IBDV is an icosahedral virus from the Birnaviridae family, whose two dsRNA segments (A and B) are organized as ribonucleoprotein complexes. An ORF of segment A encodes a polyprotein, NH₂-pVP2-VP4-VP3-COOH, which is self-cleaved by the protease VP4 (243 residues) rendering pVP2 and VP3. Segment B encodes the RNA polymerase VP1. VP1-VP4 and the genomic dsRNA associate into three assemblies crucial during the viral life cycle: T=13 capsids, VP4 helical tubes, and the RNPs.

VP4 is naturally found as helical assemblies, ~25 nm in diameter, in IBDV-infected cells. VP4 atomic structures are conserved in birnaviruses, but no structural data are available for these in vivo assemblies. We determined the cryo-EM structure of VP4 helix at 2.4 Å resolution. The basic building blocks are VP4 dimers, arranged in a right-handed three-stranded helix, with an axial rise of 18.4 Å and an azimuth angle of 40.6º. The catalytic Ser/Lys dyad, located in a surface crevice, is blocked in cis by the VP4 C-terminal end, which is also involved in intra-dimeric interactions via the 227-243 C-terminal segment. Interdimeric interactions occur via segments 209-218 and 150-155 within the same helix, and via segment 89-112 between dimers of different helices. A collection of mutants has confirmed key residues for VP4 activity, self-assembly, and infection viability. Our study suggests that once VP4 has completed proteolysis, it is securely inactivated by assembly into helical structures to prevent lethal damage to the virus or to the host components needed for virus multiplication, and offers insights into new antiviral targets.

For many structural biology research groups, access to high voltage (300 kV) cryo-electron microscopes is a limiting step in their investigations. The experiment time is often limited to a few times every year, and much time is devoted to optimizing grid preparation. Some problems that may be encountered during this process, such as preferred orientation or inadequate complex ratios, are difficult to evaluate with lower voltage (120 kV) microscopes. Here, we report a procedure to obtain, from a single grid, two distinct and high-resolution structures (2 Å) of RNA Polymerase I (Pol I) bearing a functionally relevant point-mutation. To achieve this goal, a heterogeneous sample was prepared by incubating purified Pol I with sub-stoichiometric amounts of a DNA-RNA transcription scaffold. After standard procedures for vitrification on amorphous carbon-coated carbon grids and data collection, the dataset was processed with a combination of CryoSPARC and RELION-5, using the Scipion framework, for optimized particle classification and quick refinement. Two distinct particle sets were refined separately to obtain high-resolution electron maps, which will be used to elucidate precise mechanisms behind the behavior of the point mutation.

Viruses with capsids larger than 150 nm are considered “giant viruses” (1). Prymnesium kappa virus RF02 (PkV-RF02) has a 583 kbp linear dsDNA genome and 200 nm diameter capsid (2). Using mass spectrometry, we have identified 140 different proteins in purified PkV-RF02, with nearly 70% of them having unknown function (3). We used cryo-electron microscopy (cryo-EM) single particle analysis to obtain high-resolution maps of ordered components of the PkV-RF02 icosahedral capsid, and cross-linking (XL) followed by mass spectrometry (MS) to identify protein-protein interactions (PPIs) present in both the icosahedral shell and non-icosahedral core.

Our ~3 Å resolution structure shows that PkV-RF02 has a triangulation number pseudoT=219, previously observed only for Phaeocystis pouchetii virus (PpV01) (4). The PkV-RF02 capsid is composed by more than 20 different proteins and nearly 12,000 protein copies. PkV-RF02 has novel characteristics among giant viruses: a protein channel within the virion, an unusual penton protein organization, and a multilayered stabilization network reinforced by disulfide bridges and O-linked glycosylations on the surface.

Using XL-MS, we detected 561 PPIs happening between 112 different viral proteins. These PPIs define symmetry-mismatched features escaping localization by cryo-EM, as well as numerous PPIs within the PkV-RF02 non-icosahedral core, showing highly complex functional enzymes packaged in the viral particle: bacteriorhodopsin-like protein, disulfide isomerase or RNA polymerase complex, among others. This work shows a complete structural characterization of the capsid and core components of a giant virus combining cryo-EM and XL-MS, and is the first high-resolution structure of a pT=219 virus.

Two-dimensional imaging represents a serious limitation when studying the structure of complex hierarchical materials such as bone. Three-dimensional visualization is essential for understanding the interactions that occur between the cellular network, collagen fibrils, and mineral precursors during the formation process. Over the last decade, FIB-SEM has revolutionized the study of mineralized tissues, providing not only three dimensional information in the nanometer range, but also the possibility to nanofabricate mineralized lamellae avoiding the mechanical stress caused by standard sectioning techniques.

These technical advances helped elucidate the mineral deposition in collagen-based materials, driven by a spherulitic-like crystal growth [1]. Initially, disordered mineral aggregates form in the interfibrillar spaces, and subsequently the mineral infiltrates adjacent collagen fibrils, which provide the structural framework for the formation of layered spherulites. These spherulites (also called mineral ellipsoids) imbricate forming a new hierarchical level of organization in bone termed tessellation [2]. Although the mechanism has been described in several systems [1, 2, 3], detailed data on the interaction of the organic and the mineral phases remain insufficient.

The present study combines electron tomography (FIB-SEM serial surface imaging) which provides 3D information, with the fabrication of lamellae for scanning/transmission electron microscopy (S/TEM), selected area electron diffraction (SAED) and energy dispersive spectroscopy (EDS) chemical mapping to elucidate crystal distribution and orientation throughout the collagen matrix.

The study reveals the internal structure of the forming fibrolamellar bone at nanometer resolution. A connective tissue with dispersed and non-preferentially oriented collagen fibrils seems to be deposited first, serving as a scaffold for the deposition of more aligned collagen. During embryonic development, these osteocytes initiate the mineralization process and become buried in the mineral matrix, which expands both vertically and laterally to form the nascent fibrolamellar units. At the collagen-mineral interface, a multitude of mineral spherulites proliferate and grow to confluence. Their profiles are still recognizable in the consolidated mineral layer.

Our study confirms that the formation of mineral spherulites also drives the mineral deposition in embryonic fibrolamellar bone. This fact demonstrates that this protein-mediated crystal growth mechanism occurs in different types of bone tissue and in different species, indicating that it is a common and homologous mineralization mechanism in type I collagen-based materials.

During synthesis of the ribosomal RNA precursor, RNA polymerase I (Pol I) monitors DNA integrity but its response to DNA damage remains poorly studied. Abasic sites are among the most prevalent DNA lesions in eukaryotic cells, and their detection is critical for cell survival. We report cryo-EM structures of Pol I in different stages of stalling at abasic sites, supported by in vitro transcription studies. Slow nucleotide addition opposite abasic sites occurs through base sandwiching between the RNA 3’-end and the Pol I bridge helix. Templating abasic sites can also induce Pol I cleft opening, which enables the A12 subunit to access the active center. Nucleotide addition opposite the lesion induces a translocation intermediate where DNA bases tilt to form hydrogen bonds with the new RNA base. These findings reveal unique mechanisms of Pol I stalling at abasic sites, differing from arrest by bulky lesions or from abasic site handling by RNA polymerase II.

Choline is an essential nutrient across all domains of life. In prokaryotes, it acts as a precursor to osmoprotectants such as glycine betaine, which help maintain cellular turgor under osmotic stress, and it can also be metabolized as a source of carbon and nitrogen. In humans, choline plays critical roles in key physiological processes including cell membrane building, cholinergic neurotransmission, and methylation pathways.
In cholinergic signaling, choline serves as a precursor of the primary neurotransmitter acetylcholine. After synaptic transmission, acetylcholine is hydrolyzed in the synaptic cleft, releasing choline, which must be reabsorbed by presynaptic neurons. This choline reuptake in cholinergic synapses is mediated by the human high-affinity choline transporter (hCHT) for the biosynthesis of acetylcholine to sustain neurotransmission. Mutations in hCHT are associated with several neurological disorders, highlighting its critical role in cholinergic functions.
In this study, we identified a bacterial homolog of hCHT with high sequence identity from Salimicrobium flavidum, referred to as sfCHT, to investigate the high-affinity choline transport mechanism mediated by the CHT family.
Cryo-EM structures of Na+- and Na+/choline-bound sfCHT, combined with computational structure prediction with Protein Energy Landscape Exploration (PELE) and mutational analysis, have revealed the choline translocation pathway from the substrate-binding site to the cytosol. The key residues involved in transport were found to be conserved in hCHT and essential for transport, supporting a conserved transport mechanism across the CHT lineage.

Amyloid fibrils, typically associated with neurodegenerative diseases, also play critical roles as functional assemblies in biological processes. The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) are essential for necroptosis, orchestrating the formation of amyloid-like fibrils that assemble into necrosomes. These supramolecular complexes propagate cell death signals and activate effectors like MLKL. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been resolved [1,2], the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive.

Here, we present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryo-electron microscopy and cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy. The fibrils adopt an N-shaped amyloid fold consisting of three β-sheets stabilized by the conserved IQIG RHIM motif through hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the β2-β3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. This structural feature likely contributes to the compact architecture of hRIPK1 fibrils, in contrast to the more relaxed S-shaped fold observed in hRIPK3.

These findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids.

INTRODUCTION

3D spheroid cell cultures recapitulate the tumor micro-environment[1] better than their adherent 2D counterparts[2]. Spheroid models represent a valuable cancer research tool, allowing optimized drug selection and improved tumor distribution. 3D models of breast cancer will reduce the number of animals employed and drug screening costs[3-5]. Of note, the molecular complexity of breast cancer, especially when targeting metastasis, will require combinatorial drug treatments[6-7]. Exosomes - extracellular vesicles (EVs) that play essential roles in intercellular communicators[8] - help form the pre-metastatic niche[9] and support drug resistance[10]. EV markers include tetraspanins[11]; however, current methodologies to purify exosomes remain time-consuming and challenging to translate to clinical practice. We recently optimized a quick and reliable HTS methodology[12] to identify exosome modulators that combines external signals measured by ExoScreen technology (a sensitive assay that measures protein-protein interactions) and internal exosomal markers in 2D models[13]. Our study employed MCF7 cells (Luminal A subtype).

OBJECTIVES

1. Study the effect of polymer-drug conjugates (single/combinations) on exosomes via the ExoScreen assay
2. Combine ExoScreen and Cell Painting[14] to discover exosome modulators (and other effects)

METHODOLOGY

Using low adherence plates, we cultured MCF7 mammospheres with EGF2/B27. We added free or polymer-conjugated drugs for 72 h and performed the ExoScreen assay using anti-CD9 acceptor beads and a biotinylated-anti-CD63 antibody. For Cell Painting, we employed markers of the mitochondria, endoplasmic reticulum, cell membrane, intraluminal/extracellular vesicles, and nucleus. Cell viability evaluations employed the MTS assay.

RESULTS

We identified “Drug 1” as an exosome biogenesis inhibitor (reducing ExoScreen and CD63 signals, extra- and intracellularly, respectively). “Drug 3” inhibited exosome release, manifested as a lower ExoScreen signal and accumulated intracellular CD63 signal, and modulated the endoplasmic reticulum. Both drugs mimic the behavior previously observed in a 2D model[13].

CONCLUSION

Employing this screening technique in 3D spheroids ensured the homogeneous distribution of labeling in a preclinically relevant model. We combined analysis of exosomal intracellular markers (CD63) with morphological features to create a multiplexed approach. We employed our approach to validate the antitumor/antimetastatic properties of a polypeptide-based-drug combination conjugates[7,15], with findings correlating with in vivo data. The following steps include applying suitable image analysis software and artificial intelligence tools to enhance intracellular signal quantification of different markers and establishing correlations with therapeutic outputs[7].

Eukaryotic cells use multiprotein complexes to make a copy of their chromosomes. Many of those complexes assemble on Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimer that embraces the DNA duplex. The enzyme DNA Methyltransferase 1 (DNMT1) methylates cytosine bases in the daughter DNA strand, replicating the methylation pattern of the parental one. DNMT1 is recruited to the replication fork by the regulatory protein p15 when it is doubly monoubiquitinated in its N-terminal disordered tail, and the central region of p15 binds to the front face of the PCNA ring.

We have reconstituted the complex formed by DNMT1, a DNA duplex with a methylated cytosine, doubly monoubiquitinated p15, and PCNA from the isolated components. A complex with equimolar stoichiometry is detected in solution by mass photometry, and particles with the expected size are observed in negatively stained electron micrographs and with DNMT1 on the back side of the PCNA ring. A higher resolution structure was not attainable because the complex dissociates upon vitrification.

The structure of the complex predicted with AlphaFold is consistent with the available experimental information, providing a plausible model for DNMT1 action anchored to PCNA, and suggests that methylation of the newly synthesized DNA strand can occur in concert with lagging strand replication by DNA polymerase d.

Cryo-electron microscopy (cryo-EM) has revolutionized the field of structural biology by allowing researchers to resolve macromolecular structures at near-atomic resolutions (better than 3 Å) without requiring crystallization. This technique is especially valuable for analyzing large, heterogeneous complexes in environments that closely simulate their native conditions. Despite these advantages, the process of sample preparation remains a significant challenge. Biological samples frequently encounter problems such as adopting preferred orientations or undergoing partial to complete denaturation during the vitrification process. These complications are largely due to interactions with the hydrophobic air–water interface (AWI), which particles can rapidly encounter. Such interactions may result in the disassembly of complexes, unfolding of proteins, or aggregation. Various approaches have been introduced to counter these issues, including chemical crosslinking, the addition of detergents or surfactants, and the use of grids functionalized with graphene oxide. However, these methods often present difficulties in optimization and reproducibility. An alternative approach involves the use of streptavidin affinity grids, a technology pioneered by Robert Glaeser at Berkeley and further refined by Eva Nogales’ lab [1]. These specialized grids offer an effective means to minimize sample denaturation and reduce the occurrence of preferred orientations caused by the AWI.

Streptavidin affinity grids exploit the strong binding between streptavidin and biotin (Kd ≈ 10⁻¹⁴ M) to selectively capture biotinylated molecules. The preparation of these grids involves growing two-dimensional streptavidin crystals on a biotinylated lipid monolayer, which is then applied to standard holey-carbon cryo-EM grids. This method not only enables the stable immobilization of biotinylated samples but also helps concentrate proteins present in low abundance and can even facilitate the on-grid purification of target protein complexes.

In this study, we have focused on optimizing streptavidin affinity grids to enhance the preparation and structural analysis of proteins such as 14-3-3 and tyrosine hydroxylase (TH). Our results demonstrate the potential of these grids to overcome challenges associated with imaging small and dynamic protein complexes.

Rab7a is a key small GTPase associated with late endosomal membranes, 
where it regulates critical trafficking events such as endosome 
maturation, cargo sorting, and autophagy. Its functional cycle is 
tightly controlled by nucleotide binding, post-translational 
modifications, and interactions with effector proteins. Among these, 
the retromer complex, a heterotrimer composed of Vps26, Vps29, and 
Vps35, plays a central role in cargo recognition and endosomal 
recycling. The GTPase-activating protein (GAP) TBC1d5 has been shown 
to interface with both Rab7a and retromer, suggesting a coordinated 
mechanism for temporal regulation of membrane association and 
function. In this study, we explore the structural basis and 
regulatory dynamics of the Rab7–retromer–TBC1d5 axis. Using a 
combination of structural biology approaches and biochemical assays, 
we examine how TBC1d5 engages with retromer and how this interaction 
may influence retromer’s function. Additionally, we investigate the 
impact of Rab7 on its capacity to associate with retromer and 
regulatory partners. Our findings provide new molecular insights into 
the interplay between Rab7 retromer and TBC1d5 for the spatiotemporal 
control of membrane transport pathways in the endosomal system.

Cornelia de Lange syndrome (CdLS) is a developmental disorder most commonly caused by mutations in the cohesin loader NIPBL. Previous studies have shown that these mutations lead to reduced NIPBL occupancy at high GC content regions and altered cohesin distribution across the genome, potentially impairing chromatin organization and gene regulation. To explore whether NIPBL mutations affect its dynamic association with chromatin, we are employing inverse Fluorescence Recovery After Photobleaching (iFRAP) to monitor the mobility of NIPBL in living cells under physiological conditions. In addition, we study NIPBL/Scc2 localization using in vivo time-lapse microscopy combined with high-resolution imaging strategies. This approach will allow us to determine whether disease-associated mutations in NIPBL alter its chromatin binding kinetics, potentially leading to aberrant cohesin behavior and disrupted chromatin dynamics. Insights from this analysis may help elucidate how changes in NIPBL-chromatin interactions contribute to the disruption of genome organization and transcriptional regulation in CdLS.

High Resolution High Angle Annular Dark Field imaging has boosted the structural analysis of so-called Single Atom Catalysts. These are materials constituted by a metallic active phase in the form of ultra-highly dispersed species (either subnanometric sized clusters or even isolated atoms) [1]. The unique capabilities of directly imaging isolated metal species distributed on the surface or bulk of a second material which acts as a carrier is in fact at the roots of the development of this particular type of supported metal catalysts. In fact, STEM-HAADF images are routinely used to evidence the success of the synthetic routes used to prepare this type of materials, mainly by detecting contrasts in the images which can be specifically assigned to the ultradispersed metallic component. Figure 1 shows an example corresponding to a 0.1 wt% Pd/MgO catalyst. The dot-type bright contrasts in this image would correspond to location of isolated Pd atoms.

However, the analysis of the results obtained using this technique is still mainly based on a manual approach, which severely limits both its reliability and potential to reveal the ultimate structural analysis of this specific type of catalysts. In most papers some Single Atom (SA) contrasts are just encircled on the images, without any further analysis. Moreover, image simulation studies supporting contrasts interpretation is usually lacking. Clearly, such an approach is quite weak, prone to user bias, and very limited in terms of statistical significance. It also oversees other information underlying the local distribution of contrasts in the images, particularly that related to the actual nature of the sites in which the isolated atoms locate, a question particularly relevant to model and understand the actual nature of metal-support interactions.

In this scenario it becomes necessary to develop new tools which allow processing large sets of experimental STEM images to automatically detect SA species and determine their exact location on crystalline supports. Here, we present an approach to directly quantify the detailed structural nature of metal sites in single-atom, high surface area, powder catalysts from the analysis of large sets of images. By combining advanced high-resolution High Angle Annular Dark Field scanning-transmission electron microscopy (HAADF-STEM) imaging, HAADF-STEM image simulation, deep learning and density functional theory calculations, we determine, with statistical significance, the exact location and coordination environment of Pd single-atoms supported on MgO nanoplates.

We will illustrate the analysis performed on experimental images and demonstrate how, not only the contrasts (brighter spots) associated, in agreement with simulated images, to the isolated Pd atoms are perfectly and automatically detected and segmented (separated from the irregular support background) but also how it is also possible to determine which of these atoms locate on sites corresponding to Mg in the oxide structure, i.e. as substitution of Mg²⁺ cations [2].

Importantly, a more in-depth analysis, with higher spatial resolution, of the local contrasts in the sites corresponding to the Pd atoms, involving also the use of neural networks, revealed a preferential interaction of Pd single-atoms with cationic vacancies (V-centers), followed by occupation of anionic defects on the {001} MgO surface. The former interaction results in stabilization of PdO species within V-centers, while partially embedded Pd states are found in F-defects. Therefore, this methodology opens a route to the ultimate structural analysis of metal-support interaction effects, key in the design of advanced nanocatalysts for sustainable and energy-efficient processes.

