Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice.

The potential usefulness of intravital two-photon microscopy for fate mapping is limited by its inability to track cells beyond the confines of the imaging volume. Therefore, we have developed and validated a novel method for in vivo photolabelling of spatially-restricted cells expressing the Kaede optical highlighter by two-photon excitation. This has allowed us to optically mark a cohort of follicular B cells and track their dissemination from the original imaging volume in the lymph node to the spleen and contralateral lymph node. We also present the first demonstration, to our knowledge, of in vivo photoconversion of a freely moving single cell in a live adult animal. This method of `discontinuous' cell tracking therefore significantly extends the fate mapping capabilities of two-photon microscopy to delineate the spatiotemporal dynamics of cellular processes that span multiple anatomical sites at the single cell level.