Quantification of oxidized and reduced cytochrome-c-oxidase by combining discrete-wavelength time-resolved and broadband continuous-wave near-infrared spectroscopy

Quantification of cytochrome-c-oxidase (CCO) can directly inform about cerebral metabolic capacity and function, but limited options currently exist for its in vivo assessment. Near-infrared spectroscopy (NIRS) has the potential to quantify CCO and its redox states, but hyperspectral absorption measurements are required due to their broad absorption profiles and low concentrations relative to hemoglobin. While this may be achieved with continuous-wave broadband NIRS (bNIRS), separating the signal contributions of absorption and scattering remains a challenge. Alternatively, time-resolved NIRS (trNIRS) can directly disentangle absorption and scattering but is typically constrained to a few wavelengths. This work aimed to develop an approach for quantifying absolute CCO concentration using discrete-wavelength trNIRS to calibrate bNIRS, yielding calibrated bNIRS (cbNIRS). Monte-Carlo simulations were conducted to validate the algorithm. Subsequently, a hybrid cbNIRS system was assembled, and tissue-mimicking phantoms were prepared with blood, Intralipid, and either yeast or sodium dithionite for validation. The simulations demonstrated that the algorithm can accurately measure absorption across the spectral range (error = 0.8 ± 0.4%). Further, the concentrations of CCO and its different redox states were estimated with an error of 7.9% or less. In the phantom experiments, the measured HbT concentration increased with the addition of blood, but not yeast nor sodium dithionite, and the value agreed with the expected concentration estimated from the packed cell volume of blood. A large increase in total CCO was measured only after the addition of yeast (1.8 ± 0.4 <i>µ</i>M). Transitions in the oxygenation state of hemoglobin and redox state of CCO followed the expected trends as the phantom was deoxygenated and reoxygenated. Additionally, the sodium dithionite experiments confirmed that the COO signal measured with cbNIRS is not a result of crosstalk with the hemoglobin signal. This work demonstrates that absolute concentrations of both redox states of CCO can be quantified with high accuracy using cbNIRS. Future work will assess the feasibility of in vivo CCO measurements.