Spectroscopic studies of mitochondrial NADH fluorescence signals in brain slices

Dysfunction of mitochondria links a variety of central nervous system (CNS) disorders and other neurodegenerative diseases. The primary respiratory chain substrate reduced form nicotinamide adenine dinucleotide (NADH) is an important regulator of respiratory chain function in mitochondria and, because of its fluorescent properties, has been used to assess mitochondrial pathophysiology in cells and tissues. However, assessment of changes in tissue NADH has been limited to qualitative analysis primarily because hemoglobin (Hb) interferes with NADH fluorescence measurements by absorbing both excitation and emission light. This study presents a computer-assisted approach to estimate brain tissue NADH and Hb concentrations quantitatively at the same time. The method is based on a two-dimensionally interpolated database model that is calibrated by fluorescence emission spectra with known-value standard chemical solutions. Quantitative concentrations for NADH and Hb can be determined by the corresponding known-value spectral data that have the minimum error to the sample spectrum obtained from an experiment. Repeatability and reliability tests are also presented in this report. Results demonstrate that this method can feasibly quantify the NADH content regardless of the Hb background in living hippocampal cells during hypoxia, suggesting that it has potential to be applied to in vivo experiments in the future.