Nanoscale Resolution Research Articles

Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy

Sphingomyelin is a key molecule of sphingolipid metabolism, and its enzymatic breakdown is associated with various infectious diseases. Here, we introduce trifunctional sphingomyelin derivatives that enable the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes. We demonstrate this by determining the activity of a bacterial sphingomyelinase on the plasma membrane of host cells using a combination of Förster resonance energy transfer and expansion microscopy. We further use our trifunctional sphingomyelin probes to visualize their metabolic state during infections with Chlamydia trachomatis and thereby show that chlamydial inclusions primarily contain the cleaved forms of the molecules. Using expansion microscopy, we observe that the proportion of metabolized molecules increases during maturation from reticulate to elementary bodies, indicating different membrane compositions between the two chlamydial developmental forms. Expansion microscopy of trifunctional sphingomyelins thus provides a powerful microscopy tool to analyze sphingomyelin metabolism in cells at nanoscale resolution.

A deep learning method that identifies cellular heterogeneity using nanoscale nuclear features

Cellular phenotypic heterogeneity is an important hallmark of many biological processes and understanding its origins remains a substantial challenge. This heterogeneity often reflects variations in the chromatin structure, influenced by factors such as viral infections and cancer, which dramatically reshape the cellular landscape. To address the challenge of identifying distinct cell states, we developed artificial intelligence of the nucleus (AINU), a deep learning method that can identify specific nuclear signatures at the nanoscale resolution. AINU can distinguish different cell states based on the spatial arrangement of core histone H3, RNA polymerase II or DNA from super-resolution microscopy images. With only a small number of images as the training data, AINU correctly identifies human somatic cells, human-induced pluripotent stem cells, very early stage infected cells transduced with DNA herpes simplex virus type 1 and even cancer cells after appropriate retraining. Finally, using AI interpretability methods, we find that the RNA polymerase II localizations in the nucleoli aid in distinguishing human-induced pluripotent stem cells from their somatic cells. Overall, AINU coupled with super-resolution microscopy of nuclear structures provides a robust tool for the precise detection of cellular heterogeneity, with considerable potential for advancing diagnostics and therapies in regenerative medicine, virology and cancer biology.

Vertical Current Transport in Monolayer MoS<sub>2</sub> Heterojunctions with 4H-SiC Fabricated by Sulfurization of Ultra-Thin MoO<sub>x</sub> Films

In this paper, we report on the growth of highly uniform MoS2 films, mostly consisting of monolayers, on SiC surfaces with different doping levels (n- SiC epitaxy, ~1016 cm-3, and n+ SiC substrate, ~1019 cm-3) by sulfurization of a pre-deposited ultra-thin MoOx films. MoS2 layers are lowly strained (~0.12% tensile strain) and highly p-type doped (<Nh>≈4×1019 cm−3), due to MoO3 residues still present after the sulfurization process. Nanoscale resolution I-V analyses by conductive atomic force microscopy (C-AFM) show a strongly rectifying behavior for MoS2 junction with n- SiC, whereas the p+ MoS2/n+ SiC junction exhibits an enhanced reverse current and a negative differential behavior under forward bias. This latter observation, indicating the occurrence of band-to-band-tunneling from the occupied states of n+ SiC conduction band to the empty states of p+ MoS2 valence band, is a confirmation of the very sharp hetero-interface between the two materials. These results pave the way to the fabrication of ultra-fast switching Esaki diodes on 4H-SiC.

Super‐Resolution Imaging in Collagen‐Abundant Thick Tissues

Expansion microscopy (ExM) has gained increasing popularity for 3D ultrastructural imaging of cultured cells and tissue slices at nanoscale resolution using conventional microscopes via physical expansion of biological tissues. However, its application to collagen‐abundant thick tissues is still challenging. Herein, a new method, collagen ExM (ColExM), optimized for expanding tissues containing more than 70% collagen, is demonstrated. ColExM succeeds in 4.5‐fold linear expansion with minimal structural distortion of corneal and skin tissues. It is compatible with immunostaining, allowing super‐resolution visualization of 3D neural structures innervating hair follicles, corneas, and pancreatic tumors with high stromal collagen content. The method succeeds in identifying individual mitochondria and previously unrecognized dendritic spinelike structures of corneal nerves. It also enables fine mapping of structural rearrangement of tight junctions and actin cytoskeletons. Therefore, ColExM can facilitate the exploration of 3D nanoscale structures in collagen‐rich tissues.

