title: Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping.
abstract: Cellular responses to oxygen tension have been studied extensively. Oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescence sensor. The simultaneous usage of FLIM of coenzymes as NAD(P)H and FAD(+) and PLIM of oxygen sensors could provide information about correlation of metabolic pathways and oxygen tension. We investigated correlative NAD(P)H-FLIM and oxygen sensing-PLIM for simultaneously analyzing cell metabolism and oxygen tension. Cell metabolism and pO2 were observed under different hypoxic conditions in squamous carcinoma cell cultures and in complex ex vivo systems. Increased hypoxia induced an increase of the phosphorescence lifetime of Ru(BPY)3 and in most cases a decrease in the lifetime of NAD(P)H which is in agreement to the expected decrease of the protein-bound NAD(P)H during hypoxia. Oxygen was modulated directly in the mitochondrial membrane. Blocking of complex III and accumulation of oxygen could be observed by both the decrease of the phosphorescence lifetime of Ru(BPY)3 and a reduction of the lifetime of NAD(P)H which was a clear indication of acute changes in the redox state of the cells. For the first time simultaneous FLIM/PLIM has been shown to be able to visualize intracellular oxygen tension together with a change from oxidative to glycolytic phenotype.
authors.affiliation: Ulm University, Core Facility Confocal and Multiphoton Microscopy, N24, Albert Einstein Allee 11, 89081, Ulm, Germany. sviatlana.kalinina@uni-ulm.de.
authors.forename: Sviatlana
authors.initials: S
authors.lastname: Kalinina
authors.affiliation: Ulm University, Core Facility Confocal and Multiphoton Microscopy, N24, Albert Einstein Allee 11, 89081, Ulm, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Breymayer
authors.affiliation: Zentrum biomedizinische Forschung (ZBF), Ulm University, Institute of Neurology, Helmholtzstr. 8/1, 89081, Ulm, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Schäfer
authors.affiliation: Institut für Anästhesiologische Pathophysiologie und Verfahrensentwicklung, Universitätsklinikum Ulm, Helmholtzstr. 8/1, 89081, Ulm, Germany.
authors.forename: Enrico
authors.initials: E
authors.lastname: Calzia
authors.affiliation: Becker & Hickl GmbH, Nahmitzer Damm 30, 12277, Berlin, Germany.
authors.forename: Vladislav
authors.initials: V
authors.lastname: Shcheslavskiy
authors.affiliation: Becker & Hickl GmbH, Nahmitzer Damm 30, 12277, Berlin, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Becker
authors.affiliation: Ulm University, Core Facility Confocal and Multiphoton Microscopy, N24, Albert Einstein Allee 11, 89081, Ulm, Germany. angelika.rueck@uni-ulm.de.
authors.forename: Angelika
authors.initials: A
authors.lastname: Rück
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D006801:Humans; D018613:Microscopy, Confocal; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*
chemical_list: D009249:NADP; D010100:Oxygen
keywords: FLIM; NAD(P)H; PLIM; antimycin A; cell metabolism; oxygen tension; redox state; respiratory chain