Testing ARCOS with optogenetically induced collective ERK activation. (A) Optogenetic actuator/biosensor circuit comprises the optogenetic actuator OptoRAF tagged with mCitrine, the ERK biosensor (ERK-KTR) tagged with mRuby2, and a nuclear marker (H2B) tagged with miRFP703. (B) Schematic of a spatially constrained stimulation of the 2D epithelium with a DMD. (C) Repeated, pulsed optogenetic stimulation of the MCF10A WT epithelium with a growing circular region using DMD. Upper row: Inverted raw images from selected time points. The circle outlines the stimulation region. Pink dots on the timeline indicate the timing of a 100-ms-long optogenetic pulse. Lower row: Quantification of ERK activity from image segmentation for selected time points. Black dots indicate active cells identified from detrended and binarized ERKATS; black lines indicate a convex hull around collective activation. (D) Single-cell ERK activity in different regions of the FOV from panel C. Vertical dashed lines indicate the timing of the optogenetic stimulation. Cells in the center are stimulated at all stimulation times after 10’; cells farther away are stimulated only with pulses that occur after 35’, etc. (E) Spaghetti plot of collective ERK activation: an x-axis projection of single cells from panel C during their participation in a CE. Vertical dashed lines indicate the timing and position of the optogenetic stimulation. The green solid line is the Moran’s I measure of global spatial autocorrelation calculated from single-cell ERKATS. (F) Repeated, pulsed optogenetic stimulation of a circular region moving clockwise along a circle. Legend as in panel C. (G) Spaghetti plot and global Moran’s I of CEs from panel F. (H and I) Same as F and G but the optogenetic stimulation is a circular region that splits horizontally over time.