Advances in in vivo optical imaging have enabled observation of diverse biological phenomena across different organs and organisms at single cell resolution. In the central nervous system (CNS), such imaging can be used to explore the connection between cell-resolved neural activity and behavior, a key capability for gaining insight on CNS function and dysfunction.The spinal cord of vertebrates serves as the conduit that shuttles sensory and motor information back and forth between the brain and the periphery that governs fundamental behaviors across species such as locomotion and somatosensory function. Because dense myelin covers the dorsal aspect of the spinal cord, imaging neuronal, vascular, and immune function deep inside the grey matter is challenging. The focus of my thesis is to utilize three-photon excited fluorescence (3PEF) microscopy to overcome this imaging challenge and investigate neuronal function, blood flow, and inflammatory cell behavior at an unprecedented depth in the spinal cord of awake and behaving mice. Here, I present using 3PEF microscopy combined with adaptive optics as a promising tool to image patterns of neuronal activity from deep spinal cord layers in an awake, locomoting mice. These tools also enabled measurement of blood flow in individual vessels across the vascular hierarchy and vessel types, as well as neuronal dieback and microglial dynamics after focal spinal cord ischemia, opening the door to cell-resolved studies of structure and function of the spinal cord in health and disease.