Correcting for physical distortions in visual stimuli improves reproducibility in zebrafish neuroscience

Breakthrough technologies for monitoring and manipulating single-neuron activity provide unprecedented opportunities for whole-brain neuroscience in larval zebrafish^1–9. Understanding the neural mechanisms of visually guided behavior also requires precise stimulus control, but little prior research has accounted for physical distortions that result from refraction and reflection at an air-water interface that usually separates the projected stimulus from the fish^10–12. Here we provide a computational tool that transforms between projected and received stimuli in order to detect and control these distortions. The tool considers the most commonly encountered interface geometry, and we show that this and other common configurations produce stereotyped distortions. By correcting these distortions, we reduced discrepancies in the literature concerning stimuli that evoke escape behavior^13,14, and we expect this tool will help reconcile other confusing aspects of the literature. This tool also aids experimental design, and we illustrate the dangers that uncorrected stimuli pose to receptive field mapping experiments.