Scanning Helium Ion Microscopy Research Articles

Contributions of Chinese hamster ovary cell derived extracellular vesicles and other cellular materials to hollow fiber filter fouling during perfusion manufacturing of monoclonal antibodies.

Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell-derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO-derived small extracellular vesicles (sEVs) with 50-200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV-like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF-TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X-ray spectroscopy-based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small-scale HF-TFF model, addition of CHO-specific sEVs in CHO culture simulated filter fouling behaviors in a concentration-dependent manner. Based on these results, we proposed a mechanism of HF-TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane.

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

Nanoarchitecture of the ventral disc of Giardia intestinalis as revealed by high-resolution scanning electron microscopy and helium ion microscopy

The parasitic protozoan Giardia intestinalis, the causative agent of giardiasis, presents a stable and elaborated cytoskeleton, which shapes and supports several intracellular structures, including the ventral disc, the median body, the funis, and four pairs of flagella. Giardia trophozoite is the motile form that inhabits the host small intestine and attaches to epithelial cells, leading to infection. The ventral disc is considered one important element of adhesion to the intestinal cells. It is adjacent to the plasma membrane in the ventral region of the cell and consists of a spiral layer of microtubules and microribbons. In this work, we studied the organization of the cytoskeleton in the ventral disc of G. intestinalis trophozoites using high-resolution scanning electron microscopy or helium ion microscopy in plasma membrane-extracted cells. Here, we show novel morphological details about the arrangement of cross-bridges in different regions of the ventral disc. Results showed that the disc is a non-uniformly organized structure that presents specific domains, such as the margin and the ventral groove region. High-resolution scanning electron microscopy allowed observation of the labeling pattern for several anti-tubulin antibodies using secondary gold particle-labeled antibodies. Labeling in the region of the emergence of the microtubules and supernumerary microtubules using an anti-acetylated tubulin antibody was observed. Ultrastructural analysis and immunogold labeling for gamma-tubulin suggest that disc microtubules originate from a region bounded by the bands of the banded collar and merge with microtubules formed at the perinuclear region. Actin-like filaments and microtubules of the disc are associated, showing an interconnection between elements of the cytoskeleton of the trophozoite.

Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy.

The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley (Hordeum vulgare) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.

Bio-imaging with the helium-ion microscope: A review.

Scanning helium-ion microscopy (HIM) is an imaging technique with sub-nanometre resolution and is a powerful tool to resolve some of the tiniest structures in biology. In many aspects, the HIM resembles a field-emission scanning electron microscope (FE-SEM), but the use of helium ions rather than electrons provides several advantages, including higher surface sensitivity, larger depth of field, and a straightforward charge-compensating electron flood gun, which enables imaging of non-conductive samples, rendering HIM a promising high-resolution imaging technique for biological samples. Starting with studies focused on medical research, the last decade has seen some particularly spectacular high-resolution images in studies focused on plants, microbiology, virology, and geomicrobiology. However, HIM is not just an imaging technique. The ability to use the instrument for milling biological objects as small as viruses offers unique opportunities which are not possible with more conventional focused ion beams, such as gallium. Several pioneering technical developments, such as methods to couple secondary ion mass spectrometry (SIMS) or ionoluminescence with the HIM, also offer the possibility for new and exciting research on biological materials. In this review, we present a comprehensive overview of almost all currently published literature which has demonstrated the application of HIM for imaging of biological specimens. We also discuss some technical features of this unique type of instrument and highlight some of the new advances which will likely become more widely used in the years to come.

