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

Optical microscopy, when applied to livinganimals, provides a powerful means of studying cell biology in the mostphysiologically relevant setting. The ability of two-photon microscopyto collect optical sections deep into biological tissues has opened upthe field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way inwhich two-photon microscopy can be applied to intravital studies ofkidney physiology. Because the kidney is easily externalized withoutcompromising its function, microscopy can be used to evaluate variousaspects of renal function in vivo. These include cell vitality andapoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes andsimultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of imagescontaining uncharacterized autofluorescence. The studies described heredemonstrate the way in which two-photon microscopy can provide a levelof resolution previously unattainable in intravital microscopy,enabling kinetic analyses and physiological studies of the organs ofliving animals with subcellular resolution.