A method to simultaneously measure oxygenation in vascular, intracellular, and mitochondrial spaces from optical spectra acquired from muscle has been developed. In order to validate the method, optical spectra in the visible and near-infrared regions (600–850 nm) were acquired from solutions of myoglobin, hemoglobin, and cytochrome oxidase that included Intralipid as a light scatterer. Spectra were also acquired from the rabbit forelimb. Three partial least squares (PLS) analyses were performed on second-derivative spectra, each separately calibrated to myoglobin oxygen saturation, hemoglobin oxygen saturation, or cytochrome <i>aa3</i> oxidation. The three variables were measured from in vitro and in vivo spectra that contained all three chromophores. In the in vitro studies, measured values of myoglobin saturation, hemoglobin saturation, and cytochrome <i>aa3</i> oxidation had standard errors of 5.9%, 7.4%, and 12.2%, respectively, with little cross-talk between the in vitro measurements. In the progression from normal oxygenation to ischemia in the rabbit forelimb, hemoglobin desaturated first, followed by myoglobin, while cytochrome <i>aa3</i> reduction occurred last. The ability to simultaneously measure oxygenations in the vascular, intracellular, and mitochondrial compartments will be valuable in physiological studies of muscle metabolism and in clinical studies when oxygen supply or utilization are compromised.

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