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Abstract: Dragonfly- and cicada-wing-inspired titanium nanostructured surfaces exhibit promising bactericidal properties. However, direct visualization of the bacteria–material interface under hydrated conditions remains limited, restricting experimental interrogation of how bacteria interact with nanostructured surfaces. This limitation reflects a long-standing methodological gap, as visualization of bacteria–nanostructure interfaces has largely relied upon indirect or dehydrated imaging approaches. Cryo-electron tomography (cryo-ET) enables 3D visualization of cellular ultrastructure in a native hydrated environment, but imaging bacteria attached to metallic nanostructures by cryo-ET requires preparation of thin electron-transparent lamellae. Obtaining such lamellae from vitrified bacteria on titanium substrates is technically challenging because cryo-focused ion beam (cryo-FIB) milling must simultaneously section soft biological material and hard metal whilst bacteria embedded in vitreous ice are not directly visible. Here, we establish a correlative cryogenic workflow enabling targeted extraction and cryo-ET imaging of defined bacterium–nanopillar interfaces. Individual bacteria interacting with titanium nanopillars are identified by correlative cryo-fluorescence microscopy, followed by targeted cryo-FIB lift-out, transfer to a receiver grid, and thinning into electron-transparent lamellae. The data presented demonstrate that bacteria–nanostructure interfaces can be targeted, extracted, and structurally analysed in situ under fully hydrated conditions. This workflow provides a methodological framework for future cryo-ET studies of bacteria–nanotopography interactions.

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Abstract: Hepatitis C virus (HCV) infection induces extensive rearrangements of host cytoplasmic membranes, leading to the formation of multiple membranous structures that facilitate RNA replication. Current knowledge of these membranous structures has largely relied on correlative light and electron microscopy techniques using chemical fixation and resin embedding. To overcome these limitations, cryo-preserved cells were prepared using cryo-focused ion beam (cryo-FIB) milling and cryo-ultramicrotomy. For the first time, the contents within the membranous structures have been observed in situ using cryo-electron tomography (cryo-ET) performed on lamellae (prepared via cryo-FIB) and on ultrathin sections (prepared via cryo-ultramicrotomy) from HCV subgenomic replicon-harbouring cells. Observations from 112 cryo-electron tomograms of cryo-FIB-derived samples revealed the presence of densities within the inner vesicles of a subset of single- and double-membrane vesicles, as well as within multi-vesicular bodies, which are consistent with the presence of the viral genome replication machinery. Notably, this study also presents the first direct visualization of densities within a multi-membrane vesicle observed by cryo-electron microscopy of vitreous sections. The cryo-ET methodologies established here lay the groundwork for future investigations into the architecture of the HCV replication complex, leveraging advanced computational tools for deeper structural and functional analyses.

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Abstract: Direct visualization of HIV-1 nuclear import through the nuclear pore complex (NPC) presents a technical challenge due to the rarity of this process. To enable systematic investigation, we developed a robust in situ system that mimics HIV-1 nuclear import in a near-native context using isolated HIV-1 virus like particles (VLP) cores and permeabilized CD4 + T lymphocyte (CEM) cells. This approach supports docking and translocation of abundant viral cores through nuclear pores into the nucleus. For high-resolution visualization, we implemented an integrated correlative approach to guide cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging, enabling precise targeting and structural characterization of individual nuclear import events. Using this workflow, we visualized 510 HIV-1 VLP cores at distinct stages of nuclear import, capturing key snapshots of the full progression of nuclear import. Subsequent statistical and structural analyses allow classification of core morphologies and identification of translocation-associated remodeling in nuclear pores. This work provides a methodological foundation for dissecting HIV-1 and potentially other viruses nuclear import processes and post-entry events in a controlled and quantitative manner.

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Abstract: Inorganic minerals that form via regulated biological processes exhibit remarkable properties. This is due to the involvement of macromolecules that control biomineralization. Even though the interactions of these biopolymers with solid mineral phases are intensely studied, not much is known about their involvement in the preceding steps of intracellular transport of the mineral building blocks. In this work, the model system of coccolith calcite crystallization is utilized to address the role of mineral-associated polysaccharides in the transport of calcium ions. State-of-the-art cryo-electron tomography is used to image in situ ion-rich dense phases in the wild-type and in two mutant strains, defected in coccolith production. The results show that the abundance and solubility of the calcium-rich condensates need to be finely tuned for proper crystallization. When the native macromolecular assemblage is compromised, calcium is still present in the calcifying fluid as a solute, but this is not sufficient for coccolith development. These results suggest that biomineralizing systems achieve superior regulation of crystallization due to the use of dense macromolecule-rich phases.