Single-disc optical visualization in photoreceptors uncovers protein architecture and compartmentalized pathology

Photoreceptor discs are densely stacked membranes housing visual pigments, yet their tight 35 nm spacing has kept molecular organization beyond the reach of optical microscopy. Here, we employ iterative ultrastructure expansion microscopy (iU-ExM) to achieve the first optical visualization of proteins within individual photoreceptor discs at 12 nm effective resolution through 20-fold expansion. We demonstrate that rhodopsin occupies 92% of the disc radial extent in situ, substantially exceeding the ∼50% area fraction reported from atomic force microscopy of isolated membranes. We provide the first optical detection of peripherin-2 within disc incisures and resolve connecting cilium and centriolar appendage architecture in three dimensions. Application to the RCS rat model of retinitis pigmentosa reveals compartmentalized pathology: outer segment disc spacing increased 29% while centriolar architecture remained preserved, suggesting intact protein trafficking during early degeneration. By bridging molecular identification with ultrastructural context, this work opens optical access to individual membrane compartments within densely packed cellular architectures previously resolved only by electron microscopy.