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A central characteristic of the immune system is the constantly changing location of most of its constituent cells. Lymphoid and myeloid cells circulate in the blood, and subsets of these cells enter, move, and interact within, then leave organized lymphoid tissues. When inflammation is present, various hematopoietic cells also exit the vasculature and migrate within non-lymphoid tissues, where they carry out effector functions that support host defense or result in autoimmune pathology. Effective innate and adaptive immune responses involve not only the action of these individual cells but also productive communication among them, often requiring direct membrane contact between rare antigen-specific or antigen-bearing cells. Here, we describe our ongoing studies using two-photon intravital microscopy to probe the in situ behavior of the cells of the immune system and their interactions with non-hematopoietic stromal elements. We emphasize the importance of non-random cell migration within lymphoid tissues and detail newly established mechanisms of traffic control that operate at multiple organizational scales to facilitate critical cell contacts. We also describe how the methods we have developed for imaging within lymphoid sites are being applied to other tissues and organs, revealing dynamic details of host-pathogen interactions previously inaccessible to direct observation. © 2008 The Authors.

The past few years have seen the application of confocal and especially two-photon microscopy to the dynamic high-resolution imaging of lymphocytes and antigen presenting cells within organs such as lymph nodes and thymus. After summarizing some of the published results obtained to date using these methods, we describe our view of how this technology will develop and be applied in the near future. This includes its extension to a wide variety of non-lymphoid tissues, to the tracking of functional responses in addition to migratory behavior, to the analysis of molecular events previously studied only in vitro, to dissection of the interplay between hematopoietic and stromal elements, to visualization of a wider array of cell types including neutrophils, macrophages, NK cells, NKT cells and others, and to the interaction of the host with infectious agents. Reaching these goals will depend on a combination of new tools for genetic manipulations, novel fluorescent reporters, enhanced instrumentation, and better surgical techniques for the extended imaging of live animals. The end result will be a new level of understanding of how orchestrated cell movement and interaction contribute to the physiological and pathological activities of the immune system.