Scanning Transmission Ion Microscopy Research Articles

Ionic-resolution protoacoustic microscopy: A feasibility study.

Visualizing micro- and nano-scale biological entities requires high-resolution imaging and is conventionally achieved via optical microscopic techniques. Optical diffraction limits their resolution to ∼200 nm. This limit can be overcome by using ions with ∼1 MeV energy. Such ions penetrate through several micrometers in tissues, and their much shorter de Broglie wavelengths indicate that these ion beams can be focused to much shorter scales and hence can potentially facilitate higher resolution as compared to the optical techniques. Proton microscopy with ∼1 MeV protons has been shown to have reasonable inherent contrast between sub-cellular organelles. However, being a transmission-based modality, it is unsuitable for in vivo studies and cannot facilitate three-dimensional imaging from a single raster scan. Here, we propose proton-induced acoustic microscopy (PrAM), a technique based on pulsed proton irradiation and proton-induced acoustic signal collection. This technique is capable of label-free, super-resolution, 3D imaging with a single raster scan. Converting radiation energy into ultrasound enables PrAM with reflection mode detection, making it suitable for in vivo imaging and probing deeper than proton scanning transmission ion microscopy (STIM). Using a proton STIM image of HeLa cells, a coupled Monte Carlo+k-wave simulations-based feasibility study has been performed to demonstrate the capabilities of PrAM. We demonstrate that sub-50 nm lateral (depending upon the beam size and energy) and sub-micron axial resolution (based on acoustic detection bandwidth and proton beam pulse width) can be obtained using the proposed modality. By enabling visualization of biological phenomena at cellular and subcellular levels, this high-resolution microscopic technique enhances understanding of intricate cellular processes.

Polymeric Membranes Doped with Halloysite Nanotubes Imaged using Proton Microbeam Microscopy.

Polymeric membranes are useful tools for water filtration processes, with their performance strongly dependent on the presence of hydrophilic dopants. In this study, polyaniline (PANI)-capped aluminosilicate (halloysite) nanotubes (HNTs) are dispersed into polyether sulfone (PES), with concentrations ranging from 0.5 to 1.5 wt%, to modify the properties of the PES membrane. Both undoped and HNT-doped PES membranes are investigated in terms of wettability (static and time-dependent contact angle), permeance, mechanical resistance, and morphology (using scanning electron microscopy (SEM)). The higher water permeance observed for the PES membranes incorporating PANI-capped HNTs is, finally, assessed and discussed vis-à-vis the real distribution of HNTs. Indeed, the imaging and characterization in terms of composition, spatial arrangement, and counting of HNTs embedded within the polymeric matrix are demonstrated using non-destructive Micro Particle Induced X-ray Emission (µ-PIXE) and Scanning Transmission Ion Microscopy (STIM) techniques. This approach not only exhibits the unique ability to detect/highlight the distribution of HNTs incorporated throughout the whole thickness of polymer membranes and provide volumetric morphological information consistent with SEM imaging, but also overcomes the limits of the most common analytical techniques exploiting electron probes. These aspects are comprehensively discussed in terms of practical analysis advantages.

Breaking the 10 nm barrier using molecular ions in nuclear microprobes

The spatial resolution plays a crucial role in determining the performance of a nuclear microprobe. However, the formation of spatial resolutions below 10 nm remains a challenge in nuclear microprobes. Here, we propose novel technologies (near-axis scanning transmission ion microscopy and double-fragment scattering) utilizing molecular ions to address this challenge and demonstrate a H2+ molecular beam with 6.0 × 10 nm2 lateral resolution and monolayer thickness resolution respectively. Using the improved nuclear microprobe, we directly demonstrate that the ionization of a H2+ can be efficiently achieved using one single layer graphene, and also that single and few layers of freestanding graphene can be clearly differentiated and identified. The precise control of fast molecular ions at sub-10 nm scales has the potential to unlock new avenues of applications.

On quantitative off-axis Scanning Transmission Ion Microscopy (STIM)

Spurred by a need to determine major (C, H, N and O) contents in biological tissue sections, a formalism for quantitative off-axis Scanning Transmission Ion Microscopy (OA-STIM) has been established. This can be used with, or without, simultaneous Elastic Backscattering Spectroscopy (EBS) to provide quantitative major element composition and thickness information. As part of the work, an empirical predictor with one free parameter for the proton–proton differential scattering cross section was implemented. The predictor values were in extremely close agreement with high accuracy literature data. For 2 MeV p–12C elastic scattering at forward angles ≤45°an interpolation procedure was used to determine the relative deviations from the Rutherford cross sections were determined to be ≤6.4%. The interpolation was based on a Coulomb field, angular momentum quantum number and nuclear structure dependent nuclear penetration factor. Finally, the quantitative combination of simultaneous OA-STIM and EBS data is discussed.

