The fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) including flavin adenine dinucleotide (FAD) in the respiratory chain are sensitive indicators of intracellular metabolic states and have been applied to the studies of mitochondrial function with energy-linked processes. The redox scanner, a three-dimensional (3D) low temperature imager previously developed by Chance et al., measures the in vivo metabolic properties of tissue samples by acquiring fluorescence images of NADH and Fp. The redox ratios, i.e. Fp/(Fp+NADH) and NADH/(Fp+NADH), provided a sensitive index of the mitochondrial redox state and were determined based on relative signal intensity ratios. Here we report the further development of the redox scanning technique by using a calibration method to quantify the nominal concentration of the fluorophores in tissues. The redox scanner exhibited very good linear response in the range of NADH concentration between 165–1318μM and Fp between 90–720μM using snap-frozen solution standards. Tissue samples such as human tumor mouse xenografts and various mouse organs were redox-scanned together with adjacent NADH and Fp standards of known concentration at liquid nitrogen temperature. The nominal NADH and Fp concentrations as well as the redox ratios in the tissue samples were quantified by normalizing the tissue NADH and Fp fluorescence signal to that of the snap-frozen solution standards. This calibration procedure allows comparing redox images obtained at different time, independent of instrument settings. The quantitative multi-slice redox images revealed heterogeneity in mitochondrial redox state in the tissues.