Intravital 3-photon microscopy of leukocyte dynamics in the splenic white pulp

Intravital imaging has been instrumental in uncovering the complex behaviors of immune cells during immune responses against viruses and tumors. The spleen is a major hub for systemic immune responses and immune memory. However, imaging of the spleen remains technically challenging, limiting our understanding of the dynamics of cellular interactions within specialized spleen niches, including the white pulp. Here, we present a method to stabilize the spleen for intravital imaging and to capture cell dynamics of the white pulp using 3-photon microscopy. We detail how to process images, quantify cell migration, and integrate behavior with structural information, revealing stromal guidance of T cell migration within deep T cell zones. This method of intravital 3-photon imaging of splenic white pulp supports in-depth investigation of cell dynamics and interactions in health and disease.