Quantification of Astrocytic Sodium Signals Using Fluorescence Lifetime Imaging Microscopy (FLIM)

Intensity-based imaging with fluorescent indicators is a widely used method for monitoring ion transients, including Ca²⁺ or Na⁺ signals, in astrocytes. However, using this technique, changes in ion concentrations cannot always be reliably separated from simultaneous changes in the concentration of the ion-sensitive fluorophores, e.g., due to changes in cell volume. An alternative approach is fluorescence lifetime imaging microscopy (FLIM), which is based on the time the fluorophore remains in the excited state rather than determining its emission intensity. Here, we describe the use of the chemical fluorescent indicator dyes ION Natrium Green 2 and CoroNa Green for FLIM of intracellular Na⁺ in astrocytes. We also present different strategies for analyzing the FLIM data obtained and demonstrate a procedure for their calibration. Overall, Na⁺-FLIM provides a reliable quantitative determination of changes in astrocyte Na⁺ concentrations independent of changes in fluorophore concentrations.