One-dimensional (1D) van der Waals (vdW) magnets, such as halide-based CrX₃ (X = I, Cl), are emerging as promising candidates for nanoscale spintronic applications due to their intrinsic anisotropy, low damping, and confined magnetic textures. However, their extreme air sensitivity and low dimensionality present significant challenges for structural and functional characterization. In this work, we investigate the atomic and electronic structures of CrX₃ nanotubes and nanorods encapsulated within multi-walled carbon nanotubes (MWCNTs) using advanced electron microscopy techniques. Low-energy loss electron energy loss spectroscopy (EELS) is used to probe the dielectric environment and identify signatures of excitonic, interband, and collective electronic excitations. Simultaneously, 4D-STEM measurements allow for the mapping of local strain fields and charge redistribution at the CrX₃/MWCNT interface, providing insights into confinement-induced property modulation. This study builds upon our recent demonstration of monolayer CrI₃ encapsulation in carbon nanotubes, extending the methodology to other halide variants. The results aim to deepen the understanding of how curvature, interface coupling, and quantum confinement influence the physical properties of 1D vdW magnets.

Misfit layered compounds (MLCs) have garnered considerable attention due to their fascinating chemistry and properties [1]. The structural foundation of MLCs is based on the alternating stacking of the two different layered components MX with a distorted rocksalt structure and TX₂ with a hexagonal structure, resulting in a composite or intergrowth structure [1,2]. The properties of MLCs are determined by the chemical and structural interplay between MX and TX₂.

            MLC nanotubes (NTs) synthesized via the chemical vapor technique (CVT) offer potential applications in thermoelectrics [1,3]. Recently, a modified synthesis method of MLC-NTs has permitted the introduction of additional elements to form a quaternary compound starting from LaS-TaS₂ [3,4]. These quaternary MLCs are obtained by the partial substitution of one of the elements in the ternary MLC lattice, enabling precise manipulation of charge carrier densities through substitutional doping and alloying. Our study presents a detailed electron microscopy analysis of one such quaternary (Sm_xY_1-x)S-TaS₂ nanostructure family [5].

The (Sm_xY_1-x)S-TaS₂ NTs were synthesized via CVT technique [1-3] by varying the precursor proportions of Sm_x vs Y_1-x between x=0 and x=1. The samples were designated by the Sm percentage such as Sm20 which corresponds to (Sm_0.2Y_0.8)S-TaS₂ and so on. We used TEM techniques, including HR(S)TEM imaging, selected area electron diffraction (SAED), electron energy loss spectroscopy (EELS), energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD) to analyze these NTs. Raman spectroscopy was also utilized for further characterization.

The detailed analysis of the quaternary MLCs suggests that (Sm_xY_1-x)S-TaS₂ NTs were successfully synthesized. The experimental results reveal that the Sm atoms in the SmS subsystem could be replaced by Y atoms for all studied values of x. SAED patterns reveal the c-axis periodicity of pure SmS-TaS2 to be 1.13nm while the c-axis periodicity of pure YS-TaS2 to be 1.10nm. Due to the very similar crystal structure of SmS and YS, the commensurate MLC lattice parameter ‘b’ is found to shrink by only 0.7% for x = 1 compared to x = 0, while the incommensurate (Sm,Y)S lattice parameter a(Sm_1−xY_xS) shows a linear decrease by 2.7%. The incommensurate TaS₂ lattice parameter a(TaS₂) shows an increase by 0.5%, which is attributed to strain relaxation and which leaves the TaS₂ unit cell area in the a–b plane constant. The non-linear change of the MLC lattice parameters with x and a weak superstructure reflection possibly indicate an incommensurate–commensurate phase transition for the range of Y doping ratios x = 0.2–0.6. The (Sm_xY_1-x)S-TaS₂  NT layers mostly adopted a superstructure, where adjacent stacks along the c-axis exhibited a fixed relative rotation by 30º with respect to each other. The EDS results show that the composition is homogenous within the cross section of the individual NTs, irrespective of the diameter (and the radius of curvature) of the wall.  Spectroscopic analysis by EELS and Raman scattering indicate a complex interplay between the alloying degree on side and the charge transfer and thus the electronic structure as well as the phonon configuration on the other side, giving the possibility to fine tune the MLC properties by alloying the metal in the rock-salt unit of the MLC. Spectroscopic and structural analyses suggest that the characteristics of the quaternary compound is dominated by the heavy samarium atoms even for higher contents of yttrium in the MLC.

The active material of organic thin-film transistors (TFTs) is a thin, crystalline semiconductor layer of conjugated organic molecules. The TFT performance is predominantly determined by the mobility of carriers in the first few nanometers of the organic-semiconductor layer [1-2]. The orientation and crystallinity of the organic molecules critically influence this mobility, requiring a precise analysis of the structure of the employed organic molecule and the used synthesis conditions to improve the organic TFT performance. As the active channel of the organic semiconductor is confined to the first few nanometers above the dielectric, an analysis by microscopy with high spatial resolution is required to gain meaningful insights in the OTFT devices. In this work, transmission electron microscopy (TEM) and electron diffraction is applied to analyze organic crystals and organic TFTs prepared from state-of-the-art organic semiconductors [1-3], with the aim to resolve the relationship between the performance of the devices and the structure of the organic semiconductor.

Organic crystals were fabricated by thermal vacuum sublimation of the molecules, including pentacene and 2,9-diphenyl-dinaphtho[2,3-b:20,30-f]thieno[3,2-b]thiophene (DPh-DNTT).[3] The employed molecules possess a long axis that agrees with the [001] direction of the crystal. The crystals were deposited both on TEM grids with ultrathin amorphous carbon films and on the gate dielectric of TFT devices (Si substrate, Al gate electrode, AlO_x gate dielectric, organic semiconductor and Au source/drain contacts). A focused ion beam (FIB) was used to prepare cross-section lamellae for TEM analyses of the OTFT devices. TEM measurements and selected-area electron diffraction (SAED) were performed in an image-corrected Titan microscope (Thermo Fisher Scientific) operated at 300 keV with typical electron doses of below 2 e^-A^-2s^-1.

SAED analyses performed on the different organic crystals allow to identify their crystal structure. SAED patterns acquired from pentacene thin films with a thickness of 50 nm reveal that the pentacene crystallized in the ‘bulk’ phase (COD #4109834). The molecules in the organic crystals are found to 'stand' on the substrate, meaning that the long axis of the molecules, i.e., the [001] direction of the corresponding crystal, agrees with the normal of the substrate. An exception is DPh-DNTT, where a significant amount of the thin film can be found with the long axis being parallel to the substrate surface, indicating that a different growth mechanism can be found.

A TEM analysis of a TEM lamella cross-section prepared from a DPh-DNTT device (see TOC graphic) reveals the expected layers with Si substrate, Al gate electrode, AlO_x gate dielectric, the active organic semiconductor and Au source/drain contacts. Fringes can be seen in the DPh-DNTT layer, whose periodicity is 2.4 nm, corresponding to the [001] interplanar distance of the crystal, thus confirming that the molecules are standing in the first layer of the organic semiconductor, which is the preferred orientation for lateral field-effect transistors.

The TEM analyses reveal considerable differences in the crystal structure of the organic crystals, which is crucial information to understand the carrier mobility in the organic-semiconductor layer and the organic TFT device performances. [4]

2-dimensional electron gas (2DEG) in GaAs/AlGaAs are one of the most studied systems in condensed matter physics. However, the dopants in such systems acts as scattering centers and impair the mobility.[1] Thus using undoped 2DEG improves the uniformity, in turn decreasing such impurity scattering events.[2] Undoped GaAs/Al0.35Ga0.65As/GaAs heterostructure were grown by Molecular Beam Epitaxy (MBE). An epitaxial layer of Al (with an intended thickness of 10nm) was grown in situ in the MBE chamber itself to act as the gate for inducing and controlling a 2DEG at the GaAs/AlGaAs interface. Clean oxygen was introduced into the MBE loadlock to oxidize the top layer of Aluminium. Parts of this Al layer were selectively etched, and Hall bar devices were fabricated with Ti/Au contacts to characterize the induced 2DEG. However, during electrical characterization, the Al gate was found to be leaky. 

Failure analysis was done on the growth sample and device structures using scanning transmission electron microscopy (STEM) after preparing lamellae using a Ga focused ion beam (FIB). The STEM characterization involved various characterization techniques like high angle annular dark field (HAADF) imaging, bright field STEM imaging, energy dispersive X-ray (EDX) mapping, electron energy loss spectroscopy (EELS) mapping. In addition to the GaAs/Al0.35Ga0.65As/GaAs heterostructure, we found a broken layer of metallic Ga above the top GaAs layer, up to 9 nm in thickness. This was observed not just in the device, but also on the growth sample implying the Ga sheet formation was not due to any high temperature processing during the device fabrication, but was an issue related to the heterostructure growth.           
                                                     
On the basis of EDX, high loss EELS and EELS plasmon peak we also found that the aluminium layer was completely oxidized, leaving no metallic Al to act as the top gate. The Aluminium oxide thickness was consistently less than 5 nm in thickness, implying that the MBE deposited Al thickness was much lesser than the expected 10 nm. This indicates that if a thicker Al layer is grown perhaps there would be some metallic Al remaining in the sample even after the controlled oxidation. The project is in the initial stage currently, but more details of how the growth and device fabrication was improved on the basis of STEM investigation will be presented. 

Proton exchange membrane fuel cells (PEMFCs) are electrochemical devices capable of generating electricity by oxidizing H2, reformate (H2 rich gas with carbon monoxide (CO) impurities) or other fuels. In recent times, metallic core-shell nanoparticles (NPs) (M@Pt, M=Ru, Rh…) have attracted a big interest as anode catalysts of reformate fed PEMFCs [1,2]. The high catalytic activity of Pt towards the HOR, together with the CO poisoning tolerance introduced by the accompanying metal make them ideal for heavy-duty applications. Furthermore, since in M@Pt NPs the less stable metal (Rh or Ru) is not directly exposed to the electrolyte, their stability is expected to be higher than in the corresponding alloyed NPs, which commonly suffer from dissolution and dealloying [1]. However, M@Pt can still suffer from degradation under fuel cell conditions by processes that are yet not fully understood, which hinders the design of more stable and durable catalysts.
We investigated the degradation behavior of Rh@Pt NPs by means of identical location-scanning transmission electron microscopy (IL-STEM). This quasi in-situ technique allows to overcome the limitations of the ex-situ techniques, in which only statistical general insights are possible, since in IL-STEM the changes of individual particles are tracked between potential cycles. In particular, we characterized the Rh@Pt NPs after 0, 1000, 4000 and 10000 potential cycles (0.06-0.8V, 0.1V/s). Furthermore, since many of the degradation phenomena take place in 3D (e.g., particle migration and corresponding aggregation), selected regions were reconstructed in 3D by means of electron tomography.
We observed particle migration on the carbon support in all the stages of the potential cycling. However, no widespread particle aggregation was observed, even after 10000 potential cycles. A slight Rh dissolution (up to 5 at.%) during the cycles was detected, which decreased as the number of cycles increased. Even though some small particles dissolved during the first 1000 cycles, the main degradation mechanism responsible for the loss of electrochemically active surface area was found to be particle detachment.
Our results indicate that the investigated Rh@Pt NPs present a remarkable stability, and show how IL-STEM can be used for studying the degradation of catalyst NPs.

This study presents a detailed morphological analysis of nickel-iron-phosphide (NiFeP) coated nickel (Ni) felt before and after oxygen evolution reaction (OER) using scanning electron microscopy (SEM) and focused ion beam-scanning electron microscopy (FIB-SEM). The investigation focuses on the surface morphology, coating uniformity, elemental distribution, and structural changes induced by electrochemical cycling. SEM imaging of the pristine Ni felt reveals a three-dimensional network of nickel fibers with varying diameters. The structure is highly porous and interconnected, providing a large surface area for catalyst deposition and efficient electron transport pathways. The smooth surface morphology of the fibers ensures uniform coating adhesion, making it a suitable substrate for electrocatalytic applications. The SEM images of NiFeP-coated Ni felt confirm the successful deposition of a well-dispersed NiFeP nanosheet layer. The coating maintains the porous and interconnected nature of the Ni felt, which is essential for maximizing active surface area and catalytic performance. Elemental mapping via energy-dispersive X-ray spectroscopy (EDX) shows a homogeneous distribution of nickel (Ni), iron (Fe), and phosphorus (P), indicating uniform catalyst coverage. Cross-sectional FIB-SEM imaging further validates the strong adhesion of the NiFeP layer to the Ni felt fibers. The coating is continuous and well-integrated with the substrate, ensuring structural stability under electrochemical conditions.

The global transition from fossil fuels to renewable energy is pivotal for combating climate change, with electrochemical CO₂ reduction (CO₂RR) emerging as a promising strategy for carbon mitigation. By coupling renewable electricity with efficient CO₂ utilization, CO₂RR enables the direct conversion of carbon dioxide into value-added chemicals and fuels. However, the selective generation of complex multi-carbon products remains a formidable challenge, rooted in the intricate interplay between catalyst structure, composition, and reactivity^1,2,3. Recent breakthroughs—such as harnessing spin polarization effects via external magnetic fields or chiral molecular environments —have demonstrated remarkable enhancements in catalytic performance, particularly for copper-based systems^4,5,6.

In this research, we focus on the design and investigation of chiral CuOx nanostructures, which uniquely combine the catalytic versatility of copper with the tunable properties of oxide interfaces. Cu is nearly the only metallic catalyst capable of promoting C–C coupling through CO* dimerization, making it an ideal platform for exploring spin-dependent effects and selective CO₂RR.

Advanced electron microscopy techniques, especially (Scanning) Transmission Electron Microscopy ((S)TEM), are employed to unravel the atomic-scale features of these chiral CuOx catalysts. Correlative (S)TEM (performed before and after reaction), three-dimensional tomography, and (integrated) differential phase contrast ((i)DPC) imaging enable visualization of active sites and tracking of structural evolution. These insights are crucial for understanding degradation mechanisms, optimizing catalyst design, and ultimately improving selectivity and efficiency.

By integrating state-of-the-art electron microscopy with the synthesis of robust, intrinsically chiral CuOx electrodes, this research paves the way for the rational design of next-generation electrocatalysts.

Structural heterogeneity presents a major challenge in Cryo-Electron Microscopy (Cryo-EM), where macromolecular complexes often exist in multiple compositional or conformational states. We propose a novel method that exploits the natural similarity of neighboring projections to perform localized dimensionality reduction on directionally clustered subsets of the data. This dimensionality reduction captures structural variability within narrow angular regions while mitigating the effects of the pose diversity. To integrate the resulting local analyses into a coherent global model, we employ Orthogonal Group Synchronization -an emergin technique from applied mathematics- that aligns local analises in a globally consistent manner. The method is designed to be user-friendly, requiring only a few parameters with clear physical and statistical interpretations, thus reducing manual tuning. Additionally, it is computationally efficient, significantly outperforming traditional heterogeneity analysis approaches in terms of runtime. We demonstrate the effectiveness of our approach on both synthetic and experimental datasets, recovering meaningful structural variations with high fidelity.

EMhub is a web framework designed to support the data management needs of scientific facilities (e.g., instrument bookings, user management, data transfer, and reporting). This application has been in use for several years at the Swedish National CryoEM Facility and, more recently, in some core centers within the Structural Biology department at St. Jude Children's Research Hospital. EMhub can be customized to meet different requirements and exposes a REST API that allows external processes to interact with the application. This feature has been leveraged to establish a fully automated CryoEM on-the-fly processing pipeline that can be monitored through the web interface. Here, we present the latest development of the framework, which enables the export of initial results from the pre-processing pipeline to facilitate continued data processing in other software packages, such as Relion or CryoSparc. Several new web tools have been integrated into the application to facilitate data curation and quality evaluation, providing a valuable complement to existing programs. These tools have already proven helpful at St. Jude during the initial tomography data processing efforts for instrument validation and software benchmarking, assessing the required computational resources. 

Pyrroline-5-carboxylate synthetase (P5CS) is a bifunctional single-polypeptide protein encoded by the ALDH18A1 gene, comprising two enzymatic domains, glutamate 5-kinase (G5K) and glutamyl-5-phosphate reductase, which catalyze, respectively, the first and second steps of de novo synthesis of proline and ornithine/arginine. Two clinical syndromes, a neurocutaneous (NC) early-onset severe one and a later-onset complicated spastic paraplegia (SPG9), are associated with mutations in ALDH18A1, having recessive or dominant character, with mutation specificity for presentation and dominance or recessivity. 

We concluded (Marco-Marin et al., J Inher Metab Dis 2012 and 2020) that all ALDH18A1 pathogenic mutations cause loss of P5CS function, including dominant mutations, which would act by a negative dominance mechanism. To understand how is this possible, we determined the cryoEM structure of P5CS (3.4 Å), revealing a homotetrameric building block having point group 222 symmetry. The tetramer is formed by a central G5K planar tetramer, and by two peripheral globular blebs formed by pairs of G5PR domains. Anvil-like protrusions emerge perpendicularly to the G5K plane at its center on both faces of the plane. Two tetramers stack via the anvil platforms to form octamers (3.8 Å) which could allow endless extension into approximately straight fibers defined by a torque rotation of the tetramer 60º around the fiber axis per step along the fiber, so that the same fiber orientation is seen every 180º. In fact, we have also observed two types of dodecamers (4.0 and 4.3 Å) of which one which could also be extended to the same linear fiber would represent three steps along the fiber, while the second would introduce bends in the fiber. Interactions between tetramers in the octamer and dodecamer are mediated by the central anvils and by contacts between G5PR domains of adjacent tetramers. Residues involved in these connections appear to be those causing dominance, giving the most severe presentation (NC) those affecting the anvil or residues at the interface between G5K domains within the tetramer. Thus, the ability to form fibers appears crucial for activity, and fiber distortion or abolition may decrease or abolish activity. As expected, mutations that cause misfolding and protein loss or kinetic aberrations that do not cause fiber distortion would be expected to be recessive. Experiments are under way to correllate structural derrangements with activity levels. The observed stacking in octamers and dodecamers is compatible with the filamentous assemblies reported for P5CS from Drosophila melanogaster or with optical microscopy observation in cells. It also resembles although with a different fiber torque the fiber of Arabidopsis thaliana P5CS, supporting the value of the plant enzyme as a potential model for human P5CS.

Influenza A virus replication relies on the activity of viral ribonucleoprotein complexes (vRNPs), which consist of a segmented RNA genome encapsidated by nucleoproteins (NP) and associated with a heterotrimeric RNA-dependent RNA polymerase (RdRp). This multifunctional polymerase, composed of the PB1, PB2, and PA subunits, meadiates both transcription and replication of the viral genome. PB1 serves as the catalytic core for RNA synthesis, PB2 binds host-derived capped primers, and PA harbors the endonuclease activity essential for the cap-snatching mechanism. Given its critical role in viral propagation, the polymerase is a prime target for antiviral development. Using cryo-electron microscopy (cryo-EM), we resolved the structure of the polymerase in complex with the endonuclease inhibitor baloxavir acid. The dataset revealed distinct conformational states, including classes where the PA endonuclease domain, harboring the drug-binding site, is well defined.

