We generated a matrix of the distances between all detected mRNA particles and all detected ribosome particles, and computed the corresponding histogram . We then normalized the distance histogram to account for the fact the area covered by each distance bin grows with , and plotted the resulting normalized distribution of , equivalent to the average density of ribosome spots observed as a function of distance from an mRNA detection. If mRNAs and ribosomes do not colocalize, we would randomly detect ribosomes at all positions in the cell without regard for mRNA positions and therefore we would expect to observe a flat distribution for . In the case of mRNA/ribosome colocalization, we would expect an enrichment of short distances corresponding to comoving trajectories. Left panel: distribution of distances in the vicinity of focal adhesions observed in untreated cells (dataset from Figure 4). The distribution for the entire dataset (gray histogram) consists of a peak of short values (<3 pixels) above a flat baseline. The peak at short distances is the signature of colocalized mRNA/Ribosome trajectories, while the baseline reflects the concentration of fluorescent ribosomes non-associated with detected Actin mRNA molecules. We overlaid the distribution of distances generated from the colocalized trajectories selected by our co-movement algorithm (red line). The algorithm efficiently selects the peak of colocalized trajectories. Right panel: distribution of distances observed in cells treated with puromycin (dataset from Figure 2). The distribution for the entire dataset (gray histogram) consists of essentially a flat baseline, with a modest increase at short distances. This result reflects the almost entire dissociation of ribosomes from mRNAs following puromycin treatment. The co-movement algorithm accordingly selects a very small fraction of colocalized trajectories.