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At least 19 records

Interior soft x-ray tomography with sparse global sampling

To investigate the feasibility of interior imaging reconstruction in soft X-ray tomography for higher-resolution cellular imaging, including whole-cell imaging, we develop an alignment and reconstruction algorithm that combines a small number of sparse whole-cell images with a high-resolution local interior scan. Based on numerical simulations, we demonstrate that combined reconstructions mitigate the depth-of-field limitation in high-resolution scans, enable radiation dose optimization, and yield quantitative X-ray absorption values with sparse sampling. We further validate our numerical approach using experimental data from two different cell types and show that the combined reconstruction reliably provides high spatial resolution within an interior region of interest of a whole cell. The resulting sparse reconstruction framework offers robust, faithful visualization of cellular organelles in soft X-ray tomography. This mesoscale imaging strategy allows one to ‘scout’ and zoom into selected subcellular volumes of interest, enabling increased spatial resolution without sacrificing larger-volume imaging and providing information on the relative positions of all organelles within a cell.

3D imaging↗

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.

59 BASIC BIOLOGICAL SCIENCES↗

Soft X-Ray Tomography Has Evolved into a Powerful Tool for Revealing Cell Structures

Over the past three decades, soft X-ray tomography (SXT) has rapidly evolved from a proof-of-concept microscopy method into a high-throughput quantitative imaging modality. This advancement enables researchers to address central questions in cell biology. Despite its relatively short developmental period compared to light and electron microscopy, SXT has emerged as a powerful imaging technology. It enables measuring chemical changes in cellular organelles, analyzing three-dimensional structures of whole cells and creating digital cellular models to study cell motility. We discuss the unique nature of SXT to visualize cells without fixation or labeling, enabling quantitative analyses of organelle chemical composition. We explore SXT microscopes available worldwide, SXT segmentation software, and the diverse cell types studied using this technique. We conclude with emerging directions in SXT imaging, including a brief discussion of recent discoveries that are highly influential and likely to become integral to cell biology textbooks.

Weinhardt, Venera↗

Automated segmentation of soft X-ray tomography: Native cellular structure with submicron resolution at high-throughput for whole-cell quantitative imaging in yeast

Soft X-ray tomography (SXT) is an invaluable tool for quantitatively analyzing cellular structures at suboptical isotropic resolution. However, it has traditionally depended on manual segmentation, limiting its scalability for large datasets. Here, we leverage a deep learning-based autosegmentation pipeline to segment and label cellular structures in hundreds of cells across three Saccharomyces cerevisiae strains. This task-based pipeline uses manual iterative refinement to improve segmentation accuracy for key structures, including the cell body, nucleus, vacuole, and lipid droplets, enabling high-throughput and precise phenotypic analysis. Using this approach, we quantitatively compared the three-dimensional (3D) whole-cell morphometric characteristics of wild-type, VPH1-GFP, and vac14 strains, uncovering detailed strain-specific cell and organelle size and shape variations. We show the utility of SXT data for precise 3D curvature analysis of entire organelles and cells and detection of fine morphological features using surface meshes. Our approach facilitates comparative analyses with high spatial precision and statistical throughput, uncovering subtle morphological features at the single-cell and population level. This workflow significantly enhances our ability to characterize cell anatomy and supports scalable studies on the mesoscale, with applications in investigating cellular architecture, organelle biology, and genetic research across diverse biological contexts.

Chen, Jianhua [Lawrence Berkeley National Laborato↗

Subcellular Feature-Based Classification of α and β Cells Using Soft X-ray Tomography

The dysfunction of α and β cells in pancreatic islets can lead to diabetes. Many questions remain on the subcellular organization of islet cells during the progression of disease. Existing three-dimensional cellular mapping approaches face challenges such as time-intensive sample sectioning and subjective cellular identification. To address these challenges, we have developed a subcellular feature-based classification approach, which allows us to identify α and β cells and quantify their subcellular structural characteristics using soft X-ray tomography (SXT). We observed significant differences in whole-cell morphological and organelle statistics between the two cell types. Additionally, we characterize subtle biophysical differences between individual insulin and glucagon vesicles by analyzing vesicle size and molecular density distributions, which were not previously possible using other methods. These sub-vesicular parameters enable us to predict cell types systematically using supervised machine learning. We also visualize distinct vesicle and cell subtypes using Uniform Manifold Approximation and Projection (UMAP) embeddings, which provides us with an innovative approach to explore structural heterogeneity in islet cells. This methodology presents an innovative approach for tracking biologically meaningful heterogeneity in cells that can be applied to any cellular system.

