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The fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) such
as flavin adenine dinucleotide (FAD) in the respiratory chain are sensitive indicators of intracellular redox states and
have been applied to the studies of mitochondrial function with energy-linked processes. The redox scanner, a threedimensional
(3D) redox cryo-imager previously developed by Chance et al., can quantitatively determine the metabolic
properties of tissue samples by acquiring the fluorescence images of NADH and Fp. The redox ratios, i.e.,
Fp/(Fp+NADH) and NADH/(Fp+NADH), obtained on the basis of relative signal intensity ratios, provide a sensitive
index of steady-state of the mitochondrial metabolism that has been determined for a variety of biological tissues. This
paper presents the further development of the instrument by establishing a calibration method to quantify the
concentration of the fluorophores and facilitate the comparison of redox images obtained at different time or with
different instrument functions. Calibration curves of both NADH and Fp have been obtained using snap-frozen standard
references with NADH concentration ranging from 150-1400 μM and Fp from 80-720 μM. Snap-freeze tissue samples
such as human breast tumors xenografted in mice, normal mouse pancreases and spleens were imaged. The NADH and
Fp concentrations as well as the redox ratios in the tissue samples were quantified based on the adjacent solution
standards of NADH and Fp. The obtained multi-slice redox images revealed high heterogeneity of the tissue samples
which can be quantitatively interpreted.
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