Biology subjects

Sviben, S.

Publications and source records attributed to Sviben, S..

10 recordsLinked to original sources

bioRxiv2026

Shaping cryogenic 3D volume imaging by pFIB ion species and milling voltage

Cryogenic plasma focused ion beam-scanning electron microscopy (cryo-pFIB-SEM) enables nanoscale volume imaging of vitrified cells and tissues, but the fidelity of each newly exposed block-face depends on how ions interact with compositionally heterogeneous biological material. Using xenon, argon, oxygen, and nitrogen plasma beams across a 30-2 kV voltage range, we examined how ion species and accelerating voltage influence block-face quality and ultrastructural fidelity. Curtaining imposed a species-dependent lower limit on accelerating voltage, with xenon and argon supporting uniform milling to 10 kV and oxygen and nitrogen to 15 kV. Monte Carlo simulations predicted substantially reduced ion penetration and atomic displacement under these species-specific low-kV conditions. Experimentally, lowering milling voltage suppressed charging and topographic artifacts, with the largest effects observed at heterogeneous interfaces. Oxygen and nitrogen produced greater membrane sharpness and organelle contrast than the noble gases, and these differences influenced machine-learning-based recognition of fine organelle features in 3D datasets. Together, these findings show that cryo-pFIB-SEM image quality cannot be optimized by accelerating voltage or ion species independently. Instead, milling performance reflects a tradeoff among surface fidelity, charging, contrast, and curtaining that depends on local specimen composition. Species-specific low-kV milling therefore provides a practical framework for matching cryo-pFIB-SEM acquisition conditions to the structural and biophysical properties of the biological target.

cell biology↗

bioRxiv2026

Depth-resolved analysis reveals target-dependent cryo-FIB damage profiles

Cryogenic focused ion beam (cryo-FIB) milling enables cellular cryo-electron tomography but can compromise high-resolution structural information near milled surfaces. Here we establish recombinant human apoferritin (apoF) as a reporter of depth-dependent structural preservation in cellular lamellae. ApoF yielded a 2.05 [A] in situ reconstruction. Apparent FIB damage was most pronounced within the first 15 nm for xenon, 30 nm for gallium, 45 nm for argon and remained detectable at least 75 nm from oxygen-milled surfaces under the workflows tested. Endogenous 70S ribosomes in the same xenon-milled tomograms showed a broader 30-45 nm profile, whereas {beta}-galactosidase showed a similar narrow xenon-associated profile in a different specimen. Xenon-milled lamellae 125-150 nm thick yielded the highest resolution among the thickness groups examined, with lower resolution in the thinner and thickest groups. These findings show structural preservation is constrained by both surface-associated damage and lamella thickness, with the apparent extent of damage varying by molecular reporter. Previous ribosome-based surface-exclusion estimates may therefore be overly conservative for some molecular targets.

molecular biology↗

bioRxiv2025

Contextual High-throughput 3D Volume Electron Microscopy Data Acquisition Using Artificial Intelligence

One of the greatest challenges when generating large and/or high-resolution three-dimensional (3D) volume electron microscopy (vEM) datasets is long acquisition times. We developed a method that leverages Artificial Intelligence (AI) algorithms to increase acquisition throughput for 3D datasets by creating an AI-derived mask for the targeted region of interest, referred to as Adaptive Scanning. This allowed for specific structures to be imaged at high resolution, with the surrounding area captured at lower resolution, without artificially generating or enhancing any raw data. We demonstrate that this dynamic-resolution scanning approach significantly reduced volume acquisition time across a diverse array of organisms and tissues, including brains, parasites, cultured cells, and plants. This multi-resolution strategy has the potential to enhance Focused Ion Beam Scanning Electron Microscopy and other forms of vEM, by increasing time savings by up to 2-fold or more, enabling routine generation of multiple and/or larger vEM datasets, more efficiently and cost-effectively, allowing more data collection for increase statistical power for comparative studies.

cell biology↗

bioRxiv2024

Immuno-Scanning Electron Microscopy of Islet Primary Cilia

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that contain specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immuno-labeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the 3D ultrastructure of the cilium. Here we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT) 88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immuno-labeling, and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

cell biology↗

bioRxiv2023

Endogenous recapitulation of Alzheimers disease neuropathology through human 3D direct neuronal reprogramming

