Calibration of CCD-based redox imaging for biological tissues

Clinically-translatable redox imaging methods developed in the Chance laboratory have been used for imaging mitochondrial metabolic states in tissues. The fluorescence of reduced pyridine nucleotide (PN or NADH) and oxidized flavoproteins (Fp) in the respiratory chain is sensitive to intracellular redox states. The redox ratios, i.e., Fp/(Fp+NADH) and NADH/(Fp+NADH) provide important metabolic information in living tissues. Usually the higher the metabolic flux, the less NADH, the more oxidized Fp, and the higher Fp redox ratio. Snap-freezing tissue samples under the liquid nitrogen condition preserves the tissue metabolic state in vivo. Here we report our work on the calibration of a homebuilt Charged Coupled Device (CCD) cryogenic redox imager using a series of snap-frozen solution standards of NADH and Fp. The NADH concentration ranged from 0-1318 μM and Fp from 0-719 μM. The sensitivity ratio of NADH and Fp channels was determined from the slope ratio of the two calibration curves and was used to correct the redox ratio of a human melanoma mouse xenograft. The NADH and Fp reference standards were placed adjacent to the tissue samples and their emission intensities were used to quantitatively determine the concentrations of NADH and Fp in a mouse xenograft of a human breast cancer line. Our method of imaging tissue samples along with reference NADH and Fp standards should facilitate the comparison of redox images obtained at different times or with different instrument parameters.