Transmission electron microscopy (TEM) provides nanometer-scale resolution, which is essential for ultrastructural analysis of biological tissue. However, its application for large tissue areas is limited by a restricted field of view and observer-dependent sampling. In this study, we present a workflow that combines wide-field montage TEM-via "network tele-microscopy"-with correlative light and electron microscopy to enable large-area ultrastructural analysis while preserving synapse-level resolution. We demonstrate this approach in the glomerular layer and tyrosine hydroxylase-positive neurons in mouse olfactory bulb glomeruli, which exhibit dense, heterogeneous synaptic organization. Light and confocal laser scanning microscopy was first used for orientation in regions of interest, allowing the generation of continuous wide-field montage TEM datasets. This approach allowed systematic identification and quantification of synapses across an entire glomerulus while maintaining spatial relationships among ultrastructural elements. This study demonstrates a technically feasible platform for the development of TEM and the future integration of network tele-microscopy with computational methods.
Polyvinylidene fluoride (PVDF) is a polymer with excellent piezoelectric properties. The close relationship between structure and properties necessitates structural investigation. The PVDF structure has been investigated using techniques such as X-ray diffraction, Fourier transform infrared spectroscopy, and polarized light microscopy. However, these methods only provide average structure information about the sample as a whole. To obtain local structure information, transmission electron microscopy should be used. Herein, the nanocrystals constituting the lamellar structure of heat-elongated PVDF were investigated using phase plate scanning transmission electron microscopy. Notably, the length of the nanocrystals increased with the increase in the elongation ratio, with no substantial change in its periodicity, and the nanocrystals exhibited a tendency to align with the lamellar structure orientation. This suggests that (1) the molecular chains within the nanocrystals elongate and tilt owing to the shear stress applied on them during elongation and (2) the entire lamellar structure rotates. To our knowledge, this is the first detailed study of nanocrystals in PVDF, which may further develop functional polymers.
Metabolic dysfunction-associated steatotic liver disease is defined by hepatic lipid overload resulting in a metabolic shift and subsequent mitochondrial impairment. Diagnosis currently relies on tissue biopsy and non-invasive tests. However, these have drawbacks, including subjective histology scoring and relatively low sensitivity, highlighting the need for more robust and reproducible methodologies. Fluorescence lifetime imaging microscopy visualises the metabolic state of cells by measuring the autofluorescence lifetime of metabolites, effectively avoiding the need for exogenous labelling. This technique was applied to a broad range of models, spanning from a hepatocyte cell line to a human tissue slice model, to investigate metabolic changes across disease conditions. Here, by utilising the metabolic dysfunction associated with steatotic liver disease, we propose a time-efficient method and introduce an index as a quantitative output to assess the metabolic state of human liver biopsies. The index encapsulates features of metabolic dysfunction that directly report on the disease state. These findings using lifetime imaging are substantiated by extensive analysis of structural and functional mitochondrial dysfunction. Measuring fluorescence lifetime can capture features of metabolic change that standard histological methods do not. Correlating the results to established techniques of histological evaluation highlights the potential of this method to enhance characterisation and speed of biopsy results in metabolically implicated diseases. Metabolic liver disease, caused by a build-up of fat in the liver, affects millions of people worldwide and can progress to serious liver damage if not detected early. Current tests are flawed and can miss early changes or take a long time to deliver results. This study aimed to explore a faster, more reliable way to assess liver disease by measuring liver cell metabolism. A microscopy technique was used that detects natural signals from cells, without adding dyes, to measure their metabolic state. By applying this method to human liver samples, we identified clear changes linked to liver damage that are not picked up by current standard tests. This approach has the potential to improve diagnosis, speed up clinical decisions, and help track treatment responses in the future.
