Extended Data Fig. 2. Constrained non-negative matrix factorization (CNMF) based Ca²⁺ data extraction workflow and output.

a, Raw image of an GCaMP6f infected Cx+GE organoid (left) and CNMF based identification of fluorescently active (spiking) GCaMP regions of interest (right). b-d, Identification and analysis of individual neuronal Ca²⁺ spiking data. b, Changes in GCaMP6f fluorescence (normalized ΔF/F) for each neuron in a displayed as individual spike trains (left) or the same data displayed as a colorized amplitude plot (right). Individual spiking data are then used to determine various measures of spiking behavior including overall synchronicity based on a threshold level determined following spike shuffling c and calculation of interspike intervals d. e, Simultaneous to b-d, Ca²⁺ spiking data are categorized into neuronal microcircuits (clusters) based on correlations between individual Ca²⁺ spikes. f, during initial analyses, alternative clustering approaches including cross-correlation was used and the neural microcircuits resulting from multiple approaches were compared to determine the optimal clustering paradigm.