Fig. 3

a The microfluidic (µfluidic) module is integrated with a scanning light sheet microscope for whole-brain imaging in larval zebrafish. Inset image/plot sets show characterization of the excitation and detection arms (see “Methods”). Each set includes an image showing one example profile and a plot of profiles (gray: individual; black: mean), for the lateral (x–y) and axial (y–z) point spread functions (PSFs, scale bars: 2 μm), and the light sheet side profile (scale bar: 50 μm). b Example image planes. For each set, the two images on the right are enlarged and contrast-adjusted from the outlined areas of the corresponding larger images. Scale bars: 100 μm for the larger images and 10 μm for the zoomed-in images. Arrows indicate example neurons at various brain locations. c Upper panel: temporally overlaid tail images of an example larva (scale bar: 1 mm). Lower panel: spontaneous tail flipping frequency of 9 larvae that were behaviorally active. Horizontal lines indicate mean ±1 SD. d Workflow of the custom-developed data analysis pipeline. e Brainwide spontaneous activity heatmap and region-of-interest (ROI) maps (projected to coronal, transverse, and sagittal planes) from an example larva, with 3768 extracted ROIs (each corresponding to a neuron and color-coded by ROI number (#)). Simultaneously acquired tail flipping recording and an example tail flipping event are shown below. Horizontal scale bars for the heatmap: 5 seconds. Scale bars for the event plot: 50 milliseconds (horizontal) and 10° (vertical). Scale bars for the brain maps: 50 μm in Z-Brain atlas space. f Left panel: The calcium signal traces of 48 example neurons. Scale bar: 5 seconds. Right panel: a maximum intensity projection (MIP) and the time-series images of the first 30-second interval at a hindbrain region with 6 highlighted ROIs (green: ROI masks). Arrows indicate the locations and times of each ROIs near a calcium event’s peak. Brain region abbreviations: OE olfactory epithelium, OB olfactory bulb, Pa pallium, sPa subpallium, Hb habenula, PO preoptic area, Di diencephalon, Me mesencephalon, Ce cerebellum, Rh rhombencephalon. Scale bar for the MIP and time-series images: 50 μm. Source data are provided as a Source Data file.