Quantitative, Dynamic Detection of Neuronal Na<sup>+</sup> Transients Using Multi-photon Excitation and Fluorescence Lifetime Imaging (FLIM) in Acute Mouse Brain Slices

Fluorescence lifetime imaging microscopy (FLIM) is a highly valuable technique in the fluorescence microscopy toolbox because it is essentially independent of indicator concentrations. Conventional fluorescence microscopy analyzes changes in emission intensity. In contrast, FLIM assesses the fluorescence lifetime, which is defined as the time a fluorophore remains in an excited state before emitting a photon. This principle is advantageous in experiments where fluorophore concentrations are expected to change, e.g., due to changes in cell volume. FLIM, however, requires collecting a substantial number of photons to accurately fit distribution plots, which constrains its ability for dynamic imaging. This limitation has recently been overcome by rapidFLIM, which utilizes ultra-low dead-time photodetectors in conjunction with sophisticated rapid electronics. The resulting reduction in dead-time to the picosecond range greatly enhances the potential for achieving high spatio-temporal resolution. Here, we demonstrate the use of multi-photon-based rapidFLIM with the sodium indicator ION NaTRIUM Green-2 (ING-2) for the quantitative, dynamic determination of Na⁺ concentrations in neurons in acute rodent brain tissue slices. We describe the loading of the dye into neurons and present a procedure for its calibration in situ. We show that rapidFLIM not only allows the unbiased determination of baseline Na⁺ concentrations but also allows dynamic imaging of changes in intracellular Na⁺, e.g., induced by inhibition of cellular ATP production. Overall, rapidFLIM, with its greatly improved signal-to-noise ratio and higher spatio-temporal resolution, will also facilitate dynamic measurements using other FLIM probes, particularly those with a low quantum yield. Key features • RapidFLIM of the sodium indicator ING-2 enables the intensity-independent recording of neuronal Na⁺ transients at unparalleled full frame rates of 0.5-1 Hz. • RapidFLIM is essentially independent of dye concentrations and therefore not affected by dye bleaching. • Full in situ calibrations enable the quantification of intracellular Na⁺ changes at high spatio-temporal resolution. • RapidFLIM of ING-2 allows unbiased determination of cellular Na⁺ loading also in conditions of strong cell swelling.