The invention of two-photon excitation microscopes widens the potential application of intravital microscopy (IVM) to the broad field of experimental pathology. Moreover, the recent development of fluorescent protein-based, genetically encoded biosensors provides an ideal tool to visualize the cell function in live animals. We start from a brief review of IVM with two-photon excitation microscopes and genetically encoded biosensors based on the principle of Förster resonance energy transfer (FRET). Then, we describe how IVM using biosensors has revealed the pathogenesis of several disease models.