Significance To move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes’ optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes’ whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies’ depth-perception dynamics, limits, and visual behaviors.

Inside compound eyes, photoreceptors contract to light changes, sharpening retinal images of the moving world in time. Current methods to measure these so-called photoreceptor microsaccades in living insects are spatially limited and technically challenging. Here, we present goniometric high-speed deep pseudopupil (GHS-DPP) microscopy to assess how the rhabdomeric insect photoreceptors and their microsaccades are organised across the compound eyes. This method enables non-invasive rhabdomere orientation mapping, whilst their microsaccades can be locally light-activated, revealing the eyes’ underlying active sampling motifs. By comparing the microsaccades in wild-type Drosophila’s open rhabdom eyes to spam-mutant eyes, reverted to an ancestral fused rhabdom state, and honeybee’s fused rhabdom eyes, we show how different eye types sample light information. These results show different ways compound eyes initiate the conversion of spatial light patterns in the environment into temporal neural signals and highlight how this active sampling can evolve with insects’ visual needs.

Neural mechanisms behind stereopsis, which requires simultaneous disparity inputs from two eyes, have remained mysterious. Here we show how ultrafast mirror-symmetric photomechanical contractions in the frontal forward-facing left and right eye photoreceptors give Drosophila super-resolution 3D-vision. By interlinking multiscale in vivo assays with multiscale simulations, we reveal how these photoreceptor microsaccades - by verging, diverging and narrowing the eyes' overlapping receptive fields - channel depth information, as phasic binocular image motion disparity signals in time. We further show how peripherally, outside stereopsis, microsaccadic sampling tracks a forward flying fly's optic flow field to better resolve the world in motion. These results change our understanding of how insect compound eyes work and suggest a general dynamic stereo-information sampling strategy for animals, robots and sensors.

Absence of screening pigment in insect compound eyes has been linked to visual dysfunction. We investigated how its loss in a white-eyed mutant (W-E) alters the photoreceptor electrophysiological properties, opsin gene expression and the behavior of the cockroach, Periplaneta americana. Whole-cell patch-clamp recordings of green-sensitive photoreceptors in WE cockroaches gave reduced membrane capacitance, absolute sensitivity to light and light-induced currents. Decreased low-pass filtering increased voltage bump amplitudes in WE photoreceptors. Intracellular recordings showed that angular sensitivity of WE photoreceptors had two distinct components: a large narrow component with the same acceptance angle as wild-type, plus a relatively small wide component. Information processing was evaluated using Gaussian white-noise modulated light stimulation. In bright light, WE photoreceptors demonstrated higher signal gain and signal power than wild-type photoreceptors. Expression levels of the primary UV-and green-sensitive opsins were lower and of the secondary green-sensitive opsin significantly higher in WE than in wild-type retinae. In behaviour experiments, WE cockroaches were significantly less active in dim green light, consistent with the relatively low light sensitivity of their photoreceptors. Overall, these differences can be related to the loss of screening pigment function and to a compensatory decrease in the rhabdomere size in WE retinae.

Inside compound eyes, photoreceptors contract to light changes, sharpening retinal images of the moving world in time. Current methods to measure these so-called photoreceptor microsaccades in living insects are spatially limited and technically challenging. Here, we present goniometric high-speed deep pseudopupil (GHS-DPP) microscopy to assess how the rhabdomeric insect photoreceptors and their microsaccades are organised across the compound eyes. This method enables non-invasive rhabdomere orientation mapping, whilst their microsaccades can be locally light-activated, revealing the eyes' underlying active sampling motifs. By comparing the microsaccades in wild-type Drosophila's open rhabdom eyes4 to spam-mutant eyes, reverted to an ancestral fused rhabdom state, we show how two different eye types sample light information. These results show different ways how vision converts space into time, and highlight how compound eyes and their active sampling can evolve with insects' visual needs.