To complement the structural data, we assembled recombinant ribonucleoprotein complexes (RNPs) carrying a reporter RNA encoding nanoluciferase, enabling functional assessment of polymerase activity and inhibition. 

These results highlight the utility of cryo-EM in capturing conformational dynamics relevant to drug binding and support structure-guided strategies for antiviral discovery against influenza A.

Pore-forming proteins exemplify the remarkable versatility of biological molecules. Initially produced as monomeric, water-soluble entities, they spontaneously assemble into multimeric integral membrane proteins upon encountering suitable target lipids [1]. Their functions span apoptosis, cell signaling, immunity, and inter-organismal attack and defense systems [2]. Among these are actinoporins, a family of pore-forming toxins from sea anemones that kill target cells by perforating their plasma membranes. Here, we present the structures of two such toxins, fragaceatoxin C and sticholysin II, in a complex membrane environment, determined using cryogenic electron microscopy. The structures reveal how dozens of lipid molecules interact in an orderly fashion, becoming integral components of the pore. We also isolated distinct pore-forming intermediates, in which only a subset of monomers is incorporated, forming non-closed, arc-shaped assemblies. Based on these structures, we propose a mechanism of action whereby sequential monomer assembly onto the membrane, accompanied by conformational changes, drives pore formation and membrane perforation. Our findings provide new insights into the transformative capacity of these proteins, which are increasingly recognized for their diverse biotechnological applications [3,4].

AMPA receptors (AMPARs), part of the ionotropic glutamate receptor family, function as ligand-gated cation channels that mediate fast excitatory synaptic transmission and contribute to synaptic plasticity. These receptors form tetrameric assemblies composed of GluA1–GluA4 subunits. The presence or absence of the GluA2 subunit critically influences calcium permeability: GluA2-containing receptors are impermeable to Ca²⁺, while those lacking GluA2 are permeable. AMPARs also interact with auxiliary proteins such as TARPs, CKAMPs, GSG1L, and CNIH, which modulate their functional properties, including gating dynamics and pharmacological profiles. Given their central role in synaptic function, AMPAR complexes represent important targets for the treatment of neurological diseases.

Here, we employed cryo-EM to analyse the structure of GluA4-containing AMPARs, both alone and in complex with the auxiliary subunit TARP-2, across three different conformational states. In the resting conformation, the GluA4 core exhibits a modular, Y-shaped structure and forms a dimer-of-dimers arrangement, consistent with previous observations in GluA2-based receptors. Notably, conformational changes associated with TARP-2 binding and channel gating reveal key structural deviations. In particular, we detected disruption of LBD dimer interfaces in a subset of the resting and desensitized particles, a feature reminiscent of GluA1-containing receptors. Additionally, our analysis uncovered a previously uncharacterized regulatory site implicated in TARP-2–mediated modulation.

Laser treatments are extensively employed in various applications, including surface modification, material processing, and additive manufacturing. However, the oxidation generated during these processes, whether intentional or unintentional, necessitates careful control due to its potential impact on the material's properties. Beyond alterations in surface topography, laser irradiation can induce significant microstructural and chemical changes, particularly when performed in non-protective atmospheres. This study presents a comprehensive in-situ scanning electron microscopy (SEM) characterization of both the surface topography and the oxidation map resulting from laser single-line scans on a WC-10%Co alloy, a material widely used in cutting tools and tooling where enhanced tribological properties without compromising mechanical integrity are crucial.

Measurements were conducted within the SEM chamber, allowing for direct observation and analysis of the laser-treated surfaces. Surface topography was quantitatively assessed through stereoscopic reconstructions derived from sequential SEM images acquired at different tilt angles [1]. Concurrently, in-situ energy-dispersive X-ray spectroscopy (EDX) analysis was performed to generate elemental maps, specifically focusing on oxygen distribution. Laser treatments consisted of single-line scans with systematically varied fluence parameter.

The integrated in-situ SEM and EDX approach enabled the simultaneous determination of the ablation diameter, a commonly studied parameter in laser processing  [2], and the presence of laser-induced oxidation. An exploration of the relationship between the laser process and surface oxidation can be found in [3]. A novel methodology is proposed for estimating the oxidation diameter based on the spatial distribution of oxygen as revealed by the EDX maps.

The results demonstrate a clear correlation between the laser processing parameters and both the ablation and oxidation diameter. Notably, the spatial profile of the oxidation distribution, exhibited a Gaussian-like profile, mirroring the intensity distribution of the laser beam and the trend observed for the ablation diameter. This suggests that similar models used to predict ablation dimensions can potentially be adapted to estimate the extent of oxidation. The in-situ characterization provides valuable insights into the complex interplay between laser parameters, surface morphology evolution, and oxidation mechanisms in WC-Co alloys, contributing to a better understanding and control of laser surface treatments for this important class of materials.

The In Situ Correlative Facility for Advanced Energy Materials (InCAEM) project aims to establish a cutting-edge open-access research infrastructure dedicated to advanced energy materials science. Launched in October 2022, InCAEM is a collaborative initiative led by four partner institutions — ALBA Synchrotron, ICN2, ICMAB, and PIC-IFAE — and is expected to be fully operational by the end of 2025. The facility will integrate state-of-the-art instrumentation, infrastructure, and expert personnel to support world-class research aligned with the European Green Deal.

InCAEM will enable true multi-modal and multi-length-scale characterization of functional materials by integrating operando sample environments with complementary characterization tools and advanced data analysis. While scanning transmission electron microscopy (STEM) and scanning probe microscopy (SPM) provide high spatial resolution and diverse contrast mechanisms, X-ray-based techniques offer efficient, highly specific chemical, structural, electronic, and magnetic insights, allowing the study of larger fields of view, thicker samples, and faster processes.

This presentation will introduce the InCAEM project and highlight the opportunities it offers to the scientific user community. We will focus on showcasing new STEM capabilities being installed at the Joint Electron Microscopy Center at ALBA (JEMCA), including a double-aberration-corrected STEM and in situ S/TEM holder systems. These systems enable biasing and temperature control from -160 °C to 800 °C, in situ gas-phase studies up to 2 bar for catalysis, and liquid environments for electrochemical measurements with simultaneous biasing. We will also present the adaptations being developed for various ALBA beamlines, enabling correlative workflows that integrate electron and X-ray microscopy and spectroscopy.

Lithium cobalt oxide (LCO) is a widely used cathode material in lithium-ion batteries [1, 2]. While aqueous electrolytes offer improved safety and lower cost compared to organic counterparts, LCO undergoes rapid degradation in aqueous environments, limiting its commercial viability.  The mechanisms by which the LCO degrades remain poorly understood [3, 4].

We use in situ scanning/transmission electron microscopy (S/TEM) to directly observe the LCO failure mechanisms in aqueous conditions and their implication during charge-discharge cycling. This real-time approach reveals that degradation is spontaneous and primarily driven by surface dissolution, initiated at fracture sites and regions of increased surface roughness. A porous coating layer forms around LCO particles as they dissolve into the surrounding electrolyte. The rate of dissolution is faster on smaller LCO particles. The dissolution occurs even in the absence of externally applied electrochemical cycling, indicating a chemically driven process [5].  Replacing water by ethanol or IPA eliminated/drastically reduced spontaneous LCO dissolution.

LCO particles expand and contract upon lithiation and delithiation, respectively, which can also be accompanied by shape changes.  Larger and thicker particles show milder susceptibility to cycling degradation. Using an aqueous electrolyte and a cathode material without significant air/moisture sensitivity simplified protocol development while allowing us to obtain in-depth understanding of the challenges associated with in situ S/TEM battery studies. This study paves the way for advanced studies of other relevant battery systems in commercially relevant conditions, such organic electrolytes or sensitive air/moisture battery components.

Research on plasmonic noble metal nanomaterials (NMNs) has surged in recent years due to their exceptional optical and electronic properties, which differ markedly from their bulk counterparts. These materials can generate energetic (hot) electrons and holes upon interaction with electromagnetic radiation, enabling them to drive surface chemical reactions under light or electron beam excitation—an ability that holds promise for replacing fossil-fuel-based catalytic processes. [1-3] Among NMNs, bimetallic systems have attracted considerable interest for their enhanced catalytic activity and stability, particularly in heterogeneous reactions.

Au@Pd core–shell nanoparticles and Au–Pd nanoalloys have demonstrated high efficiency in electrocatalytic applications such as ethanol oxidation and hydrogen peroxide (H₂O₂) production, outperforming monometallic Pd catalysts. Au is highly effective in converting alcohols to aldehydes, whereas Pd is superior in oxygen reduction reactions. Alloying Au and Pd can overcome the individual limitations of each metal. However, the resulting alloys often exhibit reduced electrochemical potentials compared to their monometallic counterparts, which can limit catalytic efficiency. [4]

To address this, a bimetallic nanostructure with intrinsic phase separation, wherein oxidation and reduction reactions are spatially segregated across distinct domains of a single nanoparticle. Such phase-separated systems preserve the intrinsic catalytic properties of each metal while enabling synergistic interactions at their interface. This architecture is known to enhance both activity and long-term stability. However, forming phase-separated Au–Pd nanoparticles is challenging due to their complete miscibility across all compositions and temperatures, as predicted by the bulk phase diagram. Phase separation in bimetallic systems typically requires a lattice mismatch >5%, which is absent in Au–Pd combinations. [5]

Although theoretical studies have suggested a potential miscibility gap in the nanoscale regime, direct experimental evidence for temperature-induced phase separation in Au–Pd nanostructures remains scarce. Prior studies, including those by Okamoto et al. and Wu et al., have reported alloy formation across a wide composition range, even under high-temperature annealing. [6-7] Other reports, such as that by Precot et al., suggest size-dependent segregation behavior driven by Ostwald ripening, yet clear in situ observations of phase separation within individual Au–Pd nanoparticles are lacking. [8]

In this work, we synthesize Au nanotriangles (AuNTs) via a seedless method and coat them with Pd layers of varying thickness by adjusting the concentration of H₂PdCl₄. In situ high-resolution transmission electron microscopy (HRTEM) and selected area diffraction (SAD) were performed using an image-corrected FEI TITAN at 300 kV, while elemental mapping and composition analysis were conducted using high-resolution STEM and EDS on a probe-corrected system. For in-situ heating, we used a DENS Solutions Wildfire MEMS-based double-tilt holder with SiₓNᵧ membranes, capable of reaching 1300 °C.

A detailed investigation of AuNT and AuNT@Pd nanostructures revealed various interfacial defects—such as stacking faults and dislocations—within the Pd shell. Upon in situ annealing, distinct melting behavior was observed: the melting point increased with Pd content, confirming Pd’s stabilizing effect. Most significantly, we observed temperature-induced phase separation within Au–Pd alloy nanotriangles, forming Au-rich and Pd-rich domains. This was verified by HRTEM and EDS mapping. At elevated temperatures, Pd atoms migrated outward due to their higher mobility, while Au atoms moved inward, leading to a stable, phase-separated configuration. [9]

This phase-separated state remained stable even after prolonged storage at room temperature, as confirmed by EDS spectra collected a year post-synthesis. These findings offer critical insight into the thermodynamic behavior of nominally miscible bimetallic systems at the nanoscale and pave the way for designing phase-engineered nanocatalysts with enhanced bi-functional activity and long-term durability.

The demand for real-time, nanoscale materials characterization has long challenged the capabilities of Transmission Electron Microscopy (TEM). Conventionally, TEM has been confined to the analysis of solid-state specimens, providing high-resolution imaging in dry, high-vacuum environments. This vacuum requirement precludes the direct investigation of liquid-phase systems, making it difficult to capture dynamic processes occurring in solution. Consequently, in-situ/operando observation of liquid-based phenomena has remained a persistent limitation in TEM applications. The advent of liquid cell in-situ TEM holders has transformed this landscape, enabling the direct visualization of samples in liquid environments [1, 2, 3]. These specialized holders sandwich a thin liquid layer between electron-transparent membranes (SiN), maintaining the liquid state even under vacuum. Recent innovations in liquid cell technology have greatly broadened the scope of TEM applications in areas, i.e., electrochemistry [1], photochemistry, corrosion, electroplating [3], electrocatalysis [2], battery and fuel cell materials [4], as well as biomedical applications.  Furthermore, integrating analytical techniques like Energy-Dispersive X-ray Spectroscopy (EDS) [1] and Electron Energy Loss Spectroscopy (EELS) [4] within the liquid cell environment provides valuable insights into the elemental composition and electronic structure of materials, significantly enhancing the analytical capabilities of in-situ TEM. In this presentation, we employed both conventional and in-situ electrochemical testing on faceted high-entropy alloy (HEA) CoFeNiRuZr-Pt nanoparticles decorated on graphene flakes. These HEA nanoparticle-decorated graphene flakes were deposited onto a microfabricated chip designed specifically for in-situ liquid electron microscopy. Cyclic voltammetry (CV) was conducted both outside the microscope under ambient conditions and within the TEM itself. This approach enabled a direct correlation between the nanoparticles' electrochemical behaviour and their structural and compositional evolution at the nanoscale. In addition, morphological changes in the faceted HEA nanoparticles were also examined during the in-situ observations. The HEA nanoparticles were observed to undergo a phase transformation when an electric potential was applied. At the same time the particles are also been observed to undergo a morphological transition.  

The design of rooms to host transmission electron microscopes (TEM) became a critical concern with the commercialization of the first aberration corrected instruments. [1] Suddenly, the limiting factor for performance was no longer the spatial resolution of the microscope but its sensitivity to the surrounding environment. Today, a top-performing instrument must be housed in specialized facilities, and any leading research work requires careful cradle-to-grave consideration of the equipment.

In parallel, the development of in-situ/operando TEM has been remarkable with a proliferation of methods and instrumentation, leading to a large variety of externally controlled stimuli now available, to cause a response from TEM specimens. Given the rapid advances in this field, it is prudent to start planning for the harmonization of protocols by addressing critical issues, such as safety.

One perfect example is the work of dynamical experiments performed in a gaseous environment, normally using a specialized TEM sample holder. Depending on the brand, the entire setup may include a station to accommodate the gas cylinders that feed the holder. In the simpler configuration, it is up to the microscopist to arrange the feeding lines, gas cylinders and other diverse ancillary gears that provide a connection to the holder. In such a case, gas cylinders may be positioned just a few steps from the operator, while various gases, some potentially toxic and/or flammable, may pose a potential risk of leakage to the room.

At INL, we have conceived a permanent solution in order to integrate the design of gas lines, with the building and the microscope to carry in situ gas TEM experiments. For that, a multi-departmental team composed of microscopists, specialists in Health and Safety, lab maintenance/ installation and specialized gas engineers worked together to provide a design that allows for the safe operation of TEM gas holders, along with the remote storage of gas tanks, irrespective of their size, toxicity and/or flammability level. Progress towards this goal is communicated in this paper.

Hydrogen production through water electrolysis is a critical process for the renewable energy transition. The efficiency and durability of the electrocatalysts used in proton exchange membrane (PEM) water electrolysis, particularly for the oxygen evolution reaction (OER), are central to advancing this technology. Iridium oxide (IrO₂) is one of the most effective catalysts for OER, but its high cost and limited availability hinder large-scale applications. This study aims to investigate the degradation mechanisms of IrO₂ catalysts under real-world PEM electrolysis conditions using scanning transmission electron microscopy (STEM) techniques. Specifically, in-situ STEM will be employed to observe the structural and morphological changes of IrO₂ catalysts during electrochemical cycling. This technique will allow us to track particle migration, coalescence, Ostwald ripening, dissolution, and redeposition under working conditions. Key factors in this study will involve selecting high-quality liquid cell chips; for this observation, we used Indium Tin Oxide, and minimizing electron beam damage by employing low-dose imaging methods during in-situ characterization to prevent beam-induced damage and ensure accurate imaging without interference. Ultimately, this study allows tuning the optimal operating conditions for further research into developing cost-effective and stable electrocatalysts for hydrogen production, particularly under realistic electrochemical conditions that closely simulate those in large-scale PEM water electrolysis systems.

The SARS-CoV-2 genome encodes eleven accessory proteins, many of which remain poorly characterized but are increasingly implicated in viral pathogenesis. Among them, ORF3a, the largest, is a controversial viroporin known to impair immune responses and promote vesicle formation.

We have combined sections and soft-X-ray tomography studies to elucidate the molecular mechanism underlying ORF3a’s impact on the host cell. In both A549 epithelial cells and differentiated THP1 dendritic cells, we observed a striking increase in intracellular vesicles, accompanied by fragmentation of the endoplasmic reticulum and Golgi apparatus. ORF3a localized to vesicles displaying distinctive inner tubular structures, whose origin appear variable based on co-immunostaining with fluorescent markers.

In addition, we detected an unusual accumulation of lipid droplets, which correlates with a shift in lipid metabolism as revealed by integrated transcriptomics, lipidomic and proteomic analyses. Further, our results explain previous reports showing lipids bound to ORF3a inner channels. Tomographic reconstructions also revealed altered mitochondria with abnormal cristae, consistent with recent findings linking ORF3a to an increase in low-activity mitochondria exhibiting enhanced motility and reduced displacement.

Together, our findings demonstrate that ORF3a alone is sufficient to profoundly remodel cellular architecture, supporting its potential role in the formation of SARS-CoV-2 viral factories through lipid mobilization.

Positive-strand RNA viruses, such as hepatitis C virus (HCV), West Nile virus (WNV), and coronaviruses, hijack host cellular membranes to create specialized replication compartments [1]. Despite variations in membrane origin and structure, these compartments often share conserved features—most notably, the formation of double-membrane vesicles (DMVs) and invaginated vesicles (IVs) coronavirus [2]. In the case of WNV, a defining ultrastructural hallmark is the presence of convoluted membranes (CMs), which are intimately involved in the viral life cycle [3]. Traditional microscopy methods face significant limitations in visualizing such features within whole, unstained, and unperturbed cellular environments.

To overcome these challenges, we apply a correlative cryogenic imaging strategy—Cryo correlative Light and soft X-ray Tomography (CLXT)—that combines Structured Illumination Microscopy (SIM) with cryo soft X-ray tomography (cryo-SXT). This multimodal approach is designed to probe the 3D ultrastructure of infected cells at nanometric resolution while preserving native cellular architecture. Cryo-SXT operates in the water window energy range (520 eV), where carbon-rich structures absorb soft X-rays more strongly than their oxygen-rich, hydrated surroundings. This contrast mechanism allows label-free, quantitative imaging of whole vitrified cells with lateral resolutions of 50nm and depths up to 10 µm. Tilt series of X-ray projection images are reconstructed into 3D volumes that reveal the internal organization of infected cells with high structural fidelity.

Structured Illumination Microscopy (SIM), a super-resolution light microscopy technique, complements this by providing specific molecular context. SIM achieves resolutions below 100 nm using conventional fluorescent dyes and is compatible with cryo workflows. By projecting sinusoidal light patterns and reconstructing Moiré fringes, SIM reveals fine cellular details while preserving fluorescence signals. This makes it ideal for identifying and localizing fluorescently labeled structures in a cryogenic environment.

In this study, CLXT was employed to analyze the morphological alterations induced by WNV in whole cryo-preserved cells. We successfully identified convoluted membranes, characteristic of WNV replication, in the 3D reconstructed SXT volumes and verified their presence using SIM-based localization. This correlative framework allowed us to spatially correlate ultrastructural features with fluorescent markers, significantly enhancing the interpretability and specificity of the dataset.

