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Resonance Raman (RR) spectroscopy is a well-established technique that has been used in a variety of different applications. Shown here is its ability to investigate the underlying mechanisms of photobiomodulation. Isolated mitochondria from porcine cardiac tissue was used as the sample, and its cytochrome c redox state was probed by the RR technique. By injecting a substrate, such as succinate, into the mitochondrial sample, cytochrome c becomes reduced until the succinate is consumed. Without new freeelectrons from succinate, a lack of Complex III activity allows all the molecules of cytochrome c to become re-oxidized. A measurement of this activity provides information that can be used to determine electron transport chain kinetics. Using this RR technique, changes in kinetics within isolated cardiac mitochondria were explored using low irradiance visible light. A novel result occurred when mitochondria were irradiated with higher irradiance 532 nm light (402 W/cm2) where it was found that the substrate was consumed at a quicker rate than with a comparatively lower irradiance light (170 W/cm2). It was also determined that the rate at which cytochrome c was reduced after the substrate was introduced was larger. This increase in electron transport chain kinetics was not due to temperature and was hypothesized to be a photochemical effect. It was also found that lower irradiances of 520 nm light (30 mW/cm2) did not have an effect on the kinetics, therefore a higher irradiance is needed when imaging isolated mitochondria using resonance Raman spectroscopy.
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