Figure 3

Figure 3

Optimisation of the OKR assay using infrared (IR) illumination to enhance behavioural response robustness. (A) Representative eye movement traces of 5 dpf wild-type zebrafish larvae exposed to full-field moving sinusoidal gratings under two lighting conditions: standard visible light (top) and IR illumination (bottom). Vertical blue dashed lines mark transitions in spatial frequency and direction of stimulus motion. Larvae under IR illumination exhibit clearer, higher slow-phase velocity optokinetic responses across stimulus epochs. Black overlays on the waveform represent the average slow phases and the red line represents the quick phases. Underlying eye movement trace is represented by light blue line. Orange waveforms represent the velocity trace. The bar below each panel illustrates the temporal sequence of stationary and moving gratings, with increasing spatial frequency (in cycles/degree (CPD) from left to right) and directional changes (clockwise (CW) and counterclockwise (CCW)). (B) Mean slow-phase eye velocity of the left eye as a function of spatial frequency under IR (yellow) and visible light (purple) conditions. IR illumination led to significantly enhanced eye velocities, particularly at intermediate spatial frequencies. Error bars represent standard error of the mean. (C) Box plots comparing overall eye movement velocity under IR and visible light conditions. Larvae tested under IR illumination showed significantly higher tracking velocities (p < 0.05), supporting the effectiveness of IR backlighting in enhancing OKR behavioural responses by reducing background noise and improving stimulus contrast.