This work illustrates the power of correlative cryogenic multimodal imaging to investigate virus-induced cellular remodeling. The CLXT approach bridges the resolution gap between fluorescence and electron-based techniques while maintaining the contextual advantages of whole-cell imaging. By enabling the label-free, volumetric visualization of infection-related structures and correlating them with specific fluorescent markers, CLXT offers a robust and scalable platform for structural virology and cell biology research.

The SERMET (Transmission Electron Microscopy Service) at the University of Cantabria holds two electron microscopes to service both Material Sciences and Structure Biology needs. In addition, the service also holds a cryo-ultra-microtome to fulfill the requirements for both communities. The microscope for Material Science is a Jeol Jem 2100 (200 kV) equipped with a STEM unit, a XEDS microanalysis system and a CCD camera.  On the Structural Biology side, the service hosts a Tundra Cryo-TEM (100kV), equipped with a high brightness XFEG gun, semi-automated sample loading, computerized stage with single-axis tilt holder, high resolution objective lens (SP-Twin) optimized for SPA (Single Particle Analysis) and a Thermo Scientific Ceta-F camera optimized for low dose imaging. This microscope has been designed following the ideas of Richard Henderson and Christopher Russo (1-2) on how a lower acceleration voltage could benefit the image contrast formation and reduce the dose electron damage in a cryo-electron microscope.

The Tundra Cryo-TEM setup at the SERMET also includes a cryo-loading workstation, a Vitrobot Mk IV for sample preparation, a PELCO glow discharge unit, and all the equipment necessary for sample preparation and preservation. Sample grids screened with the Tundra Cryo-TEM can be rescued for later use in top-end microscopes such as the Titan-Krios.

The microscope is setup with MiCo software and data acquisition is performed with EPU-2 software. It also includes Athena software to centralize and organize imaging data and experimental workflows.

The microscopes at the SERMET are open to be used for all the scientific community at reasonable competitive cost.

Controlling ciliary beating is essential for motility and signaling in eukaryotes. This process relies on the regulation of various axonemal proteins that assemble in stereotyped patterns onto individual microtubules of the ciliary structure. Additionally, each axonemal protein interacts exclusively with determined tubulin protofilaments of the neighboring microtubule to carry out its function. While it is known that tubulin post-translational modifications (PTMs) are important for proper ciliary motility, the mode and extent to which they contribute to these interactions remain poorly understood. Currently, the prevailing understanding is that PTMs can confer functional specialization at the level of individual microtubules. However, this paradigm falls short of explaining how the tubulin code can manage the complexity of the axonemal structure where functional interactions happen in defined patterns at the sub-microtubular scale. Here, we combine immuno-cryo-electron tomography (cryo-ET), expansion microscopy, and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nanopatterns at a sub-microtubular scale. These nanopatterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are indispensable for their regulation during ciliary beating. Our findings offer a new paradigm for understanding how different tubulin PTMs, such as glycylation, glutamylation, acetylation, tyrosination, and detyrosination, can coexist within the ciliary structure and specialize individual protofilaments for the regulation of diverse protein complexes. The identification of a ciliary tubulin nanocode by cryo-ET suggests the need for high-resolution studies to better understand the molecular role of PTMs in other cellular compartments beyond the cilium.

Herpes simplex virus type 1 (HSV-1) remodels the host chromatin structure and induces a host-to-virus transcriptional switch during lytic infection. We combined super-resolution imaging (STORM and PAINT) and chromosome-capture technologies to identify the mechanism of remodeling. We show that the host chromatin undergoes massive condensation caused not by epigenetic changes, but rather by the hijacking of RNA polymerase II (RNAP II) and topoisomerase I (TOP1). In addition, HSV-1 hijacking of cohesin results in the loss of topologically associating domains (TADs) and loops, although the A/B compartments are maintained in the host. The position of viral genomes and their association with RNAP II and cohesin was determined nanometrically. Strikingly, we reveal specific host–HSV-1 genome interactions and enrichment of upregulated human genes in the most contacting regions. This viral mechanism of host chromatin rewiring sheds light on the role of transcription in chromatin architecture and in viral infection.

The intensive use of plastic, combined with its reduced and/ or extremely slow recyclability, leads to its accumulation in the environment, becoming a persistent pollutant. The plastic discarded in the environment originates by different mechanisms such as photo-degradation, high-temperature degradation, physical erosion and microbial degradation smaller particles known as microplastics (MPs ≤ 5mm) and nanoplastics (NPs≤ 1µm) that accumulate in the soil, water and organisms causing ecotoxicological issues. Microbial degradation of plastic is a sustainable remediation process for transforming and removing that is gaining growing attention. Biofilms can play an important role in this process but can also function as an environmental reservoir for potential human pathogens and antimicrobials resistance.

Here we studied the interaction between four MPs derived from some of most used plastic polymers, namely low-density polyethylene (LDPE), polypropylene (PP), polyethylene terephthalate (PET) and polystyrene (PET) and a consortium of four bacteria (e.g. E. coli, Aeromonas sobria, Klebsiela pneumoniae and Enterobacter cloacae) isolated from surface water over 46 days either in close or open systems. Biofilm assembly was assayed using crystal violet assay and colony forming units (CFU) enumeration. In addition, FISH and SEM were used to monitored biofilm assembly overtime. Bacteria were able to assemble biofilm on all plastics following a similar kinetic. SEM allowed the identification of heterogeneity in biofilm assembly on the MPs surface for the open system; as well as, differences between the two systems with enhanced biofilm formation in the open system.

Bacteria recovered from biofilm identification showed that E. coli was the less predominant bacteria. Bacterial susceptibility to antibiotics changed over the time course of the experiment. Since selection factors were not added to the media, we hypothesize that horizontal gene transfer between different isolates might be responsible for this result. Nevertheless, more studies must be performed to identify the genes responsible for the observed antibiotic susceptibility profiles.

Understanding the precise atomic configuration of active sites is pivotal for unraveling catalytic mechanisms in lithium–sulfur (Li–S) batteries. Particularly, dual-atom catalysts (DACs) have emerged as promising candidates due to their synergistic electronic interactions and high catalytic activity.[1] However, direct observation and unambiguous identification of dual-atom configurations remain challenging due to their low loading and potential overlap with background signals.
Here, we present an advanced atom detection strategy combining aberration-corrected scanning transmission electron microscopy (AC-STEM) with statistical atom counting and machine learning-assisted image analysis to reliably detect and distinguish dual metal sites within a sulfur-host framework. Utilizing high-angle annular dark-field (HAADF) imaging, we achieve sub-angstrom resolution that enables the visualization of isolated and paired atomic columns. The approach is validated on a Co–Bi dual-atom catalyst embedded in nitrogen-doped carbon, designed to regulate the polysulfide redox reaction in Li–S batteries.
Our results reveal the spatial correlation and bonding environment of dual atoms, with electronic structure insights supported by electron energy loss spectroscopy (EELS) and density functional theory (DFT). This work offers a robust pathway for detecting and characterizing dual-atom motifs, facilitating the rational design of next-generation sulfur cathode catalysts and advancing the atomic-level understanding of complex energy systems.
 

Atomic-Scale Insights into Ba-Doped LiOCl₃ Anti-Perovskite Electrolytes for Solid-State Batteries

Maurya Sandeep Prdaeepkumar¹, Meera Mohan¹, M. Helena Braga², Francis Leonard Deepak¹

¹International Iberian Nanotechnology Laboratory (INL), Braga-4715-330, Portugal

²Faculty of Engineering, University of Porto, 4200-465, Porto, Portugal

Solid-state batteries offer significant advantages, but their commercial adoption is limited by the poor ionic conductivity of solid electrolytes at practical temperatures and their inadequate stability with lithium metal. In the present work, Ba doped Li_2.99Ba_0.005OCl a glassy electrolyte with anti-perovskite structure, exhibiting ultra-fast ionic conductivity, was synthesized and investigated [1]. To study the effect of Ba doping in Li₃OCl, aberration-corrected scanning/transmission electron microscopy (AC-S/TEM) was employed to observe lattice distortions caused by the larger ionic radius of Ba²⁺ compared to Li⁺, which may induce phase instability, defect formation, or secondary phase precipitation if the doping limit is exceeded.

Initial TEM imaging was performed under low-dose conditions; however, radiation damage was still observed. To mitigate this, a cryo-holder was employed in combination with low-dose techniques, significantly reducing beam-induced damage. This enabled the successful acquisition of high-resolution TEM (HR-TEM) images, where lattice fringes were clearly resolved. High-resolution STEM (HRSTEM) images were obtained using an aberration-corrected STEM (AC-STEM) instrument, which has clear evidence of lattice distortion. Selected area electron diffraction (SAED) confirmed that Li_2.99Ba_0.005OCl adopts a cubic structure with space group Pm-3m, indicating that some clusters are single crystalline. However, SAED patterns revealed that most clusters are polycrystalline, with multiple grain orientations present within individual cluster. TEM images further confirmed that these clusters are approximately micron sized. STEM–EDX mapping revealed the successful incorporation of Ba into the Li₃OCl matrix. Additionally, electron energy loss spectroscopy (EELS) provided insights into the chemical environment and bonding characteristics of Ba within the Li₃OCl structure.

[1]       M.H. Braga, J.A. Ferreira, V. Stockhausen, J.E. Oliveira, A. El-Azab, Novel Li 3 ClO based glasses with superionic properties for lithium batteries, J. Mater. Chem. A 2 (2014) 5470–5480. https://doi.org/10.1039/C3TA15087A.

Introduction

Organic-metal halide perovskites, with a metal-halide octahedral framework separated by organic cations, offer tunable electronic properties, making them appealing for optoelectronic applications [1]. Their soft crystal lattice allows greater tolerance to lattice mismatch, making them promising for heterostructure formation [2]. Fabrication of in-plane heterostructures with varying halide composition in 2D lead-halide perovskites forms spatially separated halide phases with different band gaps, enabling additional control on charge carrier mobility [1].

Objectives

To understand how strain and composition in 2D-layered perovskite lateral heterostructures affect electronic structure and material stability, we combine compositional data from STEM-EDX, and strain maps from 4DSTEM diffraction patterns. We focus on how lattice parameters evolve across the heterostructure, as the composition transitions from chloride to bromide to iodide phases. Insight into the local crystallography will enable optimization of structural and electronic properties for optoelectronic applications.

Materials & Methods

Heterostructures were prepared via a solution-based process in microcrystalline phenethylammonium lead-halide 2DLPs, leading to PEA₂PbCl₄–PEA₂PbBr₄–PEA₂PbI₄ heterostructures. Four-dimensional scanning transmission electron microscopy (4D-STEM) datasets were acquired in microprobe mode on an aberration-corrected ThermoFisher Spectra 300 S/TEM, operated at 300 kV and a semi-convergence angle of 0.5 mrad. Strain mapping was performed using Digital Micrograph by analyzing the 4D-STEM dataset. Compositional information was extracted from Energy-Dispersive X-ray (EDX) signal collected on a Dual-X system.

Results

EDX revealed that the bromide phase exhibits an almost pure composition, with a Pb:Br ratio close to PbBr₄. However, compositional line profiles identified a degree of intermixing in the other two phases (chloride-rich core and iodide-rich shell); we also identified a gradual interface between the chloride core and bromide phase but a sharp interface between the bromide and iodide layers (Fig 1a-g).

Low dose (~e/A2) 4D-STEM measurements showed that the crystallinity was preserved across the entire heterostructure, with a change in diffraction patterns upon transition from the interfaces (Fig 1h-k). Furthermore, the strain mapping results revealed a distinct variation in lattice constants across the heterostructure, reflecting the composition-dependent lattice mismatch. Taking the bromide phase as the strain-free reference, the chloride phase exhibited a compressive lattice distortion relative to the bromide phase. Conversely, the iodide phase showed an expansion (Fig 2a-c). These findings were qualitatively consistent with the relative lattice constants expected for the three pure phases, where the chloride phase has a smaller d-spacing and the iodide phase has a larger d-spacing compared to the bromide phase (Fig 2d-f). However, the reduced difference in plane spacings compared to the relaxed phase-pure values suggests that local alloying occurs selectively and moderates lattice mismatch.

Conclusion

We mapped local d-spacing variations against a bromide-phase reference to visualize strain across the heterostructure. Combining 4D-STEM strain mapping with EDX spectroscopy revealed structural evolution and its link to composition. These insights clarify how heterostructures achieve tunable band gaps.

There is an urgent need to implement the energy sector with earth abundant and sustainable strategies to minimize the environmental impact of current approaches. In this direction, electrochemical devices have a primary role for both producing and storing energy, such as supercapacitors that demonstrated to have high power density, although effort needs to be devoted to improve their energy density [1,2]. Here, nanostructured materials are promising candidate being able to increase the specific capacitance of the system by increasing the active surface area. Furthermore, the use of earth abundant elements is mandatory to ensure an all-in-one solution, avoiding side and undesired effects/products that could be harmful for the sustainability of our planet. Transition metal based devices  demonstrated to be the optimal choice thanks to their optoelectronic properties and the possibility to easily prepare them in form of nanostructures with green chemistry approaches [3]. In this work, we will describe the preparation and in-depth investigation of Zn-based nanostructures, with particular attention to the correlation of the measured electrochemical performance with their structure, composition and optical properties. We will demonstrate that Zn-based electrodes are a promising material to face the energy transition in the near future. 

[1] He, G. et al, Joule, 5, 2, 2021, Pages 379-392 

[2] Wu, Z., et al. (2016). Transition Metal Oxides as Supercapacitor Materials. Nanomaterials in Advanced Batteries and Supercapacitors. Nanostructure Science and Technology. Springer, Cham 

[3] Strano, V. et al, J. Phys. Chem. C 2014, 118, 48, 28189−28195 

DNA transposition plays a central role in genome evolution and adaptation, contributing to the spread of antibiotic resistance and virulence factors. The IS21 family of bacterial transposons encodes a streamlined two-component system: IstA, the transposase, and IstB, a AAA+ ATPase that regulates transposition. Using cryogenic electron microscopy (cryo-EM), we captured key intermediates in the transposition process, providing insights into how IstA and IstB form autoinhibited DNA-bound assemblies and how ATP-driven remodeling of these complexes leads to catalytic activation and strand transfer.

Our structural analysis reveals how IstB oligomerization and nucleotide-dependent conformational changes drive site selection, transposase recruitment, and activation. These findings offer a molecular framework for understanding how AAA+ ATPases regulate transposition and related biological processes.

In parallel, we explored the potential of current structure prediction methods to model this dynamic system. This comparison highlights both the opportunities and the limitations of computational tools when applied to large, multi-component protein–DNA assemblies. Together, our work provides a platform for dissecting the mechanisms of regulated transposition and evaluating integrative approaches in structural biology.

Congenital disorders of glycosylation (CDGs) are a growing group of genetic metabolic disorders caused by defects in protein glycosylation mediated by specific GlycosylTransferases (GTs). Among CDGs, structural heart defects and renal anomalies (SHDRA; OMIM 617478) syndrome is associated with early childhood mortality and features such as corpus callosum agenesis and persistent truncus arteriosus (PTA). Biallelic mutations in TMEM260 (also known as C14orf101) have been identified in SHDRA syndrome cases. Notably, TMEM260-related congenital heart diseases may now be the leading cause of PTA in Japan, surpassing DiGeorge syndrome. We recently discovered human TMEM260 to be an endoplasmic reticulum (ER)-resident integral membrane O-mannosyltransferase. Three families of O-mannosyltransferases have been found in animal cells. They all localize to the ER and specialize in the transfer of α-mannose via O-linkage from the membrane embedded sugar donor dolichyl-phosphate-β-mannose (Dol-P-Man) to specific serine/threonine residues of their different protein substrate targets. TMEM260 is the founding member of the newly discovered third family and specializes in O-mannosylation (O-Man) of Immunoglobulin-like, Plexin, Transcription-factors (IPT) domains in cell-surface receptors, including plexins and receptor tyrosine kinases MET and RON. These receptors are implicated in intercellular communication and extracellular matrix signaling. SHDRA syndrome-linked TMEM260 mutations disrupt O-Man of the IPT domains of these receptors, leading to defects in their maturation and abnormal epithelial morphogenesis in 3D cell models. Despite its role in biology and disease the structure of TMEM260 is unknown. Here, we will present the cryo-electron microscopy (cryo-EM) structure of human TMEM260 in ternary complex (3.0-Å resolution) with the natural donor Dol-P-Man and an acceptor peptide derived from the IPT1-domain of the physiological substrate plexin-B2 semaphorin receptor. The structure reveals a new pGT architecture, the basis for IPT domain recognition and the mechanism of O-Man reaction. In addition, the structure serves as a framework to understand how disease-linked mutations impair TMEM260 function.

Pollen grains contain complex proteomes. A large proportion (between a quarter and a third) of these proteins have been predicted and even more, experimentally demonstrated to be modified through S- and Tyr- nitration. Both types of PTMs could represent key mechanisms in the regulation of the activity of enzymes involved in the primary metabolism of pollen, mainly through oxidation-reduction and transfer reactions, as well as in the modulation of the hydrolytic activity of cell wall enzymes of the pollen tube. Moreover, S-nitration could increase the allergenic potential of some proteins secreted by pollen grains.

In this context, the development of methods to visualize and quantify the extent of protein nitration is of paramount importance to stablish correlations between these parameters and microscopically-determined characteristics of pollen physiology like pollen germinability and the presence of phenotypic alterations affecting morphology and growth pattern of the pollen tube.

In this work, we have adapted fluorescence-based procedures to assess the level of nitration in pollen grains at the cellular level, by using confocal fluorescence microscopy.

As a first approach, germinated pollen grains were subjected to partial digestion of the cell wall by using cellulolytic and pectinolytic enzymes, in order to enhance the penetration of anti-Cys-NO and anti-Tyr-NO antibodies, which were then detected by immunofluorescence microscopy with Alexa488-labelled secondary antibodies.

In a different approach, we have adapted a protocol for the fluorescence-based localization and quantification of NO, released from protein Cys and Tyr nitrated residues after UV treatment of the samples [1], which has been adapted to the use of the DAF-2DA fluorophore, specific to identify NO molecules.

Both methods have been successfully assayed in pollen grains from olive tree (Olea europaea) and Eastern lily (Lilium longiflorum), cultured under standard conditions, as well as in the presence of NO donors such as SNP and GSNO, and NO scavengers like CPTIO.

Biomolecules have varying degrees of conformational flexibility and compositional heterogeneity that complicates the image processing of large sets of cryo-electron microscopy images, especially when flexibility is continuous or there are many compositional options. New approaches like HetSIREN [1] and the Zernike3D family of algorithms [2] are capable of generating conformational landscapes representing complex heterogeneity cases while also producing higher resolution maps of the different states. However, the increasing pool of new algorithms approaching the conformational variability problem introduces a new challenge in comparing and assessing their estimations' reliability to extract more accurate landscapes and conformations.

In this work, we introduce FlexConsensus [3] as a new approach to comparing and validating conformational landscapes based on the estimations of different heterogeneity algorithms. This new method relies on a neural network designed and trained to learn how to merge different conformational landscapes into a common and meaningful consensus space. This consensus space leverages the preservation of the overall and local features of the input landscapes and their common characteristics.

