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Modern science is dependent on imaging on the nanoscale, often achieved through processes that detect secondary electrons created by a highly focused incident charged particle beam. Multiple types of measurement noise limit the ultimate trade-off between the image quality and the incident particle dose, which can preclude useful imaging of dose-sensitive samples. Existing methods to improve image quality do not fundamentally mitigate the noise sources. Furthermore, barriers to assigning a physically meaningful scale make the images qualitative. Here, we introduce ion count-aided microscopy (ICAM), which is a quantitative imaging technique that uses statistically principled estimation of the secondary electron yield. With a readily implemented change in data collection, ICAM substantially reduces source shot noise. In helium ion microscopy, we demonstrate 3x dose reduction and a good match between these empirical results and theoretical performance predictions. ICAM facilitates imaging of fragile samples and may make imaging with heavier particles more attractive. [ABSTRACT FROM AUTHOR]

Abstract Background Investigations into the growth and self-organization of plant roots is subject to fundamental and applied research in various areas such as botany, agriculture, and soil science. The growth activity of the plant tissue can be investigated by isotope labeling experiments with heavy water and subsequent detection of the deuterium in non-exchangeable positions incorporated into the plant biomass. Commonly used analytical methods to detect deuterium in plants are based on mass-spectrometry or neutron-scattering and they either suffer from elaborated sample preparation, destruction of the sample during analysis, or low spatial resolution. Confocal Raman micro-spectroscopy (CRM) can be considered a promising method to overcome the aforementioned challenges. The substitution of hydrogen with deuterium results in the measurable shift of the CH-related Raman bands. By employing correlative approaches with a high-resolution technique, such as helium ion microscopy (HIM), additional structural information can be added to CRM isotope maps and spatial resolution can be further increased. For that, it is necessary to develop a comprehensive workflow from sample preparation to data processing. Results A workflow to prepare and analyze roots of hydroponically grown and deuterium labeled Zea mays by correlative HIM-CRM micro-analysis was developed. The accuracy and linearity of deuterium detection by CRM were tested and confirmed with samples of deuterated glucose. A set of root samples taken from deuterated Zea mays in a time-series experiment was used to test the entire workflow. The deuterium content in the roots measured by CRM was close to the values obtained by isotope-ratio mass spectrometry. As expected, root tips being the most actively growing root zone had incorporated the highest amount of deuterium which increased with increasing time of labeling. Furthermore, correlative HIM-CRM analysis allowed for obtaining the spatial distribution pattern of deuterium and lignin in root cross-sections. Here, more active root zones with higher deuterium incorporation showed less lignification. Conclusions We demonstrated that CRM in combination with deuterium labeling can be an alternative and reliable tool for the analysis of plant growth. This approach together with the developed workflow has the potential to be extended to complex systems such as plant roots grown in soil.

Viruses come in various shapes and sizes, and understanding their morphology is central to understanding their activity and function. The need for fast recognition and real-time fingerprinting methods for pathogenic viruses is a critical bottleneck in implementing many diagnostic and therapeutic techniques. In this work, nanopore tomography (NT) is implemented for fast measurements of the characteristic dimensions of viruses and the optimal operating conditions are explored. Using a small filamentous bacteriophage as a model, it is demonstrated that NT can detect geometrical features in a few-nanometer regime, with high throughput and accuracy, in aqueous conditions. The instrumental parameters are optimized to obtain virus diameter measurements that are robust to the uncertainties of the external parameters. Furthermore, NT is critically compared to various single-particle imaging techniques, with a particular emphasis on emerging helium ion microscopy (HIM). It is shown that, with proper operating procedures, HIM can reach a nanometer-scale resolution in viral metrology, while retaining a high throughput second only to NT. The high throughput of both techniques can foster sufficient statistics for a precise exploration of viral heterogeneity. [ABSTRACT FROM AUTHOR]

Atomic layer engineering is investigated to tailor the morphotropic phase boundary (MPB) between antiferroelectric, ferroelectric, and paraelectric phases. By increasing the HfO2 seeding layer with only 2 monolayers, the overlying ZrO2 layer experiences the dramatic phase transition across the MPB. Conspicuous ferroelectric properties including record‐high remanent polarization (2Pr ≈ 60 µC cm−2), wake‐up‐free operation, and high compatibility with advanced semiconductor technology nodes, are achieved in the sub‐6 nm thin film. The prominent antiferroelectric to ferroelectric phase transformation is ascribed to the in‐plane tensile stress introduced into ZrO2 by the HfO2 seeding layer. Based on the high‐resolution and high‐contrast images of surface grains extracted precisely by helium ion microscopy, the evolution of the MPB between tetragonal, orthorhombic, and monoclinic phases with grain size is demonstrated for the first time. The result indicates that a decrease in the average grain size drives the crystallization from the tetragonal to polar orthorhombic phases. [ABSTRACT FROM AUTHOR]

Abstract Atomic layer engineering is investigated to tailor the morphotropic phase boundary (MPB) between antiferroelectric, ferroelectric, and paraelectric phases. By increasing the HfO2 seeding layer with only 2 monolayers, the overlying ZrO2 layer experiences the dramatic phase transition across the MPB. Conspicuous ferroelectric properties including record‐high remanent polarization (2Pr ≈ 60 µC cm−2), wake‐up‐free operation, and high compatibility with advanced semiconductor technology nodes, are achieved in the sub‐6 nm thin film. The prominent antiferroelectric to ferroelectric phase transformation is ascribed to the in‐plane tensile stress introduced into ZrO2 by the HfO2 seeding layer. Based on the high‐resolution and high‐contrast images of surface grains extracted precisely by helium ion microscopy, the evolution of the MPB between tetragonal, orthorhombic, and monoclinic phases with grain size is demonstrated for the first time. The result indicates that a decrease in the average grain size drives the crystallization from the tetragonal to polar orthorhombic phases.