3D cell mapping↗

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.

59 BASIC BIOLOGICAL SCIENCES↗

Soft X-ray tomography reveals variations in B. subtilis biofilm structure upon tasA deletion

Bacterial biofilms are complex cell communities within a self-produced extracellular matrix, crucial in various fields but challenging to analyze in 3D. We developed a "biofilm-in-capillary" growth method compatible with full-rotation soft X-ray tomography, enabling high-resolution 3D imaging of bacterial cells and their matrix during biofilm formation. This approach offers 50 nm isotropic spatial resolution, rapid imaging, and quantitative native analysis of biofilm structure. Using Bacillus subtilis biofilms, we detected coherent alignment and chaining of wild-type cells towards the oxygen-rich capillary tip. In contrast, the ΔtasA genetic knock-out showed a loss of cellular orientation and changes in the extracellular matrix. Adding TasA protein to the ΔtasA strain restored matrix density and led to cell assembly compaction, but without the chaining observed in wild-type biofilms. This scalable and transferable approach opens new avenues for examining biofilm structure and function across various species, including mixed biofilms, and response to genetic and environmental factors.

59 BASIC BIOLOGICAL SCIENCES↗

Classifying biophysical subpopulations of insulin secretory granules using quantitative whole-cell structure analysis

Pancreatic beta cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft X-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole beta cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts toward mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary beta cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

dense-core granules↗

Secretory stimuli distinctly regulate insulin secretory granule maturation through structural remodeling

Insulin secretory granule (ISG) maturation is a crucial aspect of insulin secretion and glucose homeostasis. The regulation of this maturation remains poorly understood, especially how secretory stimuli affect ISG maturity and subcellular localization. In this study, we used soft X-ray tomography (SXT) to quantitatively map ISG morphology, density, and location in single INS-1E and mouse pancreatic β cells under the effect of various secretory stimuli. We found that the activation of glucokinase (GK), gastric inhibitory polypeptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP-1R), and G protein-coupled receptor 40 (GPR40) promotes ISG maturation. Each stimulus induces unique structural remodeling in ISGs, by altering size and density, depending on the specific signaling cascades activated. These distinct ISG subpopulations mobilize and redistribute in the cell, altering the overall cellular structural organization. Our results provide insight into how current diabetes and obesity therapies impact ISG maturation and may inform the development of future treatments that target maturation specifically.

insulin granule maturation↗

An intensity-based post-processing tool for 3D instance segmentation of organelles in soft X-ray tomograms

Investigating the 3D structures and rearrangements of organelles within a single cell is critical for better characterizing cellular function. Imaging approaches such as soft X-ray tomography have been widely applied to reveal a complex subcellular organization involving multiple inter-organelle interactions. However, 3D segmentation of organelle instances has been challenging despite its importance in organelle characterization. Here we propose an intensity-based post-processing tool to identify and separate organelle instances. Our tool separates sphere-like (insulin vesicle) and columnar-shaped organelle instances (mitochondrion) based on the intensity of raw tomograms, semantic segmentation masks, and organelle morphology. We validate our tool using synthetic tomograms of organelles and experimental tomograms of pancreatic β -cells to separate insulin vesicle and mitochondria instances. As compared to the commonly used connected regions labeling, watershed, and watershed + Gaussian filter methods, our tool results in improved accuracy in identifying organelles in the synthetic tomograms and an improved description of organelle structures in β -cell tomograms. In addition, under different experimental treatment conditions, significant changes in volumes and intensities of both insulin vesicle and mitochondrion are observed in our instance results, revealing their potential roles in maintaining normal β -cell function. Our tool is expected to be applicable for improving the instance segmentation of other images obtained from different cell types using multiple imaging modalities.

