Automated, unbiased optoretinography reveals comprehensive nanoscopic dynamics of the outer retina in rodents

Phototransduction involves changes in concentration of ions and other solutes within photoreceptors and in subretinal space, which affect osmotic pressure and the associated water flow. Corresponding expansion and contraction of cellular layers can be imaged using optoretinography (ORG), based on phase-resolved optical coherence tomography (OCT). Until now, ORG could reliably detect only photoisomerization and phototransduction in photoreceptors, primarily in cones under very bright stimuli. By employing a novel subpixel bulk motion correction algorithm, which enabled imaging of the nanometer-scale tissue dynamics during minute-long recordings, and unsupervised learning of spatiotemporal patterns, we discovered optical signatures of the other retinal structures’ response to visual stimuli. These include inner and outer segments of rod photoreceptors, retinal pigment epithelium, and subretinal space in general. High sensitivity of our technique enabled detection of the retinal responses to very dim stimuli: down to 0.01% bleach level, corresponding to natural levels of scotopic illumination. We also demonstrated that with a single flash, the optoretinogram can map retinal responses across a 12°field of view, potentially replacing multifocal electroretinography, with its long acquisition time and low spatial resolution. This new technique expands the diagnostic capabilities and practical applicability of optoretinography, providing a more complete replacement of electroretinography, while combining structural and functional retinal imaging in the same OCT machine.