Light stimuli were produced by a digital light synthesis engine (OneLight Spectra), conveyed via a fiber optic cable, and then presented to the animal within a custom-made, MRI-compatible, circular eyepiece with an approximately 26° uniform visual field.
16 Intermittent calibration of the device and regular measurements of the spectral properties of the stimuli was performed with a spectroradiometer (PhotoResearch PR-670). During data collection, the eyepiece was held with an articulated plastic arm and positioned approximately 5 mm from the corneal surface of the stimulated eye. The fellow eye was uncovered during data collection. During the pupillometry sessions, an infrared video camera (640 × 480 pixel resolution at 60 Hz interlaced; LiveTrack, Cambridge Research Systems, Rochester, Kent, UK) was used to record the pupil response from the nonstimulated eye.
The stimuli for both experiments targeted particular photoreceptor classes using the method of silent substitution.
17,18 Stimuli were designed to stimulate or silence the L cone (also termed the L/M or ML cone in dichromatic animals), S cone, rod, and melanopsin photopigments (
Fig. 1b). The spectral sensitivity of these photoreceptor classes was modeled using the Govardovskii nomogram,
19 with a lambda max of 555, 429, 506, and 480 nm, for the L, S, rhodopsin, and melanopsin photopigments, respectively. The spectral transmittance of the canine crystalline lens was included in the calculation to account for prereceptoral filtering.
20 A nonlinear search across device settings was used to construct spectral modulations that had the desired property of silencing some photoreceptors while maximizing contrast on targeted photoreceptors (
Fig. 1c). The modulations were presented around a common, half-on spectral background. Neutral density filters were placed in the light path to bring the background into the desired luminance range. The mean ± standard deviation human luminance of the stimulus across experiments was 438 ± 83 cd/m
2 for pupillometry, and 305 ± 92 cd/m
2 for MRI, which corresponds with a corneal irradiance of 0.29 ± 0.05 and 0.20 ± 0.06 watts/m
2 respectively. Spectroradiometric measurements of the stimuli were made immediately before and after each data collection session.
Supplementary Table S2 provides the calculated contrast on the targeted and nominally silenced photoreceptors.