Selective recruitment of blood-borne leukocytes to tissues and their proper positioning within them is crucial for the many integrated functions of the immune system. Intravital microscopy (IVM) techniques have been employed for more than a century to study these events at the single-cell level in living animals. Conventional video-based IVM allows the visualization of extremely rapid adhesion events at the interface between blood and tissue. Multiphoton IVM is a relatively new tool for imaging the slower dynamics of cell migration and cell–cell interactions in the extravascular space in three dimensions. Fueled by the burgeoning development of sophisticated fluorescent markers and increasingly powerful imaging tools, we are currently witnessing the emergence of a new field in immuno-imaging, in which leukocyte function and cell–cell communication is explored in a truly physiological context.