Chromatin has a complex 3D structure and diverse binding proteins that coordinate the genome's most essential functions. Many microscopy and genomics technologies that map chromatin proteins and modifications rely on the diffusion of antibodies (Abs) to target epitopes within whole nuclei. Here, we reveal a critical flaw in such methods that arises when Abs become trapped at the edge of nuclear structures and fail to reach internally positioned epitopes. This "Ab-trapping" results in artifactual peripheral signal that fundamentally distorts the apparent positions of chromatin features across the genome and nucleus. Using computational modeling and experimental validation, we demonstrate that Ab-trapping is caused by a combination of three compounding factors-high epitope abundance, high Ab affinity, and low Ab diffusion rates. Ab-trapping can thus systematically misrepresent the localization of many prevalent chromatin features like histone modifications, transcription factors, nucleolar proteins, and protein tags. We also show that this artifact manifests in multiple technologies, including immunofluorescence microscopy, more recent CUT&Tag-seq, and likely any method relying on Ab diffusion. Finally, we outline readily implementable strategies to identify and mitigate Ab-trapping. Combined, our work presents a previously unrecognized yet prevalent artifact in Ab-based chromatin mapping methods and the means to resolve it.
Focused ion beam (FIB) processing is widely used for preparing transmission electron microscopy (TEM) specimens of polymer materials. However, it often introduces damaged layers that hinder structural analysis. In this study, we demonstrate the effectiveness of gas cluster ion beam (GCIB) irradiation in reducing such damaged layers in polyethylene (PE) extracted from a PE/polyamide (PA) multilayer film, used here as a representative single-component polymer. GCIB treatment successfully removed the FIB-induced damaged layers and enabled access by ruthenium tetroxide (RuO4), which allowed clear visualization of the lamellar structure in PE. Electron tomography confirmed that GCIB not only eliminated damaged layers but also thinned the specimen. These findings suggest that GCIB is a promising technique for preparing ultra-thin, low-damage TEM specimens of polymer materials and can facilitate more accurate structural and chemical analyses.
Cryogenic electron tomography (cryoET) offers unparalleled views into the molecular architecture of cells. As no stains or fixation are used, electrons scatter off the native atoms, and all molecules contribute to the final tomogram. As a result, it can be challenging to identify proteins of interest, especially inside a crowded cellular environment. Recent developments in molecular tags for cryoET provide several options for identifying proteins in reconstructed tomograms, but these are often not appropriate for finding an area of interest when collecting data. To increase the utility and throughput of cryoET, future approaches should combine correlative light and electron microscopy (CLEM) with tagging, so that a single modification can be used at small and large spatial scales. Automation of the detection of tags in tomograms and correlation between imaging modalities using machine learning methods will help increase the throughput of these methods, making them more suitable for rare events or structure determination by sub-tomogram averaging.
Positive surgical margins (PSMs) after robot-assisted radical prostatectomy (RARP) increase the risk of prostate cancer recurrence, often requiring salvage treatments that may compromise functional recovery. Intraoperative frozen section can detect PSM but is not feasible as a routine approach in most settings. Ex vivo fluorescence confocal microscopy (FCM) provides a rapid, real-time alternative, though evidence remains limited and knowledge gaps persist. This international Delphi consensus aimed to define expert recommendations on the clinical application of and workflow and research priorities for the safe integration of FCM during RARP. A modified Delphi process was conducted following the RAND/UCLA Appropriateness Method and reported according to the ACCORD checklist. In total, 32 international experts in urology and pathology participated in a hybrid consensus meeting after structured literature review and preparatory online sessions. Forty-four evidence-based multiple-choice items across patient selection, image acquisition, and surgical management domains were developed and anonymously voted on via a secure online platform. Consensus was predefined as ≥80% agreement among respondents. Consensus was achieved for 24 of 44 items (55%). Key agreements included a risk-adapted use of FCM (97%), its role as an adjunct to magnetic resonance imaging for nerve-sparing decisions (87%), adoption of en-face imaging as the preferred FCM technique (82%), and a maximum reporting time of 30 min (85%). The panel endorsed formal certification for image interpretation (83%), standardized reporting of positive margin length (82%), and feasibility of remote intra-institutional reporting (84%). Larger (>3 mm) PSM in high-risk cases warranted complete neurovascular bundle resection (96%). This consensus provides a structured framework for the intraoperative use of FCM during RARP, defining its indications, workflow standards, and training requirements. Future multicentre studies are needed to assess its oncological and functional impact and to establish standardized implementation pathways.
