imaging, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK"}]}],"member":"1968","published-online":{"date-parts":[[2016,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.taap.2013.01.012","article-title":"A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity","volume":"268","author":"Kostadinova","year":"2013","journal-title":"Toxicol. 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