High-Throughput Combinatorial Analysis of the Spatiotemporal Dynamics of Nanoscale Lithium Metal Plating.

The development of Li metal batteries requires a detailed understanding of complex nucleation and growth processes during electrodeposition. In situ techniques offer a framework to study these phenomena by visualizing structural dynamics that can inform the design of uniform plating morphologies. Herein, we combine scanning electrochemical cell microscopy (SECCM) with in situ interference reflection microscopy (IRM) for a comprehensive investigation of Li nucleation and growth on lithiophilic thin-film gold electrodes. This multimicroscopy approach enables nanoscale spatiotemporal monitoring of Li plating and stripping, along with high-throughput capabilities for screening experimental conditions. We reveal the accumulation of inactive Li nanoparticles in specific electrode regions, yet these regions remain functional in subsequent plating cycles, suggesting that growth does not preferentially occur from particle tips. Optical-electrochemical correlations enabled nanoscale mapping of Coulombic Efficiency (CE), showing that regions prone to inactive Li accumulation require more cycles to achieve higher CE. We demonstrate that electrochemical nucleation time (tnuc) is a lagging indicator of nucleation and introduce an optical method to determine tnuc at earlier stages with nanoscale resolution. Plating at higher current densities yielded smaller Li nanoparticles and increased areal density, and was not affected by heterogeneous topographical features, being potentially beneficial to achieve a more uniform plating at longer time scales. These results enhance the understanding of Li plating on lithiophilic surfaces and offer promising strategies for uniform nucleation and growth. Our multimicroscopy approach has broad applicability to study nanoscale metal plating and stripping phenomena, with relevance in the battery and electroplating fields.

Fast imaging of millimeter-scale areas with beam deflection transmission electron microscopy

Serial section transmission electron microscopy (TEM) has proven to be one of the leading methods for millimeter-scale 3D imaging of brain tissues at nanoscale resolution. It is important to further improve imaging efficiency to acquire larger and more brain volumes. We report here a threefold increase in the speed of TEM by using a beam deflecting mechanism to enable highly efficient acquisition of multiple image tiles (nine) for each motion of the mechanical stage. For millimeter-scale areas, the duty cycle of imaging doubles to more than 30%, yielding a net average imaging rate of 0.3 gigapixels per second. If fully utilized, an array of four beam deflection TEMs should be capable of imaging a dataset of cubic millimeter scale in five weeks.

Towards using fluorescent nanodiamonds for studying cell migration

Since wound healing requires cells to repopulate a damaged area, cell migration is essential. In addition, migration plays a crucial role in cancer metastasis. Whether tumour cells can invade tissue and metastasize is a crucial factor determining their malignancy or in other words a hallmark of cancer (Lazebnik in Nat Rev Cancer 10: 232–233, 2010, https://doi.org/10.1038/nrc2827). Nanodiamonds potentially offer a powerful tool to investigate these migration processes. Due to their unprecedented photostability, they can function as long-term fluorescent labels. Besides, nanodiamonds are robust quantum sensors that can reveal, for instance, the temperature or the concentration of certain chemicals with nanoscale resolution. However, to utilise nanodiamonds to study cell migration, it is essential to understand if and how the presence of nanodiamonds influences cell migration. Here, we investigate this process for the first time. We found that nanodiamonds do not alter the speed at which HeLa cells populate a scratch at any tested concentrations. Furthermore, we tested cell attachment by quantifying focal adhesion points. Oxygen-terminated fluorescent nanodiamonds influence the cell spreading, the number of focal adhesions and the size of focal adhesion points. Interestingly, this is different for other types of nanodiamonds in the literature. For these particles, it has been described in the literature that they hinder cell migration. Our results support that fluorescent nanodiamonds do not influence cell migration strongly and thus can be used in labelling and sensing migrating cells.Graphical

Simultaneous Mapping of the Nanoscale Organization and Redox State of Extracellular Space in Living Brain Tissue.

