Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells
- 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 Kr341In 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.…
Verfasserangaben: | Karolina Jahn, Volker Buschmann, Carsten HilleORCiDGND |
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DOI: | https://doi.org/10.1038/srep14334 |
ISSN: | 2045-2322 |
Titel des übergeordneten Werks (Englisch): | Scientific Reports |
Verlag: | Nature Publishing Group |
Verlagsort: | London |
Publikationstyp: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Erstveröffentlichung: | 22.09.2015 |
Erscheinungsjahr: | 2015 |
Veröffentlichende Institution: | Universität Potsdam |
Datum der Freischaltung: | 20.10.2015 |
Freies Schlagwort / Tag: | Confocal microscopy; Fluorescence imaging; Fluorescence spectroscopy; Fluorescent probes |
Ausgabe: | 5 |
Seitenanzahl: | 13 |
Fördernde Institution: | Universität Potsdam, Publikationsfonds |
Fördernummer: | PA 2015_24 |
Organisationseinheiten: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie |
DDC-Klassifikation: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
Peer Review: | Referiert |
Fördermittelquelle: | Publikationsfonds der Universität Potsdam |
Publikationsweg: | Open Access |
Lizenz (Deutsch): | CC-BY - Namensnennung 4.0 International |
Externe Anmerkung: | Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 202 |