SIGAA: signaling automated analysis: a new tool for Ca²⁺ signaling quantification using ratiometric Ca²⁺ dyes

Astrocytes are non-neural cells, restricted to the brain and spinal cord, whose functions and morphology depend on their location. Astrocyte-astrocyte and astrocyte-neuron interactions occur through cytoplasmic Ca2+ level changes that are assessed to determine cell function and response (i.e., drug testing). The evaluation of alterations in intracellular Ca2+ levels primarily relies on fluorescence imaging techniques, performed through video recording of cells incubated with Ca2+-sensitive dyes. By observing ion concentration shifts over time in a delimited region of interest (ROI) encompassing a single cell, it is possible to draw conclusions on cell responses to specific stimuli. Our work describes a tool named SIGAA-signaling automated analysis, for astrocyte ROI-based fluorescent imaging. This tool is specifically tailored for two wavelengths excited dyes by using two inputs of Ca2+ signaling recorded frames/videos and outputting a set of features relevant to the experiment's conclusions and cell characterization. SIGAA performs automatic drift correction for the two recorded videos with a template matching algorithm, followed by astrocyte identification (ROI) using morphological reconstruction techniques. Subsequently, SIGAA extracts intracellular Ca2+ evolution functions for all identified ROIs detects function transients, and estimates a set of features for each signal. These features closely resemble those obtained through traditional methods and software used thus far. SIGAA is a new fully automated tool, which can speed up hour-long studies and analysis to a few minutes, showing reliable results as the validity tests indicate.