Multi-photon microscopy has taken hold as a widely used technique in immunology, allowing for imaging of the kinetics of immune cell motility and cell-cell interactions, but what have we learned from this technique about the processes involved in peripheral tolerance and autoimmunity? Various studies have now looked at the dynamics of several mechanisms of peripheral T cell tolerance and efforts to examine the dynamics of the autoimmune response at the disease site are also under way. Here, we will discuss the findings of studies that use multi-photon microscopy to examine the dynamics of T cell tolerance in the lymph nodes and of the autoimmune processes involved in models of type 1 diabetes and multiple sclerosis. An emerging theme from these studies is that short T cell-antigen presenting cell interactions can lead to tolerance, and that autoreactive T cell restimulation at the disease site can play an important role in autoimmune disease exacerbation.