Supplementary Figure 5: Characterization of the spatial extent of photostimulation during high-speed piezo-based volumetric imaging of photoactivateable-GFP-expressing larval zebrafisha

a, Dorsal-view maximum intensity projections and lateral-view slices of anatomy reference stacks acquired before and after photostimulation of targets in the brains of PA-GFP-expressing larval zebrafish. Targets A and B were photoactivated in the first specimen, and targets C and D were photoactivated in a second specimen. The depths of targets A, B, C, D relative to the surface of the brain were 20.5 μm, 99.5 μm, 101.5 μm and 19 μm, respectively.b, Dorsal- and lateral-view maximum intensity projections of the photoactivated regions A, B, C and D.c, Intensity profiles of photoactivated regions along lateral and axial directions and corresponding FWHM size. Overall, the spatial extent of activated regions (6-7 µm axially and 4-6 µm laterally) was found to be uniform across the brain and consistent with the resolution characterization based on a florescent dye shown in Fig. 5. The slightly smaller axial size of activated regions compared to the fluorescein-based resolution benchmark could arise from additional non-linear effects (with respect to laser power density) involved in the photoactivation of PA-GFP. The larger lateral size is the result of GFP diffusion in the cell’s cytoplasm following photoactivation. The experiment shown in this figure was repeated independently three times with similar results (for similar depths in the fish brain). Scale bars, 100 µm (a), 5 µm (b).