Tritrichomonas foetus: New structures by high-resolution scanning helium ion microscopy

Tritrichomonas foetus: New structures by high-resolution scanning helium ion microscopy

Liquid crystalline ordered arrays of gold nanoparticles in evaporative deposits

Gold nanoparticles assembly have been studied using two different evaporation techniques. To evaluate the influence CTAB molecules on self-assembly behavior 0.1 M CTAB suspension have been imaged via scanning helium ion microscopy (HIM) and then by SEM. By using immersion methode, dumbbell-like nanoparticles crystallizes over the substrate in island-like arrays showing no specific ordered structures whereas gold nanorods crystallizes in ordered arrays within the deposited islands. However, droplet drying showed aligned arrays of gold nanorods all over the coffee-stain ring. For purified suspensions, domain-like superstructures observed away from the outer-region of the coffee stain. These crystalline arrays grows into domain-like superstructures hydrodynamically.

Acoustic attenuation spectroscopy and helium ion microscopy study of rehydration of dairy powder

Complete hydration is essential for the production of structured dairy products from powders. It is essential that the ingredients used hydrate completely. Determination of an end point of rehydration is non-trivial, but ultrasound-based methodologies have demonstrated potential in this area and are well suited to measuring bulk samples in situ. Here, acoustic attenuation spectroscopy (AAS) is used to monitor rehydration of skim milk powder, and recombined systems of micellar casein isolate with lactose and whey protein isolate. Dynamic light scattering, zeta-potential measurements and AAS as a function of pH characterise each component around its isoelectric point to assess its functionality. Scanning helium ion microscopy was used to image the dry powders, without any conductive coating, producing resolution equivalent to scanning electron microscopy, but with much larger focal lengths and fewer imaging artefacts. Imaging the powders provides information on particle size and morphology which can affect dissolution behaviour. Reconstituted skim milk powder and recombined samples were monitored showing there are changes occurring over several hours. Attenuation coefficients are shown to predict the end point of hydration. Model fitting is used to extract volume fractions and average particle sizes of large and small particle populations in recombined samples over time. AAS is demonstrated to be capable of tracking the dynamics in rehydrating dispersions over time. Physical parameters such as the volume fraction and particle size of the dispersed phase can be determined.

Deposition and distribution of gold nanoparticles in a coffee-stain ring on the HOPG terraces

Cetyltrimethylammonium bromide (CTAB)-stabilized low-aspect ratio gold nanorods have been synthesized using seed-mediated wet chemical approach. Freshly cleaved highly oriented pyrolytic graphite (HOPG) surface is used as a substrate for the deposition of nanoparticles via the droplet drying technique. Owing to the presence of CTAB molecules in suspension, nanoparticles on the non-wetting HOPG surface get deposited in the form of a coffee-stain ring. Such deposits at various positions of the coffee-stain ring have been characterized by scanning helium ion microscopy (HIM) and atomic force microscopy (AFM). Besides the assembly of nanoparticles, AFM is specially employed to investigate the deposition of CTAB molecules. The deposition and distribution of nanoparticles are studied and quantified at various positions of the coffee-stain ring. On flat HOPG terraces, HIM shows merely the deposits of the gold nanostructures whereas AFM reveals both nanoparticles and CTAB deposits over the surface. This research study primarily focused on the nanoparticle deposits within the coffee-stain ring formed over the non-wetting HOPG terraces.

The structural organization of Giardia intestinalis cytoskeleton.

Giardia intestinalis, the causative agent of giardiasis, has complex cytoskeleton organization with structures involved in motility, adhesion, cell division, and cell differentiation. Microtubules are key components of the cytoskeleton and are the main elements of the ventral disc, median body, funis, in addition to four pairs of flagella. These cytoskeletal elements are basically stable microtubule arrangements. Although tubulins are the main proteins of these elements, molecular and biochemical analyses of Giardia trophozoites have revealed the presence of several new and not yet characterized proteins in these structures, which may contribute to their nanoarchitecture (mainly in the ventral disc). Despite these findings, morphological data are still required for understanding the organization and biogenesis of the cytoskeletal structures. In the study of this complex and specialized network of filaments in Giardia, two distinct and complementary approaches have been used in recent years: (a) transmission electron microscopy tomography of conventionally processed as well as cryo-fixed samples and (b) high-resolution scanning electron microscopy and helium ion microscopy in combination with new plasma membrane extraction protocols. In this review we include the most recent studies that have allowed better understanding of new Giardia components and their association with other filamentous structures of this parasite, thus providing new insights in the role of the cytoskeletal structures and their function in Giardia trophozoites.