Coherence of electron waves is central to transmission electron microscopy. However, the mechanism underlying partial loss of coherence during inelastic scattering has been a perplexing problem for many decades. Here we show that the inelastic collision time is the key parameter governing coherence. Large energy transfers have short collision times and an inversely decaying coherence length. It is also shown that the inelastic coherence volume is highly elongated along the electron beam direction. Plasmon excitation and inter-band transition events therefore have only a minor effect on dynamical diffraction, a fact confirmed by energy filtered measurements on [001]-oriented SrTiO3.
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A recipe for producing carbon extraction replicas from twin-jet electropolished disks is described. This technique allows tracking the analysed region with respect to the bulk sample. An example use of the method as applied to a reduced activation ferritic martensitic (RAFM) steel, 'UK-RAFM', is reported. The RAFM steel described originates from a ∼5 tonne heat produced via electric-arc furnace (EAF) and was intended to match Eurofer-97 in chemistry. Here, a detailed analysis of the precipitates within the UK-RAFM is provided, with particular emphasis to show the variability in chemistry of second-phase precipitates (SPPs). Comparisons to literature reported compositions of Eurofer-97 SPPs indicate they are in reasonable agreement; comparisons are also made against thermodynamic models.
In situ tensile testing in a transmission electron microscope (TEM) is a powerful tool for investigating polymer fracture at the nanoscale, but the stochastic nature of crack initiation has long hindered systematic observation. We report a specialized diamond knife, co-developed with Syntek Co., Ltd., that introduces a controlled V-shaped notch into a trimmed specimen block prior to cryo-ultramicrotomy. Ultrathin films (∼100 nm) of a copolymer film containing a well-defined notch were prepared and observed using in situ tensile TEM. Cracks initiated reproducibly from the notch tip and propagated predictably, providing new experimental control for nanoscale fracture studies.
The technology of volume electron microscopy (vEM), which enables three-dimensional (3D) observation of organelles, cells and even tissues at the nanoscale, is advancing and being applied to various fields of life science. As the demand for vEM grows, there is an increasing need for Volume Correlative Light and Electron Microscopy (Volume CLEM) to capture targets without missing the region of interest (ROI) or to observe the localization of molecules (often proteins) at the exact location of the fluorescent signals. This mini review provides an overview of the current state of Volume CLEM and introduces several unique approaches. My aim is to share the diverse fundamental technologies and broad application scope of this technique. Volume electron microscopy (vEM) provides nanoscale imaging for life science applications. Correlation with Light and Electron Microscopy (CLEM), is very powerful for linking molecular localization to ultrastructure. This mini review outlines advances, distinctive approaches, and its broad technological and application scope.
The secretory pathway is a central and evolutionarily conserved feature of eukaryotic cells, responsible for protein and lipid trafficking, membrane biogenesis, signalling, and cellular homeostasis. Its complexity, dynamic behaviour, and nanoscale organisation have made it a longstanding target of microscopy-driven investigation. In this review, we trace the parallel evolution of our understanding of the secretory pathway and imaging technologies, with a particular emphasis on plant cells, where unique architectural and functional features have challenged and enriched mechanistic models. We highlight the foundational role of electron microscopy (EM) in defining the ultrastructural organisation of secretory organelles and establishing directional models of intracellular transport, followed by the fluorescence microscopy revolution that enabled direct visualisation of cargo flux and organelle dynamics in living cells. The advent of super-resolution fluorescence techniques bridged the long-standing resolution gap between light microscopy and EM, revealing nanoscale compartmentalisation, membrane contact sites, and trafficking intermediates previously inaccessible in living cells. More recently, the integration of functional assays, optogenetics, and artificial intelligence-driven segmentation, denoising, and adaptive imaging now enables quantitative and high-throughput analysis of secretory architecture. Together, these advances have transformed the secretory pathway from a static morphological concept into a dynamic, increasingly mechanistically defined system. We conclude by discussing emerging integrative strategies, particularly correlative and AI-enhanced approaches that promise to unify ultrastructural precision with molecular specificity and temporal resolution in future studies of endomembrane organisation.
