Figure 8—figure supplement 1.

Figure 8—figure supplement 1. Method for scoring calcium signalling in time and space and control for background signalling.

(A to F) Analysis of control larva expressing β-actin:GCamps (cyan) and Opto-ASC (magenta), yellow line: ROI; Scale bar in all panels: 20 μm (A) Imaged region with the cell of interest (which is not activated in this control) is in the center. The yellow line shows the line ROI at 0° along which fluorescence intensity is measured. (B) Top: Cartoon of the periderm. The circle around the cell of interest outlines the area that is analyzed in the kymograph. Bottom: A line ROI is drawn across the center of the cell. This line is swept by 360 degrees in 1° degree steps to cover a 82.19 μm diameter circle surrounding the center of the cell. (C) Screen shot taken during the experimental course of measurement (at the 56° measurement). The fluorescence intensity at each point along the diameter is measured at each angle. (D) Fluorescence intensities along the diameter (y-axis) at the starting angle, 0°, over time (x-axis). The image in panel A represents t=8 min. (*) represents the time point in C and D. (E, F) The intensity measurements at each point of the diameter are averaged over all angles, that is over the entire circumference at each distance from the center for each time point. Since the diameter sweeps across each point twice (once with each radius), this results in a symmetrical representation, of which only one half is shown in the final kymograph. (F) Resulting two-dimensional (kymograph) of calcium signaling in time and space. The vertical cyan lines in the graph represent the calcium signal in periderm and basal cells with the distance from the central cell represented along the y-axis.