Extended Data Fig. 5: SUNS accurately mapped the spatial extent of each neuron even if the spatial footprints of neighboring cells overlapped.

SUNS segmented active neurons within each individual frame, and then accurately collected and merged the instances belonging to the same neurons. We selected two example pairs of overlapping neurons from the ABO video 539670003 identified by SUNS, and showed their traces and instances when they were activated independently. a, The SNR images of the region surrounding the selected neurons. The left image is the maximum projection of the SNR video over the entire recording time, which shows the two neurons were active and overlapping. The right images are single-frame SNR images at two different time points, each at the peak of a fluorescence transient where only one of the two neurons was active. The segmentation of each neuron generated by SUNS is shown as a contour with a different color. The scale bar is 3 μm. b, The temporal SNR traces of the selected neurons, matched to the colors of their contours in (a). Because the pairs of neurons overlapped, their fluorescence traces displayed substantial crosstalk. The dash markers above each trace show the active periods of each neuron determined by SUNS. The colored triangles below each trace indicate the manually-selected time of the single-frame images shown in (a). c-d, are parallel to (a-b), but for a different overlapping neuron pair. e, We quantified the ability to find overlapped neurons for each segmentation algorithm using the recall score. We divided the ground truth neurons in all the ABO videos into two groups: neurons without and with overlap with other neurons. We then computed the recall scores for both groups. The recall of SUNS on spatially overlapping neurons was not significantly lower (and was numerically higher) than the recall of SUNS on non-spatially overlapping neurons (P > 0.8, one-sided Wilcoxon rank-sum test, n = 10 videos; n.s.l. – not significantly lower). Therefore, the performance of SUNS on overlapped neurons was at least equally good as the performance of SUNS on non-overlapped neurons. Moreover, the recall scores of SUNS in both groups were comparable to or significantly higher than that of other methods in those groups (**P < 0.005, n.s. – not significant; two-sided Wilcoxon signed-rank test, n = 10 videos; error bars are s.d.). The gray dots represent the scores on the test data for each round of cross-validation.