Fig. 3: High-speed volumetric voltage imaging.

a Schematic of the pipeline for volumetric voltage imaging data acquisition and signal extraction. 8 frames are acquired during the downward and another 8 during the upward stroke of the refocusing cycle. Frames at each z-position are then motion corrected in x-y and the time-average frame for each z-position is aligned with frames above and below to generate one single volume (see methods for details). This volume is then used for 3D segmentation. The obtained 3D ROIs are then used as the basis for the extraction of the fluorescent time traces (see methods for details). b Average fluorescence of 8 planes spanning the entire volume of 50 μm of spinal cord tissue of a 4 dpf zebrafish larva. animals are expressing UAS:Voltron2-ST under the control of HuC:Gal4 and are stained with JF-526 dye. Footprints of select neurons shown in (c). Most footprints are visible over several z-sections but numbered only once. Shown is a representative example of an experiment with 4 fish. c Fluorescent traces corresponding to the spatial footprints shown in b (z-scored), ordered by z-position. Several neurons show clear single spikes while others show distinct oscillations likely corresponding to fictive swimming activity. Color of footprints in b and traces in c denote z-position in the volume. d Average bleaching of baseline fluorescence in recordings from 3 fish (n_cells per fish = 28-114). e Closeup of several spikes of one example neuron (trace 6 in c) at the beginning (0 s –2.5 s) and end (18.5 s – 20 s) of the recording. Visible is the fast timing of individual spikes and only a small reduction in SNR at the end of the recording.