Figure 4

Water-coupled SCAPE revealed sub-cellular structure and dynamic calcium activity of larval zebrafish. (a) Three-dimensional rendering of one side of a zebrafish forebrain expressing GCaMP6s revealed the dense connections within the optic tectum. The scale bars along the axes are 50 µm. The volume was obtained with 0.5 µm between layers, and an exposure time of 30 ms per layer. Bottom right inset: We show the fish viewed from the frontal direction, along with the relative orientations of the primary imaging objective and excitation light sheet (blue). (b) Individual planes color matched to the planes marked in (a) revealed sub-cellular structure. This zebrafish line localized GCaMP to the extranuclear space, and neurons appeared as rings in the zoomed-in insets. The scale bar in the large images are 50 µm, while the scale bar in the insets are 10 µm. The green outlines in the insets highlight a clear neuron with ring-like expression pattern. The color profiles show the fluorescence intensity along the straight lines; the arrows note the FWHM of the profiled feature. (c) A maximum projection at one imaged slice in a zebrafish over two minutes of imaging shows active neurons. Green outlines represent individual neurons found by our analysis pipeline. We obtained these images with 2.6 µm between layers, and an exposure time of 4 ms per layer. (d) Activity from heavy green outlines in (c) revealed dynamic and independent activity from individual neurons with high peak to background ratio. The volumetric acquisition rate was 10 Hz. The scale bar is 50 µm. (e) Left: The average of the maximum projections of fluorescence from all layers of the same fish as in (d) showed all neurons simultaneously. Outlines showed all neurons from all layers simultaneously. The scale bar is 50 µm. Right: The correlation between neurons revealed clusters of neurons that had similar activity, as highlighted by the block diagonal structure of high correlation. The outlines over each block are color matched to the neuron outlines of the left panel. We observed that the cluster of purple neurons localized to the forebrain, and likely resulted from spontaneous visual response. The other two clusters, teal and yellow, localized to the cerebellum and hindbrain.