Scientific goals and objectives: Cellularly preserved Proterozoic microfossils often contain intracellular structures that have been variously interpreted as nuclei, pyrenoids, lipid vesicles, degraded cytoplasm, encysted cytoplasm, or demosponge spicules. These interpretations, however, have been contested, largely because the microchemistry and microstructures of these intracellular structures have not been adequately characterized. Correctly interpreting the origin of these intracellular structures has important implications for the phylogenetic affinities of Proterozoic microfossils. As a pilot study, we have recently carried out a detailed investigation of two populations of nucleus-like structures, hosted in three-dimensionally phosphatized Doushantuo microfossils (~600 Ma) and in two-dimensionally compressed Ruyang acritarchs (1600-1780 Ma). To expand this investigation, we propose to analyze intracellular structures that have been previously interpreted as nuclei, lipid vesicles and demosponge spicules in three-dimensionally phosphatized animal embryo-like microfossils from the Ediacaran Doushantuo Formation. The nucleus and lipid vesicle interpretations make specific prediction about the presence of a membrane that would have served as a substrate for the mineral precipitation during fossilization. The demosponge spicule hypothesis predicts that the spicular structures are siliceous in composition and contain a micrometer-sized axial canal. To test these interpretations, we propose to analyze the microchemistry and micro-/ultrastructures of these intracellular structures. The ultimate goal of this project is to test the phylogenetic hypotheses about the Doushantuo fossils and their relevance to the evolution of advanced life forms such as eukaryotes and animals. Methods: In order to constrain the origin of the Doushantuo intracellular structures interpreted as nuclei, lipid vesicles and demosponge spicules, we will analyze these structures and their petrographic context using transmitted light microscopy, scanning and transmission electron microscopy, focused ion beam electron microscopy, energy-dispersive X-ray spectroscopy, and electron probe micro-analysis. Integration of the analytical results is necessary to unequivocally characterize the chemical composition and micro-/ultrastructures of Doushantuo intracellular structures, and to clarify their possible phylogenetic relationships with eukaryotes and animals. The fossil material is already in the PI's custody, and thus no field work is needed. Funding for this project will support a PhD graduate student, who will be supervised by the PI to carry out the proposed analyses. The graduate student (Anthony Drew Muscente) has been identified and has already been trained to use the advanced analytical tools, which are available in the PI's lab or at the Nanoscale Characterization and Fabrication Laboratory at Virginia Tech. Relevance: The proposed research aims at a comprehensive microanalysis of intracellular structures that have been interpreted as nuclei, lipid vesicles and demosponge spicules. These intracellular structures are hosted in Doushantuo fossils that have been controversially interpreted as animal embryos and sponges. Microstructural and microchemical characterization of these intracellular inclusions is critical to resolve these controversies. As bona fide nuclei are arguably the most tangible evidence for eukaryotic life, and siliceous sponge spicules are a definitive feature of sponge animals, the proposed research directly addresses NASA Exobiology Program's call to understand the origin of eukaryotes and the development of multicellularity on Earth.