Preparation of the mouse spinal column for single imaging using two-photon laser-scanning microscopy

Two-photon imaging of the nervous system is now used extensively for visualizing brain dynamics and signal activities. To date, scientists have focused on the analysis either of gray matter forebrain structures, such as the cortex and cerebellum, or they have investigated muscle innervation of peripheral nerves. The spinal cord is an ideal structure to use for imaging central nervous system white matter. The dorsal columns formed by myelinated sensory axons are located directly at the surface of the spinal cord underneath the pia mater. Neuronal fibers and neighboring glial cells can be imaged in transgenic mice using cell type-specific fluorescent protein expression. This protocol describes the anesthesia and surgical procedures necessary to prepare the mouse spinal column so that neurons and glia in the spinal cord can be imaged using two-photon laser-scanning microscopy (2pLSM). These procedures are ideal for single-imaging experiments in which the spinal cord needs to be imaged at optimal spatial resolution with minimal motion artifacts.