Alzheimers disease (AD) is a neurodegenerative disorder that primarily affects elderly individuals, and is characterized by hallmark neuronal pathologies including extracellular amyloid-{beta} (A{beta}) plaque deposition, intracellular tau tangles, and neuronal death. However, recapitulating these age-associated neuronal pathologies in patient-derived neurons has remained a significant challenge, especially for late-onset AD (LOAD), the most common form of the disorder. Here, we applied the high efficiency microRNA-mediated direct neuronal reprogramming of fibroblasts from AD patients to generate cortical neurons in three-dimensional (3D) Matrigel and self-assembled neuronal spheroids. Our findings indicate that neurons and spheroids reprogrammed from both autosomal dominant AD (ADAD) and LOAD patients exhibited AD-like phenotypes linked to neurons, including extracellular A{beta} deposition, dystrophic neurites with hyperphosphorylated, K63-ubiquitin-positive, seed-competent tau, and spontaneous neuronal death in culture. Moreover, treatment with {beta}- or {gamma}-secretase inhibitors in LOAD patient-derived neurons and spheroids before A{beta} deposit formation significantly lowered A{beta} deposition, as well as tauopathy and neurodegeneration. However, the same treatment after the cells already formed A{beta} deposits only had a mild effect. Additionally, inhibiting the synthesis of age-associated retrotransposable elements (RTEs) by treating LOAD neurons and spheroids with the reverse transcriptase inhibitor, lamivudine, alleviated AD neuropathology. Overall, our results demonstrate that direct neuronal reprogramming of AD patient fibroblasts in a 3D environment can capture age-related neuropathology and reflect the interplay between A{beta} accumulation, tau dysregulation, and neuronal death. Moreover, miRNA-based 3D neuronal conversion provides a human-relevant AD model that can be used to identify compounds that can potentially ameliorate AD-associated pathologies and neurodegeneration.

neuroscience↗

bioRxiv2023

Scanning electron microscopy of human islet cilia

Human islet primary cilia are vital glucose-regulating organelles whose structure remains uncharacterized. Scanning electron microscopy (SEM) is a useful technique for studying the surface morphology of membrane projections like primary cilia, but conventional sample preparation does not reveal the sub-membrane axonemal structure which holds key implications for cilia function. To overcome this challenge, we combined SEM with membrane-extraction techniques to examine cilia in native human islets. Our data show well-preserved cilia subdomains which demonstrate both expected and unexpected ultrastructural motifs. Morphometric features were quantified when possible, including axonemal length and diameter, microtubule conformations and chirality. We further describe a novel ciliary ring, a structure that may be a specialization in human islets. Key findings are correlated with fluorescence microscopy and interpreted in the context of cilia function as a cellular sensor and communications locus in pancreatic islets.

cell biology↗

bioRxiv2021

Global cerebrospinal fluid circulation mapping using gold nanoparticle enhanced X-ray microtomography reveals region-specific brain and spinal cord CSF pathways

Cerebrospinal fluid (CSF) movement within the brain interstitium is essential for the development and functioning of the brain. However, the interstitium has largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with CSF. Here, we developed a novel technique for CSF tracking, gold nanoparticle enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF pathways during development. Using this method and subsequent histological analysis, we map global CSF pathways and present novel particle size-dependent circulation patterns through the CNS. We identify an intraparenchymal CSF circulation that targets stem cell-rich and cholinergic neuronal populations. CSF solute distribution to these areas is mediated by CSF flow along projections from the basal cisterns which is altered in posthemorrhagic hydrocephalus. Our study uncovers region-specific patterns in a biologically driven CSF circulation that has implications for normal brain development and the pathophysiology of hydrocephalus and neurodegenerative disorders.

neuroscience↗

bioRxiv2020

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.

immunology↗

bioRxiv2019

Three-dimensional Structural Interrelations between Cells, Extracellular Matrix and Mineral in Vertebrate Mineralization

The spatial-temporal relationship between cells, extracellular matrices and mineral deposits is fundamental for an improved understanding mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of microns in range. These parameters are necessary to yield fine ultrastructural details while encompassing tissue domains sufficient to provide a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a novel complex cellular network in highly mineralized tendon aspects, where [~]100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of [~]40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich globules appear between the fibrils and, with time, mineral propagates along and within collagen. These close associations between tenocytes, canaliculi, small channels, collagen and mineral suggest a new concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.\n\nSignificance StatementThe basic mechanism by which vertebrate collagenous tissues are mineralized is still not fully elucidated, despite the importance of this process for skeletal formation and regeneration. Through three-dimensional imaging of the cellular network together with the extracellular matrix and mineral deposits, the present work investigates normally mineralizing avian leg tendon as a model system for vertebrates in general. The data support a mechanism where mineral ions and possible mineral precursors are initially present in interfibrillar collagen spaces and are subsequently translocated to neighboring collagen fibrils. Mineral particles then nucleate in association with collagen to form the well known collagen-mineral composite material of the skeleton.

bioengineering↗

bioRxiv2019

Epidermal cell surface structure and chitin-protein co-assembly determine fiber architecture in the Locust cuticle

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using FIB/SEM volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.

biophysics↗