For simultaneous imaging and cell-attached recording, ring-shaped ROIs were placed over the cytosolic regions of the cells. Fluorescence transients at the soma were caused by action potentials, with little contribution from subthreshold activity62 (Supplementary Fig. 15). To quantify the efficiency for detecting single APs (Fig. 3), we identified single AP events with nearby APs at least 1 s away. Fluorescence traces consisting of 10 frames (0.17 s) before and 60 frames (1 s) after the ith 1 AP event were assembled in 70-dimensional vectors, fi. Segments of noisy traces, ni were taken from periods without APs. The average of all 1 AP traces was used as a template vector, ftemplate=Σifi/N. The vector was normalized after subtraction of the mean to create a unit vector f̂template. The projection of fi or ni along the direction of f̂template was calculated to obtain a scalar fi or ni, respectively. The AP detection threshold was defined as the 99th percentile of all ni values (i.e., 1% false positive), and the percentage of the fi values above the detection threshold was the AP detection efficiency.
