In the central nervous system, changes in neuronal ionic balance can occur at various timescales from milliseconds, in synaptic events, to seconds, via membrane cotransporters. Using Fluorescence Lifetime Imaging Microscopy (FLIM), video-rate two-photon laser scanning microscopy and the MQAE chloride indicator, the shift in current polarity of the GABAA receptor during neurodevelopment in primary neuronal cultures is studied by monitoring Cl- concentration upon GABA stimulations. To accurately study the sudden variations in local ion concentrations, the accuracy and dynamic range of a fluorescence indicator using FLIM must be established. Indeed, the spatiotemporal resolution in FLIM mainly depends on the number of photons collected during the acquisition of the biological process of interest. The number of photons detected is mainly limited by the photophysical properties of the indicator (photobleaching, quantum yield, dynamic range), the expression of the marker, the laser intensity and the timescale of the biological process of interest. Increasing the laser power to detect more photons is limited as it induces photobleaching and phototoxicity. The accuracy of a FLIM measurement highly depends on the number of photons detected which can be increased by using spatial and temporal binning. Nevertheless, for a given number of photons per analysis, the spatial resolution will be directly linked to the chosen temporal resolution and both cannot be optimized concurrently. In this project, we aim at developing an intelligent analytical approach to optimize the spatiotemporal binning as well as modulating laser line scanning frequency to better reveal the fast biological processes studied. The strategy developed to optimize spatiotemporal resolution in FLIM is used with MQAE, a Cl- indicator, to study the shift in current polarity of the GABAA receptor with TCSPC, but can easily be transposed to study many other phenomena and using other FLIM analytical modalities like the phasor approach.