In Situ Lymph Node Imaging
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010.INTRODUCTION
Imaging the single-cell dynamics of the immune system within an intact environment requires the ability to look deep inside tissues and organisms with spatial and temporal resolutions adequate to track cell morphology, motility, and signaling processes, all while minimizing perturbation of the system under study. Fluorescence techniques are highly suited for this purpose, permitting both labeling of specific cells, organelles, or proteins and functional readout of physiological events, and two-photon microscopy allows these processes to be visualized within native tissue environments. Immunoimaging preparations using explanted tissues and organs maintained by superfusion with warmed oxygenated medium are more physiological than in vitro cell systems and are easy to work with. They can be cleaned of surrounding tissue, oriented as desired with respect to the microscope objective, are readily accessible for administration of pharmaceutical agents, provide a robust and relatively drift-free preparation for long-term imaging, and minimize animal welfare issues. Explant imaging can yield results indistinguishable from intravital imaging, although intravital preparations are essential if intact blood and lymphatic flow are essential for the experimental question. This protocol describes in situ imaging of lymph nodes (LNs).
