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.