Steady-state spatially resolved diffuse reflectance spectroscopy is used to reconstruct absolute visible absorption spectra (λ = 450-610 nm) of tissue-simulating phantoms that contain intact mitochondria and red blood cells in physiologically relevant proportions. The spectra are analyzed to extract independent fitted concentrations of oxy- and deoxyhemoglobin and of oxidized and reduced mitochondria. The resulting hemoglobin oxygen-dissociation and mitochondrial oxidation-reduction curves exhibit <i>p</i><sub>50</sub> parameters of 25 and 2.5 torr, respectively, in agreement with published values. These results demonstrate the potential for absolute quantitative cytochrome spectroscopy of tissue in the presence of hemoglobin in the visible wavelength region. Extension of these measurements to the <i>in vivo</i> case is discussed.
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