Correlative Microscopy in 3D: Helium Ion Microscopy-Based Photogrammetric Topography Reconstruction Combined with in situ Secondary Ion Mass Spectrometry.

The chemical or elemental analysis of samples with complex surface topography is challenging for secondary ion mass spectrometry (SIMS), if the three-dimensional structure of the sample is not taken into account. Conventional 3D reconstruction of SIMS data assumes a flat surface and uniform sputtering conditions, which is not the case for many analytical applications involving micro- and nanosized particles, composites, or patterned materials. Reliable analysis of such samples requires knowledge of the actual 3D surface structure to correctly reconstruct the SIMS 3D maps. To this end, we introduce the use of photogrammetric 3D topography reconstruction from scanning helium ion microscopy (HIM) correlated with in situ SIMS data for the reconstruction of 3D SIMS data. The HIM and SIMS data are acquired under in situ conditions in a Zeiss ORION NanoFab HIM using a novel SIMS analyzer. We successfully tested the applicability of the approach to generate 3D models of different samples and show that the combination of SIMS and 3D topography is able to provide insights into the influence of the sample topography in a single instrument and with a single ion column and hence without the need for ex-situ sample analysis or additional instrumentation. These findings offer a path toward ion-based correlative 3D spectromicroscopy (3D-HIM-SIMS) and suggest that many combinations of charged particle based P3D (SEM, HIM) and analytical microscopy techniques, such as SIMS, energy-dispersive X-ray spectroscopy (EDX), or ionoluminescence/cathodoluminescence (IL/CL), can be used for correlative microscopy in 3D.

He-Ion Microscopy as a High-Resolution Probe for Complex Quantum Heterostructures in Core-Shell Nanowires.

Core-shell semiconductor nanowires (NW) with internal quantum heterostructures are amongst the most complex nanostructured materials to be explored for assessing the ultimate capabilities of diverse ultrahigh-resolution imaging techniques. To probe the structure and composition of these materials in their native environment with minimal damage and sample preparation calls for high-resolution electron or ion microscopy methods, which have not yet been tested on such classes of ultrasmall quantum nanostructures. Here, we demonstrate that scanning helium ion microscopy (SHeIM) provides a powerful and straightforward method to map quantum heterostructures embedded in complex III-V semiconductor NWs with unique material contrast at ∼1 nm resolution. By probing the cross sections of GaAs-Al(Ga)As core-shell NWs with coaxial GaAs quantum wells as well as short-period GaAs/AlAs superlattice (SL) structures in the shell, the Al-rich and Ga-rich layers are accurately discriminated by their image contrast in excellent agreement with correlated, yet destructive, scanning transmission electron microscopy and atom probe tomography analysis. Most interestingly, quantitative He-ion dose-dependent SHeIM analysis of the ternary AlGaAs shell layers and of compositionally nonuniform GaAs/AlAs SLs reveals distinct alloy composition fluctuations in the form of Al-rich clusters with size distributions between ∼1-10 nm. In the GaAs/AlAs SLs the alloy clustering vanishes with increasing SL-period (>5 nm-GaAs/4 nm-AlAs), providing insights into critical size dimensions for atomic intermixing effects in short-period SLs within a NW geometry. The straightforward SHeIM technique therefore provides unique benefits in imaging the tiniest nanoscale features in topography, structure and composition of a multitude of diverse complex semiconductor nanostructures.

Design Rules for Oxygen Evolution Catalysis at Porous Iron Oxide Electrodes: A 1000-Fold Current Density Increase.

