title: Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging.
abstract: The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptor-mediated Ca(2+) influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer.
authors.affiliation: Luxcel Biosciences, BioInnovation Centre, University College Cork, Cork, Ireland.
authors.forename: Tomás C
authors.initials: TC
authors.lastname: O'Riordan
authors.forename: Kathleen
authors.initials: K
authors.lastname: Fitzgerald
authors.forename: Gelii V
authors.initials: GV
authors.lastname: Ponomarev
authors.forename: John
authors.initials: J
authors.lastname: Mackrill
authors.forename: James
authors.initials: J
authors.lastname: Hynes
authors.forename: Cormac
authors.initials: C
authors.lastname: Taylor
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D000968:Antimycin A / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D019169:Jurkat Cells; D007700:Kinetics; D049408:Luminescent Agents; D008163:Luminescent Measurements; D008175:Lung Neoplasms / Q000473:pathology; D008665:Metalloporphyrins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects; D010100:Oxygen / Q000032:analysis*; D019265:Spectroscopy, Near-Infrared; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; D007476:Ionophores; D049408:Luminescent Agents; D008665:Metalloporphyrins; D014634:Valinomycin; C007293:DAPI; D000968:Antimycin A; D010100:Oxygen