The spatial organization characteristics and redox status of the extracellular space (ECS) are crucial in the development of brain diseases. However, it remains a challenge to simultaneously capture dynamic changes in microstructural features and redox states at the submicron level within the ECS. Here, we developed a reversible glutathione (GSH)-responsive nanoprobe (RGN) for mapping the spatial organization features and redox status of the ECS in brain tissues with nanoscale resolution. The RGN is composed of polymer nanoparticles modified with GSH-responsive molecules and amino-functionalized methoxypoly(ethylene glycol), which exhibit exceptional single-particle brightness and excellent free diffusion capability in the ECS of brain tissues. Tracking single RGNs in acute brain slices allowed us to dynamically map spatial organizational features and redox levels within the ECS of brain tissues in disease models. This provides a powerful super-resolution imaging method that offers a potential opportunity to study the dynamic changes in the ECS microenvironment and to understand the physiological and pathological roles of the ECS in vivo.

Quantitative Three-Dimensional Imaging Analysis of HfO2 Nanoparticles in Single Cells via Deep Learning Aided X-ray Nano-Computed Tomography.

It is crucial for understanding mechanisms of drug action to quantify the three-dimensional (3D) drug distribution within a single cell at nanoscale resolution. Yet it remains a great challenge due to limited lateral resolution, detection sensitivities, and reconstruction problems. The preferable method is using X-ray nano-computed tomography (Nano-CT) to observe and analyze drug distribution within cells, but it is time-consuming, requiring specialized expertise, and often subjective, particularly with ultrasmall metal nanoparticles (NPs). Furthermore, the accuracy of batch data analysis through conventional processing methods remains uncertain. In this study, we used radioenhancer ultrasmall HfO2 nanoparticles as a model to develop a modular and automated deep learning aided Nano-CT method for the localization quantitative analysis of ultrasmall metal NPs uptake in cancer cells. We have established an ultrasmall objects segmentation method for 3D Nano-CT images in single cells, which can highly sensitively analyze minute NPs and even ultrasmall NPs in single cells. We also constructed a localization quantitative analysis method, which may accurately segment the intracellularly bioavailable particles from those of the extracellular space and intracellular components and NPs. The high bioavailability of HfO2 NPs in tumor cells from deeper penetration in tumor tissue and higher tumor intracellular uptake provide mechanistic insight into HfO2 NPs as advanced radioenhancers in the combination of quantitative subcellular image analysis with the therapeutic effects of NPs on 3D tumor spheroids and breast cancer. Our findings unveil the substantial uptake rate and subcellular quantification of HfO2 NPs by the human breast cancer cell line (MCF-7). This revelation explicates the notable efficacy and safety profile of HfO2 NPs in tumor treatment. These findings demonstrate that this 3D imaging technique promoted by the deep learning algorithm has the potential to provide localization quantitative information about the 3D distributions of specific molecules at the nanoscale level. This study provides an approach for exploring the subcellular quantitative analysis of NPs in single cells, offering a valuable quantitative imaging tool for minute amounts or ultrasmall NPs.

Unveiling enamel demineralization mechanisms by sensitive dielectric differentiation based on terahertz nanospectroscopy.

The early stage of dental caries, i.e. demineralization, has always been a topic of concern to dentists. Understanding the essential mechanism of its occurrence is of great significance for the prevention and treatment of dental caries. However, owing to limitations in resolution and the detection capabilities of diagnostic tools, the study of enamel demineralization has always been a challenge. Terahertz (THz) technology, especially the combination of scanning near-field optical microscopy (s-SNOM) and THz time-domain spectroscopy (TDS), due to its nanoscale resolution, has shown great advantages in the field of biological imaging. Here, a THz s-SNOM system is used to perform near-field imaging of enamel before and after demineralization at the nanoscale. It can be found that near-field signals decrease significantly after demineralization. This is due to the changes of the crystal lattice and the transfer of mineral ions during demineralization, which leads to a decrease in the permittivity of the enamel. The novel approach in this study reveals the essence of demineralization and lays the groundwork for additional research and potential interventions.

Nanoscale volumetric fluorescence imaging via photochemical sectioning.

Optical nanoscopy of intact biological specimens has been transformed by recent advancements in hydrogel-based tissue clearing and expansion, enabling the imaging of cellular and subcellular structures with molecular contrast. However, existing high-resolution fluorescence microscopes have limited imaging depth, which prevents the study of whole-mount specimens without physical sectioning. To address this challenge, we developed "photochemical sectioning," a spatially precise, light-based sample sectioning process. By combining photochemical sectioning with volumetric lattice light-sheet imaging and petabyte-scale computation, we imaged and reconstructed axons and myelination sheaths across entire mouse olfactory bulbs at nanoscale resolution. An olfactory-bulb-wide analysis of myelinated and unmyelinated axons revealed distinctive patterns of axon degeneration and de-/dysmyelination in the neurodegenerative mouse, highlighting the potential for peta- to exabyte-scale super-resolution studies using this approach.

