Keana C. Scott

Minimizing damage during FIB sample preparation of soft materials

Although focused ion beam (FIB) microscopy has been used successfully for milling patterns and creating ultra‐thin electron and soft X‐ray transparent sections of polymers and other soft materials, little has been documented regarding FIB‐induced damage of these materials beyond qualitative evaluations of microstructure. In this study, we sought to identify steps in the FIB preparation process that can cause changes in chemical composition and bonding in soft materials. The impact of various parameters in the FIB‐scanning electron microscope (SEM) sample preparation process, such as final milling voltage, temperature, ion beam overlap and mechanical stability of soft samples, was evaluated using two test‐case materials systems: polyacrylamide, a low melting‐point polymer, and Wyodak lignite coal, a refractory organic material. We evaluated changes in carbon bonding in the samples using X‐ray absorption near‐edge structure spectroscopy (XANES) at the carbon K edge and compared these samples with thin sections that had been prepared mechanically using ultramicrotomy. Minor chemical changes were induced in the coal samples during FIB‐SEM preparation, and little effect was observed by changing ion‐beam parameters. However, polyacrylamide was particularly sensitive to irradiation by the electron beam, which drastically altered the chemistry of the sample, with the primary damage occurring as an increase in the amount of aromatic carbon bonding (C=C). Changes in temperature, final milling voltage and beam overlap led to small improvements in the quality of the specimens. We outline a series of best practices for preparing electron and soft X‐ray transparent samples, with respect to preserving chemical structure and mechanical stability of soft materials using the FIB.

3D elemental and structural analysis of biological specimens using electrons and ions.

We demonstrate the utility of focused ion beam scanning electron microscopy combined with energy dispersive x‐ray spectrometry for 3D morphological and elemental correlative analysis of subcellular features. Although recent advances in super‐resolution light microscopy techniques and traditional transmission electron microscopy methods can provide cellular imaging at a wide range of length scales, simultaneous 3D morphological and elemental imaging of cellular features at nanometre scale can only be achieved with techniques such as focused ion beam scanning electron microscopy with energy dispersive x‐ray spectrometry capability. We demonstrate the technique by analysing the 3D silicon cell wall structure of a marine diatom, Thalassiosira pseudonana. This study also highlights the limitations of the technique in its current state and suggests several possible improvements needed for the routine use of the technique for biological specimens.

Analysis of 3D elemental mapping artefacts in biological specimens using Monte Carlo simulation.

In this paper, we present Monte Carlo simulation results demonstrating the feasibility of using the focused ion beam based X‐ray microanalysis technique (FIB‐EDS) for the 3D elemental analysis of biological samples. In this study, we used a marine diatom Thalassiosira pseudonana as our model organism and NISTMonte for the Monte Carlo simulations. We explored several beam energies commonly used for the X‐ray microanalysis to examine their effects on the resulting 3D elemental volume of the model organism. We also performed a preliminary study on the sensitivity of X‐ray analysis for detecting nanoparticles in the model. For the conditions considered in this work, we show that the X‐ray mapping performed using the 5 keV beam energy results in 3D elemental distributions that closely reflect the elemental distributions in the original model. At 5 keV, the depth resolution of the X‐ray maps is about 250 nm for the model organism. We also show that the nanoparticles that are 50 nm in diameter or greater are easily located. Although much work is still needed in generating more accurate biological models and simulating experimental conditions relevant to these samples, our results indicate that FIB‐EDS is a promising technique for the 3D elemental analysis of some biological specimens.

Effects of nanoparticle size and charge on interactions with self-assembled collagen.

HYPOTHESIS Insights into bone formation have suggested that the critical first step in the biomineralization process is the integration of small (nanometer dimension) mineral clusters into collagen fibers. Not only is such behavior of interest for understanding biomineralization but also should be important to nanotoxicology because collagen is a major component of structural tissues in the human body and accounts for more than 25% of the whole body protein content. Here, utilizing the current insights from biomineralization, we hypothesize that the binding affinity of nanoparticles to self-assembled collagen fibers is size and surface charge dependent. EXPERIMENTS We developed a self-assembled collagen substrate compatible with Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), which is very sensitive to mechanical changes of the substrate as a consequence of nanoparticle binding. QCM-D experiments were conducted with both positively and negatively charged gold nanoparticles between 2 and 10 nm in size. Complementary ex situ imaging Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) were used to confirm the QCM-D results. FINDINGS We find that both positively and negatively charged nanoparticles of all sizes exhibited binding affinity for self-assembled collagen fibers. Furthermore, the smallest particles (2 nm) mechanically integrated with collagen fibers.

Detection and speciation of brominated flame retardants in high-impact polystyrene (HIPS) polymers

Polymeric materials have been suggested as possible environmental sources of persistent organic pollutants such as flame retardants. In situ, micrometre‐scale characterization techniques for polymer matrix containing flame retardants may provide some insight into the dominant environmental transfer mechanism(s) of these brominated compounds. In this work, we demonstrate that micro X‐ray fluorescence spectroscopy (μXRF), focused ion beam scanning electron microscopy (FIB‐SEM) combined with energy dispersive X‐ray spectroscopy (EDS), and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) are promising techniques for the elemental and chemical identification of brominated fire retardant compounds (such as the deca‐congener of polybrominated diphenyl ether, BDE‐209) within polymeric materials (e.g. high‐impact polystyrene or HIPS). Data from μXRF demonstrated that bromine (Br) inclusions were evenly distributed throughout the HIPS samples, whereas FIB SEM‐EDS analysis revealed that small antimony (Sb) and Br inclusions are present, and regionally higher concentrations of Br surround the Sb inclusions (compared to the bulk material). Four prominent mass‐to‐charge ratio peaks (m/z 485, 487, 489 and 491) that correspond to BDE‐209 were identified by ToF‐SIMS and can be used to chemically distinguish this molecule on the surface of polymeric materials with respect to other brominated organic molecules. These techniques can be important in any study that investigates the route of entry to the environmental surroundings of BDE‐containing materials.

