title: Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models.
abstract: Fluorescence lifetime imaging microscopy (FLIM) can measure and discriminate endogenous fluorophores present in biological samples. This study seeks to identify FLIM as a suitable method to non-invasively detect a shift in cellular metabolic activity towards glycolysis or oxidative phosphorylation in 3D Caco-2 models of colorectal carcinoma. These models were treated with potassium cyanide or hydrogen peroxide as controls, and epidermal growth factor (EGF) as a physiologically-relevant influencer of cell metabolic behaviour. Autofluorescence, attributed to nicotinamide adenine dinucleotide (NADH), was induced by two-photon laser excitation and its lifetime decay was analysed using a standard multi-exponential decay approach and also a novel custom-written code for phasor-based analysis. While both methods enabled detection of a statistically significant shift of metabolic activity towards glycolysis using potassium cyanide, and oxidative phosphorylation using hydrogen peroxide, employing the phasor approach required fewer initial assumptions to quantify the lifetimes of contributing fluorophores. 3D Caco-2 models treated with EGF had increased glucose consumption, production of lactate, and presence of ATP. FLIM analyses of these cultures revealed a significant shift in the contribution of protein-bound NADH towards free NADH, indicating increased glycolysis-mediated metabolic activity. This data demonstrate that FLIM is suitable to interpret metabolic changes in 3D in vitro models.
authors.affiliation: Department of Women's Health, Research Institute for Women's Health, University Hospital of the Eberhard Karls University Tübingen, Tübingen, Germany.
authors.forename: Pirmin H
authors.initials: PH
authors.lastname: Lakner
authors.affiliation: Department of Women's Health, Research Institute for Women's Health, University Hospital of the Eberhard Karls University Tübingen, Tübingen, Germany.|Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.|Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland.|Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Stuttgart, Germany.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Monaghan
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.|Center for Personalised Medicine (ZPM), University Hospital of the Eberhard Karls University Tübingen, Tübingen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Möller
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.|Stuttgart Research Center Systems Biology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Monilola A
authors.initials: MA
authors.lastname: Olayioye
authors.affiliation: Department of Women's Health, Research Institute for Women's Health, University Hospital of the Eberhard Karls University Tübingen, Tübingen, Germany.|Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Stuttgart, Germany.|Department of Medicine/Cardiology, University of California Los Angeles (UCLA), Los Angeles/CA, USA.
authors.forename: Katja
authors.initials: K
authors.lastname: Schenke-Layland
mesh_terms: D000465:Algorithms; D018938:Caco-2 Cells; D018929:Cell Culture Techniques / Q000379:methods; D006019:Glycolysis; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010085:Oxidative Phosphorylation