Abstract: Recognizing that tuberculosis (TB) is still one of the leading causes of human death, the international health-community has set ambitious targets to control TB by 2050. Unfortunately, this target cannot be achieved with current tools and requires the development and use of better anti-TB drugs/vaccines. Since, Mycobacterium tuberculosis (M.tb.) adapts to a quiescent physiological state - dormancy - and successfully evades anti-TB drugs and host-immune responses for decades, understanding the kinetics of adaptive bacterial responses and the host-microenvironment is essential for developing better anti-TB drugs/vaccines. However, current tools for assessing bacterial-host kinetics in animal models are limited to analyzing postmortem tissues. Artifacts introduced during sacrifice/processing make them less reliable. Moreover, lesionspecific characteristics are generally not assessed separate from the whole organ. Since a different animal is sacrificed at every time-point, bacterial-lesion kinetics in an individual animal can also never be assessed. We have pioneered the development of imaging biomarkers to assess M.tb. bacterial burden in animal models. In this proposal, we will develop novel imaging biomarkers that will not only permit assessment of M.tb. burden but also allow monitoring and localization of both adaptive bacterial responses and the hostmicroenvironment (inflammation, hypoxia and early immunity), in the same, live animal, over several timepoints. These tools will be utilized to address fundamental controversies in TB pathogenesis that cannot be tackled using current tools: a) Are host tissue inflammation and hypoxia a sanctuary for dormant M.tb.? b) Where do dormant M.tb. reside? c) Is innate immunity required for controlling initial M.tb. infection? Knowledge gained from this proposal will provide unique insights for developing better anti-TB drugs/vaccines. By permitting cost-effective, cross-species pre-clinical assessment, these tools will also dramatically reduce the time required for bench-to-bedside translation. Finally, since these tools are easily translatable, preclinical validation will lay the groundwork for their future use in humans.