59 BASIC BIOLOGICAL SCIENCES↗

Spin Textures at Conformal Surfaces and Interfaces: CRADA Final Report

The scientific and technological exploration of artificially designed three-dimensional magnetic nanostructures opens the path to exciting novel physical phenomena, originating from the increased complexity in spin textures, topology, and frustration in three dimensions. Theory predicts that the equilibrium magnetic ground state of two-dimensional systems which reflects the competition between symmetric (Heisenberg) and antisymmetric (Dzyaloshinskii-Moriya interaction (DMI)) exchange interaction is significantly modified on curved surfaces when the radius of local curvature becomes comparable to fundamental magnetic length scales. In this CRADA we performed collaboratively an experimental study of the spin texture in an 8 nm thin magnetic multilayer with growth-induced in-plane anisotropy and DMI deposited onto the curved surface of a 1.8µm long non-magnetic carbon nanowire with a 67 nm radius. The sample was fabricated in Juelich and with collaborators at BESSY in Berlin. Using magnetic soft x-ray tomography at the ALBA synchrotron in Spain, the three-dimensional spin configuration in this nanotube was retrieved with about 30nm spatial resolution. The transition between two vortex configurations on the two ends of the nanotube with opposite circulation occurs through a domain wall that is aligned at an inclined angle relative to the wire axis. Three-dimensional micromagnetic simulations support the experimental observations and represent a visualization of the curvature-mediated DMI. They also allow a quantitative estimate of the DMI value for the magnetic multilayered nanotube.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Self-Assembled Magnetic Nanoparticle Layers: Structural Control for Reconfigurable Magnetism and Functional Applications

We advance soft X-ray vector ptychographic tomography to map the 3D magnetization field in self-assembled superparamagnetic nanoparticles at a liquid–liquid interface, revealing how layered structures influence magnetic ordering. We observe that monolayers with low coordination numbers exhibit weak magnetic order, with magnetic vortices disrupting spin alignment. In contrast, bilayers and trilayers with higher coordination numbers display long-range magnetic order with strong spin correlations across larger distances and a suppression of magnetic vortices. We further quantify the average distance for vortex–antivortex pairs as 26.0 ± 2.0 nm, while vortex–vortex and antivortex–antivortex pairs exhibit larger separations, averaging 44.9 ± 5.2 and 54.1 ± 7.4 nm, respectively. These experimental results are supported by micromagnetic Monte Carlo simulations. Our findings illustrate how layered structures enhance magnetic order and spin correlation in superparamagnetic nanoparticle assemblies, providing a promising approach for tuning magnetic properties in applications such as data storage, microrobotics, and biomedicine.

Lu, Xingyuan↗

Three-dimensional topological magnetic monopoles and their interactions in a ferromagnetic meta-lattice

Topological magnetic monopoles (TMMs), also known as hedgehogs or Bloch points, are three-dimensional (3D) nonlocal spin textures that are robust to thermal and quantum fluctuations due to the topology protection. Although TMMs have been observed in skyrmion lattices, spinor Bose–Einstein condensates, chiral magnets, vortex rings and vortex cores, it has been difficult to directly measure the 3D magnetization vector field of TMMs and probe their interactions at the nanoscale. Here, we report the creation of 138 stable TMMs at the specific sites of a ferromagnetic meta-lattice at room temperature. We further develop soft x-ray vector ptycho-tomography to determine the magnetization vector and emergent magnetic field of the TMMs with a 3D spatial resolution of 10 nm. This spatial resolution is comparable to the magnetic exchange length of transition metals, enabling us to probe monopole-monopole interactions. We find that the TMM and anti-TMM pairs are separated by 18.3±1.6 nm, while the TMM and TMM, anti-TMM and anti-TMM pairs are stabilized at comparatively longer distances of 36.1±2.4 nm and 43.1±2.0 nm, respectively. We also observe virtual TMMs created by magnetic voids in the meta-lattice. This work demonstrates that ferromagnetic meta lattices could be used as a platform to create and investigate the interactions and dynamics of TMMs. Furthermore, we expect that soft x-ray vector ptycho-tomography can be broadly applied to quantitatively image 3D vector fields in magnetic and anisotropic materials at the nanoscale.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Nondestructively Visualizing and Understanding the Mechano-Electro-chemical Origins of “Soft Short” and “Creeping” in All-Solid-State Batteries

