Supplementary Figure 12: Mesoscale volumetric functional imaging of callosal-projection neurons across two hemispheres of an awake mouse.

(a) Schematic drawings illustrating the labeling of callosal-projection neurons and imaging FOVs in two hemispheres. (b) Maximum intensity projections (MIP) of images of two hemispheres and the cortical volumes P1, P2, and P3 at 50 – 250 µm below the dura, color-coded by depth. P1 and P2: two volumes symmetrically located across the midline, 400 µm × 400 µm × 200 µm and 400 µm × 800 µm × 200 µm, respectively; P3: a more posterior volume of 400 µm × 549 µm × 200 µm. (c) Images acquired by Gaussian and Bessel focus scanning of cortical volumes P1 – P3 (pixel size: 0.5 µm; z-step size: 2 µm for Gaussian). Gaussian images are MIPs with neurons color coded by depth. Volume rate: 0.08 Hz for Gaussian, 3.1 Hz for Bessel. Post-objective power: 36 mW for Gaussian, 108 mW for Bessel. (d) Calcium transients (ΔF/F) of all 189 neurons (48 in P1, 116 in P2, 25 in P3). (e) Traces of example neurons marked by arrows in (b). Red indicates neurons highly correlated with Neuron 1. CC: Pearson’s correlation coefficient. On population level, the cross-region activity correlation was higher between P1 and P2 than between P3 and P2 (Kolmogorov–Smirnov test, p = 0.0007, # of correlation coefficients: 5,568 for P1-P2, 2,900 for P3-P2; percentage of correlation coefficients above 0.2: 8% for P1-P2, 4% for P3-P2). Experiment carried out in n = 1 mouse.