ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy
- Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+](i)), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms ofSodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+](i)), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+](i) recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+](i) rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl-cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.…
Author details: | Phillip RoderORCiD, Carsten HilleORCiDGND |
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DOI: | https://doi.org/10.1039/c4pp00061g |
ISSN: | 1474-905X |
ISSN: | 1474-9092 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/25311309 |
Title of parent work (English): | Photochemical & photobiological sciences |
Publisher: | Royal Society of Chemistry |
Place of publishing: | Cambridge |
Publication type: | Article |
Language: | English |
Year of first publication: | 2014 |
Publication year: | 2014 |
Release date: | 2017/03/27 |
Volume: | 13 |
Issue: | 12 |
Number of pages: | 12 |
First page: | 1699 |
Last Page: | 1710 |
Funding institution: | German Research Foundation [1850/30001355, DO1268/3-1]; Federal Ministry of Education and Research [03IPT517Y] |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
Peer review: | Referiert |