PROJECT SUMMARY Zinc fluctuations have recently been discovered to play a number of regulatory roles in human physiology. For example, significant zinc deployments occur during female gamete maturation, neuron function, immune response, and insulin secretion. Metal imaging techniques at the single-cell level have established the cellular and subcellular distribution and fluxes of zinc in a number of cellular models. However, few imaging tools are able to measure how ensembles of cells and organs store and mobilize zinc to carry out function. This proposal aims to develop new quantitative tools for mapping zinc in tissue. We will test the utility of new zinc imaging tools developed in this proposal by simultaneously testing the hypothesis that zinc fluctuations direct the selective activation of primordial follicles in the murine ovary. This hypothesis will be addressed using three coordinated and complementary approaches: (1) mapping total zinc levels in mouse ovaries using synchrotron X-ray fluorescence microscopy and laser ablation inductively coupled plasma time-of-flight mass spectrometry; (2) the development of zinc-responsive photoacoustic probes; (3) spatiotemporal mapping of zinc in ovarian tissue with photoacoustic microscopy. Taken together, the knowledge gained from this research project will reveal key insight into zinc physiology in reproductive health, and the tools produced here will be valuable for the study of zinc in other human physiologies and pathologies. This award will support research and professional training at the interface of chemistry, reproductive biology, and engineering. These preparations provide a strong foundation for an independent and multidisciplinary professorship at a research university. Training activities will target progress in scientific communication, lab management, mentorship, college-level teaching, and outreach.