Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Dehydration as alternative sample preparation for soft X-ray tomography.

Soft X-ray tomography (SXT) is an imaging technique to visualise whole cells without fixation, staining, and sectioning. For SXT imaging, cells are cryopreserved and imaged at cryogenic conditions. Such 'near-to-native' state imaging is in high demand and initiated the development of the laboratory table-top SXT microscope. As many laboratories do not have access to cryogenic equipment, we asked ourselves whether SXT imaging is feasible on dry specimens. This paper shows how the dehydration of cells can be used as an alternative sample preparation to obtain ultrastructure information. We compare different dehydration processes on mouse embryonic fibroblasts in terms of ultrastructural preservation and shrinkage. Based on this analysis, we chose critical point (CPD) dried cells for SXT imaging. In comparison to cryopreserved and air-dried cells, CPD dehydrated cells show high structural integrity although with about 3-7 times higher X-ray absorption for cellular organelles. As the difference in X-ray absorption values between organelles is preserved, 3D anatomy of CPD-dried cells can be segmented and analysed, demonstrating the applicability of CPD-dried sample preparation for SXT imaging. LAY DESCRIPTION: Soft X-ray tomography (SXT) is an imaging technique that allows to see the internal structures of cells without the need for special treatments like fixation or staining. Typically, SXT imaging involves freezing and imaging cells at very low temperatures. However, since many labs lack the necessary equipment, we explored whether SXT imaging could be done on dry samples instead. We compared different dehydration methods and found that critical point drying (CPD) was the most promising for SXT imaging. CPD-dried cells showed high structural integrity, although they absorbed more X-rays than hydrated cells, demonstrating that CPD-dried sample preparation is a viable alternative for SXT imaging.

Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties.

Phase separation of multivalent protein and RNA molecules underlies the biogenesis of biomolecular condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of molecules that typically organize into multilayered structures supporting the differential partitioning of molecules into distinct regions with distinct material properties. The interplay between driven (active) versus spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, we deploy systematic experiments and simulations based on coarse-grained models to show that the collective interactions among the simplest, biologically relevant proteins and archetypal RNA molecules are sufficient for driving the spontaneous emergence of multilayered condensates with distinct material properties. These studies yield a set of rules regarding homotypic and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomolecular condensates.

Identification, visualization and clonal analysis of intestinal stem cells in fish.

Recently, a stochastic model of symmetrical stem cell division followed by neutral drift has been proposed for intestinal stem cells (ISCs), which has been suggested to represent the predominant mode of stem cell progression in mammals. In contrast, stem cells in the retina of teleost fish show an asymmetric division mode. To address whether the mode of stem cell division follows phylogenetic or ontogenetic routes, we analysed the entire gastrointestinal tract of the teleost medaka (Oryzias latipes). X-ray microcomputed tomography shows a correlation of 3D topography with the functional domains. Analysis of ISCs in proliferation assays and via genetically encoded lineage tracing highlights a stem cell niche in the furrow between the long intestinal folds that is functionally equivalent to mammalian intestinal crypts. Stem cells in this compartment are characterized by the expression of homologs of mammalian ISC markers - sox9, axin2 and lgr5 - emphasizing the evolutionary conservation of the Wnt pathway components in the stem cell niche of the intestine. The stochastic, sparse initial labelling of ISCs ultimately resulted in extended labelled or unlabelled domains originating from single stem cells in the furrow niche, contributing to both homeostasis and growth. Thus, different modes of stem cell division co-evolved within one organism, and in the absence of physical isolation in crypts, ISCs contribute to homeostatic growth.

Imaging cell morphology and physiology using X-rays.

Morphometric measurements, such as quantifying cell shape, characterizing sub-cellular organization, and probing cell-cell interactions, are fundamental in cell biology and clinical medicine. Until quite recently, the main source of morphometric data on cells has been light- and electron-based microscope images. However, many technological advances have propelled X-ray microscopy into becoming another source of high-quality morphometric information. Here, we review the status of X-ray microscopy as a quantitative biological imaging modality. We also describe the combination of X-ray microscopy data with information from other modalities to generate polychromatic views of biological systems. For example, the amalgamation of molecular localization data, from fluorescence microscopy or spectromicroscopy, with structural information from X-ray tomography. This combination of data from the same specimen generates a more complete picture of the system than that can be obtained by a single microscopy method. Such multimodal combinations greatly enhance our understanding of biology by combining physiological and morphological data to create models that more accurately reflect the complexities of life.

PSF correction in soft X-ray tomography.

In this article, we introduce a linear approximation of the forward model of soft X-ray tomography, such that the reconstruction is solvable by standard iterative schemes. This linear model takes into account the three-dimensional point spread function (PSF) of the optical system, which consequently enhances the reconstruction of data. The feasibility of the model is demonstrated on both simulated and experimental data, based on theoretically estimated and experimentally measured PSFs.

Gauging low-dose X-ray phase-contrast imaging at a single and large propagation distance.

The interactions of a beam of hard and spatio-temporally coherent X-rays with a soft-matter sample primarily induce a transverse distribution of exit phase variations δϕ (retardations or advancements in pieces of the wave front exiting the object compared to the incoming wave front) whose free-space propagation over a distance z gives rise to intensity contrast gz. For single-distance image detection and |δϕ| ≪ 1 all-order-in-z phase-intensity contrast transfer is linear in δϕ. Here we show that ideal coherence implies a decay of the (shot-)noise-to-signal ratio in gz and of the associated phase noise as z(-1/2) and z(-1), respectively. Limits on X-ray dose thus favor large values of z. We discuss how a phase-scaling symmetry, exact in the limit δϕ → 0 and dynamically unbroken up to |δϕ| ∼ 1, suggests a filtering of gz in Fourier space, preserving non-iterative quasi-linear phase retrieval for phase variations up to order unity if induced by multi-scale objects inducing phase variations δϕ of a broad spatial frequency spectrum. Such an approach continues to be applicable under an assumed phase-attenuation duality. Using synchrotron radiation, ex and in vivo microtomography on frog embryos exemplifies improved resolution compared to a conventional single-distance phase-retrieval algorithm.

