39] Three-dimensional redox imaging of frozen-quenched brain and other organs

Abstract

The chapter describes a technique of a low temperature redox scanning developed in our laboratory. The redox ratio is a sensitive parameter of the status of energy metabolism of the cell. Molecular biology has focused on the downregulation of mitochondria-mediated apoptosis. The redox imaging technique can also help elucidate research into redox signaling mechanisms. The major flavoprotein fluorescence of mitochondria is the FAD of α-lipoamide dehydrogenase and Co-Q-linked flavin of the fatty acid-oxidizing system. Only the α-lipoamide dehydrogenase is considered as a main source of flavin fluorescence and is in equilibrium with the mitochondrial NAD+/nicotinamide adenine dinucleotide (NADH) couple. The content rate of the NAD-linked α-lipoamide dehydrogenase and Co-Q-linked flavin differs according to the tissue. Pyridine nucleotide (PN) signal may originate from two different redox couples—that is, cytosolic and mitochondrial components. In the brain, heart, and skeletal muscle, data suggest that the PN signal predominantly originates from mitochondrial NADH. However, in the liver the PN signal suggests an almost equal contribution from the cytosol and mitochondria. NADH and flavoprotein components can be effectively trapped at liquid nitrogen temperatures, and thus the redox state of this portion of the respiratory chain can be preserved for long periods and assayed at any time, without alteration.