Fluorescent Nanodiamonds for High-Resolution Thermometry in Biology.

Optically active color centers in diamond and nanodiamonds can be utilized as quantum sensors for measuring various physical parameters, particularly magnetic and electric fields, as well as temperature. Due to their small size and possible surface functionalization, fluorescent nanodiamonds are extremely attractive systems for biological and medical applications since they can be used for intracellular experiments. This review focuses on fluorescent nanodiamonds for thermometry with high sensitivity and a nanoscale spatial resolution for the investigation of living systems. The current state of the art, possible further development, and potential limitations of fluorescent nanodiamonds as thermometers will be discussed here.

Morphology of Copper Nanofoams for Radiation Hydrodynamics and Fusion Applications Investigated by 3D Ptychotomography.

The performance of metal and polymer foams used in inertial confinement fusion (ICF), inertial fusion energy (IFE), and high-energy-density (HED) experiments is currently limited by our understanding of their nanostructure and its variation in bulk material. We utilized an X-ray-free electron laser (XFEL) together with lensless X-ray imaging techniques to probe the 3D morphology of copper foams at nanoscale resolution (28 nm). The observed morphology of the thin shells is more varied than expected from previous characterizations, with a large number of them distorted, merged, or open, and a targeted mass density 14% less than calculated. This nanoscale information can be used to directly inform and improve foam modeling and fabrication methods to create a tailored material response for HED experiments.

Direct detection system for full-field nanoscale X-ray diffraction-contrast imaging.

Recent developments in X-ray science provide methods to probe deeply embedded mesoscale grain structures and spatially resolve them using dark field X-ray microscopy (DFXM). Extending this technique to investigate weak diffraction signals such as magnetic systems, quantum materials and thin films prove challenging due to available detection methods and incident X-ray flux at the sample. We present a direct detection method developed in conjunction with KAImaging which focuses on DFXM studies in the hard X-ray range of 10s of keV and above capable of approaching nanoscale resolution. Additionally, we compare this direct detection scheme with routinely used scintillator-based optical detection and achieve an order of magnitude improvement in exposure times allowing for imaging of weakly diffracting ordered systems.

Dynamics for High-Sensitivity Detection of Free Radicals in Primary Bronchial Epithelial Cells upon Stimulation with Cigarette Smoke Extract.

Chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide, is caused by chronic exposure to toxic particles and gases, such as cigarette smoke. Free radicals, which are produced during a stress response to toxic particles, play a crucial role in disease progression. Measuring these radicals is difficult since the complex mixture of chemicals within cigarette smoke interferes with radical detection. We used a new quantum sensing technique called relaxometry to measure free radicals with nanoscale resolution on cells from COPD patients and healthy controls exposed to cigarette smoke extract (CSE) or control medium. Epithelial cells from COPD patients display a higher free radical load than those from healthy donors and are more vulnerable to CSE. We show that epithelial cells of COPD patients are more susceptible to the damaging effects of cigarette smoke, leading to increased release of free radicals.

Three-Dimensional Label-Free Observing of the Self-Assembled Nanoparticles inside a Single Cell at Nanoscale Resolution.

Understanding the intracellular behavior of nanoparticles (NPs) plays a key role in optimizing the self-assembly performance of nanomedicine. However, conducting the 3D, label-free, quantitative observation of self-assembled NPs within intact single cells remains a substantial challenge in complicated intracellular environments. Here, we propose a deep learning combined synchrotron radiation hard X-ray nanotomography approach to visualize the self-assembled ultrasmall iron oxide (USIO) NPs in a single cell. The method allows us to explore comprehensive information on NPs, such as their distribution, morphology, location, and interaction with cell organelles, and provides quantitative analysis of the heterogeneous size and morphologies of USIO NPs under diverse conditions. This label-free, in situ method provides a tool for precise characterization of intracellular self-assembled NPs to improve the evaluation and design of a bioresponsive nanomedicine.

