PROJECT SUMMARY: This application addresses a major challenge that radiologists and other physicians encounter frequently, namely distinguishing active infection from other processes in the human body. In response to this challenge, our laboratory has developed both hyperpolarized 13C MRI and positron emission tomography (PET) tools targeting bacteria-specific metabolism. In this proposal, we employ another nuclear magnetic resonance (NMR)-observable stable isotope namely deuterium (2H) to detect living microorganisms both in vitro and in vivo. In doing so we address two major challenges of 2H magnetic resonance imaging (MRI) by (1) identifying 2H MRI-observable metabolites that have the needed chemical shift separation from HDO (water) and (2) using bacteria-specific sugar alcohols whose incorporation into microorganisms is not easily saturated. Our approach also takes advantage of two different strategies to incorporate 2H in a microorganism-specific way, and newly developed 2H MRI methods to maximize sensitivity. The two detection strategies pursued are either (1) a 2H substrate is converted to a dominant, 2H-MRI observable downstream metabolite or (2) a 2H substrate is converted to a dead-end metabolite that is accumulated as a 2H-MRI detectable species. These two concepts are highlighted by enriched variants of sorbitol, either D-[6,6'-2H2]sorbitol whose metabolism may be detected by its conversion to lactate/ethanol, or 2-deoxy-D-[2,2'-2H2]sorbitol anticipated to be “trapped” as its 6-phosphorylated adduct in bacteria. In both cases, the 2H nuclei have the needed chemical shift for in vivo 2H MRI imaging. In addition, the technologies pursued are amenable to clinical translation; our long-term goal is application of nontoxic 2H sugar alcohols to vulnerable populations including children, without the need for ionizing radiation. We will first adapt an NMR- compatible bioreactor for use in investigating 2H substrate metabolism in bacteria in vitro (Specific Aim 1). We will show then show bacteria-specific incorporation of 2H using enriched substrates, that are either metabolized to dominant 2H-MRI detectable products, or retained as dead-end molecules by pathogens (Specific Aim 2). In Specific Aim 3, we will show that 2H MRI at 14T using these tools can detect living bacteria in vivo.