Multiphoton fluorescence microscopy is a powerful, important tool in biomedical research that offers low photon toxicity and higher spatial and temporal resolution than other in vivo imaging modalities. The capability to collect images hundreds of micrometers into biological tissues provides an invaluable tool for studying cellular and subcellular processes in the context of tissues and organs in living animals. Multiphoton microscopy is based upon two-photon excitation of fluorescence that occurs only in a sub-femtoliter volume at the focus; by scanning the focus through a sample, 2- and 3-dimensional images can be collected. The complex 3-dimensional organization of the kidney makes it especially appropriate for multiphoton microscopic analysis, which has been used to characterize numerous aspects of renal physiology and pathophysiology in living rats and mice. However, the ability to collect fluorescence images deep into biological tissues raises unique problems not encountered in other forms of optical microscopy, including issues of probe access, and tissue optics. Future improvements in multiphoton fluorescence microscopy will involve optimizing objectives for the unique characteristics of multiphoton fluorescence imaging, improving the speed at which images may be collected and extending the depth to which imaging may be conducted.