Mesoscale characterization of osseointegration around an additively manufactured genistein-coated implant

Given the hierarchical nature of bone and bone interfaces, osseointegration, namely the formation of a direct bone-implant contact, is best evaluated using a multiscale approach. However, a trade-off exists between field of view and spatial resolution, making it challenging to image large volumes with high resolution. In this study, we combine established electron microscopy techniques to probe bone-implant interfaces at the microscale and nanoscale with plasma focused ion beam-scanning electron microscopy (PFIB-SEM) tomography to evaluate osseointegration at the mesoscale. This characterization workflow is demonstrated for bone response to an additively manufactured Ti-6Al-4V implant which combines engineered porosity to facilitate bone ingrowth and surface functionalization via genistein, a phytoestrogen, to counteract bone loss in osteoporosis. SEM demonstrated new bone formation at the implant site, including in the internal implant pores. At the nanoscale, scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy confirmed the gradual nature of the bone-implant interface. By leveraging mesoscale analysis with PFIB-SEM tomography that captures large volumes of bone-implant interface with nearly nanoscale resolution, the presence of mineral ellipsoids varying in size and orientation was revealed. In addition, a well-developed lacuno-canalicular network and mineralization fronts directed both towards the implant and away from it were highlighted.

In Operando Imaging Electrostatic-Driven Disassembly and Reassembly of Collagen Nanostructures.

Collagen is the most abundant protein in tissue scaffolds in live organisms. Collagen can self-assemble in vitro, which has led to a number of biotechnological and biomedical applications. To understand the dominant factors that participate in the formation of collagen nanostructures, here we study in real time and with nanoscale resolution the disassembly and reassembly of collagens. We implement a high-speed force microscope, which provides in situ high spatiotemporal resolution images of collagen nanostructures under changing pH conditions. The disassembly and reassembly are dominated by the electrostatic interactions among amino-acid residues of different molecules. Acidic conditions favor disassembly by neutralizing negatively charged residues. The process sets a net repulsive force between collagen molecules. A neutral pH favors the presence of negative and positively charged residues along the collagen molecules, which promotes their electrostatic attraction. Molecular dynamics simulations reproduce the experimental behavior and validate the electrostatic-based model of the disassembly and reassembly processes.

3D Nanoprinting of Heterogeneous Metal Oxides with High Shape Fidelity.

3D nanoprinting can significantly enhance the performance of sensors, batteries, optoelectronic/microelectronic devices, etc. However, current 3D nanoprinting methods for metal oxides are suffering from three key issues including limited material applicability, serious shape distortion, and the difficulty of heterogeneous integration. This paper discovers a mechanism in which imidazole and acrylic acid synergistically coordinate with metal ions in water. Using the mechanism, this work develops a series of metal ion synergistic coordination water-soluble (MISCWS) resins for 3D nanoprinting of various metal oxides, including MnO2, Cr2O3, Co3O4, and ZnO, as well as heterogeneous structures of MnO2/NiO, Cr2O3/Al2O3, and ZnO/MgO. Besides, the synergistic coordination effect results in a 2.54-fold increase in inorganic mass fraction within the polymer, compared with previous works, which effectively mitigates the shape distortion of metal oxide microstructures. Based on this method, this work also demonstrates a 3D ZnO microsensor with a high sensitivity (1.113 million at 200ppm NO2), surpassing the conventional 2D ZnO sensors by tenfold. The method yields high-fidelity 3D structures of heterogeneous metal oxides with nanoscale resolution, paving the way for applications such as sensing, micro-optics, energy storage, and microsystems.

Terahertz s-SNOM Imaging of a Single Cell with Nanoscale Resolution.

Terahertz scattering scanning near-field optical microscopy is a robust spectral detection technique with a nanoscale resolution. However, there are still major challenges in investigating the heterogeneity of cell membrane components in individual cells. Here, we present a novel and comprehensive analytical approach for detecting and investigating heterogeneity in cell membrane components at the single-cell level. In comparison to the resolution of the topographical atomic force microscopy image, the spatial resolution of the terahertz near-field amplitude image is 3 times that of the former. This ultrafine resolution enables the compositional distribution in the cell membrane, such as the distribution of extracellular vesicles, to be finely characterized. Furthermore, via extraction of the near-field absorption images at specific frequencies, the visualization and compositional difference analysis of cell membrane components can be presented in detail. These findings have significant implications for the intuitive and visual analysis of cell development and disease evolutionary pathways.