Live, video-rate super-resolution microscopy using structured illumination and rapid GPU-based parallel processing.

Structured illumination fluorescence microscopy is a powerful super-resolution method that is capable of achieving a resolution below 100 nm. Each super-resolution image is computationally constructed from a set of differentially illuminated images. However, real-time application of structured illumination microscopy (SIM) has generally been limited due to the computational overhead needed to generate super-resolution images. Here, we have developed a real-time SIM system that incorporates graphic processing unit (GPU) based in-line parallel processing of raw/differentially illuminated images. By using GPU processing, the system has achieved a 90-fold increase in processing speed compared to performing equivalent operations on a multiprocessor computer--the total throughput of the system is limited by data acquisition speed, but not by image processing. Overall, more than 350 raw images (16-bit depth, 512 × 512 pixels) can be processed per second, resulting in a maximum frame rate of 39 super-resolution images per second. This ultrafast processing capability is used to provide immediate feedback of super-resolution images for real-time display. These developments are increasing the potential for sophisticated super-resolution imaging applications.

Characterization of SiGe films for use as a National Institute of Standards and Technology Microanalysis Reference Material (RM 8905).

Bulk silicon-germanium (SiGe) alloys and two SiGe thick films (4 and 5 microm) on Si wafers were tested with the electron probe microanalyzer (EPMA) using wavelength dispersive spectrometers (WDS) for heterogeneity and composition for use as reference materials needed by the microelectronics industry. One alloy with a nominal composition of Si0.86Ge0.14 and the two thick films with nominal compositions of Si0.90Ge0.10 and Si0.75Ge0.25 on Si, evaluated for micro- and macroheterogeneity, will make good microanalysis reference materials with an overall expanded heterogeneity uncertainty of 1.1% relative or less for Ge. The bulk Ge composition in the Si0.86Ge0.14 alloy was determined to be 30.228% mass fraction Ge with an expanded uncertainty of the mean of 0.195% mass fraction. The thick films were quantified with WDS-EPMA using both the Si0.86Ge0.14 alloy and element wafers as reference materials. The Ge concentration was determined to be 22.80% mass fraction with an expanded uncertainty of the mean of 0.12% mass fraction for the Si0.90Ge0.10 wafer and 43.66% mass fraction for the Si0.75Ge0.25 wafer with an expanded uncertainty of the mean of 0.25% mass fraction. The two thick SiGe films will be issued as National Institute of Standards and Technology Reference Materials (RM 8905).

Giant Surface Conductivity Enhancement in a Carbon Nanotube Composite by Ultraviolet Light Exposure.

Carbon nanotube composites are lightweight, multifunctional materials with readily adjustable mechanical and electrical properties-relevant to the aerospace, automotive, and sporting goods industries as high-performance structural materials. Here, we combine well-established and newly developed characterization techniques to demonstrate that ultraviolet (UV) light exposure provides a controllable means to enhance the electrical conductivity of the surface of a commercial carbon nanotube-epoxy composite by over 5 orders of magnitude. Our observations, combined with theory and simulations, reveal that the increase in conductivity is due to the formation of a concentrated layer of nanotubes on the composite surface. Our model implies that contacts between nanotube-rich microdomains dominate the conductivity of this layer at low UV dose, while tube-tube transport dominates at high UV dose. Further, we use this model to predictably pattern conductive traces with a UV laser, providing a facile approach for direct integration of lightweight conductors on nanocomposite surfaces.

Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis.

For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested AuNPs. Both conventional inductively coupled plasma mass spectrometry (ICP-MS) and single particle (sp)ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake via high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and in vivo quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment.

Three-dimensional Microanalysis Using FIB SEM: Variations in Technique

In recent years, by combining energy dispersive x-ray spectrometry (EDS) and automated three-dimensional (3D) imaging using focused ion beam scanning electron microscopy (FIB SEM), multiple groups have demonstrated 3D microanalysis of complex microstructures [1, 2]. In addition to the usual analytical challenges associated with traditional microanalysis methods, the FIB-based 3D method comes with several additional complications. In this method, the analysis surface is prepared by removing material surrounding the feature of interest to permit access by the electron beam and to create an unobstructed view for the x-ray detector. In many cases spurious contributions to the analytical signals can arise from the base and the walls of the pit surrounding the analysis volume, complicating reliable quantitative analysis. In their recent paper, Schaffer et al. suggest lifting the volume of interest from the bulk sample and performing the EDS analysis with the volume suspended in vacuum as a possible alternative [3]. The non-optimal takeoff angle of the EDS detector in some instruments is another possible complication [4]. Performing additional stage movements and/or rotations following each milling step can mitigate the takeoff angle problem in some cases, but at the expense of increased data acquisition time.