Thanks to the properties of the consensus space, it is possible to measure each particle's stability. In this way, one can derive error histograms to easily identify particles estimated to have similar or different conformations and extract them to continue their analysis.

Additionally, FlexConsensus allows for the recovery of original spaces from the consensus space. Therefore, FlexConsensus also serves as a conversion tool, seamlessly moving from one landscape representation to another to recover and compare the structures determined by different methods.

To illustrate FlexConsensus capabilities, we tested the method's performance against a wide range of simulated and experimental datasets.

Among the simulated datasets, we analyzed a set of controlled but challenging datasets capturing a wide range of conformational variations integrated in CryoBench [4], such that a simulated disordered motion of an IgG antibody or a dataset obtained from a molecular dynamics simulation of the SARS-CoV-2 spike. In these tests, we proved a strong correlation between the location in the consensus space and the conformational states simulated in the images.

Moving to the experimental datasets, FlexConsensus was tested against the EMPIAR 10028 dataset, a realistic and well-studied set of images that illustrates the method's practical capabilities. Complementary to the EMPIAR 10028, we analyzed an experimental SARS-CoV-2 dataset capturing a wide range of the main spike motions to assess better how different methods' landscapes compare and to determine the stability of the distribution of states found by the algorithms.

FlexConsensus is publicly available through Scipion [5] and the Scipion Flexibility Hub [6].

Eukaryotic cells employ RNA polymerase III (Pol III), an enzyme constituted by 17 subunits, to synthesize tRNAs and other short and abundant RNAs which are essential for cellular functions. Transcriptional barriers or nucleotide misincorporations during DNA transcription cause Pol III pausing. Reactivation of the enzyme requires RNA cleavage, which depends on subunit C11. We used cryo-EM combined with biochemical analysis to characterize different stages of Pol III transcriptional pausing. We report three structures representing the arrested complex, the pre-cleavage complex and the reactivated complex. Our results show that paused Pol III interacts more loosely with the hybrid formed by the template DNA strand and the newly-synthetized RNA. Moreover, the C-terminal domain of subunit C11 and the Pol III catalytic center coordinate to cleave backtracked RNA nucleotides. The high resolution of our structures allows to obtain mechanistic insights into Pol III pausing and opens the avenue to understand enzyme reactivation.

Colorectal cancer (CRC) arises from perturbations in the colonic epithelium, including disruption of the epithelial cell polarity and motility. Gut epithelium maintenance relies on the fine-tuning function of the actin cytoskeleton. One of the proteins that governs actin cytoskeleton dynamics is the tumour suppressor Adenomatous Polyposis Coli (APC), well-known as the gatekeeper in colorectal cancer. In vitro work showed that APC interacts with cytoskeletal networks through its C-terminal basic domain, and this interaction has been shown to be critical for proper cell adhesion and directionality of individual cells. However, whether APC-driven actin activity contributes to any collective cell migration event in gut homeostasis and/or impacts on health is unknown.

Here, we began to decipher whether APC-driven actin nucleation plays a role in collective cell remodelling and, consequently, collective migration and invasion of colorectal cancer cells. We used cell monolayers and spheroids stably expressing either wild-type APC (APC-WT) or a separation-of-function mutant (APC-m4) incapable of nucleating actin filaments. We combined this genetic tool with different molecular and cellular biology assays and several bioimaging techniques – confocal microscopy (fixed & live imaging), scanning electron microscopy (SEM) and traction force microscopy (TFM) – a method to measure forces exerted by cells on their surrounding environment. Confocal imaging experiments using cell monolayers revealed that the lack of APC-driven actin nucleation activity perturbed the integrity of the cell-cell junctions and collective cell migration. Ultrastructural effects of APC-m4 mutation at the cell junctions were confirmed by SEM imaging. Moreover, confocal microscopy assays using APC-m4 collagen-embedded spheroids showed reduced and shorter cell finger-like protrusions emanating from spheroids compared to APC-WT spheroids. In addition, TFM experiments showed that APC-m4 spheroids exhibited lower traction forces than APC-WT spheroids, which could explain the reduced abilities of the mutant spheroids to invade.

Together, microscopical approaches across scales have been instrumental in uncovering the importance of APC-driven actin nucleation activity, not only in maintaining the integrity of epithelial monolayers but also in facilitating the force of actin finger-like protrusions that penetrate the basement membrane and invade surrounding tissues. Further studies will be needed to validate APC’s cytoskeletal functions in vivo and to exploit its potential as a therapeutic target for interventions aimed at preventing colorectal cancer progression.

Receptor Binding Proteins (RBPs) are bacteriophage proteins found at the distal end of the phage tail that mediate recognition and binding to bacterial cell surfaces by specifically recognizing structural elements such as LPS, pili, porins, teichoic acids, etc. For encapsulated bacteria, the RBPs can also present a depolymerase domain capable of digesting oligosaccharide bonds to facilitate access to the cell surface.

Phage K50PH164C1 targeting Klebsiella pneumoniae presents a depolymerase at the end of its tail fiber of interest to develop phage-derived sensing proteins for diagnostic applications. For this, our group focused on solving its structure, an elongated fiber more than 40 nm long, which is of note since few structures alike have been solved.

Working with a deletion of the N-terminal, we obtained micrographs at the 200kV Glacios microscope at ALBA Synchrotron, and 2D classes which were used to model a preliminary 3D volume at 5Å. This model confirmed our predictions about the structure of the fiber protein, and we obtained data at the 300kV Titan Krios microscope at ESRF, which we hope will provide more information when processed.

Finally, grids were prepared and screened with the full-length protein. In this run we overcame the issues we found with the first run, obtaining better quality grids that will allow for improved data collection. We are currently waiting for time to perform a full data collection on the full-length protein to finally elucidate the structure of this interesting protein at high resolution.

The bacterial flagellum is a highly sophisticated organelle primarily evolved for motility that exhibits a long whip-like filament consisting of up to 30,000 subunits of molecular building block, the protein flagellin. In 2017, our lab discovered and describe the very first enzymatically active bacterial flagella, displaying proteolytic domains on their filament surface (1). Based on this discovery, we now aim to re-engineer naturally occurring structural flagellins to encode for enzymatic activity, and to use these semi-synthetic functionalized nanomachines and bacteria for dedicated biotechnological applications.

To do so, we have first established a plasmid-based system for flagella and thus motility restoration in a flagellin-knockout E. coli strain. Next, we recreated proteolytically active flagella in E. coli – a first proof of concept of enzymatic flagellar display, and a key first step in the development of a robust flagellar surface display system for other functional domains. Over the last months, we have optimized sample preparation of intact flagellar filaments and collected several cryo-EM datasets of various semi-synthetic filaments displaying different functional domains.

We hope, this will allow us to understand how these displays affect at the molecular level the flagellum’s overall architecture, the conformation of the displayed domain, and its interaction with the filament, and enable us to develop a more rational approach for grafting functional domains onto the flagella surface.

Retina degenerative diseases where photoreceptors are progressively lost, are currently the primary cause of untreatable low vision and blindness. A promising approach to finding a treatment is harvesting healthy photoreceptors from human-derived retinal organoids and transplanting them into the diseased retina. This has already been proven effective structurally, but its impact on retinal network signaling has not yet been fully understood.

              To fill in this gap, we aim at developing a methodology to study neuronal networks in healthy, diseased and treated retinas. Currently, we are developing calcium imaging in 2D to study the retina's ganglion cell layer (GCL) under luminous LED stimulation in the visible range. To be able to follow calcium dynamics, we first need to express the GCaMP calcium reporter in the retina. We achieve this by performing an intravitreal injection of the AAV2-CAG-GCaMP5 construct in Long Evans rats. Then, we dissect the retina using dim red-light illumination and oxygenated media to keep the tissue responsive to light and alive. To avoid bleaching the retina's light responsiveness, researchers use two-photon excitation fluorescence (2PEF) microscopy to image the calcium reporter, so we follow this same approach.

              To this point, we have adapted and developed a methodology to image the healthy retinas using a custom-built 2PEF scanning microscope. Firstly, we checked the performance of our microscope by imaging single neurons with sufficient temporal resolution (5Hz) in cell culture and in the Rbfox1-GCaMP6s zebrafish larva’s brain. Then, we validated our stimulation setup by recording light stimulated responses in the neurons of the optical tectum of these same zebrafish larvae. By doing so, we have been able to perform stable long-duration recordings from retinal organoids and retinal explants, where we observe robust calcium spikes. The following step will be to retrieve light-evoked responses from retinas using adequate light-stimulation schemes.

              We have developed a steady methodology for 2D calcium imaging of retinal samples using a custom-built 2PEF microscope. In the future, we will evaluate the feasibility of using 2PEF light-sheet microscopy to perform fast volumetric imaging, capturing retinal dynamics in 3D. Connectivity maps will be constructed from the 2D and 3D datasets to analyze the functional responses of GCLs in the different retina cases and comparing the different imaging systems. This approach holds promise for advancing our understanding of retinal function and pathology.

Cerium oxide has been widely studied, mainly because of its ability to rapidly and reversibly switch between Ce³⁺ and Ce⁴⁺ oxidation states within a cubic structure, facilitating reversible oxygen exchange with its surroundings^[1]. Furthermore, the incorporation of noble metals, such as platinum, improves these redox properties by promoting the formation and mobility of oxygen vacancies^[2].

In the study of the reduction of cerium oxide based catalyst, conventional transmission electron microscopy (TEM) procedures involve treating the sample in a quartz reactor outside the microscope, and then it is exposed to the air during transfer, grid assembling and inserting into the microscope using a standard sample holder. However, this approach has several limitations; i) potential changes in the oxidation state and ii) difficulties in tracking all the structural and chemical changes having place. To overcome this, performing in-situ experiments using specialized TEM holders^[3] has emerged as a widely used, powerful and increasingly recognized technique. These experiments enable to track changes in real-time by recreating the reaction environment. This is achieved by using ultrathin silicon nitride windows to seal the sample in a tiny chamber where gases can flow while increasing the temperature, simulating real reaction conditions^[4].

Alternatively, there are complementary techniques that partially address these limitations. On the one hand, ILSTEM involves analyzing the exact same spot on a catalyst before and after a reaction^[5]. The catalyst is first loaded on a silicon nitride grid, examined under the microscope and then loaded into a quartz reactor. After the reaction, it is brought back to the microscope to study the same area again. On the other hand, another useful strategy is transferring the catalyst directly from a reactor to the microscope without exposure to air (anaerobic transfer)^[6]. This is done by sealing the reactor after the reaction, moving it inside a glovebox with an inert atmosphere and loading it on a copper microscopy grid. From there, a special transfer holder keeps the sample isolated from the atmosphere until it’s inside the microscope.

In our work, we propose to study the reduction of Pt/PrOx@CeO₂ nanocubes with the above mentioned techniques, to compare the results obtained of each approach. The oxidation state has been analyzed by means of EELS following the Ce-M_4,5 edge signal.

In the ILSTEM experiments initial surface reduction is observed at 500ºC and becomes more pronounced at 700ºC. By 900ºC, the nanocubes are fully reduced and have totally lost their original morphology. In anaerobic transfer experiments after reducing at 900ºC, most regions do not exhibit clear reduction, except for a few areas where some patches show signs of reduction and the morphology has transitioned to polyhedral particles close to octahedra. The in-situ experiments under a reducing atmosphere with H₂ reveal that surface reduction begins at 450ºC and progresses with temperature, becoming clearly visible by 850ºC. The reduction propagates from surface to bulk, creating a Ce³⁺ shell. Apart from this finding, no major morphological changes are observed except for a slight rounding of the edges.

The observed differences in reduction behavior and morphology among these approaches suggest that the interaction between the sample and the Si₃N₄ window may affect the catalyst reduction, potentially triggering a cerium diffusion or altering the local chemical environment. Overall, these results highlight how the intrinsic variables of each technique can influence the outcomes, stressing the importance of considering multiple perspectives in order to achieve accurate conclusions during in-situ experiments.

Sodium-ion batteries (SIBs) are receiving increasing attention due to the abundance of sodium resources and their wide distribution compared to their lithium counterparts. Benefiting from the environment-friendly and cost-effective manganese resources, as well as a high theoretical specific capacity of 265 mAh g^-1, P2-type layered Na_xMn_yO₂ cathode exhibits great potential in SIBs community. However, given that the complexity of the battery system, the practical cathodic reaction mechanism is inherently complicated by several factors such as mass loading, particle size, interfacial deposition, charge transfer, electrolyte and diaphragm, which hinders the application of the P2 type layered Na_xMn_yO₂ in sodium-ion batteries. Furthermore, these factors affect the cathodic reaction mechanism across multiple scales, from nano to micro scales, thereby completing the analysis. However, elucidation of the actual cathode reaction and degradation mechanisms is essential for optimizing cathode performance and developing sodium-ion batteries with high energy density and long cycle life.

In this study, we employ a comprehensive nano-to micro-to macro-length characterizations approach that combines Scanning Electron Microscopy (SEM), High Resolution-Transmission Electron Microscopy (TEM), in-situ X-ray diffraction (XRD), in-situ pair distribution function (PDF) analysis, in-situ Raman spectroscopy and in-situ differential electrochemical mass spectrometry (DEMS). This integrated characterization strategy provides a holistic understanding of the reaction mechanisms at the cathode of sodium-ion batteries. This work elucidates the degradation mechanism of the P2-type layered Na_xMn_yO2 cathode combining global structure and local-structure analysis that manganese atom migration induces severe phase transitions that lead to intense particle cracking after long-term cycling. To further analysis the origin of the manganese atom migration, HAADF, HR-TEM, in-situ Raman and in-situ DEMS techniques were employed to probe the anionic-redox reaction evolution from the local-scale to overall scale during battery cycling process. It was found that part oxygen released from Na_xMn_yO₂ at the sodium-deficient state, and these oxygen releases originate from an irreversible oxygen reaction, leading to a deterioration of its cyclic stability. Based on this finding, this work proposes a sulfuration approach to stabilize the Na_xMn_yO₂ structure, as sulfur anions are partially integrated into the oxygen sites within the lattice structure. Impressively, the oxygen redox reaction is significantly inhibited due to the higher gasification temperature of sulfur compared to oxygen. The sulfur anions within the internal lattice could reversibly participate in the muti-elements redox process and improve the integral coordination stability by mitigating undesired manganese migration. Moreover, after several cycles, there are no significant cracks in the material particles with sulfuration compared to the unmodified material. Consequently, the modified P2-Na_xMnO_2-yS_y exhibits remarkably long-term cycling stability of >1000 cycles with 98% capacity retention at 0.5 A g^-1 compared to the unmodified P2 sample which retains only 18% of its capacity after 1000 cycles. Our work not only reveals the degradation mechanism of the P2-type layered Na_xMn_yO₂ cathode but also presents a pathway for designing long-cycle-life SIB cathode materials.

Transition metal dichalcogenides (TMDs), particularly molybdenum disulfide (MoS₂), have garnered significant attention as cost-effective alternatives to platinum-based catalysts for the hydrogen evolution reaction (HER). However, their catalytic performance is often limited by low electrical conductivity and a scarcity of active sites. Recent studies have demonstrated that integrating plasmonic metal cores such as gold (Au), silver (Ag), and copper (Cu) into MoS₂ shells can enhance photoelectrocatalytic activity through localized surface plasmon resonance effects.^1,2

In this work, we present a comprehensive study on the synthesis, structural characterization, and electrochemical performance of various Metal@MoS₂ core-shell nanostructures (M=Au, Ag, Cu). By employing a multi-step synthesis approach, we successfully fabricated nanostructures with tunable morphologies and controlled defect densities in the MoS₂ shell. Defect engineering strategies included the introduction of sulfur vacancies, heteroatom doping with tungsten and vanadium, and thermal treatments under reducing atmospheres.

Advanced characterization techniques were employed to elucidate the structural and electronic properties of the synthesized nanostructures. Aberration-corrected scanning transmission electron microscopy (STEM) combined with electron energy loss spectroscopy (EELS) provided insights into shell crystallinity, interlayer spacing, and the nature of defects. In-situ transmission electron microscopy (TEM) studies under hydrogen atmospheres revealed dynamic structural changes during HER conditions. X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy further confirmed the successful incorporation of dopants and the presence of vacancies.

Electrochemical analyses, including linear sweep voltammetry (LSV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), demonstrated that defect-engineered and heteroatom-doped Metal@MoS₂ nanostructures exhibited superior HER performance compared to pristine MoS₂. Specifically, Au@MoS₂ samples after reducing termal annealing achieved an overpotential of 203 mV at a current density of 10 mA.cm⁻², with a Tafel slope of 55.mV dec⁻¹, indicating enhanced catalytic kinetics.¹ These improvements are attributed to increased active site density and improved charge transfer.

Photoelectrochemical measurements under simulated LED illumination revealed that the incorporation of plasmonic metal cores significantly enhanced photocurrent densities. Light-assisted chronoamperometry showed a prompt, steady, and reversible response during repeated on/off cycles. In addition, the variations of the photoelectrochemical responses with the wavelength highlight the influence of a plasmon-based process. STEM-EELS plasmonic studies corroborated these findings by demonstrating strong localized surface plasmon resonances in the visible range, which are instrumental in promoting charge carrier generation and separation.

Our findings underscore the critical role of controlled defect introduction and plasmonic core integration in optimizing the electrocatalytic and photoelectrocatalytic properties of Metal@MoS₂ nanostructures. The tunability of these core-shell architectures offers a versatile platform for designing efficient and sustainable catalysts for hydrogen production. This work lays the groundwork for future studies aimed at the rational design of advanced nanostructured materials for clean energy applications.

Cobalt-free, anode-less All Solid-State Batteries (ASSBs), identified as “Generation 4” in the EU Strategic Agenda on Batteries, represent a highly promising technology due to their safety, longevity, faster charging capabilities, environmental benefits, flexibility in application, and potential for high energy density. Nevertheless, their fabrication continues to face several challenges, such as interfacial contact resistance between the components and the stability of solid electrolytes in the presence of lithium metal. To evaluate this latter issue, the materials Li₁.₃Al₀.₃Ti₁.₇(PO₄)₃ and LiFePO₄, serving as solid electrolyte and cathode respectively, have been examined through a series of Transmission Electron Microscopy (TEM)-based techniques, with a focus on structural defects originating either from the fabrication process or from microstructural evolution during charge-discharge cycles.

The research has been organized into two principal areas: structural and analytical studies. From a structural perspective, High Resolution TEM has been employed to analyze the crystallinity in regions such as grain boundaries, interfaces, and cracks. Additionally, quasi-parallel precession-assisted 4D Scanning TEM¹ (4D-STEM), has been utilized to investigate the material’s texture and its role in the development defective regions. From the analytical standpoint, Energy Dispersive X-ray Spectroscopy (EDS) has been used to monitor structural evolution in terms of elemental diffusion and degradation of layer composition, while Electron Energy-Loss Spectroscopy (EELS) has provided more detailed insights into the local chemical composition and bonding around defects. Furthermore, two innovative approaches have been implemented to capture the most subtle features present in the EELS spectrum. First, by applying dimensionality reduction and clustering algorithms², local variations in the oxidation states of transition metals have been successfully identified³. Second, ab-initio simulations using density functional theory, along with simulations of the free density of states, have been applied to investigate the lithium signature within the EELS spectrum.

The integration of these diverse characterization techniques has yielded a comprehensive and unprecedented understanding of the structural characteristics and their evolution during the charging process in ASSB electrodes. This lays the groundwork for future in-operando experiments, which will enable real-time observation of microstructural changes.

