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The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of the intervertebral window does not elicit a reaction of GFP⁺ microglial cells in CX3CR1^gfp/+ mice. We next demonstrate a distinct rostrocaudal migration of GFP⁺ immune cells in the spinal cord of CXCR6^gfp/+ mice during active experimental autoimmune encephalomyelitis (EAE …