The CNS is effectively shielded from the periphery by the blood–brain barrier (BBB) which limits the entry of cells and solutes. However, in autoimmune disorders such as multiple sclerosis, immune cells can overcome this barrier and induce the formation of CNS inflammatory lesions. Recently, two-photon laser scanning microscopy (TPLSM) has made it possible to visualize autoimmune processes in the living CNS in real time. However, along with a high microscopy standard, this technique requires an advanced surgical procedure to access the region of interest. Here, we describe in detail the necessary methodological steps to visualize (auto)immune processes in living rodent tissue. We focus on the procedures to image the leptomeningeal vessels of the thoracic spinal cord during transfer experimental autoimmune encephalomyelitis in LEW rats (AT EAE) and in active EAE in C57BL/6 mice (aEAE).