All-solid-state Li-metal batteries (ASLMBs) represent a significant breakthrough in the quest to overcome limitations associated with traditional Li-ion batteries, particularly in energy density and safety aspects. However, widespread implementation is stymied due to a lack of profound understanding of the complex mechano-electro-chemical behavior of Li metal in the ASLMBs. Herein, operando neutron imaging and X-ray computed tomography (XCT) are leveraged to nondestructively visualize Li behaviors within ASLMBs. This approach offers real-time observations of Li evolutions, both pre- and post- occurrence of a “soft short”. The coordination of 2D neutron radiography and 3D neutron tomography enables charting of the terrain of Li metal deformation operando. Concurrently, XCT offers a 3D insight into the internal structure of the battery following a “soft short”. Despite the manifestation of a “soft short”, the persistence of Faradaic processes is observed. To study the elusive “soft short”, phase field modeling is coupled with electrochemistry and solid mechanics theory. The research unravels how external pressure curbs dendrite growth, potentially leading to dendrite fractures and thus uncovering the origins of both “soft” and “hard” shorts in ASLMBs. Furthermore, by harnessing finite element modeling, it dive deeper into the mechanical deformation and the fluidity of Li metal.

25 ENERGY STORAGE↗

Controlled Self-Assembly of Gold Nanotetrahedra into Quasicrystals and Complex Periodic Supracrystals

The self-assembly of shape-anisotropic nanocrystals into large-scale structures is a versatile and scalable approach to creating multifunctional materials. The tetrahedral geometry is ubiquitous in natural and manmade materials, yet regular tetrahedra present a formidable challenge in understanding their self-assembly behavior as they do not tile space. Here, we report diverse supracrystals from gold nanotetrahedra including the quasicrystal (QC) and the dimer packing predicted more than a decade ago and hitherto unknown phases. We solve the complex three-dimensional (3D) structure of the QC by a combination of electron microscopy, tomography, and synchrotron X-ray scattering. Nanotetrahedron vertex sharpness, surface ligands, and assembly conditions work in concert to regulate supracrystal structure. We also discover that the surface curvature of supracrystals can induce structural changes of the QC tiling and eventually, for small supracrystals with high curvature, stabilize a hexagonal approximant. Finally, our findings bridge the gap between computational design and experimental realization of soft matter assemblies and demonstrate the importance of accurate control over nanocrystal attributes and the assembly conditions to realize increasingly complex nanopolyhedron supracrystals.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Tomographic detection of photon pairs produced from high-energy X-rays for the monitoring of radiotherapy dosing

Measuring the radiation dose reaching a patient’s body is difficult. Here we report a technique for the tomographic reconstruction of the location of photon pairs originating from the annihilation of positron–electron pairs produced by high-energy X-rays travelling through tissue. We used Monte Carlo simulations on pre-recorded data from tissue-mimicking phantoms and from a patient with a brain tumour to show the feasibility of this imaging modality, which we named ‘pair-production tomography’, for the monitoring of radiotherapy dosing. We simulated three image-reconstruction methods, one applicable to a pencil X-ray beam scanning through a region of interest, and two applicable to the excitation of tissue volumes via broad beams (with temporal resolution sufficient to identify coincident photon pairs via filtered back projection, or with higher temporal resolution sufficient for the estimation of a photon’s time-of-flight). In addition to the monitoring of radiotherapy dosing, we show that image contrast resulting from pair-production tomography is highly proportional to the material’s atomic number. The technique may thus also allow for element mapping and for soft-tissue differentiation.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

An optical-input Maximum Likelihood Estimation feedback system demonstrated on tokamak horizontal equilibrium control

A readily parallelized Maximum Likelihood Estimation (MLE) algorithm with linear computational complexity is demonstrated in real time using only measurements from an extreme ultraviolet (EUV) diagnostic to control the horizontal position of a tokamak plasma. A set of trial emissivity profiles are parameterized by the control quantity of interest (R m ), and the MLE is identified from the profile which minimizes the signal reconstruction residual. The algorithm depends on an empirically determined likelihood function with exponential form. EUV emission (λ ≈ 15eV-1keV) is captured in a poloidal plane by four 16-channel AXUV diodes mounted at different poloidal angles with radial and angular resolution sufficient to discern plasma equilibrium evolution in HBT-EP. Calculations of the plasma major radius by the system are consistent within diagnostic uncertainty for the majority of the discharge with those of: a weighted average of vertical soft X-ray or EUV chords, magnetic sensors, and an equilibrium reconstruction. The feedback system corrects for a horizontal displacement of the major radius equal to 20% of the plasma minor radius by adjusting the vertical field produced from 40 in-vessel control coils in real time. The MLE calculation is performed on a GPU in a 15 μs cycle, with similar performance in this application to a simple weighted average of vertical chords. Finally, results demonstrate horizontal position control using magnetic actuators and an optical observer.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