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.
Highlights
Sodium ions (Na+) are important cations in living cells
Since no data is available about the application of Asante NaTRIUM Green-2 (ANG-2) for fluorescence lifetime imaging microscopy (FLIM), the purpose of this study is to provide time-resolved fluorescence data and demonstrate the feasibility of ANG-2 for quantitative [Na+]i recordings in living cells using 2P-FLIM
ANG-2 is especially suitable for measurements in living cells, because the excitation energy in the visible spectral range is not as high as that for other Na+-sensitive dyes (e.g. sodium-binding benzofuran isophthalate (SBFI)).[4]
Summary
Since cellular plasma membranes are highly impermeable to ions, integrated transport proteins play an important role in the regulation of ionic homeostasis.[1] One of the primary membrane transport proteins is the Na+/K+ATPase, which maintains a Na+ concentration gradient between the intracellular space (∼5–15 mM) and the extracellular space (∼100–150 mM). This Na+ gradient is important for a variety of functions, such as nutrient uptake in the small intestine, activity of neurons, photoreception in the retina as well as secretion processes.[1] For a better understanding of Na+dependent transport processes in epithelia under physiological and pathological conditions, the monitoring of spatiotemporal changes in the intracellular Na+ concentration ([Na+]i) is required. Most of these dyes consist of a 15-crown-5 ether, which binds to Na+ and is linked to the fluorophore, such as fluorescein
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