Closed-loop optical optimization enables patterned retinal stimulation in vivo at cellular scales.

Visual neuroscience requires precise spatiotemporal projection of optical stimulation onto the retina, especially in experimental mouse models. However, in vivo patterned stimulation in mice is profoundly hindered by the extreme optical power and severe anatomical aberrations of the eye. Consequently, visual stimulation relies mainly on unverifiable, open-loop approximations that often lack spatial precision. Here, we introduce a closed-loop, spatially modulated stimulation platform that overcomes these barriers. By integrating a digital micromirror device (DMD) with electronically tunable lenses (ETLs) and a real-time, fundus camera-guided focus optimization module, we directly verify the location of patterned stimuli on the retina while dynamically correcting for chromatic and geometric defocus. This platform delivers quantitatively verified static and dynamic patterned stimuli to the living retina with lateral resolutions as fine as 6.7 µm. Guided by ray-tracing optical analysis, our work establishes a technological foundation that enables highly reproducible, cellular-scale interrogations of the visual pathway.