Sequestration of Ca in simian nasal mucosa: Determination of Ca molarity in ex vivo tissue by simultaneous off-axis Scanning Transmission Ion Microscopy, Particle Induced X-Ray Emission and Elastic Backscattering Spectrometry

A technique to determine the molar concentration of elements in ex vivo tissue sections by Particle Induced X-ray Emission (PIXE) has been developed. The method is based on simultaneous off-axis scanning transmission ion microscopy (OA-STIM) and Elastic Backscattering Spectrometry (EBS) measurement of the sample thickness and major element (H, C, N, O) composition. The method was applied to determine the molarity of localised Ca concentration hot-spots in the outer epithelium tissue of nasal mucosa of a rhesus macaque subject infected with simian immunodeficiency virus (SIV). The results show Ca sequestration in concentration hot-spots and outer epithelial tissue that significantly exceeded the Ca concentration in the surrounding tissues. This may originate from mineralisation and/or Ca enhancement in goblet cells.

Proton beam induced degradation of Pioloform® (polyvinyl butyral (PVB)) support films used for analysis of biomedical tissue sections

Pioloform®, an often used support film for ion microprobe research is a terpolymer of polyvinyl butyral (PVB) and ≲ 18 mass % polyvinyl alcohol (PVA). Simultaneous off-axis Scanning Transmission Ion Microscopy (OA-STIM) and Elastic Backscattering Spectrometry (EBS) measurements have been used to measure the evolution of the C, H and O contents for an increasing proton fluence. The results showed that the composition at zero proton-fluence was in close agreement with the theoretical atomic composition. This strongly suggests OA-STIM measurements. With increasing proton fluences preferential loss of H and O was observed from the films.

Overview of Polyethylene Terephthalate Foils Patterned Using 10 MeV Carbon Ions for Realization of Micromembranes

Polymer membranes are conventionally prepared using high-energy particles from radioactive decay or by the bombardment of hundreds of MeVs energy ions. In both circumstances, tracks of damage are produced by particles/ions passing through the polymer, and successively, the damaged material is removed by chemical etching to create narrow pores. This process ensures nanosized pore diameter but with random placement, leading to non-uniform local pore density and low membrane porosity, which is necessary to reduce the risk of their overlapping. The present study is focused on the use of polyethylene terephthalate (PET) foils irradiated by 10.0 MeV carbon ions, easily achievable with ordinary ion accelerators. The ion irradiation conditions and the chemical etching conditions were monitored to obtain customized pore locations without pore overlapping in PET. The quality, shape, and size of the pores generated in the micromembranes can have a large impact on their applicability. In this view, the Scanning Transmission Ion Microscopy coupled with a computer code created in our laboratory was implemented to acquire new visual and quantitative insights on fabricated membranes.

Scanning Transmission Ion Microscopy Time-of-Flight Spectroscopy Using 20 keV Helium Ions.

Scanning transmission ion microscopy imaging was performed whilst using a delay-line detector to record the impact position and arrival time of transmitted ions or neutrals. The incident helium ion beam had an energy of 20 keV and the arrival time measurements were used to calculate the energy loss after transmission through the sample. The 5D dataset thus produced (2D position in the sample plane, 2D position in the detector plane, and energy) is analyzed by collection into energy spectra or images. It is demonstrated that ion energy loss maps can identify regions of identical materials in the sample plane. The behavior of the energy loss with respect to the scattering angle is calculated and these simulations agree with the experimentally measured results. This experiment demonstrates the capability of keV helium ions to be successfully used in energy loss imaging experiments. This is the first step in the development of keV scanning transmission ion microscopy energy loss techniques.

Accuracy of three-dimensional proton imaging of an inertial confinement fusion target assessed by Geant4 simulation

The Geant4 toolkit was used to perform benchmark Monte Carlo simulations of proton micro-tomography imaging. A phantom of an inertial confinement fusion (ICF) target was designed, based on experimental data. Simulations of STIM (Scanning Transmission Ion Microscopy) and PIXE (Particle Induced X-ray Emission) tomography were performed. Quantitative images were obtained from the TomoRebuild and JPIXET software packages, chosen for their ability to handle large solid angles of X-ray detection. The tomographic images were compared with the original phantom, used as a reference. For STIM-T, the accuracy of the calculated mass density was ≤ 2 % for TomoRebuild and ≤ 14 % for JPIXET. Corrections of X-ray production cross section and X-ray absorption were tested for the quantification of Ge, used as a dopant in the ICF target. The accuracy on the obtained Ge density was ≤ 2.9 % for TomoRebuild and ≤ 6.4 % for JPIXET, whereas the error was about 40 % without correction.

