title: ANG-2 for quantitative Na(+) determination in living cells by time-resolved fluorescence microscopy.
abstract: 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 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.
authors.affiliation: Physical Chemistry/Applied Laser Sensing in Complex Biosystems (ALS ComBi), Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany. hille@uni-potsdam.de.
authors.forename: Phillip
authors.initials: P
authors.lastname: Roder
authors.forename: Carsten
authors.initials: C
authors.lastname: Hille
mesh_terms: D061354:Acinar Cells / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D003058:Cockroaches; D004298:Dopamine / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D019476:Insect Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism*; D007477:Ions / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D012469:Salivary Glands / Q000187:drug effects / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D019476:Insect Proteins; D007477:Ions; D049994:Sodium Potassium Chloride Symporter Inhibitors; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; D012964:Sodium; D004298:Dopamine