a–d Simulation of the targeted stimulus injection at a volume flow rate of 0.1 µl/s with simultaneous supply of oxygen-enriched medium at 1 µl/s in a cross-sectional view in the closed a, b and open c, d NeuroExaminer versions at 0.3 and 1.5 s after the entry of the stimulus into the alignment chamber. The color-coded distribution shows the concentration (blue = 100% medium and red = 100% stimulus) in the mid-plane of the channel, and on the larval surface after impacting the larva. e, f 3D view of stimulus streamline patterns of injected stimuli (blue = 0 mm/s, red = 25 mm/s) in the closed e and open f system variant simulated assuming the same inflow volume currents as in a–d. The larva head experiences a homogeneous stimulus exposure in the closed system, whereas in the open system the stimulus is also directed outside of the device reducing the contact surface of the larva head with the stimulus solution. g 3D design of the closed system variant. h Schematic 3D view of the zebrafish larva positioned inside the fluidic chip and the light sheet microscope setup (see also Supplementary Fig. 1). i–l Illustration of the four-step loading concept. m A photo of the closed NeuroExaminer chip variant taken after thermal bonding. n The influence of the post treatment process on optical transparency illustrated by a photo comparing NeuroExaminer chips representing (from left to right) the open system before and after the combined chemical and thermal treatments and the closed system before and after the treatment. The scale bar is 1 mm throughout, except for g where it is 10 mm.

a, e A live 6 dpf Tg(elavl3:H2B-GCaMP6s); crystal larva in an open a or closed e version of the NeuroExaminer. The outer borders of the open a or closed e microfluidic device are outlined with dotted white lines; the recess area above the larva’s head in a is marked by a dotted blue line (see also Supplementary Movie 8). b, f A maximum intensity projection consisting of 21 optical sections at 5 min and 45 s (time is indicated in min:s:ms) depicting nuclear-localized GCaMP6s throughout the larva’s brain in the open b and closed f system (see also Supplementary Movies 3, 4). Dotted color-coded lines outline five major brain regions depicted on the lower right. c, d and g, h Neural activity patterns at four single optical sections spaced 60 µm apart at two indicated time points (shown in min:s:ms) in the open c, d and closed g, h NeuroExaminer (see also Supplementary Movies 5, 6). Note that optical sections in the open system are spatially well-resolved throughout the entire brain c, d, whereas in the closed system dorsal sections display a spatially better resolution than ventral sections localized deeper in the brain g, h. i–m Single-cell color-coded calcium signals (25 frames; ~84 s) for the fish in the closed system in the telencephalon (i, blue), habenula (j, green), optic tectum (k, magenta), cerebellum (l, orange), and hind brain (m, yellow; see also Supplementary Movie 7). Scale bar is 500 µm in a and e and 100 µm in b–d and f–h.