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For the investigation of these processes and quantification of intracellular Na<jats:sup>+<\/jats:sup>concentrations ([Na<jats:sup>+<\/jats:sup>]<jats:sub>i<\/jats:sub>), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach<jats:italic>Periplaneta americana<\/jats:italic>. For this, the novel Na<jats:sup>+<\/jats:sup>-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both<jats:italic>in vitro<\/jats:italic>and<jats:italic>in situ<\/jats:italic>. 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<jats:italic>in vitro<\/jats:italic>, its analysis indicates a Na<jats:sup>+<\/jats:sup>-sensitivity appropriate for recordings in living cells. The Na<jats:sup>+<\/jats:sup>-sensitivity was reduced<jats:italic>in situ<\/jats:italic>, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na<jats:sup>+<\/jats:sup>]<jats:sub>i<\/jats:sub>recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na<jats:sup>+<\/jats:sup>]<jats:sub>i<\/jats:sub>rise in cockroach salivary gland cells, which was dependent on a Na<jats:sup>+<\/jats:sup>-K<jats:sup>+<\/jats:sup>-2Cl<jats:sup>\u2212<\/jats:sup>cotransporter (NKCC) activity. 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