QUANTITATIVE REDOX SCANNING OF TISSUE SAMPLES USING A CALIBRATION PROCEDURE.

Area of Science:

• Biophysics
• Biochemistry
• Medical Imaging

Background:

• Fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) indicate intracellular metabolic states.
• These fluorophores are crucial for studying mitochondrial function and energy-linked processes.
• Existing redox scanning techniques measure in vivo metabolic properties using fluorescence imaging.

Purpose of the Study:

• To develop a calibration method for quantifying fluorophore concentrations in tissues using the redox scanner.
• To enable accurate and comparable measurements of mitochondrial redox state independent of instrument settings.
• To reveal heterogeneity in mitochondrial redox state within tissue samples.

Main Methods:

• Utilized a 3D low-temperature redox scanner to acquire fluorescence images of NADH and Fp.
• Developed a calibration method using snap-frozen solution standards for linear response quantification.
• Quantified nominal NADH and Fp concentrations and redox ratios in tissue samples (mouse xenografts, organs) by normalizing fluorescence signals to standards.

Main Results:

• The redox scanner demonstrated linear response for NADH (165-1318 μM) and Fp (90-720 μM) concentrations.
• A calibration procedure was successfully implemented for quantitative redox scanning of tissues.
• Quantitative multi-slice redox images revealed heterogeneity in the mitochondrial redox state within the analyzed tissues.

Conclusions:

• The developed calibration method allows for accurate quantification of NADH and Fp concentrations in tissues.
• This quantitative approach enables reliable comparison of redox images across different experimental conditions and time points.
• The technique provides valuable insights into the heterogeneous mitochondrial redox state within various tissue types, aiding metabolic research.