This paper describes a study where an argon cold plasma jet, generated by a dielectric‐barrier discharge (DBD), is combined with nanosecond laser ablation (248 nm, 25 ns, 10 Hz) to deposit silver particle aerosols onto the substrate at atmospheric pressure. The deposition of the particle is examined using various microscopy techniques and absorption spectroscopy for the plasma jet produced by operating DBD in the normal and reversed mode. Plasma facilitated the deposition process by delivering the particle to the substrate and significantly influenced its morphology depending on the jet interaction, length, and substrate position. In both cases, the particles are clustered; however, there is less deposit for the plasma ignited in the reverse mode. The theoretical analysis of the deposition process is performed using ANSYS software and evaluated in terms of plasma‐induced flow velocity. This study infers that the hybrid plasma‐laser deposition scheme considered is attractive for material processing and deposition, especially overextended substrate distances, and for altering the properties of the deposited particles for practical utilization in surface‐enhanced Raman spectroscopy, solar cells, and catalysis. [ABSTRACT FROM AUTHOR]

Background: Investigations into the growth and self-organization of plant roots is subject to fundamental and applied research in various areas such as botany, agriculture, and soil science. The growth activity of the plant tissue can be investigated by isotope labeling experiments with heavy water and subsequent detection of the deuterium in non-exchangeable positions incorporated into the plant biomass. Commonly used analytical methods to detect deuterium in plants are based on mass-spectrometry or neutron-scattering and they either suffer from elaborated sample preparation, destruction of the sample during analysis, or low spatial resolution. Confocal Raman micro-spectroscopy (CRM) can be considered a promising method to overcome the aforementioned challenges. The substitution of hydrogen with deuterium results in the measurable shift of the CH-related Raman bands. By employing correlative approaches with a high-resolution technique, such as helium ion microscopy (HIM), additional structural information can be added to CRM isotope maps and spatial resolution can be further increased. For that, it is necessary to develop a comprehensive workflow from sample preparation to data processing. Results: A workflow to prepare and analyze roots of hydroponically grown and deuterium labeled Zea mays by correlative HIM-CRM micro-analysis was developed. The accuracy and linearity of deuterium detection by CRM were tested and confirmed with samples of deuterated glucose. A set of root samples taken from deuterated Zea mays in a time-series experiment was used to test the entire workflow. The deuterium content in the roots measured by CRM was close to the values obtained by isotope-ratio mass spectrometry. As expected, root tips being the most actively growing root zone had incorporated the highest amount of deuterium which increased with increasing time of labeling. Furthermore, correlative HIM-CRM analysis allowed for obtaining the spatial distribution pattern of deuterium and lignin in root cross-sections. Here, more active root zones with higher deuterium incorporation showed less lignification. Conclusions: We demonstrated that CRM in combination with deuterium labeling can be an alternative and reliable tool for the analysis of plant growth. This approach together with the developed workflow has the potential to be extended to complex systems such as plant roots grown in soil. [ABSTRACT FROM AUTHOR]

Ethylene propylene diene (EPDM) rubber has gained increasing interest for use in hydrogen infrastructure due to its excellent sealing performance and low temperature properties. However, severe structural damage has been observed in EPDM O-rings after exposure to high-pressure hydrogen. The origination and propagation mechanisms of this damage are poorly understood. To address this knowledge gap, multi-scale imaging leveraging X-ray micro-computed tomography (micro-CT), helium ion microscopy (HeIM), and transmission electron microscopy (TEM) were used in this work to study a series of sulfur-cured ethylene propylene diene (EPDM) rubber materials with varying additives that were exposed to different hydrogen environments. Micro-CT captured the substantial structural damage due to hydrogen exposure; it revealed an association between zinc oxide (ZnO) particles and damage initiation. Further studies by TEM and scanning TEM with energy dispersive X-ray spectroscopy (EDS) were focused on these particles at the micro-to nano-scale range. TEM indicated that hydrogen causes void formation at the interface between ZnO and the rubber matrix. HeIM enabled imaging of surface morphology of the material at high resolution pre- and post-hydrogen exposure while providing information on chemical composition and that cannot be captured by either micro-CT or TEM. • XCT suggests that cracks are likely to occur around ZnO agglomerates after exposure to high-pressure hydrogen. • HeIM reveals that pressure cycling provokes damage growth, whereas fillers counter the damage evolution process. • Filled rubber compounds show improved resistance to hydrogen damage compared with unfilled systems. • TEM with EDS captures structural alteration around ZnO agglomerates as indicative of initiation of hydrogen damages. [ABSTRACT FROM AUTHOR]

Abstract This paper describes a study where an argon cold plasma jet, generated by a dielectric‐barrier discharge (DBD), is combined with nanosecond laser ablation (248 nm, 25 ns, 10 Hz) to deposit silver particle aerosols onto the substrate at atmospheric pressure. The deposition of the particle is examined using various microscopy techniques and absorption spectroscopy for the plasma jet produced by operating DBD in the normal and reversed mode. Plasma facilitated the deposition process by delivering the particle to the substrate and significantly influenced its morphology depending on the jet interaction, length, and substrate position. In both cases, the particles are clustered; however, there is less deposit for the plasma ignited in the reverse mode. The theoretical analysis of the deposition process is performed using ANSYS software and evaluated in terms of plasma‐induced flow velocity. This study infers that the hybrid plasma‐laser deposition scheme considered is attractive for material processing and deposition, especially overextended substrate distances, and for altering the properties of the deposited particles for practical utilization in surface‐enhanced Raman spectroscopy, solar cells, and catalysis.

