Spinal cord disorders are frequent causes of morbidity and mortality in human and veterinary medicine alike. Despite spinal cord disease affecting nearly every vertebrate species on earth, the pathophysiological mechanisms of central nervous system (CNS) disease remain poorly understood, largely due to failure of traditional ex-vivo imaging methods to capture the dynamic nature of complex neurophysiologic interactions. To address this, we have developed novel optical techniques for high-resolution, in-vivo imaging of the spinal cord in rodent models. This dissertation details novel methods for two-photon excited fluorescence (2PEF) microscopy of the spinal cord that enable direct observation of the cellular interactions occurring during health and disease. Specifically, we have developed surgical procedures that allow long term optical access in multiple regions of the mouse spinal cord, optimized labeling strategies for multicolor fluorescent in vivo imaging of cellular interactions, demonstrated successful recording of real-time calcium transients and neural activity from populations of sensory and motor neurons in the rodent spinal cord, explored and refined established models of SCI and neuropathic pain to make them suitable for serial imaging of axonal dynamics, inflammatory responses, and alterations in neural circuitry, designed and custom built a treadmill system on which a mouse can walk and run while spine-fixed under a multiphoton microscope, and implemented a recording system for capturing video of spine-fixed, running mice from which 3D spatiotemporal positioning of limbs can be measured and ultimately integrated into a comprehensive kinematic gait analysis system. Finally, we discuss the current capabilities and limitations of 2PEF microscopy for spinal cord imaging, and introduce ongoing initiatives and the inevitable transition to the next generation of multiphoton microscopy, three-photon excited fluorescence (3PEF), as a way to overcome the current challenges to 2PEF and further drive spinal ...