PROJECT SUMMARY / ABSTRACT (DESCRIPTION) Four of the top 10 worldwide causes of death are related to oxygen supply:demand mismatching,(2) and two involve infectious processes(2) which are known to impact the mitochondria,(27) the oxygen-producing organelles which generate the majority of ATP in mammals. Despite the essential function of oxidative phosphorylation (OXPHOS) in human survival,(28) and the disruption of OXPHOS that occurs in a variety of disease states, our understanding of the exact process of hypoxic cell death remains incompletely understood.(28-30) This proposal’s objective is to construct and experimentally validate a frequency-domain near infrared spectroscopy (NIRS) instrument capable of measuring absolute concentrations of oxidized and reduced forms of cytochrome aa3, the terminal component of the electron transport chain (ETC). Our long term goal is to develop a patient monitor capable of detecting organ ischemia at the level of the mitochondria (ETC dysfunction) to facilitate prevention and treatment of leading causes of death. Our central hypothesis is that combination of emerging advances in engineering, in particular the use of advanced photon detectors (e.g. silicon photomultipliers [SiPM]) as well as novel combinations of near infrared radiation (NIR) wavelengths, will allow for accurate measurement of CytOX. We will test our central hypothesis and achieve our objective via the following specific aims: Specific Aim 1: Select Wavelength Number and Frequency. Using in silico simulation, we will test all possible combinations of commercially available wavelengths of lasers that can be modulated at 100 MHz to determine the combinations which offer the most attractive tradeoff between accuracy (CytOX validated against broadband) and simplicity (least number of wavelengths), using a pre-existing dataset; Specific Aim 2: Optimize Photon Detection. In a laboratory environment, we will test combinations of near infrared laser light and extended range photomultiplier tubes to identify a combination suitable for frequency domain spectroscopy measurement of CytOX (the oxidation state of cytochrome aa3); Specific Aim 3: Construct and Test a Functioning FD-NIRS Device to Measure CytOX. We will construct and test a frequency domain instrument capable of measuring absolute concentrations of hemoglobin (oxygenated, de-oxygenated) and cytochrome aa3 (oxidized, reduced) in a quasi-biological tissue phantom.