Coupled dual-beam focused ion beam electron microscopy (FIB-EM) has gained popularity across multiple disciplines over the past decade. Widely utilized as a stand-alone instrument for micromachining and metal or insulator deposition in numerous industries, the submicron-scale ion milling and cutting capabilities of FIB-EM systems have been well documented in the materials science literature. These capacities make FIB-EM a powerful tool for in situ, site-specific transmission electron microscopy (TEM) ultrathin foil preparation. Recent advancements in the field-emission guns (FEGs) of FIB-EM systems have provided spatial resolution comparable to that of many high-end scanning electron microscopes (SEM), thus providing enhanced imaging capacities with material deposition and material removal capabilities. More recently, FIB-EM preparation techniques have been applied to geological samples to characterize mineral inclusions, grain boundaries, and microfossils. Here, we demonstrate a novel method for analyzing three-dimensional (3-D) ultrastructures of microfossils using FIB-EM. Our method, FIB-EM nanotomography, consists of sequential ion milling, or cross sectioning, and concurrent SEM imaging. This technique with coupled dual-beam systems allows for real-time, 3-D ultrastructural analysis and compositional mapping with precise site selectivity and may provide new insights in fossil ultrastructures. Using the FIB-EM nanotomography method, we investigated herkomorphic and acanthomorphic acritarchs (organic-walled microfossils) extracted from the ≥999 Ma Mesoproterozoic Ruyang Group of North China. The 3-D characteristics of such important but controversial acritarch features as processes and vesicularly enclosed central bodies are described. Through these case studies, we demonstrate that FIB-EM nanotomography is a powerful and useful tool for investigating the three-dimensionality of microfossil ultra- and nanostructures.

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