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.…
Author details: | Karolina Jahn, Volker Buschmann, Carsten HilleORCiDGND |
---|---|
DOI: | https://doi.org/10.1038/srep14334 |
ISSN: | 2045-2322 |
Title of parent work (English): | Scientific Reports |
Publisher: | Nature Publishing Group |
Place of publishing: | London |
Publication type: | Article |
Language: | English |
Date of first publication: | 2015/09/22 |
Publication year: | 2015 |
Publishing institution: | Universität Potsdam |
Release date: | 2015/10/20 |
Tag: | Confocal microscopy; Fluorescence imaging; Fluorescence spectroscopy; Fluorescent probes |
Issue: | 5 |
Number of pages: | 13 |
Funding institution: | Universität Potsdam, Publikationsfonds |
Funding number: | PA 2015_24 |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
Peer review: | Referiert |
Grantor: | Publikationsfonds der Universität Potsdam |
Publishing method: | Open Access |
License (German): | CC-BY - Namensnennung 4.0 International |
External remark: | Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 202 |