High accuracy electron microscopy simulations required for quantitative crystal structure refinements face a fundamental challenge: while physical interactions are well-described theoretically, real-world experimental effects are challenging to model analytically. To address this gap, we present a hybrid physics-machine learning framework that integrates differentiable physical simulations with neural networks. By leveraging automatic differentiation throughout the simulation pipeline, our method enables gradient-based joint optimization of physical parameters and neural network components representing experimental variables, offering superior scalability compared to traditional second-order methods. We demonstrate this framework through application to three-dimensional electron diffraction (3D-ED) structure refinement, where our approach learns complex thickness distributions directly from diffraction data rather than relying on simplified geometric models. This method achieves state-of-the-art refinement performance across synthetic and experimental datasets, recovering atomic positions, thermal displacements, and thickness profiles with high fidelity. The modular architecture proposed can naturally be extended to accommodate additional physical phenomena and extended to other electron microscopy techniques. This establishes differentiable hybrid modeling as a powerful paradigm for quantitative electron microscopy, where experimental complexities have historically limited analysis.
Scanning transmission electron microscopy (STEM) has a number of benefits over conventional parallel illumination transmission electron microscopy, such as the ability to simultaneously perform a range of imaging and spectroscopy techniques. However, one distinct disadvantage is the slow imaging speeds, a consequence of the sequential nature of the pixel acquisition, limiting imaging speeds to a few frames per second (fps). This reduces dose-rate control, increases the effect of distortions, and hinder the ability to capture dynamic events for in-situ experiments. The main obstacle to faster framerates is the inductance and hysteresis of the scanning coils that has previously only been addressable with the addition of entirely new hardware coils. Here we demonstrate a predictive scan shaping approach using conventional scanning systems, where scan input is determined based on where the beam will be instead of where the beam should be. We use this new approach to acquire fully sampled 512x512 images at 60 ns per pixel, giving a framerate of 41 fps.
The plant endoplasmic reticulum (ER) is a dynamic organelle composed of multiple distinct structural domains, such as cisternae, which are maintained by ER morphogens including the Arabidopsis thaliana Lunapark proteins (LNPs). Cisternae are typically described as sac-like structures connected by tubules. Here we challenge this assumption and propose that cisternae have a more complex structure that modifies ER functionality. This study used state-of-the-art high-resolution confocal and variable-angle epifluorescence microscopy, along with transmission electron microscopy and tomography, on high-pressure frozen Arabidopsis thaliana samples. We found that AtLNP1-stabilised ER forms cisternae composed of dense tubular matrices, whereas AtLNP2 forms cisternae with a uniform, sac-like structure. Furthermore, overexpression of AtLNP proteins alters Golgi morphology, affecting ER-to-Golgi transport and secretion. Our findings reveal that the balance between AtLNP1 and AtLNP2 is critical for ER cisternae organisation and ER functionality in protein production and secretion. This work provides new insights into ER structural plasticity and its functional implications in plant cells.
High-resolution transmission electron microscopy (HRTEM) is an important method for imaging beam sensitive materials often under cryo conditions. Electron ptychography in the scanning transmission electron microscope (STEM) has been shown to reconstruct low-noise phase data at a reduced fluence for such materials. This raises the question of whether ptychography or HRTEM provides a more fluence-efficient imaging technique. Even though the transfer function is a common metric for evaluating the performance of an imaging method, it only describes the signal transfer with respect to spatial frequency, irrespective of the noise transfer. It can also not be well defined for methods, such as ptychography, that use an algorithm to form the final image. Here we apply the concept of detective quantum efficiency (DQE) to electron microscopy as a fluence independent and sample independent measure of technique performance. We find that, for a weak-phase object, ptychography can never reach the efficiency of a perfect Zernike phase imaging microscope but that ptychography is more robust to partial coherence.