Human Neocortex Layer Features Evaluated by PIXE, STIM, and STXM Techniques.

The human neocortex has a cytoarchitecture composed of six layers with an intrinsic organization that relates to afferent and efferent pathways for a high functional specialization. Various histological, neurochemical, and connectional techniques have been used to study these cortical layers. Here, we explore the additional possibilities of swift ion beam and synchrotron radiation techniques to distinguish cellular layers based on the elemental distributions and areal density pattern in the human neocortex. Temporal cortex samples were obtained from two neurologically normal adult men (postmortem interval: 6-12h). A cortical area of 500 × 500 μm2 was scanned by a 3MeV proton beam for elemental composition and areal density measurements using particle induced x-ray emission (PIXE) and scanning transmission ion microscopy (STIM), respectively. Zinc showed higher values in cortical layers II and V, which needs a critical discussion. Furthermore, the areal density decreased in regions with a higher density of pyramidal neurons in layers III and V. Scanning transmission X-ray microscopy (STXM) revealed the cellular density with higher lateral resolution than STIM, but not enough to distinguish each cortical lamination border. Our data describe the practical results of these approaches employing both X-ray and ion-beam based techniques for the human cerebral cortex and its heterogeneous layers. These results add to the potential approaches and knowledge of the human neocortical gray matter in normal tissue to develop improvements and address further studies on pathological conditions.

A Geant4 simulation of X-ray emission for three-dimensional proton imaging of microscopic samples.

Proton computed microtomography is a technique that reveals the inner content of microscopic samples. The density distribution of the material (in g·cm-3) is obtained from proton transmission tomography (STIM: Scanning Transmission Ion Microscopy) and the element content from X-ray emission tomography (PIXE: Particle Induced X-ray Emission). A precise quantification of chemical elements is difficult for thick samples, because of the variations of X-ray production cross-sections and of X-ray absorption. Both phenomena are at the origin of an attenuation of the measured X-ray spectra, which leads to an underestimation of the element content. Our aim is to quantify the accuracy of a specific correction method that we designed for thick samples. In this study, we describe how the 3D variations in the mass density were taken into account in the reconstruction code, in order to quantify the correction according to the position of the proton beam and the position and aperture angle of the X-ray detector. Moreover, we assess the accuracy of the reconstructed densities using Geant4 simulations on numerical phantoms, used as references. The correction process was successfully applied and led, for the largest regions of interest (little affected by partial volume effects), to an accuracy ≤ 4% for phosphorus (compared to about 40% discrepancy without correction). This study demonstrates the accuracy of the correction method implemented in the tomographic reconstruction code for thick samples. It also points out some advantages offered by Geant4 simulations: i) they produce projection data that are totally independent of the inversion method used for the image reconstruction; ii) one or more physical processes (X-ray absorption, proton energy loss) can be artificially turned off, in order to precisely quantify the effect of the different phenomena involved in the attenuation of X-ray spectra.

Quantitative nanoscale imaging using transmission He ion channelling contrast: Proof-of-concept and application to study isolated crystalline defects

A newly developed microscope prototype, namely npSCOPE, consisting of a Gas Field Ion Source (GFIS) column and a position sensitive Delay-line Detector (DLD) was used to perform Scanning Transmission Ion Microscopy (STIM) using keV He+ ions. One experiment used 25 keV ions and a second experiment used 30 keV ions. STIM imaging of a 50 nm thick free-standing gold membrane exhibited excellent contrast due to ion channelling and revealed rich microstructural features including isolated nanoscale twin bands which matched well with the contrast in the conventional ion-induced Secondary Electron (SE) imaging mode. Transmission Kikuchi Diffraction (TKD) and Backscattered Electron (BSE) imaging were performed on the same areas to correlate and confirm the microstructural features observed in STIM. Monte Carlo simulations of the ion and electron trajectories were performed with parameters similar to the experimental conditions to derive insights related to beam broadening and its effect in the degradation of transmission image resolution. For the experimental conditions used, STIM imaging showed a lateral resolution close to30 nm. Dark twin bands in bright grains as well as bright twin bands in dark grains were observed in STIM. Some of the twin bands were invisible in STIM. For the specific experimental conditions used, the ion transmission efficiency across a particular twin band was found to decrease by a factor of 2.8. Surprisingly, some grains showed contrast reversal when the Field of View (FOV) was changed indicating the sensitivity of the channelling contrast to even small changes in illumination conditions. These observations are discussed using ion channelling conditions and crystallographic orientations of the grains and twin bands. This study demonstrates for the first time the potential of STIM imaging using keV He+ ions to quantitatively investigate channelling in nanoscale structures including isolated crystalline defects.

