Longitudinal Single‐Axon‐Resolution Imaging of Peripheral Nerve Injury Response in Mice Using an Optical Window Implant

ABSTRACT Chronic, high‐resolution imaging of peripheral nerve injury and regeneration remains a major technical challenge due to the optical inaccessibility of peripheral nerves embedded in soft tissue. Here, we present a broadly applicable approach for longitudinal, high‐resolution optical imaging of peripheral nerves in vivo that combines a flexible, optically transparent polydimethylsiloxane (PDMS) window along with multimodal microscopy. The PDMS window is implanted in the skin above the mouse sciatic nerve, providing stable, motion‐tolerant optical access for over one month without impairing nerve function. Using confocal, two‐photon, and second‐harmonic generation microscopy, we simultaneously visualized fluorescently labeled axons and Schwann cells, endogenous collagen, and vasculature with high spatial and temporal resolution. This approach enabled real‐time tracking of axonal degeneration and regeneration, Schwann cell dynamics, and extracellular matrix‐guided axon pathfinding across multiple nerve injury models. The platform also supports longitudinal imaging of axonal regrowth within a clinically relevant model of nerve transection gap repair. Overall, our method enables new avenues for studying peripheral nerve biology and testing regenerative therapies in vivo.