Cytochrome c is a small heme protein located within the inner membrane of the mitochondrion. It is an essential component of the electron transport chain. Its redox state is closely related to mitochondrion functions, such as ATP generation and oxygen utilization. Therefore, distinguishing the redox state of cytochrome c represents a potentially novel method for monitoring mitochondrial function in conditions where alterations in electron transport are implicated, such as diabetes, cancer and mitochondrial disease.
In our experiment, a pair of pump and probe ultrafast laser pulses, with duration of 750 fs, have been focused at the same spot in a tissue slice. The difference in arrival time of the two pulses can be adjusted with picosecond resolution. It is known that a pump pulse readily dissociates one of the amino acids attached to the central iron ion, but only in the reduced form of cytochrome. This alters the absorption spectrum of reduced cytochrome until the ligand reattaches in about 5ps. That absorption change can be detected by pump-probe microscopy and allows distinction between redox states.
By using 533nm pump and 490 nm probe, our system is able to distinguish redox state of cytochrome c in solution. We also have acquired pump-probe response from live insect muscle, selected for high cytochrome content and absence of hemoglobin and myoglobin, and are progressing towards redox imaging of tissue.