Supplementary Figure 7: AQuA features enable detailed Ca²⁺ activity plots.

(a) Spatiotemporal plot of Ca²⁺ activity from a five minute video (the first minute of which is shown in Fig. 3b). Each event is represented by a polygon proportional to the area of the event as it changes over its lifetime, and color-coded by propagation direction. (b) Example time series illustrating how propagation direction is determined (left). A propagation direction score is calculated for each event by multiplying the Euclidian distance between the event pixels’ proximity to the soma at each frame by each pixel’s intensity. The overall score is the summation of this weighted pixel intensity distance over the lifetime of the event. Therefore, if more pixels with higher intensity move toward the soma it will be classified as such (top). While some events appear in the plot as moving toward the soma, they are actually calculated as moving away from the soma (middle) since we are only displaying the minimum event proximity to the soma in the spatiotemporal plot, but calculate each pixel’s proximity to the soma when generating a propagation score. Additionally, pixel intensity is first thresholded at 0.3 dF/F. Therefore, events that move toward or away from the soma yet have pixel intensities below the threshold (bottom) appear to have a propagation direction when plotted, yet have a zero propagation direction score when calculated. Scale bar = 20μm. (c) Additional events plotted for each propagation direction category to demonstrate range of detected/plotted events. Scale is not equivalent to events shown in b, but is equivalent within entire group shown here.