Abstract This review introduces the technique of helium ion microscopy along with some unique applications of this technology in the fields of electronics and biology, as performed at the National Institute of Advanced Industrial Science and Technology, Japan, over the last several years. Observations of large-scale integrated circuits, analyses of low-dielectric-constant films with minimal damage, and assessments of copper metal in insulating films are discussed. The special characteristics of this technique are explained, including low-energy input to the material and minimal secondary electron energy resulting from helium ion irradiation. Applications to electronic materials, such as tuning the electrical conductivity of graphene films by helium ion beam irradiation and the formation of nanopore arrays on graphene films with nanometer-scale control, are presented. The use of helium ion microscopy to examine cellular tissues based on the low damage imparted by the ion beam is also evaluated.

This review introduces the technique of helium ion microscopy along with some unique applications of this technology in the fields of electronics and biology, as performed at the National Institute of Advanced Industrial Science and Technology, Japan, over the last several years. Observations of large-scale integrated circuits, analyses of low-dielectric-constant films with minimal damage, and assessments of copper metal in insulating films are discussed. The special characteristics of this technique are explained, including low-energy input to the material and minimal secondary electron energy resulting from helium ion irradiation. Applications to electronic materials, such as tuning the electrical conductivity of graphene films by helium ion beam irradiation and the formation of nanopore arrays on graphene films with nanometer-scale control, are presented. The use of helium ion microscopy to examine cellular tissues based on the low damage imparted by the ion beam is also evaluated. [ABSTRACT FROM AUTHOR]

3D nanostructures hold the key to building custom devices with special and flexible functionalities. Ion beam‐induced bending and folding are used for the fabrication of 3D nanostructures from prepared 2D nano‐patterned membranes. Tensile and compressive stresses can be introduced within the layers of a thin membrane under ion beam irradiation to manipulate and control the formation of nano‐kirigamis. Both localized and broader ion beam irradiation can be used to induce bending in the membrane with remarkable control of the bending angle and direction. In this work, an approach to fabricate nano‐kirigamis with focused ion beams (FIBs) is developed. First, helium or neon ion beams are used to mill 2D patterns into the planar membrane with good accuracy, nanoscale, and less damage. Subsequently, gallium ion beam is used to broadly and uniformly irradiate the patterned region to induce out‐of‐plane bending to form nano‐kirigamis. Both upward and downward bending are observed for different thicknesses of membranes. The bending angle is easily controlled by varying the irradiation dosage. Moreover, with FIB‐induced deposition, nanopillars, or nanowires (NWs) can be grown on the membrane before or after bending. Various complex 3D nanostructures and branched NWs can be fabricated using this approach. [ABSTRACT FROM AUTHOR]

Background: The use of image analysis to understand the structure of chromosome and chromatin is critical to the study of genetic evolution and diversification. Furthermore, a detailed chromosome map and the structure of chromatin in the nucleus may contribute to the plant breeding and the study of fundamental biology and genetics in organisms. Results: In plants with a fully annotated genome project, such as the Leguminosae species, the integration of genetic information, including DNA sequence data, a linkage map, and the cytological quantitative chromosome map could further improve their genetic value. The numerical parameters of chromocenters in 3D can provide useful genetic information for phylogenetic studies of plant diversity and heterochromatic markers whose epigenetic changes may explain the developmental and environmental changes in the plant genome. Extended DNA fibers combined with fluorescence in situ hybridization revealed the highest spatial resolution of the obtained genome structure. Moreover, image analysis at the nano-scale level using a helium ion microscope revealed the surface structure of chromatin, which consists of chromatin fibers compacted into plant chromosomes. Conclusions: The studies described in this review sought to measure and evaluate chromosome and chromatin using the image analysis method, which may reduce measurement time and improve resolution. Further, we discussed the development of an effective image analysis to evaluate the structure of chromosome and chromatin. An effective application study of cell biology and the genetics of plants using image analysis methods is expected to be a major propeller in the development of new applications. [ABSTRACT FROM AUTHOR]

Chalk is a very fine-grained carbonate and can accommodate high porosity which is a key characteristic for high-quality hydrocarbon reservoirs. A standard procedure within Improved Oil Recovery (IOR) is seawater-injection which repressurizes the reservoir pore pressure. Long-term seawater-injection will influence mineralogical processes as dissolution and precipitation of secondary minerals. These secondary minerals (<1 micrometer) precipitate during flooding experiments mimicking reservoir conditions. Due to their small sizes, analysis from traditional scanning electron microscopy combined with energy dispersive X-ray spectroscopy is not conclusive because of insufficient spatial resolution and detection limit. Therefore, chalk was analyzed with high-resolution imaging by helium ion microscopy (HIM) combined with secondary ion mass spectrometry (SIMS) for the first time. Our aim was to identify mineral phases at sub-micrometer scale and identify locations of brine–rock interactions. In addition, we wanted to test if current understanding of these alteration processes can be improved with the combination of complementary imaging techniques and give new insights to IOR. The HIM-SIMS imaging revealed well-defined crystal boundaries and provided images of excellent lateral resolution, allowing for identification of specific mineral phases. Using this new methodology, we developed chemical identification of clay minerals and could define their exact location on micron-sized coccolith grains. This shows that it is essential to study mineralogical processes at nanometer scale in general, specifically in the research field of applied petroleum geology within IOR. [ABSTRACT FROM AUTHOR]

Helium ion microscopy (HIM) offers the opportunity to obtain direct views of biological samples such as cellular structures, virus particles, and microbial interactions. Imaging with the HIM combines sub-nanometer resolution, large depth of field, and high surface sensitivity. Due to its charge compensation capability, the HIM can image insulating biological samples without additional conductive coatings. Here, we present an exploratory HIM study of SARS-CoV-2 infected Vero E6 cells, in which several areas of interaction between cells and virus particles, as well as among virus particles, were imaged. The HIM pictures show the three-dimensional appearance of SARS-CoV-2 and the surface of Vero E6 cells at a multiplicity of infection of approximately 1 with great morphological detail. The absence of a conductive coating allows for a distinction between virus particles bound to the cell membrane and virus particles lying on top of the membrane. After prolonged imaging, it was found that ion-induced deposition of hydrocarbons from the vacuum renders the sample sufficiently conductive to allow for imaging even without charge compensation. The presented images demonstrate the potential of the HIM in bioimaging, especially for the imaging of interactions between viruses and their host organisms.

