Die konventionelle histopathologische Diagnostik stößt bei der Beurteilung komplexer, dreidimensionaler Gewebearchitekturen an inhärente methodische Grenzen. Insbesondere bei heterogen zusammengesetzten Geweben wie dem Pankreas oder bei komplexen Gewebspathologien erschwert die Beschränkung auf zweidimensionale Schnittbilder die ubiquitäre Erfassung morphologischer Merkmale. Ziel dieser Studie ist es, das Potenzial der synchrotronbasierten Phasenkontrastbildgebung (SRµCT) für die hochauflösende, dreidimensionale Visualisierung verschiedener Pankreasgewebe zu demonstrieren. Anhand dreier paradigmatischer Fallbeispiele werden morphologische Parameter volumetrisch erfasst und mit korrespondierenden immunhistochemischen Markerprofilen korreliert. Gewebestanzen aus formalinfixierten, paraffineingebetteten Blöcken von humanen Pankreasgewebeproben wurden mittels SRµCT volumetrisch erfasst. Das untersuchte Probenmaterial wurde als Microarrays weiterverarbeitet. Konsekutive Schnitte und immunhistochemische Färbungen wurden mit den 3D-Datensätzen korreliert. Die Bildgebung ermöglichte die differenzierte räumliche Darstellung funktioneller Kompartimente und neoplastischer Infiltrationsmuster. Nichtneoplastisches Gewebe zeigte klar abgegrenzte Kompartimente. Ein gut differenzierter neuroendokriner Tumor präsentierte trabekuläre Binnenstrukturen. Das duktale Adenokarzinom zeigte ein infiltratives Wachstumsmuster mit diffuser, heterogener Architektur, irregulären Gangformationen und Stromadesmoplasie. Die virtuelle Schnittführung ermöglichte die Analyse in jeder Raumrichtung. Durch Korrelation mit immunhistochemischen Markerprofilen konnten morphofunktionelle Merkmale validiert werden. Die SRµCT ist eine hochsensitive Methode, die nichtinvasiv und ohne Färbung dreidimensionale Einsichten in die Gewebearchitektur des Pankreas unter Nutzung archivierter Paraffinblöcke erlaubt. Die Methode bietet neue Perspektiven für Forschung, Lehre und potenziell erweiterte Spezialdiagnostik.

BACKGROUND:Conventional histopathology faces methodological limitations when assessing complex three-dimensional tissue architectures. In particular, for heterogeneous tissues such as the pancreas or in complex tissue pathologies, restriction to two-dimensional sections hampers comprehensive recognition of morphological features. OBJECTIVE:This study aims to demonstrate the potential of synchrotron-based phase-contrast imaging (SRµCT) as a tool for high-resolution visualization of pancreatic tissue. Three representative case examples were analyzed to capture morphological parameters volumetrically and correlate them with immunohistochemical marker profiles. MATERIALS AND METHODS:Tissue cores from formalin-fixed, paraffin-embedded human pancreatic samples were volumetrically assessed using SRµCT. The investigated material was further processed as microarrays. Serial sections and immunohistochemical stains were correlated with the 3D datasets. RESULTS:SRµCT enabled detailed spatial visualization of functional compartments and neoplastic infiltration patterns. Non-neoplastic tissue revealed distinct morphological compartments. A well-differentiated neuroendocrine tumor exhibited trabecular architecture, whereas ductal adenocarcinoma displayed infiltrative growth with diffuse, heterogeneous architecture, irregular duct formations and stromal desmoplasia. Virtual slicing permitted orientation-independent analyses. Correlation with immunohistochemical profiles validated the morphofunctional findings. CONCLUSION:SRµCT is a sensitive, non-invasive technique providing label-free 3D insights into pancreatic architecture. It opens new perspectives for research, teaching, and potentially advanced diagnostic applications.