NpSCOPE: A New Multimodal Instrument for In Situ Correlative Analysis of Nanoparticles.

Over the last few decades, nanoparticles have become a key element in a number of scientific and technological fields, spanning from materials science to life sciences. The characterization of nanoparticles or samples containing nanoparticles, in terms of morphology, chemical composition, and other parameters, typically involves investigations with various analytical tools, requiring complex workflows and extending the duration of such studies to several days or even weeks. Here, we report on the development of a new unique in situ correlative instrument, allowing us to answer questions about the shape, size, size distribution, and chemical composition of the nanoparticles using a single probe. Combining various microscopic and analytical capabilities in one single instrument allows a considerable increase in flexibility and a reduction in the duration of such complex investigations. The new instrument is based on focused ion beam microscopy technology using a gas field ion source as a key enabler and combining it with specifically developed secondary ion mass spectrometry and scanning transmission ion microscopy technology. We will present the underlying concept, the instrument and its main components, and proof-of-concept studies performed on this novel instrument. For this purpose, different pure titanium dioxide nanoparticle samples were investigated. Furthermore, the distribution and localization of the nanoparticles in biological model systems were studied. Our results demonstrate the performance and usefulness of the instrument for nanoparticle investigations, paving the way for a number of future applications, in particular, nanotoxicological research.

Scanning transmission helium ion microscopy on carbon nanomembranes.

A dark-field scanning transmission ion microscopy detector was designed for the helium ion microscope. The detection principle is based on a secondary electron conversion holder with an exchangeable aperture strip allowing its acceptance angle to be tuned from 3 to 98 mrad. The contrast mechanism and performance were investigated using freestanding nanometer-thin carbon membranes. The results demonstrate that the detector can be optimized either for most efficient signal collection or for maximum image contrast. The designed setup allows for the imaging of thin low-density materials that otherwise provide little signal or contrast and for a clear end-point detection in the fabrication of nanopores. In addition, the detector is able to determine the thickness of membranes with sub-nanometer precision by quantitatively evaluating the image signal and comparing the results with Monte Carlo simulations. The thickness determined by the dark-field transmission detector is compared to X-ray photoelectron spectroscopy and energy-filtered transmission electron microscopy measurements.

Scanning transmission imaging in the helium ion microscope using a microchannel plate with a delay line detector.

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.

Alfa Laval AB, Sweden

The tomographic reconstruction of biological specimens requires robust algorithms, able to deal with low density contrast and low element concentrations. At the IST/ITN microprobe facility new GPU-accelerated reconstruction software, JPIXET, has been developed, which can significantly increase the speed of quantitative reconstruction of Proton Induced X-ray Emission Tomography (PIXE-T) data. It has a user-friendly graphical user interface for pre-processing, data analysis and reconstruction of PIXE-T and Scanning Transmission Ion Microscopy Tomography (STIM-T). The reconstruction of PIXE-T data is performed using either an algorithm based on a GPU-accelerated version of the Maximum Likelihood Expectation Maximisation (MLEM) method or a GPU-accelerated version of the Discrete Image Space Reconstruction Algorithm (DISRA) (Sakellariou (2001) [2]). The original DISRA, its accelerated version, and the MLEM algorithm, were compared for the reconstruction of a biological sample of Caenorhabditis elegans – a small worm. This sample was analysed at the microbeam line of the AIFIRA facility of CENBG, Bordeaux. A qualitative PIXE-T reconstruction was obtained using the CENBG software package TomoRebuild (Habchi et al. (2013) [6]). The effects of pre-processing and experimental conditions on the elemental concentrations are discussed.

Increased Cd2+ biosorption capability of Aspergillus nidulans elicited by crpA deletion.