Nanotubular iron(III) oxide electrodes are optimized for catalytic efficiency in the water oxidation reaction at neutral pH. The nanostructured electrodes are prepared from anodic alumina templates, which are coated with Fe2 O3 by atomic layer deposition. Scanning helium ion microscopy, X-ray diffraction, and Raman spectroscopy are used to characterize the morphologies and phases of samples submitted to various treatments. These methods demonstrate the contrasting effects of thermal annealing and electrochemical treatment. The electrochemical performances of the corresponding electrodes under dark conditions are quantified by steady-state electrolysis and electrochemical impedance spectroscopy. A rough and amorphous Fe2 O3 with phosphate incorporation is critical for the optimization of the water oxidation reaction. For the ideal pore length of 17 μm, the maximum catalytic turnover is reached with an effective current density of 140 μA cm-2 at an applied overpotential of 0.49 V.

Bladder schistosomiasis

Giardia intestinalis, the causative agent of giardiasis, has complex cytoskeleton organization with structures involved in motility, adhesion, cell division, and cell differentiation. Microtubules are key components of the cytoskeleton and are the main elements of the ventral disc, median body, funis, in addition to four pairs of flagella. These cytoskeletal elements are basically stable microtubule arrangements. Although tubulins are the main proteins of these elements, molecular and biochemical analyses of Giardia trophozoites have revealed the presence of several new and not yet characterized proteins in these structures, which may contribute to their nanoarchitecture (mainly in the ventral disc). Despite these findings, morphological data are still required for understanding the organization and biogenesis of the cytoskeletal structures. In the study of this complex and specialized network of filaments in Giardia, two distinct and complementary approaches have been used in recent years: (a) transmission electron microscopy tomography of conventionally processed as well as cryo-fixed samples and (b) high-resolution scanning electron microscopy and helium ion microscopy in combination with new plasma membrane extraction protocols. In this review we include the most recent studies that have allowed better understanding of new Giardia components and their association with other filamentous structures of this parasite, thus providing new insights in the role of the cytoskeletal structures and their function in Giardia trophozoites.

Thermal decomposition of fullerene nanowhiskers protected by amorphous carbon mask.

Fullerene nanostructures are well known for their unique morphology, physical and mechanical properties. The thermal stability of fullerene nanostructures, such as their sublimation at high temperature is also very important for studying their structures and applications. In this work, We observed fullerene nanowhiskers (FNWs) in situ with scanning helium ion microscopy (HIM) at elevated temperatures. The FNWs exhibited different stabilities with different thermal histories during the observation. The pristine FNWs were decomposed at the temperatures higher than 300 °C in a vacuum environment. Other FNWs were protected from decomposition with an amorphous carbon (aC) film deposited on the surface. Based on high spacial resolution, aC film with periodic structure was deposited by helium ion beam induced deposition (IBID) on the surface of FNWs. Annealed at the high temperature, the fullerene molecules were selectively sublimated from the FNWs. The periodic structure was formed on the surface of FNWs and observed by HIM. Monte Carlo simulation and Raman characterization proved that the morphology of the FNWs was changed by helium IBID at high temperature. This work provides a new method of fabricating artificial structure on the surface of FNWs with periodic aC film as a mask.

Residual matrix from different separation techniques impacts exosome biological activity.

Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.

Graphene engineering by neon ion beams

Achieving the ultimate limits of lithographic resolution and material performance necessitates engineering of matter with atomic, molecular, and mesoscale fidelity. With the advent of scanning helium ion microscopy, maskless He+ and Ne+ beam lithography of 2D materials, such as graphene-based nanoelectronics, is coming to the forefront as a tool for fabrication and surface manipulation. However, the effects of using a Ne focused-ion-beam on the fidelity of structures created out of 2D materials have yet to be explored. Here, we will discuss the use of energetic Ne ions in engineering graphene nanostructures and explore their mechanical, electromechanical and chemical properties using scanning probe microscopy (SPM). By using SPM-based techniques such as band excitation (BE) force modulation microscopy, Kelvin probe force microscopy (KPFM) and Raman spectroscopy, we are able to ascertain changes in the mechanical, electrical and optical properties of Ne+ beam milled graphene nanostructures and surrounding regions. Additionally, we are able to link localized defects around the milled graphene to ion milling parameters such as dwell time and number of beam passes in order to characterize the induced changes in mechanical and electromechanical properties of the graphene surface.

