title: Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.
abstract: In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.
authors.affiliation: Physical Chemistry/ALS ComBi, Institute of Chemistry, University of Potsdam, Potsdam, Germany.
authors.forename: Karolina
authors.initials: K
authors.lastname: Jahn
authors.affiliation: PicoQuant GmbH, Berlin, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Buschmann
authors.affiliation: Physical Chemistry/ALS ComBi, Institute of Chemistry, University of Potsdam, Potsdam, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Hille
mesh_terms: D000818:Animals; D003058:Cockroaches; D004298:Dopamine / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010100:Oxygen / Q000378:metabolism; D018987:Salivary Ducts / Q000166:cytology / Q000378:metabolism
chemical_list: D005182:Flavin-Adenine Dinucleotide; D010100:Oxygen; D004298:Dopamine