A novel setup for simultaneous two-photon functional imaging and precise spectral and spatial visual stimulation in Drosophila

Motion vision has been extensively characterised in Drosophila melanogaster, but substantially less is known about how flies process colour, or how spectral information affects other visual modalities. To accurately dissect the components of the early visual system responsible for processing colour, we developed a versatile visual stimulation setup to probe combined spatial, temporal and spectral response properties. Using flies expressing neural activity indicators, we tracked visual responses in the medulla, the second visual neuropil, to a projected colour stimulus. The introduction of custom bandpass optical filters enables simultaneous two-photon imaging and visual stimulation over a large range of wavelengths without compromising the temporal stimulation rate. With monochromator-produced light, any spectral bandwidth and centre wavelength from 390 to 730 nm can be selected to produce a narrow spectral hue. A specialised screen material scatters each band of light across the visible spectrum equally at all locations of the screen, thus enabling presentation of spatially structured stimuli. We show layer-specific shifts of spectral response properties in the medulla correlating with projection regions of photoreceptor terminals.