Gold Nanoparticle Quantitation by Whole Cell Tomography.

Many proposed biomedical applications for engineered gold nanoparticles require their incorporation by mammalian cells in specific numbers and locations. Here, the number of gold nanoparticles inside of individual mammalian stem cells was characterized using fast focused ion beam-scanning electron microscopy based tomography. Enhanced optical microscopy was used to provide a multiscale map of the in vitro sample, which allows cells of interest to be identified within their local environment. Cells were then serially sectioned using a gallium ion beam and imaged using a scanning electron beam. To confirm the accuracy of single cross sections, nanoparticles in similar cross sections were imaged using transmission electron microscopy and scanning helium ion microscopy. Complete tomographic series were then used to count the nanoparticles inside of each cell and measure their spatial distribution. We investigated the influence of slice thickness on counting single particles and clusters as well as nanoparticle packing within clusters. For 60 nm citrate stabilized particles, the nanoparticle cluster packing volume is 2.15 ± 0.20 times the volume of the bare gold nanoparticles.

Dopant profiling based on scanning electron and helium ion microscopy

In this paper, we evaluate and compare doping contrast generated inside the scanning electron microscope (SEM) and scanning helium ion microscope (SHIM). Specialised energy-filtering techniques are often required to produce strong doping contrast to map donor distributions using the secondary electron (SE) signal in the SEM. However, strong doping contrast can be obtained from n-type regions in the SHIM, even without energy-filtering. This SHIM technique is more sensitive than the SEM to donor density changes above its sensitivity threshold, i.e. of the order of 1016 or 1017donorscm−3 respectively on specimens with or without a p–n junction; its sensitivity limit is well above 2×1017acceptorscm−3 on specimens with or without a p–n junction. Good correlation is found between the widths and slopes of experimentally measured doping contrast profiles of thin p-layers and the calculated widths and slopes of the potential energy distributions across these layers, at a depth of 1 to 3nm and 5 to 10nm below the surface in the SHIM and the SEM respectively. This is consistent with the mean escape depth of SEs in silicon being about 1.8nm and 7nm in the SHIM and SEM respectively, and we conclude that short escape depth, low energy SE signals are most suitable for donor profiling.

Solvent-free functionalization of carbon nanotube buckypaper with amines

We demonstrate the possibility of fast and efficient solvent-free functionalization of buckypaper (BP) mats prefabricated from oxidized multiwalled carbon nanotubes (MWCNTs-ox), by using three representative amines of different structure: one monofunctional aliphatic amine, octadecylamine (ODA), one monofunctional aromatic amine, 1-aminopyrene (AP), and one aromatic diamine, 1,5-diaminonaphthalene (DAN). The functionalization procedure, which relies on the formation of amide bonds with carboxylic groups of MWCNTs-ox, is performed at 150–180°C under reduced pressure and takes about 4h including auxiliary degassing. The amine-treated BP samples (BP-ODA, BP-AP and BP-DAN, respectively) were characterized by means of a variety of analytical techniques such as Fourier-transform infrared and Raman spectroscopy, thermogravimetric and differential thermal analysis, scanning and transmission electron microscopy, scanning helium ion microscopy, and atomic force microscopy. The highest amine content was found for BP-ODA, and the lowest one was observed for BP-DAN, with a possible contribution of non-covalently bonded amine molecules in all three cases. Despite of some differences in spectral and morphological characteristics for amine-functionalized BP samples, they have in common a dramatically increased stability in water as compared to pristine BP and, on the other hand, a relatively invariable electrical conductivity.