A detection system based on a microchannel plate with a delay line readout structure has been developed to perform scanning transmission ion microscopy (STIM) in the helium ion microscope (HIM). This system is an improvement over other existing approaches since it combines the information of the scanning beam position on the sample with the position (scattering angle) and time of the transmission events. Various imaging modes, such as bright field and dark field or the direct image of the transmitted signal, can be created by post-processing the collected STIM data. Furthermore, the detector has high spatial and temporal resolution, is sensitive to both ions and neutral particles over a wide energy range, and shows robustness against ion beam-induced damage. A special in-vacuum movable support gives the possibility of moving the detector vertically, placing the detector closer to the sample for the detection of high-angle scattering events, or moving it down to increase the angular resolution and distance for time-of-flight measurements. With this new system, we show composition-dependent contrast for amorphous materials and the contrast difference between small-angle and high-angle scattering signals. We also detect channeling-related contrast on polycrystalline silicon, thallium chloride nanocrystals, and single-crystalline silicon by comparing the signal transmitted at different directions for the same data set.

We fabricate plasmonic heptamer-arranged nanohole (HNH) arrays by helium (He) focused ion beam (HeFIB) milling, which is a resist-free, maskless, direct-write method. The small He+ beam spot size and high milling resolution achieved by the gas field-ionization source used in our HeFIB allows the milling of high aspect ratio (4:1) nanoscale features in metal, such as HNHs incorporating 15 nm walls of high verticality between holes in a 55-nm-thick gold film. Drifts encountered during the HeFIB milling of large arrays, due to sample stage vibrations or He beam instability, were compensated by a drift correction technique based on in situ He ion imaging of alignment features. Our drift correction technique yielded 20 nm maximum dislocation of HNHs, with 6.9 and 4.6 nm average dislocations along the horizontal and vertical directions, respectively. The measured optical resonance spectra of the fabricated plasmonic HNH arrays are presented to support the fabrication technique. Defects associated with HeFIB milling are also discussed.

During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro− to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance.

In this work we present images of carbon fiber fractured surfaces observed for the first time by Helium Ion Microscopy (HIM) at high magnifications, aiming to capture defects ranging in size between 10 nm and 100 nm. Nominal critical defect sizes of various commercial carbon fibers were calculated from the Griffith Equation and their critical defect size distributions were calculated from their Weibull strength distributions. HIM images of the carbon fibers with the standard modulus (~230 GPa), intermediate modulus (~280 GPa), high modulus (>350 GPa) and ultra-high strength (>6.8 GPa) are shown to have contrasting microstructures. Some of the three dimensional (3D) structural features, like crystalline sheets, flakes, and sets of amorphous material, have not been previously captured by Scanning Electron Microscopy (SEM). Furthermore, a representative intermediate modulus carbon fiber was mechanically tested by the single filament test method and its strength characteristics were corelated to structural features observed by HIM. HIM imaging offers a new, reproducible, and feasible technique to analyze the original 3D nanostructures of carbon fibers.

During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro− to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance. [ABSTRACT FROM AUTHOR]

Nanoparticles occur in various environments as a consequence of man‐made processes, which raises concerns about their impact on the environment and human health. To allow for proper risk assessment, a precise and statistically relevant analysis of particle characteristics (such as size, shape, and composition) is required that would greatly benefit from automated image analysis procedures. While deep learning shows impressive results in object detection tasks, its applicability is limited by the amount of representative, experimentally collected and manually annotated training data. Here, an elegant, flexible, and versatile method to bypass this costly and tedious data acquisition process is presented. It shows that using a rendering software allows to generate realistic, synthetic training data to train a state‐of‐the art deep neural network. Using this approach, a segmentation accuracy can be derived that is comparable to man‐made annotations for toxicologically relevant metal‐oxide nanoparticle ensembles which were chosen as examples. The presented study paves the way toward the use of deep learning for automated, high‐throughput particle detection in a variety of imaging techniques such as in microscopies and spectroscopies, for a wide range of applications, including the detection of micro‐ and nanoplastic particles in water and tissue samples. [ABSTRACT FROM AUTHOR]

A dedicated transmission helium ion microscope (THIM) for sub-50 keV helium has been constructed to investigate ion scattering processes and contrast mechanisms, aiding the development of new imaging and analysis modalities. Unlike a commercial helium ion microscope (HIM), the in-house built instrument allows full flexibility in experimental configuration. Here, we report projection imaging and intensity patterns obtained from powder and bulk crystalline samples using stationary broad-beam as well as convergent-beam illumination conditions in THIM. The He+ ions formed unexpected spot patterns in the far field for MgO, BN and NaCl powder samples, but not for Au-coated MgO. The origin of the spot patterns in these samples was investigated. Surface diffraction of ions was excluded as a possible cause because the recorded scattering angles do not correspond to the predicted Bragg angles. Complementary secondary electron (SE) imaging in the HIM revealed that these samples charge significantly under He+ ion irradiation. The spot patterns obtained in the THIM experiments are explained as artefacts related to sample charging. The results presented here indicate that factors other than channeling, blocking and surface diffraction of ions have an impact on the final intensity distribution in the far field. Hence, the different processes contributing to the final intensities will need to be understood in order to decouple and study the relevant ion-beam scattering and deflection phenomena.

