Serial intravital microscopy reveals temporal dynamics of autoreactive germinal centers in the spleen.

The spleen plays a key role in clearing blood-borne infections and is involved in autoimmune and hematological disorders. It undergoes extensive remodeling during inflammation and immune reactions, but its localization in the peritoneal cavity has hampered studies of these dynamic changes. We establish a protocol for serial two-photon microscopy of the murine spleen to capture dynamic processes in the living animal over weeks. We elucidate the expansion and contraction of autoreactive germinal centers (GCs) induced by the epicutaneous application of the TLR7 agonist resiquimod. Leveraging a biocompatible abdominal imaging window, intravital labeling, and fluorescent reporters, we follow GCs for >2 weeks up to 180 μm below the capsule by tracking follicular dendritic cell networks. We also demonstrate that we can repeatedly image dynamic cell migration in the same area over a two-week period without appreciable perturbation of normal physiology, enabling a deeper understanding of the spleen and its associated disease states.