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

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.