Automated 3D cytoplasm segmentation in soft X-ray tomography.

Cells' structure is key to understanding cellular function, diagnostics, and therapy development. Soft X-ray tomography (SXT) is a unique tool to image cellular structure without fixation or labeling at high spatial resolution and throughput. Fast acquisition times increase demand for accelerated image analysis, like segmentation. Currently, segmenting cellular structures is done manually and is a major bottleneck in the SXT data analysis. This paper introduces ACSeg, an automated 3D cytoplasm segmentation model. ACSeg is generated using semi-automated labels and 3D U-Net and is trained on 43 SXT tomograms of immune T cells, rapidly converging to high-accuracy segmentation, therefore reducing time and labor. Furthermore, adding only 6 SXT tomograms of other cell types diversifies the model, showing potential for optimal experimental design. ACSeg successfully segmented unseen tomograms and is published on Biomedisa, enabling high-throughput analysis of cell volume and structure of cytoplasm in diverse cell types.

Visualisation of gene expression within the context of tissues using an X-ray computed tomography-based multimodal approach.

The development of an organism is orchestrated by the spatial and temporal expression of genes. Accurate visualisation of gene expression patterns in the context of the surrounding tissues offers a glimpse into the mechanisms that drive morphogenesis. We developed correlative light-sheet fluorescence microscopy and X-ray computed tomography approach to map gene expression patterns to the whole organism`s 3D anatomy. We show that this multimodal approach is applicable to gene expression visualized by protein-specific antibodies and fluorescence RNA in situ hybridisation offering a detailed understanding of individual phenotypic variations in model organisms. Furthermore, the approach offers a unique possibility to identify tissues together with their 3D cellular and molecular composition in anatomically less-defined in vitro models, such as organoids. We anticipate that the visual and quantitative insights into the 3D distribution of gene expression within tissue architecture, by multimodal approach developed here, will be equally valuable for reference atlases of model organisms development, as well as for comprehensive screens, and morphogenesis studies of in vitro models.

A comprehensive method to study the DNA's association with lamin and chromatin compaction in intact cell nuclei at super resolution.

Super-resolution fluorescence microscopy has revolutionized multicolor imaging of nuclear structures due to the combination of high labeling specificity and high resolution. Here we expanded the recently developed fBALM (DNA structure fluctuation-assisted binding activated localization microscopy) method by developing a stable methodological sequence that enables dual-color imaging of high-resolution genomic DNA together with an immunofluorescently labeled intranuclear protein. Our measurements of the nuclear periphery, imaging DNA and LaminB1 in biologically relevant samples, show that this novel dual-color imaging method is feasible for further quantitative evaluations. We were able to study the relative spatial signal organization between DNA and LaminB1 by means of highly specific colocalization measurements at nanometer resolution. Measurements were performed with and without the antifade embedding medium ProLong Gold, which proved to be essential for imaging of LaminB1, but not for imaging of SytoxOrange labeled DNA. The localization precision was used to differentiate between localizations with higher and lower amounts of emitting photons. We interpret high intensity localizations to be renatured DNA sections in which a high amount of Sytox Orange molecules were bound. This could give insight into the denaturation kinetics of DNA during fBALM. These results were further complemented by measurements of γH2AX and H3K9me3 signal organization to demonstrate differences within the chromatin landscape, which were quantified with image processing methods such as Voronoi segmentation.

Soft X-ray Tomography Reveals HSV-1-Induced Remodeling of Human B Cells.

Upon infection, viruses hijack the cell machinery and remodel host cell structures to utilize them for viral proliferation. Since viruses are about a thousand times smaller than their host cells, imaging virus-host interactions at high spatial resolution is like looking for a needle in a haystack. Scouting gross cellular changes with fluorescent microscopy is only possible for well-established viruses, where fluorescent tagging is developed. Soft X-ray tomography (SXT) offers 3D imaging of entire cells without the need for chemical fixation or labeling. Here, we use full-rotation SXT to visualize entire human B cells infected by the herpes simplex virus 1 (HSV-1). We have mapped the temporospatial remodeling of cells during the infection and observed changes in cellular structures, such as the presence of cytoplasmic stress granules and multivesicular structures, formation of nuclear virus-induced dense bodies, and aggregates of capsids. Our results demonstrate the power of SXT imaging for scouting virus-induced changes in infected cells and understanding the orchestration of virus-host remodeling quantitatively.

A protocol for full-rotation soft X-ray tomography of single cells.

The protocol describes step-by-step sample preparation, data acquisition, and segmentation of cellular organelles with soft X-ray tomography. It is designed for microscopes built to perform full-rotation data acquisition on specimens in cylindrical sample holders, such as the XM-2 microscope at the Advanced Light Source, LBNL; however, it might be generalized for similar sample holder designs for both synchrotron and table-top microscopes. For complete details on the use and execution of this profile, please refer to Loconte et al. (2021).

Fish primary embryonic pluripotent cells assemble into retinal tissue mirroring in vivo early eye development.

Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Mammalian organoids are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome these limitations and address species-specific cellular organization, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic pluripotent cells from medaka and zebrafish efficiently assemble into anterior neural structures, particularly retina. Within 4 days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis, and differentiation. The number of aggregated cells and genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High efficiency and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematic probing of impact of the physical environment on morphogenesis and differentiation.