Abstract Serial Block Face – Scanning Electron Microscopy (SBF-SEM) is a volume EM method suited to investigate the 3D architecture of tissues and even entire organisms at high resolution. However, imaging large volumes in their entirety is time-consuming and not always necessary. Many research projects have a focused interest in well-defined sub-regions of the samples. The targeting and acquisition of such regions of interest (ROIs) are however currently conducted in a manual way and require heavy involvement of experienced operators. We present a workflow and an original open-source software tool (iSBEM), which allow automated targeting of ROIs in a large tissue sample, based on X-ray microscopy (XRM) maps. After an initial ROI identification and registration of the XRM map with the sample mounted on the SBF-SEM stage, iSBEM takes over the control of the microscope, triggering high resolution acquisitions at defined ROI positions, with minimal user intervention. We demonstrate the approach on two biologically distinct specimens — malarial oocysts in infected mosquito midgut tissue, and immune cells in human kidney biopsies — achieving significant improvement in acquisition throughput relative to manual operations, without compromising targeting precision. We also showcase the workflow in a correlative light-Xray-electron microscopy setup, which allowed us to further improve the correct target definition.

Current understanding of anatomical structures of ascidians remains limited. This study presents multimodal imaging techniques, including Light, Thunder, and fluorescent confocal microscopy, to investigate neural structures and the tunic of Halocynthia papillosa, a common ascidian in the Mediterranean Sea. We demonstrate advanced 3D imaging methods, i.e., Magnetic Resonance Imaging, and High-Throughput Tomography (HiTT) at a synchrotron beamline. Imaging results show structural differences in the central nerve of H. papillosa compared to other ascidians and identify three distinct suborders of oral tentacles. We also document detailed autofluorescent patterns in ascidian cuticular sheds for the first time. HiTT imaging of the tunic reveals a spiralized structure emerging from cellulose layers. The state-of-the-art imaging techniques presented here encourage a broader use of HiTT to study functional anatomy in marine invertebrates. It establishes a strong foundation for future studies on solitary ascidians and highlights the need to expand research beyond model species.

X-ray phase-contrast tomography can efficiently image brain tissue at subcellular resolution. However, current sample preparation methods are not optimized to exploit the full potential of X-ray contrast mechanisms. Here we propose to replace interstitial material by air to enhance X-ray phase contrast of the ultrastructural features. Critical point drying (CPD) of heavy-metal-stained mouse brain tissue produced samples with preserved ultrastructure, a nanofoam-like material that remains compatible with follow-up conventional resin embedding. Using two synchrotron-based setups, namely, a high-throughput microtomography beamline and a nanoscale holographic tomography beamline, we found that CPD samples consistently showed 2–4× stronger phase-shift signal than samples embedded in resin. CPD offers a versatile route for preparing tissue for subcellular and ultrastructural-resolution X-ray imaging. It retains structural detail while improving signal, and is compatible with follow-up protocols involving femtosecond laser milling or electron microscopy, paving the path for biological tissue imaging beyond the mm3 scale.

Accurate diagnosis and characterization of lung disease increasingly rely on advanced imaging modalities capable of resolving fine microstructural details while minimizing radiation exposure. Phase-sensitive computed tomography (CT), particularly propagation-based imaging (PBI), offers superior soft tissue contrast but has historically been limited by the lack of compatible fixation techniques that preserve lung architecture post-excision. We present an adapted formaldehyde (FA) vapour fixation protocol designed to maintain human-sized lungs in a physiologically inflated and morphologically stable state. This approach prevents collapse of the delicate air–tissue interfaces, a major barrier to high-fidelity phase-contrast imaging and histological correlation. Our method enables high-resolution, multiscale imaging from whole-organ PBI at 67 µm voxel size to localized subcellular synchrotron PBI at 650 nm voxel size on the same specimen, with preserved spatial relationships critical for accurate validation of imaging findings. In porcine models, FA vapour fixation maintained alveolar integrity and radiological contrast without compromising histological detail, while also avoiding the artifacts associated with liquid fixation. Crucially, the protocol allows regulation of inflation and fixation dynamics, addressing longstanding challenges in ex vivo lung imaging and enabling consistent specimen preparation across studies. This fixation technique supports biosafe stabilization of freshly explanted human lungs–such as those from transplant procedures creating new opportunities for translational research on pathological tissue. By bridging high-resolution radiology and histopathology, our scalable fixation protocol establishes a standardized foundation for multimodal lung imaging and offers a critical tool for advancing both fundamental lung research and clinical diagnostics.

