Continuous assessment of metabolic activity of mitochondria using resonance Raman microspectroscopy.

Abstract

Dysfunctional mitochondrial activity can lead to a variety of different diseases. As such, there exists a need to quantify changes in mitochondria function as it relates to these specific diseased states. Here, we present the use of resonance Raman (RR) spectroscopy as a tool to determine changes in isolated mitochondrial activity. RR spectroscopy, using 532 nm as the excitation source, specifically provides information on the reduction and oxidation (RedOx) state of cytochrome c, which is determined by the activity of protein complexes in the electron transport chain (ETC). In this model, injection of the substrate succinate into the mitochondrial sample is used to drive the ETC, which causes a subsequent change in cytochrome c RedOx state. This change in RedOx state is tracked by RR spectroscopy. This tool gives real-time information on the rise and fall of the amount of reduced cytochrome c within the mitochondrial sample, providing a method for rapid assessment of mitochondrial metabolism that has broad applications in both basic science and medical research.