Compensation of x-ray mirror distortion by cooling temperature control

Synchrotron radiation is emitted from a bending magnet source in a wide ray fan which is collected by the first optical element in a beamline. In order to maximize angular acceptance, and hence flux, it is beneficial to increase the length of this mirror and optical design requirements may necessitate that the optical surface be over 1 m in length. Such mirrors also require cooling as they may be subject to high heat loads from the incident radiation. Two beamlines, B07 and B24, at Diamond Light Source, UK, use 1.4 m long toroidal mirrors which utilize a similar side-clamped cooling manifold design. While this scheme has been successful in providing effective cooling of the mirror, it has also been discovered that it introduces deformation of the radius of curvature which is sufficient to alter the focusing characteristics of the mirror. At both beamlines, the horizontal focus of the beam was found to differ by up to several meter from the design position at the exit slit which resulted in poor flux throughput, reduced energy resolution and other side effects. A pencil beam scan method has been used to diagnose this issue and infer the position of the focus and mirror shape. Through the use of a standalone chiller to alter the temperature of the water within the cooling loop, it has been possible to correct the distortion of the radius and restore the focus to its nominal position

Single-cell chemistry of photoactivatable platinum anticancer complexes

The Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)2(N3)2(OH)2] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)2(N3)2(OH)(coumarin-3-carboxylate)] (Pt2) are promising agents for photoactivated chemotherapy. These complexes are inert in the dark but release Pt(II) species and radicals upon visible light irradiation, resulting in photocytotoxicity toward cancer cells. Here, we have used synchrotron techniques to investigate the in-cell behavior of these prodrugs and visualize, for the first time, changes in cellular morphology and Pt localization upon treatment with and without light irradiation. We show that photoactivation of Pt2 induces remarkable cellular damage with extreme alterations to multiple cellular components, including formation of vacuoles, while also significantly increasing the cellular accumulation of Pt species compared to dark conditions. X-ray absorption near-edge structure (XANES) measurements in cells treated with Pt2 indicate only partial reduction of the prodrug upon irradiation, highlighting that phototoxicity in cancer cells may involve not only Pt(II) photoproducts but also photoexcited Pt(IV) species

Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress.

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress

An integrated approach to elucidate the interplay between iron uptake dynamics and magnetosome formation at the single-cell level in Magnetospirillum gryphiswaldense

Iron is a crucial element integral to various fundamental biological molecular mechanisms, including magnetosome biogenesis in magnetotactic bacteria (MTB). Magnetosomes are formed through the internalization and biomineralization of iron into magnetite crystals. However, the interconnected mechanisms by which MTB uptake and regulate intracellular iron for magnetosome biomineralization remain poorly understood, particularly at the single-cell level. To gain insights we employed a holistic multiscale approach, i.e., from elemental iron species to bacterial populations, to elucidate the interplay between iron uptake dynamics and magnetosome formation in Magnetospirillum gryphiswaldense MSR-1 under near-native conditions. We combined a correlative microscopy approach integrating light and X-ray tomography with analytical techniques, such as flow cytometry and inductively coupled plasma spectroscopy, to evaluate the effects of iron and oxygen availability on cellular growth, magnetosome biogenesis, and intracellular iron pool in MSR-1. Our results revealed that increased iron availability under microaerobic conditions significantly promoted the formation of longer magnetosome chains and increased intracellular iron uptake, with a saturation point at 300 μM iron citrate. Beyond this threshold, additional iron did not further extend the magnetosome chain length or increase total intracellular iron levels. Moreover, our work reveals (i) a direct correlation between the labile Fe2+ pool size and magnetosome content, with higher intracellular iron concentrations correlating with increased magnetosome production, and (ii) the existence of an intracellular iron pool, distinct from magnetite, persisting during all stages of biomineralization. This study offers insights into iron dynamics in magnetosome biomineralization at a single-cell level, potentially enhancing the industrial biomanufacturing of magnetosomes

CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.Funding: Wellcome Trust (091911/Z/11/Z, 096144/Z/11/Z, 105605/Z/14/Z, 107457/Z/15/Z, 203141/Z/16/Z, 209412/Z/17/Z); H2020Marie Skłodowska-Curie Actions (700184)

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 µm thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology

Single Cell Cryo-Soft X-ray Tomography Shows That Each Chlamydia Trachomatis Inclusion Is a Unique Community of Bacteria

Chlamydiae are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. Therefore, each infected cell can, be considered as a single entity where bacteria form a community within the inclusion. It remains unclear as to how the population of bacteria within the inclusion influences individual bacterium. The life cycle of Chlamydia involves transitioning between the invasive elementary bodies (EBs) and replicative reticulate bodies (RBs). We have used cryo-soft X-ray tomography to observe individual inclusions, an approach that combines 40 nm spatial resolution and large volume imaging (up to 16 µm). Using semi-automated segmentation pipeline, we considered each inclusion as an individual bacterial niche. Within each inclusion, we identifyed and classified different forms of the bacteria and confirmed the recent finding that RBs have a variety of volumes (small, large and abnormal). We demonstrate that the proportions of these different RB forms depend on the bacterial concentration in the inclusion. We conclude that each inclusion operates as an autonomous community that influences the characteristics of individual bacteria within the inclusion

Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures in mammalian cells

Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Funder: Medical Research CouncilSince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules