(a) Simplified schematic showing additional options for optical ports in NeuBtracker here shown in MicroFixed configuration. (b) Example of coupling a 488 nm laser instead of the LED as an excitation light source. Efficient excitation of a GCAMP6 tg(HuC:H2B-GCaMP6s) could also be achieved with a laser but speckle patterns were apparent (which could be removed by a rotating diffuser or alternatively used for structured illumination approaches). (c) The transparent bottom of the arena and the use of an NIR illumination ring rather than a table afford direct coupling of a video projector to provide visual stimulation to the animal. (d) A 405 nm laser was coupled via the galvanometric mirrors to provide a visual stimulus to the animal. During periods in which the laser spot was moved to repeatedly approach the head, the fish swam a longer distance than during periods in which the 405 nm laser was off. This was the case with and without a 488 nm laser turned on in the background. These data are consistent with data we obtained from fish immobilized in agar in which illumination of the head with the 405 nm laser lead to an increase in the number of specific tail deflections that can be characterized as C-turns (indicative of aversion) and J-turns by videography conducted with an IR camera. Individual counts from 6 individuals are shown with median and interquartile range. (e) In this experiment, the 405 nm laser spot was moved in a semi-circular path in a left-right-left trajectory in front of the head of a tg(gSA2AzGFF49A;UAS:GCaMP7a, 7 dpf) larva as indicated by color-coding for relative position. Neuronal activations were elicited in the optic tectum contralateral to the relative location of the laser spot (the color overlay over the co-registered frames in gray shows the ratio of the average fluorescence between frames in which the laser spot was located on the left and frames in which it was positioned on the right relative to the location of the fish head). The plot (median and interquartile range) shows a significant difference (p < 0.01, Wilcoxon matched-pairs signed rank test) of the ratio of the average fluorescence intensity of the left and right OT in frames with laser spots presented on the left (green) or right (red) of the fish respectively.