Functional studies of the kidney of living animals using multicolor 2-photon microscopy

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of 2-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to highresolution studies of the brain, lens, skin and tumors. Here we present examples of how 2-photon microscopy can be applied to intravital studies of kidney physiology. As the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow and leukocyte trafficking. Efficient 2-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters, and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate how 2-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.