Figure: a) TEM overview of the layered structure, b) in-plane orientation from 4D-STEM, with arrows marking defects. c) HAADF-STEM image from the area analysed in EELS, with the UMAP data reduced map in d) and corresponding Fe L_2,3 and Fe M_2,3 - Li K edge regions in  e) and f) respectively.

Recent interests in the PV industry have gravitated towards earth abundant materials suitable for large scale commercialization that can compete in terms of performance with their more expensive and scarce counterparts. Among various candidates, the ternary phosphide ZnSnP2 was reported to have optimal optoelectronic properties for a solar cell absorber [1]. ZnSnP₂ has a chalcopyrite structure and a tunable bandgap from 1.37-1.60 eV, depending on the order-disorder behavior of Zn and Sn atoms, which enables advanced solar cell technologies from a single material while maintaining recyclability [2]. So far, the main limitations have concerned the growth process mainly due to the poor control over the material composition and lack of a suitable substrate for epitaxial growth. Recently, ZnSnP2 was successfully grown via selective area epitaxy (SAE) on InP and Si in forms of nanopyramids. In this work, we successfully exploit annular dark field scanning TEM (HAADF-STEM) together with core-loss EELS analysis to prove the quality of the crystals in terms of defects formation, strain relaxation, and composition at the atomic level. We delve further into the composition analysis of ZnSnP2 nanostructures by focusing on the ELNES part of the EELS spectrum. The fine structure that extends approximately 50 eV after the ionization edge onset carries the information on the electronic structure in the material, which include the coordination of atoms and site symmetry in the local atomic environment. Based on the reshaping of the characteristic fine structure of both Zn and Sn from one sample to the other, we highlight the correlation between the order-disorder parameter and the growth conditions of the material. At last, we collect the low-loss interval of the EEL spectrum to retrieve the bandgap values of each sample with proper fit of the corresponding spectra [3]. The change in the bandgap values is then related to the atoms’ coordination and site symmetry in the local atomic environment. 

The immune synapse (IS) is a transient, dynamic cell-to-cell communication structure that forms at the interface of T cells and antigen-presenting cells (APCs). During this interaction, the organelles and the cytoskeleton of the T-cell polarize to the IS.

Actin and tubulin cytoskeleton are major clients of the chaperonin-containing tailless complex polypeptide 1 (CCT). Therefore, we hypothesized that CCT must be highly concentrated in this process playing an important role.

Although the CCT structure has been largely characterized using single particle analysis, there is only a few works identifying CCT in situ, opening a new line of study for the characterisation of the CCT structure within the cell.

In this work we have performed cryoelectron tomography on thin T cell projections generated during IS. Our first batches of tomograms show an abundance of actin filaments surrounded by particles whose size and shape may match CCT. We are currently working on subtomogram averaging and map fitting of CCT.

Once CCT can be certainly identified, as well as its predominant conformation, we plan to explore further the biological functions of CCT during IS.

The CryoEM Facility at the National Centre for Biotechnology (CNB-CSIC), part of the Spanish node of INSTRUCT-ERIC, is entering a new phase. This year marks a significant renewal in infrastructure, services, and the way users interact with the facility and their data. The facility has undergone technical upgrades, and we are now better equipped to meet the growing demands of structural biology in cryo-electron microscopy.

At the core of this transformation is the installation of a new Titan Krios G5, a high-end instrument that complements our existing Talos Arctica. This setup increases our flexibility and throughput, allowing us to support a broader range of experimental needs. Both microscopes are equipped with the latest dedicated software for high-throughput and high-performance image acquisition.

In addition, we now offer dedicated instrumentation for cryo-lamella preparation (FIB-SEM), and enhanced capabilities for cryo-fluorescence imaging, enabling robust cryo-correlative workflows (CLEM). These upgrades allow us to cover the entire pipeline, from sample preparation to data acquisition, across multiple cryoEM modalities.

Our current service offering includes:

• Preliminary screening and optimization of cryo-samples.
• High-throughput cryo-image acquisition for statistical analysis.
• High-resolution single particle analysis (SPA).
• Cryo-electron tomography (CryoET).
• Correlative light and electron microscopy (CLEM).
• Micro-electron diffraction (MicroED) for small crystals.
• Post-processing: Movie Alignment; Fully automated 2D/3D SPA workflows (Scipion [1]); Fully Automated tomographic reconstruction.

We remain committed to being a user-focused facility through EMHUB [2]. With this new phase, we are adopting more open access to data and post-processing resources. Users will benefit from clearer workflows, improved scheduling, and tailored support based on their level of experience, from direct access for expert users to fully supported projects for researchers new to the technique. We also aim to foster long-term scientific collaborations through training, consultation, and co-development of experimental strategies.

The CryoEM Facility (CNB-CSIC) continues to operate within the INSTRUCT-ERIC framework, providing access not only to national users but also to international researchers through transnational access programs. As part of a broader European infrastructure that promotes excellence in structural biology, we are committed to contributing high-quality data, reliable instrumentation, and expert support.

In summary, the CryoEM Facility at CNB-CSIC is entering a new chapter, one defined by advanced instrumentation, expanded capabilities, and a renewed approach to user interaction. We are prepared to enable the next generation of cryoEM research and to play an active role in driving forward structural biology

Pleomorphic Bovine Viral Diarrhea Virus (BVDV) is a positive-sense, single-stranded RNA virus belonging to the Flaviviridae family (genus Pestivirus). It infects cattle, alpacas, deer, sheep, and goats and is considered one of the most costly viral diseases affecting cattle, posing a significant burden on livestock welfare. Disease control relies on modified live and killed vaccines when prevention programs are in place. The viral envelope is composed of three BVDV glycoproteins - E^rns, E1, and E2 - which are responsible for viral entry and infection. Among these, E^rns and E2 are the primary targets of neutralizing antibodies. Although the crystal structures of the corresponding ectodomains have been determined [1-3], the overall 3D architecture and organization of the glycoproteins within the viral lipid bilayer, as well as the mechanisms of antibody recognition, remain poorly understood [4].

By integrating cryo-electron tomography (cryo-ET), proteomics, and molecular dynamics (MD) techniques, we provide new insights into the antibody recognition and assembly mechanisms of pestiviruses – essential prerequisites for the development of effective vaccines.

Biomineralization is the process by which living organisms form mineral crystals. It plays a crucial role in a wide range of biological functions, from skeletal formation to bio-mining. While the presence of mature crystals in various biomineralizing cells is well documented, the exact mechanisms underlying crystal formation often differ between organisms and remain not fully understood, especially during the early stages [1], [2].

Both the Calcium L-edge (~350 eV) and the Nitrogen K-edge (~400 eV) fall within the "water window" energy range. This spectral window enables the correlative use of cryo-tomography and cryo-spectromicroscopy to investigate early-stage Ca and N biomineralization processes in cryopreserved, frozen-hydrated whole cells.

These transmission X-ray microscopy (TXM) techniques are available at the Mistral beamline of the ALBA synchrotron with about 30 nm spatial resolution [3]. In this work examples of biomineralization studies performed at Mistral on both Ca and N in different biological cells will be presented [4], [5].   

The recent development of transmission electron microscopy has opened the door to data acquisition at atomic resolution and, consequently, to a new dimension in the research of high resolution molecular structures, both from biological and materials samples. While other regions in Spain have made a strong commitment to equip themselves with state-of-the- art equipment, in Catalonia we are far behind with these kind of infrastructures. This situation has finally changed thanks to the installation of a Glacios 200kV TEM located in the JEMCA (Joint Electron Microscopy Center at Alba), in the ALBA synchrotron. This new infrastructure, devoted to high-end transmission electron microscopy analyses, emerged thanks to the collaboration between several local and national institutions.

The IBMB-CSIC Cryo-electron Microscope Platform possess a specialized cryo-electron microscope for structural biology applications. The platform will give access to state-of-the-art cryo-EM equipment for structure determination projects using the latest technology and methods. Glacios 200kV transmission electron microscope equipped with a cryogenic sample manipulator robot and with the last generation of direct electron detector, a Falcon 4 that can take up to 400 movies per hour. Its high level of automation and user guidance of experimental settings enable scientists to efficiently unravel protein structures in 3D, as well as understand their functional context in the biological cell.

Beyond the gene editing applications of CRISPRCas, the development of nuclease-deadCas molecules offers an extension of the applications of the CRISPRCas technology without gene editing or RNAse processing. Despite Cas proteins being large and multi-domain proteins, early work showed silencing the two endonuclease domains in Cas9 via point mutations resulted in a nuclease-dead Cas9 that could still bind to DNA [1]. CRISPR-Cas13 is an RNA-guided and RNA-targeting RNAse protein. Cas13 functions similarly to Cas9, using a guide RNA (CRISPR RNA; crRNA) to encode target specificity. DeadCas13 mutants fused to EGFP have been recently successfully employed to visualize the location and dynamics of specific RNAs in live cells [2]. This is a very interesting and promising system to efficiently visualize specific mRNAs in live cells and learn how they relate to the translation machinery. This approach is currently of great interest as there is growing evidence on the existence of specialized ribosomes that translate specific mRNAs that operate in certain physiological and pathological conditions. We have recently reported by using electron tomography that ribosomes in the neurons of a Huntington’s disease (HD) knock-in mouse model show a remodelling towards a more compacted polysomal architecture, an aberrant polysomal architecture that is compatible with ribosome stalling [3]. Here we explore if deadCas13 strategies could allow the specific labelling of mRNAs coding for mutant huntingtin and image their relation to the ribosome stalling phenotype by cryo-correlative light and electron tomography. To start with we have set up the protocol for specific mRNA labelling in HEK293T cells by co-transfecting a plasmid overexpressing a deadCas13b protein fused to EGFP and a plasmid expressing specific guides for huntingtin mRNA. As a positive control we have used a guide directed against NEAT1 (a non-coding RNA present in nuclear paraspeckles) [2] and as a negative control a non-specific “non-target” guide that is not complementary to any known sequence. We will also present here the integration of the labelling protocol into the cryo-CLEM workflow.

Electron-dense granules (EDGs) are known to exist within bacteria, and play a key role as intracellular metal and polyphosphate reservoirs. We have been investigating the function of electron dense granules in bacterium Deinococcus radiodurans, which is a model organism regarding its extreme resistance to radiation. Using X-ray fluorescence nano-imaging data (ID16A-NI and ID16-B beamlines at ESRF), we analyzed the metal content in these compartments. Our results show that EDGs are elemental-rich regions, particularly with phosphorous mostly in the form of poly-P, calcium and manganese under control conditions, and that these elements are mobilized in response to stress. To gain deeper molecular and structural insights, we performed Cryo-STEM coupled with Electron Dispersive X-ray Spectroscopy (EDS) on the D. radiodurans cells. The results reveal a heterogeneous metal composition across different cells, suggesting that EDGs contribute to a dynamic stress-responsive mechanism of metal regulation within the bacterial population.

The long-noncoding RNA (lncRNA) CONCR (cohesion regulator noncoding RNA) plays a crucial role in regulating DNA replication and sister chromatid cohesion by modulating the activity of DDX11 helicase (1). Remarkably, its expression is significantly elevated in various cancer types (1,2). Recent studies highlighting the structural-functional relationships of lncRNAs prompted us to investigate the structure of CONCR and its influence on functional mechanisms, an area that remains largely unexplored. We have combined biochemistry, SHAPE-MaP (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension and Mutational Profiling), atomic force microscopy (AFM), and cryo-electron microscopy (cryo-EM) to elucidate the structure of CONCR. Our findings indicate that the lncRNA comprises multiple structural domains interconnected by flexible regions. Notably, the 3’-end of CONCR, specifically the segment encompassing nucleotides 419 through 718, forms a structural domain that efficiently interacts with DDX11 in vitro. The preliminary three-dimensional structure of the CONCR 3´-end domain, determined by cryo-EM, reveals a well-organized V-shaped architecture. Collectively, our results uncover a structurally defined domain within CONCR that is both necessary and sufficient for DDX11 binding. These findings support the idea that lncRNAs are organized into discrete structural domains with specific functional activities, interconnected within a larger flexible framework. Such organization may have important implications for understanding the regulatory roles of lncRNAs in cellular processes, particularly in cancer biology.

CryoEM has become a well-established technique for solving macromolecular complexes at high resolution [1]. By aligning and averaging thousands of individual projections of a biological specimen, single-particle analysis (SPA) allows for the generation of one or a few high-resolution structures from a single data collection. However, these static structures still represent discrete states within the complex motions that proteins typically undergo, despite the fact that the information on the dynamics of the sample is indeed contained in the data. Recently, the cryoEM community has begun to exploit this information, developing new image processing algorithms to gain insights into the conformational landscape of protein complexes and/or to correct for local motions, thereby increasing the resolution of moving subregions of a protein. Therefore, a growing demand is anticipated in the coming years from users seeking to unravel the molecular motions present in their samples of interest. We introduce FlexibilityHub [2], a new service/technology offered by the Instruct-ES centre, now available for users to utilize alongside our current SPA service. In this service, users are encouraged to provide a particle set that has undergone prior 2D and 3D classification but still exhibits a certain degree of flexibility, whether compositional or conformational. Subsequently, we will conduct a comprehensive study of the dataset's flexibility using state-of-the-art software packages such as Zernikes3D, cryoDRGN, opusDSD, cryoSPARC 3DVA, and more, in combination with methods for flexible reconstruction to correct for motions such as Relion5 - DynaMight, cryoSPARC - 3D Flex, and Xmipp - ZART. This multi-algorithmic approach is facilitated by Scipion, a cryoEM image processing framework developed by our team, which integrates various tools into a single platform. To illustrate the potential of this service, we present several key use cases that highlight its capabilities and the benefits it offers to researchers.

The Bile Salt Export Pump (BSEP), also known as ABCB11, is an ATP-binding cassette transporter expressed in hepatocytes, responsible for exporting bile salts into bile canaliculi. This process is powered by ATP hydrolysis, enabling the active transport of bile salts against their concentration gradient. Disruptions in BSEP function, due to genetic mutations or drug interactions, are commonly linked to severe liver disorders such as cholestasis. Structurally, ABCB11 comprises two transmembrane domains (TMDs), each formed by six helices, and two nucleotide-binding domains (NBDs). During its transport cycle, ABCB11 adopts multiple conformational states [1,2,3]: the inward-facing (IF) state, which permits bile salt binding from the cytoplasmic side; and the outward-facing (OF) state, triggered by the binding of two ATP molecules, which enables substrate release into the bile canaliculus. ATP hydrolysis and substrate release then reset the transporter to the IF state.

In this study, we employed cryo-electron microscopy (cryo-EM) and single-particle image analysis to investigate ABCB11 embedded in nanodiscs in the presence of ATP and chenodeoxycholic acid (CDCA), a major bile salt. Our analysis revealed two distinct inward-facing (IF) conformations: IFwide, characterized by an autoinhibitory loop that hinders substrate access; and IFnarrow, where the NBDs are closely opposed with two ATP molecules bound, and no substrate present, suggesting a post-translocation state. These structural insights expand our understanding of ABCB11's dynamic mechanism and its critical role in bile salt transport.

The recent development of fast pixelated detectors has enabled routine collection of four-dimensional scanning transmission electron microscopy (4D‑STEM) datasets, expanding our ability to investigate and understand material structures and even their functional properties at the atomic level [1]. In particular, 4D-STEM enables phase imaging methods such as differential phase contrast and ptychography, which are capable of probing electric and magnetic field structures in materials [2]. This capability is particularly interesting for the study of complex oxide heterostructures in which a wealth of exotic interfacial phenomena can arise due to the interplay between the physical and electronic structures of their constituent materials [3]. In this work, we present an analytical approach to the investigation of an artificial multiferroic tunnel junction system consisting of ferromagnetic La_0.7Sr_0.3MnO₃ (LSMO) electrodes separated by a thin ferroelectric BaTiO₃ (BTO) barrier, with interest for future applications such as non‑volatile ferroelectric memories. In such heterostructures the domain organization resulting from the imposed electrostatic and mechanical boundary conditions [4] can lead to the formation of charged domain walls that modulate its functional properties [5]. Here, a combination of phase imaging techniques – namely, center-of-mass (CoM) and ptychographic reconstructions – are used to determine the configuration of our system, defining an approach that simultaneously reveals both its structural (local atomic arrangements) and functional (longer range field properties) aspects, culminating in the identification of a charged domain wall within the BTO layer. In addition, the conclusions of this investigation are supported by density functional theory calculations, which also help in further understanding the polar nature of the structure. In this way, we show how correlation between different methods allows for the exploration of charged atomic-scale features, enabling a direct connection between structural rearrangements and the resulting heterostructure functionality.

A current cornerstone of EELS research features the possibility to use STEM-EELS for oxidation state quantification by analyzing the fine structure of the elemental ionization edges [1,2]. Advances in the last decade include oxidation state mapping at the atomic level [3] as well as 3D tomography via EELS tilt-series reconstruction [4].

To date, most oxidation state analyses rely on ionization edges with onset energies >100 eV (e.g., Fe-L_2,3 ionization edges at ~708 eV). However, the small ionization cross-section of these edges challenges studies of beam-sensitive samples.

The advent of highly sensitive Direct Electron Detectors (DEDs) has expanded possibilities in EELS and microscopy. Their high dynamic range allows probing edges that could not be properly exploited before. While most research focuses on high-energy core-loss edges, this work explores a new avenue enabled by hybrid pixel DEDs: quantitative analysis using low-energy core-loss edges (LE-CLEELS) (50-100 eV).

We demonstrate this approach by performing EELS-bonding tomography on previously studied beam-sensitive FeO/Fe₃O₄ core-shell nanocubes[4]. These tomographic EELS studies have exploited the Fe-M_2,3 ionization edges, situated at an energy of  54 eV.  The 2D and 3D acquisitions were analyzed using a combination of SVD decomposition, blind source separation, and curve fitting EELS quantification.

The results of this study, which can be seen in the TOC graphic, show a successful tomographic reconstruction enabled by the greatly enhanced signal-to-noise ratio (SNR) of the Fe-M_2,3 edges. After quantification, we have found out to need 50 times less electron dose when comparing to the widely used Fe-L_2,3 edges. Furthermore, this higher SNR has allowed us to perform this experiment with a much higher resolution with respect to previous studies on these materials, down to 1 nm.

This work challenges the conventional notion that EELS is mostly suited to the chemical analysis of light elements. This also highlights the potential of LE-CLEELS for low- dose analysis, opening new possibilities for advanced materials characterization on beam-sensitive materials.

Full Field Transmission X-ray Microscopy (TXM) is a powerful synchrotron-based imaging technique that combines nanoscale spatial resolution (down to tens of nanometers) with spectroscopic sensitivity in the soft X-ray range (between 200–2000 eV). By acquiring energy-resolved transmission images across absorption edges, TXM allows high-fidelity mapping of elemental distributions, oxidation states, and chemical bonding environments. However, the high dimensionality and noise inherent to TXM spectral datasets present significant challenges for conventional analysis workflows, which often rely on manual analysis or linear classical decomposition methods, as multiple linear least squares fittings.