The P-type ATPase CrpA is an important Cu2+ /Cd2+ pump in the Aspergilli, significantly contributing to the heavy metal stress tolerance of these ascomycetous fungi. As expected, the deletion of crpA resulted in Cu2+ /Cd2+ -sensitive phenotypes in Aspergillus nidulans on stress agar plates inoculated with conidia. Nevertheless, paradoxical growth stimulations were observed with the ΔcrpA strain in both standard Cu2+ stress agar plate experiments and cellophane colony harvest (CCH) cultures, when exposed to Cd2+ . These observations reflect efficient compensatory mechanisms for the loss of CrpA operating under these experimental conditions. It is remarkable that the ΔcrpA strain showed a 2.7 times higher Cd biosorption capacity in CCH cultures, which may facilitate the development of new, fungal biomass-based bioremediation technologies to extract harmful Cd2+ ions from the environment. The nullification of crpA also significantlychanged the spatial distribution of Cu and Cd in CCH cultures, as demonstrated by the combined particle-induced X-ray emission and scanning transmission ion microscopy technique. Most important, the centers of gravity for Cu and Cd accumulations of the ΔcrpA colonies shifted toward the older regions as compared with wild-type surface cultures.

A Geant4 simulation for three-dimensional proton imaging of microscopic samples

Proton imaging can be carried out on microscopic samples by focusing the beam to a diameter ranging from a few micrometers down to a few tens of nanometers, depending on the required beam intensity and spatial resolution. Three-dimensional (3D) imaging by tomography is obtained from proton transmission (STIM: Scanning Transmission Ion Microscopy) and/or X-ray emission (PIXE: Particle Induced X-ray Emission). In these experiments, the samples are dehydrated for under vacuum analysis. In situ quantification of nanoparticles has been carried out at CENBG in the frame of nanotoxicology studies, on cells and small organisms used as biological models, especially on Caenorhabditis elegans (C. elegans) nematodes. Tomography experiments reveal the distribution of mass density and chemical content (in g.cm−3) within the analyzed volume. These density values are obtained using an inversion algorithm. To investigate the effect of this data reduction process, we defined different numerical phantoms, including a (dehydrated) C. elegans phantom whose geometry and density were derived from experimental data. A Monte Carlo simulation based on the Geant4 toolkit was developed. Using different simulation and reconstruction conditions, we compared the resulting tomographic images to the initial numerical reference phantom. A study of the relative error between the reconstructed and the reference images lead to the result that 20 protons per shot can be considered as an optimal number for 3D STIM imaging. Preliminary results for PIXE tomography are also presented, showing the interest of such numerical phantoms to produce reference data for future studies on X-ray signal attenuation in thick samples.

Imaging and Analytics on the Helium Ion Microscope.

The helium ion microscope (HIM) has emerged as an instrument of choice for patterning, imaging and, more recently, analytics at the nanoscale. Here, we review secondary electron imaging on the HIM and the various methodologies and hardware components that have been developed to confer analytical capabilities to the HIM. Secondary electron-based imaging can be performed at resolutions down to 0.5 nm with high contrast, with high depth of field, and directly on insulating samples. Analytical methods include secondary electron hyperspectral imaging (SEHI), scanning transmission ion microscopy (STIM), backscattering spectrometry and, in particular, secondary ion mass spectrometry (SIMS). The SIMS system that was specifically designed for the HIM allows the detection of all elements, the differentiation between isotopes, and the detection of trace elements. It provides mass spectra, depth profiles, and 2D or 3D images with lateral resolutions down to 10 nm.

Performance and application of heavy ion nuclear microbeam facility at the Nuclear Physics Institute in Řež, Czech Republic

The Tandetron Laboratory of the Nuclear Physics Institute of the Czech Academy of Sciences is equipped with five beam lines associated with a 3 MV tandem electrostatic accelerator model 4130 MC from High Voltage Engineering Europa B.V. This accelerator is coupled with two duoplasmatron sources and a single sputter ion source and provides ions from hydrogen to gold. One of these lines is a nuclear microbeam facility, utilizing ion beams of micro- and sub-micro sizes for materials research by use of particle induced x-ray emission spectroscopy, particle induced gamma emission, Rutherford back-scattering spectroscopy, and scanning transmission ion microscopy methods as well as for ion beam writing. The major advantage of the presented microprobe is a possibility of 3D structure creation not only in polymer materials using light ions but also in other materials such as glass, ceramics, etc. by use of heavy ions. The focusing system allows focusing of charged particles with a maximum rigidity of 11 MeV amu/q2. The usual resolution in high and low current modes is 2 × 3 µm2 for a 100 pA and 0.3 × 0.5 µm2 for the 2000 ions/s of 2 MeV protons, respectively. A detailed facility description is given in the paper. The applications of focused beams of heavy ions as well as examples of light ions utilizing are also presented in the article.