Label-free tracking of cytochrome C oxidation state in live cells by resonance Raman imaging

Free interconversion of cytochrome C (CytC) between native ferrous (Cyt-Fe^II) and oxidized ferric (CytC-Fe^III) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC-Fe^III to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt-Fe^II to CytC-Fe^III within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear-cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label-free detection of ferrous and ferric heme proteins.