The composition, conformation, and function of most macromolecular complexes depend on their cellular context and must be studied inside cells. Few cells are sufficiently thin to permit direct imaging with cryoelectron microscopy (cryo-EM). Focused ion beam milling enables cryo-EM to visualize macromolecules in cells at high resolution by generating thin sections of frozen-hydrated cells. We show how thin cellular sections can be prepared in a controlled fashion using an integrated light microscope coincident with electron and ion beams. The procedure provides live feedback on the thickness and uniformity of the prepared lamella, reducing complexity and increasing the success rate. Combined with its ability for fluorescence-based targeting, our procedure paves the way toward an automated workflow that allows for control over lamella quality, thickness, and target inclusion, facilitating the routine fabrication of frozen-hydrated cellular sections.@en

TiO₂ nanoparticles (NPs) are extensively used in various applications, highlighting the importance of ongoing research into their effects. This work belongs among rare whole-body inhalation studies investigating the effects of TiO₂ NPs on mice. Unlike previous studies, the concentration of TiO₂ NPs in the inhalation chamber (130.8 μg/m³) was significantly lower. This 11-week study on mice confirmed in vivo the presence of TiO₂ NPs in lung macrophages and type II pneumocytes including their intracellular localization by using the electron microscopy and the state-of-the-art methods detecting NPs' chemical identity/crystal structure, such as the energy-dispersed X-ray spectroscopy (EDX), cathodoluminescence (CL), and detailed diffraction pattern analysis using powder nanobeam diffraction (PNBD). For the first time in inhalation study in vivo, the alterations in erythrocyte morphology with evidence of echinocytes and stomatocytes, accompanied by iron accumulation in spleen, liver, and kidney, are reported following NP's exposure. Together with the histopathological evidence of hyperaemia in the spleen and kidney, and haemosiderin presence in the spleen, the finding of NPs containing iron might suggest the increased decomposition of damaged erythrocytes. The detection of TiO₂ NPs on erythrocytes through CL analysis confirmed their potential systemic availability. On the contrary, TiO₂ NPs were not confirmed in other organs (spleen, liver, and kidney); Ti was detected only in the kidney near the detection limit.

@en

A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub-micrometer amorphous specimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIB-SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam-sensitive materials.

@en