The bacterial flagellum is a protein-based rotary machine that drives bacterial motility. It comprises the bacterial flagellar motor (BFM), consisting of a stator which is anchored to the cell wall and a rotor in the cytoplasmic membrane, linked via the flagellar rod to the extracellular hook and filament. We observe passive rotational diffusion of six individual Escherichia coli flagella lacking torque-generating units via polarization microscopy of single gold nanorods attached to the hook, sampled at 250 kHz. Transitions across energy barriers of the 26-fold symmetric LP-ring/rod flagellar bearing exhibit highly non-Poissonian kinetics spanning four orders of magnitude in time scale. At sub-millisecond timescales we observe anomalous ultra-slow diffusion typically associated with disordered systems, despite the ordered crystalline atomic structure of the bearing revealed by cryo-Electron Microscopy. Over longer periods, we observe dynamic shifts in the preferred angular positions, indicating that the bearing's energy landscape evolves over time.
Age-specific patterns of malaria are well-established for children aged  < 5 years. Less understood is the epidemiology of malaria in older children and adults, and the influence of granular environmental risk. We analyzed data from SchistoTrack, a community-based cohort in rural Uganda. We studied 4308 participants aged 5 to 90 years from 52 villages across three lakeside districts of Mayuge, Buliisa, and Pakwach, with enrollment between January 2022 to February 2024. The primary outcome was malaria infection status by rapid diagnostic test (RDT). Secondary outcomes included microscopy-confirmed infection with parasite density quantification and self-reported fever within the past month. We fitted a generalized additive mixed model (GAMM) with adaptive age smoothing, adjusting for sociodemographic factors, household characteristics, healthcare access, and environmental exposures. Environmental exposure was quantified using the Normalized Difference Vegetation Index (NDVI) derived from Sentinel-2 satellite imagery (10 m resolution), processed through hexagonal aggregation with Gaussian neighborhood smoothing and validated against field malacology surveys and participatory community mapping. Overall RDT prevalence was 41.2% (1776/4308), with microscopy prevalence at 32.3% (1363/4219), which was predominantly Plasmodium falciparum (83.1%; 1133/1363). Most infections were low-density ( < 999 parasites/μL; 71.6%; 976/1363). Malaria prevalence showed non-linear age patterns, peaking at 10 to 11 years then declining through adolescence before stabilizing in adulthood. Among RDT-positive individuals, fever prevalence decreased with age from 30.8% in children (aged 5 to 10 years) to 11.2% in adults (aged ≥20 years). Dense vegetation (per unit NDVI increase: Odds Ratio (OR) 3.25, 95% Confidence Interval (CI) 1.33-7.96) and greater distance from government health centers (per log-km: OR 1.87, 95% CI 1.34-2.59) increased the odds of infection. Proximity to vegetated water bodies increased the odds of infection compared to beaches: ponds/swamps (OR 1.65, 95% CI 1.19-2.28), river/river marsh (OR 1.63, 95% CI 1.16-2.31), lake marsh (OR 1.40, 95% CI 1.07-1.83). Malaria prevalence remains high in older children and adults, though with fewer febrile cases, and is influenced by the local environment. Our findings support age-specific interventions targeting school-aged children while maintaining adult surveillance, and using validated environmental indices to guide sub-district resource allocation in high-risk areas.
We present the design, fabrication, and characterization of continuous phase Fresnel zone plates (FZPs) using two-photon polymerization direct laser writing in a polymerizable nematic liquid crystal (LC) confined between glass substrates. Unlike conventional binary LC diffractive elements, our devices exhibit a smooth, continuous three-dimensional phase profile. Two devices were demonstrated with wrapped phase profiles of 2π and 4π radians, respectively. Polarized optical microscopy and digital holographic microscopy confirm that the polymerized regions follow the intended spatially varying phase distribution. Far field measurements show that the 2π rad FZP generates a strong focal spot at 0 Vpp and switches off at higher voltages. In contrast, the 4π rad FZP exhibits varifocal behavior, switching between two focal lengths: 24 mm at 0 Vpp and 48 mm at an intermediate voltage of 2.1 Vpp. At higher voltages, the focus disappears entirely. Compared to a binary FZP of equal size and focal length, the continuous phase design nearly doubles the focusing efficiency and enables switchable, compact, vari-focal, and energy-efficient optical components. This approach offers new opportunities for advanced applications such as augmented and virtual reality, adaptive optics, and other next-generation photonic systems.