Background: The application of superparamagnetic particles as biomolecular transporters in microfluidic systems for lab-on-a-chip applications crucially depends on the ability to control their motion. One approach for magnetic-particle motion control is the superposition of static magnetic stray field landscapes (MFLs) with dynamically varying external fields. These MFLs may emerge from magnetic domains engineered both in shape and in their local anisotropies. Motion control of smaller beads does necessarily need smaller magnetic patterns, i.e., MFLs varying on smaller lateral scales. The achievable size limit of engineered magnetic domains depends on the magnetic patterning method and on the magnetic anisotropies of the material system. Smallest patterns are expected to be in the range of the domain wall width of the particular material system. To explore these limits a patterning technology is needed with a spatial resolution significantly smaller than the domain wall width.Results: We demonstrate the application of a helium ion microscope with a beam diameter of 8 nm as a mask-less method for local domain patterning of magnetic thin-film systems. For a prototypical in-plane exchange-bias system the domain wall width has been investigated as a function of the angle between unidirectional anisotropy and domain wall. By shrinking the domain size of periodic domain stripes, we analyzed the influence of domain wall overlap on the domain stability. Finally, by changing the geometry of artificial two-dimensional domains, the influence of domain wall overlap and domain wall geometry on the ultimate domain size in the chosen system was analyzed.Conclusion: The application of a helium ion microscope for magnetic patterning has been shown. It allowed for exploring the fundamental limits of domain engineering in an in-plane exchange-bias thin film as a prototypical system. For two-dimensional domains the limit depends on the domain geometry. The relative orientation between domain wall and anisotropy axes is a crucial parameter and therefore influences the achievable minimum domain size dramatically.

Chalk is a very fine-grained carbonate and can accommodate high porosity which is a key characteristic for high-quality hydrocarbon reservoirs. A standard procedure within Improved Oil Recovery (IOR) is seawater-injection which repressurizes the reservoir pore pressure. Long-term seawater-injection will influence mineralogical processes as dissolution and precipitation of secondary minerals. These secondary minerals (

For future nanoelectronic devices – such as room-temperature single electron transistors – the site-controlled formation of single Si nanocrystals (NCs) is a crucial prerequisite. Here, we report an approach to fabricate single Si NCs via medium-energy Si+ or Ne+ ion beam mixing of Si into a buried SiO2 layer followed by thermally activated phase separation. Binary collision approximation and kinetic Monte Carlo methods are conducted to gain atomistic insight into the influence of relevant experimental parameters on the Si NC formation process. Energy-filtered transmission electron microscopy is performed to obtain quantitative values on the Si NC size and distribution in dependence of the layer stack geometry, ion fluence and thermal budget. Employing a focused Ne+ beam from a helium ion microscope, we demonstrate site-controlled self-assembly of single Si NCs. Line irradiation with a fluence of 3000 Ne+/nm2 and a line width of 4 nm leads to the formation of a chain of Si NCs, and a single NC with 2.2 nm diameter is subsequently isolated and visualized in a few nanometer thin lamella prepared by a focused ion beam (FIB). The Si NC is centered between the SiO2 layers and perpendicular to the incident Ne+ beam.

Helium-ion-induced swelling and blistering of single-crystal tungsten is investigated using a Helium Ion Microscope for site-specific dose-controlled irradiation (at 25 keV) with analysis by Helium Ion Microscopy, Atomic Force Microscopy and Transmission Electron Microscopy (cross-sectioning by Focused Ion Beam milling). We show that the blister cavity forms at a depth close to the simulated helium peak and that nanobubbles coalesce to form nanocracks within the envelope of the ion stopping range, swelling the blister shell. These results provide the first direct experimental evidence for the interbubble fracture mechanism proposed in the framework of the gas pressure model for blister formation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Spent hens are by-product of poultry and potential feed stock of lipids. • Lipids extracted using supercritical CO 2 and parameters investigated. • Extracted lipids epoxidized in solvent free conditions. Spent hens are by-product of egg and hatching egg production and a potential source of renewable compounds. In this study, lipids were extracted using supercritical carbon dioxide (SC−CO 2) at 50 − 70 °C, 30 − 50 MPa, and constant CO 2 flow rate of 1 L/min. The maximum yield of total lipid 37 ± 0.4 % (w/w) with 91.4% recovery was obtained at 50 MPa/70 °C. Fatty acid compositional analysis was performed using gas chromatography with flame ionization detector (GC-FID). Helium ion microscopy (HIM) was used to assess the morphological changes before and after extraction. Furthermore, epoxidation of the extracted lipids was conducted with and without the use of a solvent, where the solvent-free epoxidation was completed within 20 min and the yield was comparable. The reaction progress was monitored by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy analysis, which showed the conversion rates of 59.8, 84.2 and 100% at 5, 10, and 20 min, respectively. The findings suggest that an alternative bio-epoxy can be produced using SC−CO 2 extraction and solvent-free oxidation of extracted lipids from poultry industry waste/by-product. [ABSTRACT FROM AUTHOR]

