Deciphering spatio-temporal fluorescence changes using multi-threshold event detection (MTED)

Recent achievements in indicator optimization and imaging techniques promote the exploration of Ca 2+ activity patterns as a main second messenger in many organs. Astrocytes are important regulators of brain activity and well known for their Ca 2+ -dependent modulation of neurons. However, standardized methods to analyze and interpret Ca 2+ activity recordings are missing and hindering global comparisons. Here, we present a biophysics-based concept to analyze Ca 2+ signals, which includes multiple thresholds and provides the experimenter with a comprehensive toolbox for a differentiated and in-depth characterization of Ca 2+ signals. We analyzed various ex vivo and in vivo imaging datasets and verify the validity of our multi-threshold event detection (MTED) algorithm across Ca 2+ indicators, imaging setups, and model systems from primary cell culture to awake, head-fixed mice. Applying our MTED concept enables standardized analysis and advances research using optical readouts of cellular activity to decrypt brain function. It allowed us to obtain new insights into the complex dependence of Ca 2+ activity patterns on temperature and neuronal activity. Highlights → We present a robust pixel-based algorithm to analyze multidimensional fluorescence data. → Automated multiple-threshold analysis accurately quantifies changes in fluorescence across magnitudes. → It characterizes the complexity of dynamic and overlapping activity patterns of Ca 2+ activity of astrocytes in vitro, in situ , and in vivo . → Its application provides quantitative parameters how temperature and neuronal activity determine astrocytic Ca 2+ activity.