In 2021, Blanco-Portals et al. proposed an innovative analysis strategy for Electron Energy-Loss Spectroscopy (EELS) based on unsupervised learning techniques, combining UMAP (Uniform Manifold Approximation and Projection) for nonlinear dimensionality reduction and HDBSCAN (Hierarchical Density-Based Spatial Clustering of Applications with Noise) for density-aware clustering. Their approach outperformed traditional methods, like Principal Component Analysis (PCA), Non-Matrix Factorization (NMF) or K-Means, in segmenting complex spectral data and identifying faint characteristics [1].

I In this work, we adapt the UMAP-HDBSCAN methodology for TXM spectral imaging demonstrating that this methodology preserves the physical information in spectral features while enhancing the accuracy and interpretability of spatial classification. Unlike linear techniques, UMAP effectively captures the nonlinear manifold structure of spectral variations, and HDBSCAN provides flexible, noise-robust clustering without requiring predefined component numbers, that is, number of clusters. This is illustrated in Figure 1, where UMAP–HDBSCAN is applied to a TXM spectromicroscopy measurement at the Ca L edge acquired at the Mistral beamline of the ALBA synchrotron [2], revealing distinct spatial regions with clear different Ca chemical state (Hydroxyapatite in orange, Calcite in blue). The results highlight the capacity of this methodology to identify complex spectral features and spatially map it. These results support the generalizability of this framework across other spectroscopic imaging techniques and support its expansion and implementation in X-ray Spectromicroscopy.

Figure 1. Application of the UMAP-HDBSCAN clustering strategy to STXM spectral data to study calcite (in orange) and hydroxyapatite (in blue) regions in a composite calcium-based material. (a) Spatial distribution of the two clusters identified by HDBSCAN on the UMAP-reduced dataset. (b) UMAP 2D embedding of the spectral dataset, showing the density-based clustering in the reduced manifold space. (c) Centroid spectra corresponding to each cluster.

Membrane distillation (MD) is an emerging desalination technology that offers high salt rejection and the ability to treat hypersaline feedwaters, while operating at relatively low pressures and potentially utilizing low-grade or waste heat sources. However, despite these advantages, MD systems are hindered by limitations in permeate flux and overall energy efficiency. A critical challenge, particularly during extended operation, is membrane fouling and pore wetting, which adversely affect separation performance and reduce membrane lifespan [1].

The incorporation of nanomaterials represents a promising strategy to improve the anti-fouling performance of membrane systems [2]. In this study, composite membranes were fabricated using nanoadsorbents of metal-organic frameworks (MOFs) grown on graphene oxide (GO), resulting in MOF@GO/polyethylene membranes [3]. This composite membrane design leverages the tuneable porosity, ease of functionalization, and thermal and chemical stability of MOFs, along with the high specific surface area and aspect ratio of GO nanosheets. Strong interfacial interactions between MOFs and GO promote uniform MOF growth on the basal planes of GO, improving dispersion and reinforcing the synergistic properties of both components, rendering the membranes highly effective for anti-fouling applications.

To investigate the mechanisms underlying membrane fouling and wetting, membrane autopsy analyses were performed using advanced microscopy techniques on both pristine and fouled membranes [4][5]. Membrane characteristics, such as surface roughness, adhesion, electrical and interaction tendencies, polymer elasticity, mechanical strength, hydrophilicity, and adsorption behaviour (including porous structure and distribution of hybrids) have been identified as key factors influencing filtration performance.

In this context, Atomic Force Microscopy (AFM) is a high-resolution technique well-suited for investigating the surface topography of various membrane materials. It enables the acquisition of three-dimensional surface images, valuable for gaining detailed insights into the ultrastructure of polymeric membranes. As a result, a more accurate and detailed representation of the membrane surface and profile can be achieved. To complement the topographical information provided by AFM, Scanning Electron Microscopy (SEM) will be employed to check the layout of the membrane and the possible presence and distribution of salts and other foulants, and Transmission Electron Microscopy (TEM) to assess the distribution and crystallinity of the MOF@GO hybrid nanostructures within the membrane matrix.

When in-depth profiling and three-dimensional structural analysis are required, meticulous sample preparation protocols may be necessary. These can include serial sectioning via ultramicrotomy and contrast enhancement through heavy metal staining.

Josephson junctions (JJs) are foundational elements in superconducting quantum devices, where their operation is governed by complex interplays between superconductivity, quantum confinement, and Coulomb interactions. A precise understanding of these junctions at the nanoscale is essential for enhancing device coherence and reliability [1]. Advanced transmission electron microscopy techniques, including atomic resolution high angle annular dark field scanning transmission electron microscopy (HAADF STEM) imaging, energy-dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) analysis, were employed to investigate the structural and compositional properties of the JJs. Special attention was paid to the thin oxide barrier (AlOx) formed between the superconducting aluminum electrodes. We assessed the oxide thickness, uniformity, and growth conformity over the bottom Al layer, which are parameters that critically influence tunneling properties, trap state formation, and overall junction performance [2]. This research employs aberration corrected atomic resolution HAADF STEM and EELS to elucidate the structural and electronic intricacies of Josephson junctions at the nanoscale [3]. We investigated the ways in which material interfaces, defects, and nonuniformities could possibly affect the properties of the junction. These observations provide valuable information on Josephson junctions and critical insights for the fabrication and optimization of superconducting quantum circuits.

Chaperones assist in the de novo protein folding and prevent protein aggregation [1]. One of the most important chaperone families are the chaperonins (Hsp60s), which are organized as two oligomeric back-two-back rings generating a cavity in each ring where the substrate is placed for its folding [2]. The most complex and important of all chaperonins is the eukaryotic CCT (Chaperonin Containing TCP-1) whose structure and the folding mechanism are key for nanotechnological applications [3].

The main aim of this project is to build a stable synthetic cylindrical structure capable of encapsulating chemical reagents or small proteins. It has been shown that CCT5 is able to self-oligomerize. When compared to the eukaryotic CCT, poly-CCT5 is easier to purify, can be genetically modified in all subunits and has potential to be biocompatible [4]. These capabilities enable poly-CCT5 to act as a nanocontainer delivering molecules to specific targets (See Figure a).

Our group used negative staining EM to assess the encapsulation of various nanoparticles inside synthetic poly-CCT5. VENOFER, an iron-sucrose coating NP, produced the best results overall and was chosen for Cryoelectron microscopy (CryoEM) analysis. We first generated a 3.2 Å 3D reconstruction of the NP-bound poly-CCT5, with the NP presumably held by CCT5 apical domains (See Figure b). Subsequently, our efforts shifted towards designing and structurally characterizing poly-CCT5 mutants, aimed at rearranging the charge distribution within the cavity to minimize undesired interactions (See Figure c). This approach led to the generation of an initial 5 Å 3D reconstruction of NP-bound poly-CCT5, demonstrating improved nanoparticle internalization. Currently, we are focusing on inducing the closure of the cavity to rearrange the N- and C-terminal, eliminating steric impediments and thereby achieving complete nanoparticle encapsulation.

Figure. Summary of the obtained results. a) Proof of concept illustrating the delivery of the Poly-CCT5:NP:Drug complex through the bloodstream, including tumor cell recognition, Poly-CCT5 aperture, and drug release mechanism..  b) 3D reconstruction of the poly-CCT5 complex (blue) with nanoparticle (red) at 3.16 Å resolution. c) Helical protrusion of the CCT5 subunit, with shaded areas and arrows highlighting the lysines selected for mutation.

Protein homeostasis is sustained through a finely tuned balance of protein synthesis, folding, trafficking, assembly, and degradation, processes that are essential for proper cellular function¹. Molecular chaperones act as key regulators within this proteostasis network, with their functional outcomes determined by specific co-chaperones. These co-chaperones modulate the Hsp70:substrate complex, directing it either towards folding or degradation via the ubiquitin-proteasome system (UPS) or autophagy². Bag1 (Bcl-2-associated athanogene 1) is one such co-chaperone, serving as a nucleotide exchange factor (NEF) for Hsp70 and containing a ubiquitin-like (UBL) domain that mediates its interaction with the proteasome³.

In this study, we demonstrate a strong interaction between Bag1 and the proteasomal subunit Rpn1. The ternary complex (Rpn1:Bag1:Hsp70) was successfully isolated and characterized through cryo-electron microscopy (cryo-EM) and cross-linking mass spectrometry, offering valuable mechanistic insights into the process of substrate transfer from Hsp70 to the proteasome.

A high-resolution cryo-EM map (3.6 Å) of the 26S proteasome in complex with Bag1 revealed binding of the UBL domain to the toroidal region of Rpn1 (Fig. 1a). This interaction induces significant structural rearrangements within the 19S AAA-ATPase, particularly at the Rpt4–Rpt5 interface, quite different from known structures (Fig. 1b-d). Such conformational changes may facilitate the positioning of Hsp70 near the proteasomal gate, promoting efficient substrate transfer. Furthermore, biochemical assays suggest that Bag1 binding induces an alternative open-gate conformation of the proteasome (Fig. 1e), enabling translocation and degradation of unfolded proteins in an ATP- and ubiquitin-independent manner.

Figure 1. Structural Reorganization of the 26S Proteasome Induced by Bag1 Binding. (a) Cryo-EM reconstruction of the 26S proteasome in the S_BAG1 conformation (EMDB:52097) at 3.6 Å resolution, revealing only the UBL domain of Bag1 (Bag1_UBL). The inset highlights the interaction interface between Rpn1 and Bag1_UBL. (b) Cross-sectional view of the cryo-EM maps of S_BAG1 and S_D4 (EMDB: 32282; PDB: 7W3K) shows a distinct alignment of the OB, ATPase, and 20S rings in S_BAG1, indicating structural rearrangements upon Bag1 binding. (c, d) Segmentation of the OB-ATPase interface (c) and ATPase ring (d) reveals that Bag1 disrupts the ATPase ring symmetry, generating a large central cavity and inducing conformational shifts in Rpt3 (green) and Rpt4 (pink). These changes suggest an alternative substrate entry pathway (red arrow in (c)) into the 20S proteasome. (e) Differences in 20S gate opening between S_D4 and S_BAG1. In S_BAG1, only Rpt2, Rpt3, and Rpt6 insert into the α-ring, yet the 20S gate remains open, suggesting a non-canonical activation mechanism. The S_D4 conformation serves as a reference due to its structural similarity to S_BAG1 in both 19S and 20S.

Many adeno-associate virus (AAV) serotypes are being investigated in preclinical and clinical trials. A few have been already approved by the US and European regulatory drug agencies. A major challenge in the AAV gene therapy applications is the large-scale production of a good-manufacturing-practice (GMP) AAV product that fulfils international regulations for clinical release. 

Here, we investigated the capsid thermostability and transgene release of purified AAV vectors via differential scanning calorimetry, and by performing potency assays. We also determined by high-resolution cryogenic electron microscopy the 3D structure of AAV6 – a valuable tool for genome editing of hematopoietic cells– at different pHs.

We demonstrate that genome leakage occurs at temperatures below capsid unfolding temperatures and that acidic pH led to conformational changes detrimental to particle stability. These results agreed with the detected difference in cells transduction efficiency. These biophysical and structural analyses of AAV support the idea that sample handling might not only impact viral vector yields and purity but also modulate residues’ interactions thus potentially impacting product potency.

Molecular chaperones are key proteins in cellular homeostasis. One of the most important chaperones is CCT (chaperonin containing TCP-1), a large chaperonin composed of 16 subunits with ATP-dependent activity, which is responsible for the folding of ~10% of newly synthesized [1-4]. Multiple studies highlight the role of this macromolecular complex in cancer, where it is generally up-regulated, and in neurodegenerative diseases, where it is down-regulated [5-9]. For that reason, the aim of this project is to find potential therapeutic modulators of CCT.

A Differential Scanning Fluorimetry (DSF) Screening allowed us to identify a battery of small molecules that interact with CCT. Among these, two compounds -Drugs 1 and 7- stood out for their strong binding affinities and similar chemical structure. Cell viability assays have shown that both of these compounds selectively inhibit the growth of cancer cells over healthy epithelial cells. Proteome Integral Solubility Alteration (PISA) assays and immunofluorescence imaging have been used to reinforce these results, proving that Drugs 1 and 7 target CCT in cells and compromise the integrity of the actin cytoskeleton.

Cryo-electron microscopy (CryoEM) was used to investigate the binding site of Drug 7 on the chaperonin CCT, since is is the small molecule that showed more selectivity towards cancer cells. Our CryoEM maps revealed additional densities in the ATP binding pocket of the CCT6 and CCT8 subunits. The extra density observed in CCT6 is likely attributable to the presence of ADP. In contrast, the density in the CCT8 subunit can be confidently modeled with Drug 7, along with a magnesium ion that appears to coordinate the phosphate groups of ADP. These observations are further supported by in silico docking analyses, which suggest that Drug 7 preferentially binds to the CCT8 subunit. Collectively, our data support the formation of a CCT–Drug 7–ADP ternary complex. However, limitations in the resolution of the apical domains currently prevent us from assessing potential Drug 7 binding at those sites. Further studies are necessary to elucidate the mechanism by which Drug 7 selectively inhibits the proliferation of cancer cells.

Understanding material properties requires access to their atomic structure. Traditional diffraction methods, which rely on sharp Bragg reflections, are often ineffective for nanocrystalline and amorphous materials due to the absence of long-range order. In such cases, Pair Distribution Function (PDF) analysis—using X-rays (xPDF), neutrons (nPDF), or electrons (ePDF)—provides critical insight into short- and medium-range atomic arrangements [1].

ePDF analysis in a Transmission Electron Microscope (TEM) offers several advantages over xPDF, including higher spatial resolution, faster data acquisition, and reduced sample requirements. While conventional ePDF approaches—such as Selected Area Electron Diffraction (SAED) or Nanobeam Diffraction (NBD)—require acquiring one pattern at a time, our method leverages automated workflows for more efficient data collection and analysis.

We introduce a user-friendly software solution capable of processing both single-pattern data and complete datasets acquired via Scanning Precession Electron Diffraction (SPED) or precession-enhanced 4D-STEM. These techniques employ a focused electron probe (1–10 nm) that scans the sample with fine step sizes (1–3 nm), enabling structural mapping at ~1 nm spatial resolution [2]. At each scan point, the software computes the ePDF and generates correlation maps, analyzing peak positions, widths, and integrated areas to reveal nanoscale structural variations [3].

This methodology is broadly applicable to a range of disordered materials, including semiconductors, glasses, catalysts, amorphous dispersions, and polymers. In one case study, ePDF mapping resolved two distinct amorphous layers in a semiconductor: a Si₃N₄ layer (first peak at 1.65 Å) and a SiO₂ layer (first peak at 1.56 Å, slightly shorter than expected), the latter likely affected by carbon incorporation during processing.

In summary, ePDF mapping provides a robust and versatile platform for high- spatial resolution local structural analysis of amorphous materials, uncovering variations beyond the reach of conventional diffraction techniques. 

Beam-sensitive materials with mixed carbon sp² and sp³ hybridizations exhibit subtle variations in local bonding environments, which are critical to their electronic and structural properties. In this study, we develop an optimized methodology for analyzing the σ* and π* components in the C-K edge using electron energy loss spectroscopy (EELS). STEM with an energy filter and monochromator is employed to minimize beam damage and preserve sample integrity. Low-dose acquisition protocols are implemented, and the resulting spectra are processed using principal component analysis (PCA) and multiple linear least squares (MLLS) fitting to improve signal-to-noise ratios and isolate bonding-specific features.

  Reference spectra from standard materials such as graphite and diamond-like carbon are used for calibration and validation. Additionally, initial machine learning-based approaches, such as unsupervised clustering and denoising algorithms, are explored to assist in identifying bonding-specific spectral features. These methods show potential for enhancing interpretation quality, especially in low-dose and low-SNR conditions.

  This approach enables reliable σ/π bonding analysis in electron beam-sensitive systems and lays the groundwork for future studies on local bonding structures with high spatial resolution and chemical sensitivity.

The process of discovering and refining new materials, as well as enhancing existing ones for a variety of uses, including quantum applications, is a complex and multifaceted endeavour. This involves identifying needs, reviewing existing literature, proposing materials, engineering devices, characterizing materials, and testing applications. However, the process can be hindered by its time-intensive and costly nature, especially when precision at the atomic level is necessary to comprehend the functionality of materials and heterostructured devices.

In this digital age and with quantum supremacy in the horizon, semiconductor heterostructures within a chip have become indispensable and ubiquitous, propelling significant industrial value chains. They facilitate progress in both emerging sectors like quantum applications and established ones. For that, the trend of miniaturization, which is now reaching nanoscale dimensions and nearing the atomic limit, is a key driver of progress which demands special characterisation needs. To answer to these needs, in this work, we present a revolutionary analytical framework aimed at the comprehensive characterization of device heterostructures, with a particular focus on quantum devices and their materials.

The characterisation that must answer these demands is high-resolution Transmission Electron Microscopy (TEM), as the most optimal way to access structural information at the atomic level. We propose a workflow capable of processing both parallel illumination TEM and Scanning TEM (STEM) data, although it is specially designed for the latter. In addition, it can extract compositional information from Electron Energy Loss Spectroscopy (EELS), which is used to make the characterisation an exhaustive and complete process. Importantly, the workflow we suggest, combines traditional algorithmics, with unsupervised and supervised machine learning algorithms. This way, we ensure that model-based computing and artificial intelligence work hand by hand to provide the comprehensive and material-independent solution we seek for.

Our pioneering workflow autonomously determines material composition, crystallographic phase, and spatial orientation across various regions of analysed (S)TEM-based images or image datasets through detailed model comparison. It is completed with automated strain analysis, enabling a comprehensive characterization of the device's structural properties. Eventually, we incorporate the extracted knowledge to automate the creation of models that facilitate simulations and provide vital physical and chemical insights necessary for understanding the device's performance in practical applications.

Although the method is highly versatile, we focus on quantum computing devices to autonomously optimize their functional properties. In particular, we prove it with SiGe quantum well heterostructures that proved outstanding quantum performance for spin qubits generation. However, the main advantage placing our workflow beyond state of the art is its generalisation capabilities. It works for any material configuration and not only does it answer the pressing automation need, but unlocks getting physical models and simulations of complex devices with unprecedented accuracy.

3DSEM is a technique that combines a series of secondary of backscattered electron (SE or BSE) microscopy images with photogrammetry methods to obtain a 3D surface model of a sample under investigation, which can be further analyzed digitally to extract quantitative features [1,2]. The technique can be used in any SEM microscope, is non-destructive, and can be applied with minimal preparation to many samples with the possible exception of those requiring conductive coating for reducing charging effects. Moreover, the reconstructed three-dimensional surface can be textured with physicochemical information obtained from different detectors available in a SEM. For example, 3DSEM combined with energy-dispersive X-ray spectometry (EDXS) has been applied to study adhesion wear in cutting tools, or carbon segregation in catalytic bodies [3,4].

In this work, we show to the best of our knowledge, the first application of 3DSEM combined with cathodoluminescence (CL), that is a technique extensively used in geology and ore mining as it allows researchers to distinguish different mineralogical phases inside a rock sample [5]. One difficulty found for the application of CL is sample preparation. CL in SEM often requires samples prepared with perfectly planar surfaces, as irregularities ocurring in fragments of a sample (i.e. a rock) could hinder the differentiation of phases due to shadowing of the CL signal by the sample itself. Sample preparation is a lengthy procedure which, furthermore, can introduce artefacts that can be misinterpret as real features.