The determination of the negative ion yield of 2′-chloro-1,1′-biphenyl (2-Cl-BP), 2′-bromo-1,1′-biphenyl (2-Br-BP) and 2′-iodo-1,1′-biphenyl (2-I-BP) upon dissociative electron attachment (DEA) at an electron energy of 0 eV revealed cross section values that were more than ten times higher for iodide loss from 2-I-BP than for the other halogenides from the respective biphenyls (BPs). Comparison with dissociative ionization mass spectra shows that the ratio of the efficiency of electron impact ionization induced fragmentation of 2-I-BP, 2-Br-BP, and 2-Cl-BP amounts to approximately 1:0.7:0.6. Inspired by these results, self-assembled monolayers (SAMs) of the respective biphenyl-4-thiols, 2-Cl-BPT, 2-Br-BPT, 2-I-BPT as well as BPT, were grown on a Au(111) substrate and exposed to 50 eV electrons. The effect of electron irradiation was investigated by X-ray photoelectron spectroscopy (XPS), to determine whether the high relative DEA cross section for iodide loss from 2-I-BPT as compared to 2-Br-BP and 2-Cl-BP is reflected in the cross-linking efficiency of SAMs made from these materials. Such sensitization could reduce the electron dose needed for the cross-linking process and may thus lead to a significantly faster conversion of the respective SAMs into carbon nanomembranes (CNMs) without the need for an increased current density. XPS data support the notation that DEA sensitization may be used to achieve more efficient electron-induced cross-linking of SAMs, revealing more than ten times faster cross-linking of 2-I-BPT SAMs compared to those made from the other halogenated biphenyls or from native BPT at the same current density. Furthermore, the transfer of a freestanding membrane onto a TEM grid and the subsequent investigation by helium ion microscopy (HIM) verified the existence of a mechanically stable CNM created from 2-I-BPT after exposure to an electron dose as low as 1.8 mC/cm2. In contrast, SAMs made from BPT, 2-Cl-BPT and 2-Br-BPT did not form stable CNMs after a significantly higher electron dose of 9 mC/cm2.

Highly uniform samples of carbon nanofoam from hydrothermal sucrose carbonization were studied by helium ion microscopy (HIM), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Foams with different densities were produced by changing the process temperature in the autoclave reactor. This work illustrates how the geometrical structure, electron core levels, and the vibrational signatures change when the density of the foams is varied. We find that the low-density foams have very uniform structure consisting of micropearls with ≈2–3 μm average diameter. Higher density foams contain larger-sized micropearls (≈6–9 μm diameter) which often coalesced to form nonspherical μm-sized units. Both, low- and high-density foams are comprised of predominantly sp2-type carbon. The higher density foams, however, show an advanced graphitization degree and a stronger sp3-type electronic contribution, related to the inclusion of sp3 connections in their surface network.

The defect emission from h-BN at 1.55 eV is interesting as it enables optical readout of spins. It is necessary to identify the nature of the relevant point defects for its controlled introduction. However, it is challenging to engineer point defects in h-BN without changing the local atomic structure. Here, we controllably introduce boron vacancies in h-BN using an ultrahigh spatial resolution and low-energy He + ion beam. By optimizing the He + ion irradiation conditions, we control the quantity and location of defects spatially and along the depth of h-BN to achieve a robust photoluminescence emission at 1.55 eV from 10 K to room temperature. We show that as-generated defects activate an additional Raman mode at 1295 cm -1 . Electron energy loss spectroscopy confirms introduction of boron vacancies without modification of the local h-BN crystal structure. Our results provide a deterministic strategy to create scalable boron vacancy emitters in h-BN for quantum photonics.

The molecular fingerprints of yeasts Saccharomyces cerevisiae , Dekkera bruxellensis , and Wickerhamomyces anomalus (former name Pichia anomala) have been examined using surface-enhanced Raman spectroscopy (SERS) and helium ion microscopy (HIM). The SERS spectra obtained from cell cultures (lysate and non-treated cells) distinguish between these very closely related fungal species. Highly SERS active silver nano-particles suitable for detecting complex biomolecules were fabricated using a simple synthesis route. The yeast samples mixed with aggregated Ag nanoparticles yielded highly enhanced and reproducible Raman signal owing to the high density of the hot spots at the junctions of two or more Ag nanoparticles and enabled to differentiate the three species based on their unique features (spectral fingerprint). We also collected SERS spectra of the three yeast species in beer medium to demonstrate the potential of the method for industrial application. These findings demonstrate the great potential of SERS for detection and identification of fungi species based on the biochemical compositions, even in a chemically complex sample. Unlabelled Image • High quality, i.e. sharp, reproducible, and excellent signal to noise ratio, SERS spectra of yeast microbes ((SC (wild yeast) , DB and WA obtained • SERS detection from very closely related fungal species (lysed and non-treated) • Detection of the three species in complex beer medium • High-resolution helium ion microscope (HIM) images give insight into the surface morphology of the yeast cells attached to AgNP • SERS to be used for reliable detection and identification of the species in biosensing applications. [ABSTRACT FROM AUTHOR]

By combining a focused inert-gas ion beam instrument and a custom magnetic-sector mass spectrometer, high spatial resolution imaging and chemical analysis are provided within a single instrument. Sub-nanometer image resolution is achieved by secondary electron (SE) imaging, limited only by the probe-size of the primary beam, while the spatial resolution for chemical mapping obtained via secondary ion mass spectrometry (SIMS) is limited mainly by beam-sample interactions to about 10 nm. This article introduces the background behind this development, describes the instrument and its various operating modes, and presents examples of its applications. [ABSTRACT FROM AUTHOR]

