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.

We present a catheter-based optical diffusion and fluorescence (ODF) probe to study the functional changes of the brain in vivo. This ODF probe enables the simultaneous detection of the multi-wavelength absorbance and fluorescence emission from the living rat brain. Our previous studies, including a transient stroke experiment of the rat brain as well as the brain response to cocaine, have established the feasibility of simultaneously determining changes in cerebral blood volume (CBV), tissue oxygenation (S_tO₂) and intracellular calcium ([Ca²⁺]i, using the fluorescence indicator Rhod₂). Here, we present our preliminary results of somatosensory response to electrical forepaw stimulation obtained from the rat cortical brain by using the ODF probe, which indicate that the probe could track brain activation by directly detecting [Ca²⁺]_i along with separately distinguishing CBV and S_tO₂ in real time. The changes of CBV, S_tO₂ and [Ca²⁺]_i are comparable with the blood-oxygen-level-dependent (BOLD) response to the stimulation obtained using functional magnetic resonance imaging (fMRI). However, the high temporal resolution of the optical methodology is advanced, thus providing a new modality for brain functional studies to understand the hemodynamic changes that underlie the neuronal activity.