Synchrotron-based tomographic phase-contrast X-ray imaging (SRµCT or SRnCT) is a versatile isotropic three-dimensional imaging technique that can be used to study biological samples spanning from single cells to human-sized specimens. SRµCT and SRnCT take advantage of the highly brilliant and coherent X-rays produced by a synchrotron light source. This enables fast data acquisition and enhanced image contrast for soft biological samples owing to the exploitation of phase contrast. In this Review, we provide an overview of the basics behind the technique, discuss its applications for biologists and provide an outlook on the future of this emerging technique for biology. We introduce the latest advances in the field, such as whole human organs imaged with micron resolution, using X-rays as a tool for virtual histology and resolving neuronal connections in the brain.

Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer’s disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology.

Here, high-throughput tomography (HiTT), a fast and versatile phase-contrast imaging platform for life-science samples on the EMBL beamline P14 at DESY in Hamburg, Germany, is presented. A high-photon-flux undulator beamline is used to perform tomographic phase-contrast acquisition in about two minutes which is linked to an automated data processing pipeline that delivers a 3D reconstructed data set less than a minute and a half after the completion of the X-ray scan. Combining this workflow with a sophisticated robotic sample changer enables the streamlined collection and reconstruction of X-ray imaging data from potentially hundreds of samples during a beam-time shift. HiTT permits optimal data collection for many different samples and makes possible the imaging of large sample cohorts thus allowing population studies to be attempted. The successful application of HiTT on various soft tissue samples in both liquid (hydrated and also dehydrated) and paraffin-embedded preparations is demonstrated. Furthermore, the feasibility of HiTT to be used as a targeting tool for volume electron microscopy, as well as using HiTT to study plant morphology, is demonstrated. It is also shown how the high-throughput nature of the work has allowed large numbers of `identical' samples to be imaged to enable statistically relevant sample volumes to be studied.

AbstractProtein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer’s disease (AD), which is characterized by self-assembly and accumulation of Amyloid-beta in the brain. Furthermore, impaired proteostatic mechanisms have been implicated in NDs. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell. Using a model of AD neurodegeneration where toxic amyloid beta oligomers are applied exogenously to primary hippocampal neurons, we have previously established that oligomers are endocytosed and trafficked to lysosomes where they accumulate and prevent further endocytosis. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state. We demonstrate that lysosomes in oligomer treated neurons are less X-ray dense suggesting they contain less carbon-rich material than untreated cells. Furthermore, we observe an increase in carbon dense lipid vesicles in oligomer treated cells. This work provides unprecedented and critical visual information on the changes to neuronal architecture inflicted by amyloid beta oligomers using advanced methods in structural cell biology.

Bioimaging data have significant potential for reuse, but unlocking this potential requires systematic archiving of data and metadata in public databases. We propose draft metadata guidelines to begin addressing the needs of diverse communities within light and electron microscopy. We hope this publication and the proposed Recommended Metadata for Biological Images (REMBI) will stimulate discussions about their implementation and future extension.

Cryo-soft X-ray tomography is an imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for staining or chemical modification. It allows the imaging of cellular ultrastructure to a resolution of 25–40 nm and can be used in correlation with other imaging modalities, such as electron tomography and fluorescence microscopy, to further enhance the information content derived from biological samples. An overview of the technique, discussion of sample suitability and information about sample preparation, data collection and data analysis is presented here. Recent developments and future outlook are also discussed.

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.

