Impact of noise on optical redox ratio estimation

Optical redox ratio (ORR) is a commonly used metric to quantify the metabolic state of biological samples based on autofluorescence of NAD(P)H and FAD. Despite its relevance and popularity, little work has been done to quantify the estimation error of ORR, especially in low-photon count data. Here, we provide an analytical solution to determine ORR variability in the presence of shot noise and dark counts. We then validate this analytical model with simulated and experimental data. This work will enable researchers to quantify the uncertainty in their ORR measurements, aiding in more accurate and efficient experimental design and data analysis.