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Supplemental Document (Supplement 1)

Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics. Deutsche Forschungsgemeinschaft FP7 Ideas: European Research Council

Multifunctional composite nanoprobes, Ag–Magnetite and Au–Magnetite, were manipulated in fibroblast cells and characterized using SERS, LA-ICP-MS, and nanotomography., Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag–Magnetite and Au–Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surface-enhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag–Magnetite and Au–Magnetite nanostructures that is very similar to that of other composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.

Resistive random access memory (RRAM) is considered an attractive candidate for next generation memory devices due to its competitive scalability, low-power operation and high switching speed. The technology however, still faces several challenges that overall prohibit its industrial translation, such as low yields, large switching variability and ultimately hard breakdown due to long-term operation or high-voltage biasing. The latter issue is of particular interest, because it ultimately leads to device failure. In this work, we have investigated the physicochemical changes that occur within RRAM devices as a consequence of soft and hard breakdown by combining full-field transmission x-ray microscopy with soft x-ray spectroscopic analysis performed on lamella samples. The high lateral resolution of this technique (down to 25 nm) allows the investigation of localized nanometric areas underneath permanent damage of the metal top electrode. Results show that devices after hard breakdown present discontinuity in the active layer, Pt inclusions and the formation of crystalline phases such as rutile, which indicates that the temperature increased locally up to 1000 K.

Summary Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM., Graphical Abstract, Highlights • Multimodal imaging reveals mechanism of vesicle formation at inner nuclear membrane • Nucleo-cytoplasmic cargo vesicle coat in situ comprises two distinct lattices • Lattices are formed by hexameric building blocks made of the nuclear egress complex • Induction of membrane curvature based solely on heterodimeric interactions, A multi-modal live-cell and cryo-imaging approach reveals how vesicles assemble at the inner nuclear membrane for transport to the cytoplasm during herpesvirus maturation. This also suggests a functional model for counterparts in uninfected cells that mediate nuclear egress of large cargo like ribonucleoprotein particles.