Fig. 4

Functional imaging of a 5 dpf larval zebrafish with pan-neuronal fluorescent calcium indicators, Tg(elavl3:H2b-GCaMP6s). Spontaneous brain activity, over a volume ~600 × 600 × 100 µm³ (depth), was recorded at 1 volume s⁻¹, with SVIM, in either 1- or 2-photon excitation mode (1p-SVIM or 2p-SVIM, respectively), or conventional 1p wide-field LFM. Cellular-resolution representations of active neurons were found with standard methodology based on spot segmentation of the time-domain standard deviation of the 3D time-series data (Methods section). a–c Images shown are depth color-coded xy- or xz-projections, of the time-domain standard deviation projection of the recorded brain activity over a time window of 100 s. Colored ellipsoids represent active neurons. Dashed box in the xy-projection image represent the region that produces the corresponding xz-projection image. Activity traces of segmented neurons are shown in d–f, revealing that the most number of neurons were found with 2p-SVIM (1104 cells), then with 1p-SVIM (796 cells), both of which were several-fold higher than with conventional wide-field LFM (263 cells). Scale bars, 100 µm.