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Segmentation of biological volumes is a crucial step needed to fully analyse their scientific content. Not having access to convenient tools with which to segment or annotate the data means many biological volumes remain under-utilised. Automatic segmentation of biological volumes is still a very challenging research field, and current methods usually require a large amount of manually-produced training data to deliver a high-quality segmentation. However, the complex appearance of cellular features and the high variance from one sample to another, along with the time-consuming work of manually labelling complete volumes, makes the required training data very scarce or non-existent. Thus, fully automatic approaches are often infeasible for many practical applications. With the aim of unifying the segmentation power of automatic approaches with the user expertise and ability to manually annotate biological samples, we present a new workbench named SuRVoS (Super-Region Volume Segmentation). Within this software, a volume to be segmented is first partitioned into hierarchical segmentation layers (named Super-Regions) and is then interactively segmented with the user's knowledge input in the form of training annotations. SuRVoS first learns from and then extends user inputs to the rest of the volume, while using Super-Regions for quicker and easier segmentation than when using a voxel grid. These benefits are especially noticeable on noisy, low-dose, biological datasets.

Journal Article Soft X-Ray Tomography: Filling the Gap Between Light and Electrons for Imaging Hydrated Biological Cells Get access Lucy M Collinson, Lucy M Collinson The Francis Crick Institute, 1 Midland Road, London, UK Search for other works by this author on: Oxford Academic Google Scholar Marie-Charlotte Domart, Marie-Charlotte Domart The Francis Crick Institute, 1 Midland Road, London, UK Search for other works by this author on: Oxford Academic Google Scholar Raffaella Carzaniga, Raffaella Carzaniga The Francis Crick Institute, 1 Midland Road, London, UK Search for other works by this author on: Oxford Academic Google Scholar Minoo Razi, Minoo Razi The Francis Crick Institute, 1 Midland Road, London, UK Search for other works by this author on: Oxford Academic Google Scholar Peter Guttmann, Peter Guttmann Helmholtz-Zentrum Berlin fur Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, Berlin, Germany Search for other works by this author on: Oxford Academic Google Scholar Gerd Schneider, Gerd Schneider Helmholtz-Zentrum Berlin fur Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, Berlin, Germany Search for other works by this author on: Oxford Academic Google Scholar Eva Pereiro, Eva Pereiro ALBA Synchrotron Light Source, 08290 Cerdanyola del Valles, Barcelona, Spain Search for other works by this author on: Oxford Academic Google Scholar Sharon A Tooze, Sharon A Tooze The Francis Crick Institute, 1 Midland Road, London, UK Search for other works by this author on: Oxford Academic Google Scholar Elizabeth Duke Elizabeth Duke Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxon, UK Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 23, Issue S1, 1 July 2017, Pages 986–987, https://doi.org/10.1017/S1431927617005591 Published: 04 August 2017

Cryo-soft X-ray tomography (cryo-SXT) is a synchrotron-hosted imaging technique used to analyze the ultrastructure of intact, cryo-prepared cells. Correlation of cryo-fluorescence microscopy and cryo-SXT can be used to localize fluorescent proteins to organelles preserved close to native state. Cryo-correlative light and X-ray microscopy (cryo-CLXM) is particularly useful for the study of organelles that are susceptible to chemical fixation artifacts during sample preparation for electron microscopy. In our recent work, we used cryo-CLXM to characterize GFP-LC3-positive early autophagosomes in nutrient-starved HEK293A cells (Duke et al., 2013). Cup-shaped omegasomes were found to form at "hot-spots" on the endoplasmic reticulum. Furthermore, cryo-SXT image stacks revealed the presence of large complex networks of tubulated mitochondria in the starved cells, which would be challenging to model at this scale and resolution using light or electron microscopy. In this chapter, we detail the cryo-CLXM workflow that we developed and optimized for studying adherent mammalian cells. We show examples of data collected at the three European synchrotrons that currently host cryo-SXT microscopes, and describe how raw cryo-SXT datasets are processed into tomoX stacks, modeled, and correlated with cryo-fluorescence data to identify structures of interest.

One of the ultimate aims of imaging in biology is to achieve molecular localisation in the context of the structure of cells in their native state. Here, we review the current state of the art in cryo-soft X-ray tomography (cryo-SXT), which is the only imaging modality that can provide nanoscale 3D information from cryo-preserved, unstained, whole cells thicker than 1 μm. Correlative cryo-fluorescence and cryo-SXT adds functional information to structure, enabling studies of cellular events that cannot be captured using light, electron or X-ray microscopes alone.