Since the dual-wavelength spectrophotometer was developed, it has been widely used for studying biological samples and applied to extensive investigations of the electron transport in respiration and redox cofactors, redox state, metabolic control, and the generation of reactive oxygen species in mitochondria. Here, we discuss some extension of dual-wavelength approaches in our research to study the physiological and functional changes in intact hearts and in vivo brain. Specifically, we aimed at (1) making nonratiometric fluorescent indicator become ratiometric fluorescence function for investigation of Ca²⁺ dynamics in live tissue; (2) eliminating the effects of physiological changes on measurement of intracellular calcium; (3) permitting simultaneous imaging of multiple physiological parameters. The animal models of the perfused heart and transiently ischemic insult of brain are used to validate these approaches for physiological applications.