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Liquid phase electron microscopy has emerged as a powerful technique forin-situobservation of material formation in liquid. However, the interaction of electrons with an aqueous environment leads to the decomposition of water molecules into multiple radicals (radiolysis), which can affect the formation process. Graphene’s ultra-conductive properties have made it an efficient way to mitigate this problem, as it acts as an electron scavenger when used as a window material. However, finding accessible and reliable protocols for the formation of graphene liquid cell (GLC) has been an enormous challenge. Even though loop-assisted transfer has recently been demonstrated as an effective method, the liquid cells are randomly formed and the process of interest is initiated when the GLCs are sealed. This means that the process cannot be imaged at early timepoints, because searching for their location is a time-consuming effort during which the sample is exposed to electrons.Here we report a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses some of the limitations of the graphene liquid cells, combining the advantages of fluorescence and electron microscopy. Fluorescence microscopy (FM) is used to locate the GLCs without exposure to electrons and electron microscopy to capture dynamics with nanoscale resolution. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at the moment of interest. We demonstrate the workflow by observing non-classical crystallization pathways, but also highlight its potential application in biological systems.

Despite the enormous significance of malaria parasites for global health, some basic features of their ultrastructure remain obscure. In this study, we apply high-resolution volumetric electron microscopy to examine and compare the ultrastructure ofPlasmodium falciparumgametocytes of both genders and in different stages of development as well as the more intensively studied asexual blood stages revisiting previously described phenomena in 3D. In doing so, we challenge the widely accepted notion of a single mitochondrion by demonstrating the presence of multiple mitochondria in gametocytes. We also provide evidence for a gametocyte-specific cytostome variant. Furthermore, we generate, among other organelles, the first 3D reconstructions of endoplasmic reticulum (ER), Golgi apparatus, and extraparasitic structures in gametocytes. Assessing interconnectivity between organelles, we find frequent structural appositions between the nucleus, mitochondria, and apicoplast. We provide evidence that the ER is a promiscuous interactor with numerous organelles and the trilaminar membrane of the gametocyte. Public availability of these volumetric electron microscopy resources of wild-type asexual and sexual blood-stage malaria parasites will facilitate reinterrogation of this global dataset with different research questions and expertise. Taken together, we reconstruct the 3D ultrastructure ofP. falciparumgametocytes in high detail and shed light on the unique organellar biology of these deadly parasites.

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state, in particular for in combination with cryo-electron tomography (cryoET). In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating 3D cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging and cryo-lift out for cryoTEM.

Development 149(9), dev200198 (2022). doi:10.1242/dev.200198, Published by The Company of Biologists, Cambridge

Horizontal gene transfer in bacteria is widely believed to occur via three main mechanisms: conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. We present here a new mechanism of DNA uptake that is independent of canonical DNA uptake machineries and is used by bacteria that live without a cell wall. We show that the cell wall-deficient bacteria engulf extracellular material, whereby intracellular vesicles are formed, and DNA is internalized. This mechanism is not specific to DNA, and allows uptake of other macromolecules and even 125 nm lipid nanoparticles (LNPs). Uptake was prevented by molecules known to inhibit eukaryotic endocytosis, suggesting this to be an energy-dependent process. Given that cell wall-deficient bacteria are considered a model for early life forms, our work provides a possible mechanism for primordial cells to acquire new genetic material or food before invention of the bacterial cell wall.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID., Graphical Abstract, Jansen, Reimer, Nagai et al report that SARS-CoV-2 infects kidney cells and is associated with kidney fibrosis in patients. Using single cell transcriptomics of infected kidney organoids, they show that SARS-CoV-2 causes kidney injury and stimulates pro-fibrotic signaling. Viral infection in organoids was inhibited by a recently developed protease blocker.