To overcome this drawbacks, we have measured simultaneously SE images and RGB and panchromatic-CL maps on a grain of fluorite, a material known for displaying strong CL and a lack of phosphorescence. No sample preparation beyond cutting the sample to fit inside the microscope and gold coating was performed in this study. The 3D surface was reconstructed and textured with the CL signal with a commercial photogrammetry software package.

Our results demonstrate the feasibility of 3DSEM-CL as a characterization tool to be added to gelogist's toolbox and, in general, for materials science valid for research as well as scientific dissemination.  

Twistronics is an emerging area of research focused on how rotating layers of 2D materials relative to each other affects their electronic behavior. By controlling the twist angle, researchers can engineer quantum materials with tailored properties. The formation of Moiré superlattices strongly influence the local electrostatic potential, charge distribution, and electric fields in 2D materials. Analyzing these Moiré patterns is essential for understanding their underlying properties. In this study, we used a JEOL JEM-2100 transmission electron microscope operating at 200 kV, to achieve atomic resolution of Moiré patterns in graphene. Twist angles were quantitatively extracted through fast Fourier transform (FFT) analysis of the TEM images, by identifying the relative orientation of diffraction spots corresponding to each atomic lattice. This method enables precise determination of interlayer rotation and provides critical insight into the resulting Moiré superlattice geometry. Multislice computer simulations were employed to complement the experimental observations by generating 4D-STEM images, enabling the calculation of the projected electric field (eCOM), charge distribution (dCOM), and electrostatic potential (iCOM). The integration of TEM imaging and advanced computational modeling provides a detailed framework for investigating the electrostatic properties of twisted layers of 2D materials.

*amayoral@unizar.es

Zeolites are microporous materials with important industrial applications as desiccants and heterogeneous catalysts. Their structural features have traditionally been revealed through a combination of methods, with X-ray diffraction playing a central role by enabling the resolution of numerous new topologies. However, since the 1990s, transmission electron microscopy (TEM) has emerged as a powerful tool in zeolite science, as it enables the direct visualization of the framework—and more recently—even the various species incorporated into the porous network.

Unfortunately, despite their often high and tunable crystallinity, zeolites are highly sensitive to electron beam damage, which has limited the use of TEM for obtaining atomic-resolution information. This beam sensitivity results in a diminished image resolution due to a low signal-to-noise ratio (SNR), itself a consequence of both the limited number of electrons used and the low detection efficiency of electron detectors.

Since 2010, with careful control of the electron dose, spherical aberration-corrected scanning transmission electron microscopy (STEM) has been successfully used to obtain atomic-resolution information on various zeolitic materials and even metal-organic frameworks (MOFs)[2]. Depending on the detector employed, complementary information can be extracted, and therefore, a complete dataset often requires the combination of multiple imaging techniques. Annular dark field (ADF) imaging is sensitive to atomic number and offers relatively straightforward interpretation, but it suffers from low SNR due to limited electron collection. Annular bright field (ABF) imaging provides enhanced spatial resolution and is better suited to light elements, though data collection and interpretation are more complex and also constrained by low detector efficiency. More recently, integrated differential phase contrast (iDPC) STEM has emerged as an effective low-dose imaging technique for visualizing both zeolite frameworks and guest species[3]. Four-dimensional (4D) STEM, which involves collecting 2D convergent beam electron diffraction (CBED) patterns at 2D scan positions, provides high-SNR data under low-dose conditions. It also enables extensive post-acquisition data analysis to retrieve various image contrasts. Among 4D-STEM techniques, ptychography—a phase contrast method based on a series of diffraction patterns—offers enhanced spatial resolution, high SNR, and dose efficiency. Although electron ptychography has been previously reported, its application to beam-sensitive materials remains underexplored.

We have developed a code that enables phase contrast imaging via ptychography with outstanding SNR under low-dose conditions (<500 e⁻/Ų). This method was applied to the titanosilicate ETS-10, which crystallizes as a mixture of two polymorphs and contains several structural defects. The experimental data were compared with simulations generated using the abTEM software, demonstrating the method’s potential to enhance spatial resolution, improve SNR, and enable both average structure analysis and direct visualization of crystal surfaces without artefacts. The feasibility of this approach was validated using MFI zeolite (Figure 1)[4] and also applied to the structural elucidation of a highly complex zeolite NU-88, whose structure remained unknown since its discovery 20 years ago.

Serial electron diffraction is emerging as a powerful approach for structure determination from nanocrystals, combining the benefits of electron microscopy with the serial data acquisition strategy of X-ray free electron lasers. In this work, we present the successful implementation of a Serial electron diffraction pipeline for small organic molecules (e.g. carbamazepine), based on the collection of single-shot diffraction patterns from randomly oriented crystals dispersed on TEM grids. By collecting hundreds of patterns and merging them, we were able to reconstruct complete datasets and solve crystal structures at high resolution, with minimal radiation damage.

We have since successfully applied the method to data obtained from protein nanocrystals (e.g. lysozyme). In the future, we plan to combine it with beam precession to increase the number of spots in each diffraction pattern. This strategy could offer a low-dose, high-throughput alternative for protein structure determination using standard transmission electron microscopes. Our results support serial electron diffraction as a versatile technique, potentially bridging the gap between organic and biomolecular crystallography in electron microscopy.

Figure 1. Workflow of data collection using serial electron diffraction. (A) An overview image of the grid. (B) A close-up of a grid square containing crystals. (C) The diffraction pattern from one of these crystals.

Immunogenic cell death (ICD) is a regulated form of cell death capable of activating an adaptive immune response. It is gaining relevance as a therapeutic mechanism in cancer, as cells undergoing ICD in vitro can function as vaccines promoting tumour clearance in vivo. ICD is characterized by a range of plasma membrane modifications and changes in the surrounding microenvironment. It is associated with endoplasmic reticulum stress and the release of damage-associated molecular patterns, including calreticulin, ATP, HMGB1, HSP70, and HSP90. In some instances, tumour cells undergoing ICD can release extracellular vesicles carrying bioactive molecules that modulate dendritic cell function^[1]. However, there is still no definitive hallmark to unambiguously identify ICD.

Oxaliplatin is a third-generation platinum-based chemotherapeutic widely used in the treatment of colorectal cancer. Its primary mechanism of action involves the formation of DNA crosslinks that disrupt replication and transcription, ultimately leading to cell death. Unlike other platinum compounds such as cisplatin or carboplatin, it is also able to induce ICD^[2]. Despite its clinical relevance, the cellular and subcellular mechanisms underlying oxaliplatin-induced ICD remain incompletely understood.

In this study, we employed a correlative multimodal microscopy approach to investigate ICD induced by oxaliplatin in cancer cells. We combined fluorescence microscopy, cryo-electron tomography (cryo-ET), and cryo soft X-ray tomography (cryo-SXT) to visualize the morphological and molecular changes associated with ICD at high resolution and under near-native conditions. MC38 cells, either untreated or treated with oxaliplatin or cisplatin, were cultured in vitro and vitrified for imaging. Fluorescence microscopy was used to monitor dynamic changes in cellular compartments and ICD markers in real time. Cryo-ET provided nanometer-scale three-dimensional insights into intracellular organelle remodelling, while cryo-SXT enabled label-free, whole-cell imaging of structural transformations during ICD. This work highlights the power of integrated imaging platforms to dissect complex cell pathways.

Phenylalanine hydroxylase (PAH) is the key metabolic enzyme responsible for the catabolism of phenylalanine. Mutations in the PAH gene cause phenylketonuria (PKU), a genetic disorder that, if left untreated, leads to brain damage and intellectual disability. While some patients benefit from supplementation with synthetic formulations of the natural cofactor tetrahydrobiopterin (BH4), which increases the activity of the PAH variants, more effective treatments are still needed. Our understanding of PAH activation and regulation remains incomplete, and advancing this knowledge is critical for the development of more effective therapeutic strategies.

Here, we will present and discuss four novel cryo-EM structures of full-length human PAH (hPAH) bound to phenylalanine at stimulatory concentrations, and in the presence of cofactors and inhibitors. These structures reveal how phenylalanine regulates PAH activity through conformational changes of its regulatory domain, providing essential insights into the enzyme’s allosteric control and the molecular basis of disease associated mutations found in patients.

The influenza virus is a paradigm for understanding viral infections, providing critical insights into the complex host-pathogen interaction. As an opportunistic invader, influenza exploits the cellular endocytic machinery for infection, demonstrating a remarkable ability to hijack host processes. Our study focuses on a crucial stage of this viral journey: the release and transport of the virus from endosomes to the nucleus.

The objectives of this work start with the optimization of cryo-correlative light and electron microscopy (cryo-CLEM) techniques for the study of influenza virus structure and infection processes, with the aim of providing new insights into viral biology at the structural and cellular levels.

To solve this, we focused on critical aspects of the cryo-CLEM workflow, including:

• Virus purification to achieve higher viral titre and enable high Multiplicity of Infection (MOI).
• Infection process on Electron Microscopy (EM) grids.
• Vitrification by plunge freezing with cryoprotectants.
• Lamellae preparation by cryoFIB/SEM.
• Tomography by cryoTEM.

Additionally, we are developing fluorescent labeling techniques for the virus to enable precise correlation of the obtained tomograms.

Our optimizations resulted in improved sample preparation and imaging protocols for cryo-CLEM studies of influenza.

These advances in cryo-CLEM methodology for influenza research enable the collection of high-quality data on viral structure and host-cell interactions. This optimized approach has the potential to reveal new aspects of influenza virus biology and contribute to the development of improved prevention and treatment strategies against this significant global health problem.

The study of high-resolution structures of biological macromolecular complexes by cryo-electron microscopy (cryo-EM) has undergone remarkable progress over the past decade. The improvement in resolution achieved by this technique has been driven by the development of new automated data acquisition methodologies, increased detector sensitivity, and the implementation of novel image processing algorithms.

Unfortunately, access to state-of-the-art electron microscopes remains limited and extremely costly, as only a few institutions in Spain have secured sufficient funding to acquire instruments equipped with the best source of illumination (FEGs) and direct electron detectors.

Given the limited access structural biologists have to these high-end instruments, it is crucial to optimize sample preparation and screening prior to high-resolution analysis. Low-voltage (120 kV) electron microscopes can be employed for this purpose through the use of both negative staining and cryo-EM techniques.

In this work, we present the results obtained using a Talos L120C G2 cryo-electron microscope, equipped with a 16-megapixel Ceta-F camera. This microscope is capable of autonomously acquiring data from macromolecular complexes and assessing whether the sample is suitable for analysis on high-voltage electron microscopes.

Among other parameters, it allows for the evaluation of ice quality, particle concentration and distribution, the number of suitable data collection areas on the grid, and angular distribution of views. Image processing of datasets collected with this type of microscope can provide insight, not only into the presence or absence of preferred orientation, but also offer preliminary information on the biochemical and structural homogeneity of the sample.

The study of standard samples, such as apoferritin, using low-voltage microscopes has shown that these instruments can reach resolutions below 3 Å when paired with direct electron detectors (1). In our work, we have explored the performance of the Ceta-F camera, a non-direct detector capable of frame acquisition, under different imaging conditions. Although this setup does not match the performance of direct detectors, it supports motion correction during acquisition and has enabled reconstructions reaching resolutions around 4.5 Å in favorable cases.

This level of resolution enables the identification of secondary structure elements, the assessment of proper protein folding, determination of the number of components in the complex, and much other critical information for evaluating the feasibility of structural biology projects and making informed decisions about accessing high-voltage electron microscopes.

The endosomal system is responsible for receiving, classifying and redistributing hundreds of proteins in the cell. Proteins that enter in the endosomal pathway could follow two different destinations: be degraded in lysosomes or be recycled to continue developing their functions. A key component in the retrograde pathway is the retromer complex. Retromer is a highly conserved protein complex that mediates retrograde transport of hundreds of transmembrane proteins, known as cargos, from the endosomes to the trans-Golgi network and plasma membrane. To select cargo, retromer is recruited by members of the Sortin nexin (SNX) family to the endosomal membrane. Here, retromer promotes the emerging of tubulo-vesicular carriers that allow cargo to exit the endosome and avoid lysosomal degradation. Given its impact in the maintenance of cellular proteostasis, retromer machinery is hijacked of several intracellular pathogens to support their survival and replication. One of them is the Human Papillomavirus (HPV), that hijack retromer complex through the minor capsid protein L2 which in turn interacts with viral DNA. This interaction promotes the retrograde transport of the viral genome into the cell nucleus where its replicated.

This study combines biochemical and functional assays with X-ray crystallography and cryo-electron microscopy to establish the mechanism by which L2 subverts retromer function. Furthermore, the integration of Cryo-ET and STA has allowed to define for the first time the spatial organization of the retromer-SNX12 membrane coat around tubular vesicles.  

ZEISS Volutome is an in-chamber ultramicrotome for ZEISS field emission scanning electron microscopes (FE-SEM) designed to image the ultrastructure of biological, resin-embedded samples in 3D over a large area.

A diamond knife cuts away sections from the sample block and the newly exposed surface is imaged with the backscattered electron detector, ZEISS Volume BSD, which is specifically designed for serial block-face imaging. The cutting and imaging process is repeated thousands of times in an automatic, autonomous process. The increased sensitivity of the detector allows fast image acquisition with low acceleration voltages, protecting your sample from beam damage and mitigating charging effects.

By activating the ZEISS patented Focal Charge Compensation (Focal CC), even the most charge-prone samples can be investigated without degrading image quality. Focal CC neutralizes charges directly at the block face with no compromise in signal-to-noise ratio or resolution.

ZEISS Volutome is an end-to-end solution from hardware to software including image processing, segmentation, and visualization. The ultramicrotome can be easily replaced by a conventional SEM stage, converting your 3D FE-SEM into a standard, multipurpose FE-SEM, making your system adaptable to a multipurpose environment.

Precise atomic arrangement and defects in materials critically influence their physical properties and emergent phenomena such as superconductivity, ferroelectricity or metal-insulator transitions [1]. Even small variations in local symmetry, chemical or electronical doping can lead to drastic changes in these physical properties, making it necessary to characterize materials at the nanoscale for understanding and engineering these functionalities.

Recent advances in four-dimensional scanning transmission electron microscopy (4D-STEM) have created new opportunities to tackle this challenge [2]. These rich datasets have been employed in numerous modalities including nano-diffraction, ptychography, and differential phase contrast imaging. However, the large size and noise characteristics of 4D-STEM data present significant computational challenges. Moreover, data analysis workflows typically require customization for each material system or experimental condition.

To address these issues, machine learning and unsupervised clustering methods have increasingly been explored to extract meaningful structural and chemical information from 4D-STEM datasets [3]. For example, convolutional neural networks have been used to denoise data at low electron doses, and clustering algorithms have identified crystalline phases and grain orientations from nanobeam electron diffraction patterns [4]. However, automated identification of atomic column sites directly from atomic-resolution 4D-STEM data—particularly distinguishing subtle differences within unit cells—has remained unexplored.

In this work, we present a computational pipeline combining dimensionality reduction and hierarchical clustering to automatically detect and classify atomic columns within the unit cell from atomic-resolution 4D-STEM data. We apply this approach to three representative materials with distinct structural and compositional characteristics: monolayer MoS₂, the semiconductor GaN, and the complex oxide SrTiO₃. These materials span a broad range of atomic weights, symmetries, and column types, including heavy and light atoms, posing challenges for automated identification.

Our three-stage cascaded clustering strategy effectively distinguishes atomic columns from interatomic background regions across all tested materials, resolving even columns with closely related scattering profiles. This approach provides a new opportunity for information extraction, with potential live application at the microscope for data driven experiments, opening pathways for detailed defect analysis, phase identification, and strain mapping in future electron microscopy studies.

A key step in enabling hydrogen (H₂) as an efficient and scalable energy carrier is improving the oxygen evolution reaction (OER), crucial in water electrolysis for green H₂ production. However, the inherently sluggish four-electron transfer mechanism of the OER limits efficiency and typically requires noble metal-based catalysts such as IrO₂ and RuO₂ [1]. The scarcity and high cost of these materials remain major obstacles to widespread adoption of H₂ technologies.

First-row transition metal oxides offer a prospective alternative due to their high earth abundance and promising activity in acidic OER. Yet, their poor stability, especially under acidic conditions, remains a critical limitation [2]. One strategy to overcome this involves incorporating high-valence sacrificial elements such as W into a MWO₄-type crystal structure. Upon selective removal through a water/hydroxide - WO₄²⁻ anion exchange process, these structures can trap hydroxide and water species within a defective metal oxide framework [3]. However, this delamination–activation mechanism is not equally effective across all first-row transition metals. Among them, cobalt tungstate (CoWO₄) has shown exceptional OER performance [4]. Upon activation, CoWO₄ achieves activity comparable to IrO₂, along with significantly improved durability. When implemented in water electrolysis systems, CoWO₄-based anodes achieved a threefold improvement over Ir- and Ru-free alternatives, maintaining stability for over 600 hours at 1 A cm⁻².

To understand the role of the delamination process across different MWO₄ compounds, advanced characterization techniques with atomic-scale imaging resolution and spectroscopic information are essential [5]. In this work, scanning transmission electron microscopy (STEM) was employed to monitor structural and compositional transformations during activation. Integrated differential phase contrast (iDPC-STEM) enabled direct imaging of oxygen atoms within the MWO₄ lattices, facilitating assessment of potential Jahn–Teller distortions in the MO₆ octahedra. Electron energy loss spectroscopy (EELS) provided insights into the elemental evolution and oxidation states changes at various activation stages. High-angle annular dark field (HAADF-STEM) imaging confirmed the formation of surface vacancies in CoWO₄, features directly linked to its improved catalytic durability. Finally, post-OER STEM analysis revealed the remarkable structural stability of the activated material. These findings were supported by complementary spectroscopic analyses, offering a comprehensive picture of the structure–property relationships that govern catalytic performance.

By revealing the mechanisms behind CoWO₄'s high activity and durability, this work contributes to the rational design of next-generation, earth-abundant OER catalysts. These insights pave the way for scalable, cost-effective solutions in hydrogen production and support the broader transition toward sustainable, zero-emission energy systems.

In recent decades, we have witnessed the development of new analytical tools in the frame of STEM/TEM characterization such as, Differential Phase Contrast (DPC) or 4D-STEM technique, which have changed the paradigm of its uses. The core idea behind DPC is to measure the differential phase shift between two orthogonally polarized beams of light transmitted through a sample. One of the most interested applications in semiconductors is the detection of local electric fields [1]. In 4D-STEM, a highly collimated electron beam scans over the sample, resulting in a diffraction pattern recorded for each scanning position. This creates a dataset that includes information about position (2D) and diffraction pattern (2D), resulting in a 4D dataset. It allows strain calculations or orientation mapping for nanoparticles, among other possibilities [2]. In this work, we present some preliminary results carried out on III-V nanowires as an example to show the potential of these novel techniques. We show HRSTEM and DPC data carried out at the surface of the nanowire revealing the existence of a local electric field which can be due to the formation of a core. Regarding 4D-STEM, by proper data analysis we present normal strain maps (e_xx and e_yy) carried out on the GaInP section of the nanowire using open scrips [3]. These first results reveal compression in growth direction (axial direction) and tension in y direction (radial direction). Preliminary EDS results reveal oscillations in Ga/In atomic composition. The data presented validate the 4D-STEM and DPC measurements and show the potential of these analytical tools.