Non-contact confocal calcium imaging of <i>in vivo</i> murine corneal nerves

Abnormal corneal nerve function and associated disease is a significant public health concern. It is associated with prevalent ocular surface diseases, including dry eye disease. Corneal nerve dysfunction is also a common side effect of refractive surgeries, as well as a symptom of diseases that cause peripheral neuropathies. Here, we demonstrate <i>in vivo</i> calcium imaging of mouse corneal nerves expressing GCaMP6f, a genetically encoded calcium indicator. A custom fluorescence imaging and stereotactic system was designed, allowing for non-contact imaging of the mouse cornea with an air objective. Dynamic imaging of neuronal activity is demonstrated in the various layers of the cornea and in response to local anesthetic administration. This approach demonstrates a less invasive means of assessing corneal nerve function than has been previously used, and has significant potential for studying the effects of ocular diseases, refractive surgeries, and peripheral neuropathies on corneal nerve function, as well as the effectiveness of various therapies to treat corneal nerve dysfunction.