Abstract Addition of biochar into a soil changes its water retention properties by modifying soil textural and structural properties. In addition, internal micrometer-scale porosity that is able to directly store readily plant available water affects soil water retention properties. This study shows how precise knowledge of the internal micrometer-scale pore size distribution of biochar can deepen the understanding of the biochar-water interactions in soils. The micrometer-scale porosity of willow biochar was quantitatively and qualitatively characterized using X-ray tomography, 3D image analysis and Helium ion microscopy. The effect of biochar application on clay soil water retention was studied by conventional water retention curve approach. The results indicate that the internal pores of biochar, with sizes of at 50 and 10 μm (equivalent pore diameter), increased soil porosity and the amount of readily plant available water. After biochar addition, changes in soil porosity were detected at pore size regimes 5–10 and 25 μm, i.e. biochar pore sizes multiplied by factor 0.5. The detected pore size distribution of biochar does not predict directly (1:1 compatibility) the changes observed in the soil moisture characteristics. It is likely that biochar chemistry and pore morphology affect biochar-water interactions via e.g. surface roughness and contact angle. In addition, biochar induced changes in soil structure and texture affected soil moisture characteristics. However, the approach presented is an attractive pathway to more generalized understanding on how and why biochar internal porosity affects soil moisture characteristics. Graphical abstract Image 1 Highlights • Biochar porosity was studied using X-ray tomography and Helium ion microscopy. • Biochar had bimodal pore structure with local maxima at 50 and 10 μm. • Detected biochar porosity affected soil water retention via direct mechanism. • Soil porosity at regime of 25 and 5–10 μm increased due to biochar addition. • The role of micrometer-scale pores of biochar on soil water retention was highlighted. [ABSTRACT FROM AUTHOR]

The main goal of this study was to apply magnetic bead surface functionalization in the form of immunomagnetic separation (IMS) combined with real-time polymerase chain reaction (qPCR) (IMS-qPCR) to detect Human mastadenovirus species C (HAdV-C) and F (HAdV-F) in water samples. The technique efficiency was compared to a nonfunctionalized method (ultracentrifugation) followed by laboratory detection. Tests were carried out to standardize IMS parameters followed by tests on 15 water samples concentrated by IMS and ultracentrifugation. Microscopic analyses detected a successful beads–antibody attachment. HAdV was detected up to dilutions of 10−6 by IMS-qPCR, and samples concentrated by IMS were able to infect cell cultures. In water samples, HAdV-C was detected in 60% (monoclonal) and 47% (polyclonal) by IMS-qPCR, while 13% of samples concentrated by ultracentrifugation gave a positive result. HAdV-F was positive in 27% of samples by IMS-qPCR (polyclonal) and ultracentrifugation and 20% by IMS-qPCR (monoclonal). The rate of detection varied from 4.55 × 102 to 5.83 × 106 genomic copies/L for IMS-qPCR and from 2.00 × 102 to 2.11 × 103 GC/L for ultracentrifugation. IMS showed to be a more effective concentration technique for HAdV than ultracentrifugation, improving the assessment of infectious HAdV in water resources.

The analysis of polymers by secondary ion mass spectrometry (SIMS) has been a topic of interest for many years. In recent years, the primary ion species evolved from heavy monatomic ions to cluster and massive cluster primary ions in order to preserve a maximum of organic information. The progress in less-damaging sputtering goes along with a loss in lateral resolution for 2D and 3D imaging. By contrast the development of a mass spectrometer as an add-on tool for the helium ion microscope (HIM), which uses finely focussed He+ or Ne+ beams, allows for the analysis of secondary ions and small secondary cluster ions with unprecedented lateral resolution. Irradiation induced damage and depth profiling capabilities obtained with these light rare gas species have been far less investigated than ion species used classically in SIMS. In this paper we simulated the sputtering of multi-layered polymer samples using the BCA (binary collision approximation) code SD_TRIM_SP to study preferential sputtering and atomic mixing in such samples up to a fluence of 1018 ions/cm2. Results show that helium primary ions are completely inappropriate for depth profiling applications with this kind of sample materials while results for neon are similar to argon. The latter is commonly used as primary ion species in SIMS. For the two heavier species, layers separated by 10 nm can be distinguished for impact energies of a few keV. These results are encouraging for 3D imaging applications where lateral and depth information are of importance.

Secondary ion mass spectrometry (SIMS) on the helium ion microscope (HIM) promises higher lateral resolution than on classical SIMS instruments. However, full advantage of this new technique can only be obtained when the interaction of He+ or Ne+ primary ions with the sample is fully controlled. In this work we investigate how He+ and Ne+ bombardment influences roughness formation and preferential sputtering for polymer samples and how they compare to Ar+ primary ions used in classical SIMS by combining experimental techniques with Molecular Dynamics (MD) simulations and SD_TRIM_SP modelling. The results show that diffusion coefficients for He, Ne and Ar in polymers are sufficiently high to prevent any accumulation of rare gas atoms in the polymers which could lead to some swelling and bubble formation. Roughness formation was also not observed. Preferential sputtering is more of a problem, with enrichment of carbon up to surface concentrations above 80%. In general, the preferential sputtering is largely depending on the primary ion species and the impact energies. For He+ bombardment, it is more of an issue for low keV impact energies and for the heavier primary ion species the preferential sputtering is sample dependent. For He+ steady state conditions are reached for fluences much higher than 1018 ions/cm2. For Ne+ and Ar+, the transient regime extends up to fluences of 1017–1018 ions/cm2. Hence, preferential sputtering needs to be taken into account when interpreting images recorded under He+ or Ne+ bombardment on the HIM.

Physisorbed water originating from exposure to the ambient can have a strong impact on the structure and chemistry of oxide nanomaterials. The effect can be particularly pronounced when these oxides are in physical contact with a solid substrate such as the ones used for immobilization to perform electron or ion microscopy imaging. We used helium ion microscopy (HIM) and investigated morphological changes of vapor-phase-grown MgO cubes after vacuum annealing and pressing into foils of soft and high purity indium. The indium foils were either used as obtained or, for reference, subjected to vacuum drying. After four days of storage in the vacuum chamber of the microscope and at a base pressure of p < 10−7 mbar, we observed on these cubic particles the attack of residual physisorbed water molecules from the indium substrate. As a result, thin magnesium hydroxide layers spontaneously grew, giving rise to characteristic volume expansion effects, which depended on the size of the particles. Rounding of the originally sharp cube edges leads to a significant loss of the morphological definition specific to the MgO cubes. Comparison of different regions within one sample before and after exposure to liquid water reveals different transformation processes, such as the formation of Mg(OH)2 shells that act as diffusion barriers for MgO dissolution or the evolution of brucite nanosheets organized in characteristic flower-like microstructures. The findings underline the significant metastability of nanomaterials under both ambient and high-vacuum conditions and show the dramatic effect of ubiquitous water films during storage and characterization of oxide nanomaterials.

The next generation optical, electronic, biological, and sensing devices as well as platforms will inevitably extend their architecture into the 3rd dimension to enhance functionality. In focused ion beam induced deposition (FIBID), a helium gas field ion source can be used with an organometallic precursor gas to fabricate nanoscale structures in 3D with high-precision and smaller critical dimensions than focused electron beam induced deposition (FEBID), traditional liquid metal source FIBID, or other additive manufacturing technology. In this work, we report the effect of beam current, dwell time, and pixel pitch on the resultant segment and angle growth for nanoscale 3D mesh objects. We note subtle beam heating effects, which impact the segment angle and the feature size. Additionally, we investigate the competition of material deposition and sputtering during the 3D FIBID process, with helium ion microscopy experiments and Monte Carlo simulations. Our results show complex 3D mesh structures measuring ~300 nm in the largest dimension, with individual features as small as 16 nm at full width half maximum (FWHM). These assemblies can be completed in minutes, with the underlying fabrication technology compatible with existing lithographic techniques, suggesting a higher-throughput pathway to integrating FIBID with established nanofabrication techniques.

Normal corn starch granules are imaged using a helium ion microscope (HIM). This relatively new imaging technique produces high‐resolution images of both the interior and exterior of the granules that allow features to be observed which had previously not been seen with scanning electron microscope. Of particular interest is the mesh‐like structure which is observed in the interior when starch granules are sectioned with a metal blade, a structure not currently part of existing models of starch architecture. HIM imaging provides a new way to characterize starch in the ongoing quest to elucidate its internal structure. [ABSTRACT FROM AUTHOR]

Carbon nanomembranes (CNMs) prepared from aromatic self-assembled monolayers constitute a recently developed class of 2D materials. They are made by a combination of self-assembly, radiation-induced cross-linking and the detachment of the cross-linked SAM from its substrate. CNMs can be deposited on arbitrary substrates, including holey and perforated ones, as well as on metallic (transmission electron microscopy) grids. Therewith, freestanding membranes with a thickness of 1 nm and macroscopic lateral dimensions can be prepared. Although free-standing CNMs cannot be imaged by light microscopy, charged particle techniques can visualize them. However, CNMs are electrically insulating, which makes them sensitive to charging. We demonstrate that the helium ion microscope (HIM) is a good candidate for imaging freestanding CNMs due to its efficient charge compensation tool. Scanning with a beam of helium ions while recording the emitted secondary electrons generates the HIM images. The advantages of HIM are high resolution, high surface sensitivity and large depth of field. The effects of sample charging, imaging of multilayer CNMs as well as imaging artefacts are discussed.

While it is well established that the composition and organisation of articular cartilage dramatically change during skeletal maturation, relatively little is known about how this impacts the mechanical properties of the tissue. In this study, digital image correlation was first used to quantify spatial deformation within mechanically compressed skeletally immature (4 and 8 week old) and mature (1 and 3 year old) porcine articular cartilage. The compressive modulus of the immature tissue was relatively homogeneous, while the stiffness of mature articular cartilage dramatically increased with depth from the articular surface. Other, well documented, biomechanical characteristics of the tissue also emerged with skeletal maturity, such as strain-softening and a depth-dependent Poisson’s ratio. The most significant changes that occurred with age were in the deep zone of the tissue, where an order of magnitude increase in compressive modulus (from 0.97 MPa to 9.4 MPa for low applied strains) was observed from 4 weeks postnatal to skeletal maturity. These temporal increases in compressive stiffness occurred despite a decrease in tissue sulphated glycosaminoglycan content, but were accompanied by increases in tissue collagen content. Furthermore, helium ion microscopy revealed dramatic changes in collagen fibril alignment through the depth of the tissue with skeletal maturity, as well as a fivefold increase in fibril diameter with age. Finally, computational modelling was used to demonstrate how both collagen network reorganisation and collagen stiffening play a key role in determining the final compressive mechanical properties of the tissue. Together these findings provide a unique insight into evolving structure-function relations in articular cartilage.

Graphical abstract Highlights • Ar gas cluster ion beams create nanostructured InP surfaces. • Helium ion microscopy is well-suited to investigating GCIB etch craters on InP. • Metallic In nanoparticles form on the surface immediately after oxide removal. • In nanoparticles mask the underlying InP creating nanopillars. • Nanopillars oriented along the axis of the incident gas cluster ion beam. Abstract Indium phosphide (InP) surfaces develop a pronounced nanostructured texture upon irradiation by energetic ion beams. We have observed the mechanism of nanostructure evolution of InP under irradiation by an Ar gas cluster ion beam (GCIB) using helium ion microscopy (HIM). Initially, metallic indium nanoparticles form on the surface after removal of the top-most oxide layer. These nanoparticles form a mask which shadows the underlying InP. As the ion dose is increased, the masking effect results in substantial nanostructured topography in the form of pillars or nanocones, oriented along the axis of the incident GCIB. The surface sensitivity and high resolution of the HIM facilitates the direct observation of the metallic indium cap at the top of the pillars. [ABSTRACT FROM AUTHOR]

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii , Tritrichomonas foetus , Giardia intestinalis , and Trypanosoma cruzi , compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities. [ABSTRACT FROM AUTHOR]