Quantification Challenges for Atom Probe Tomography of Hydrogen and Deuterium in Zircaloy-4.

Analysis and understanding of the role of hydrogen in metals is a significant challenge for the future of materials science, and this is a clear objective of recent work in the atom probe tomography (APT) community. Isotopic marking by deuteration has often been proposed as the preferred route to enable quantification of hydrogen by APT. Zircaloy-4 was charged electrochemically with hydrogen and deuterium under the same conditions to form large hydrides and deuterides. Our results from a Zr hydride and a Zr deuteride highlight the challenges associated with accurate quantification of hydrogen and deuterium, in particular associated with the overlap of peaks at a low mass-to-charge ratio and of hydrogen/deuterium containing molecular ions. We discuss possible ways to ensure that appropriate information is extracted from APT analysis of hydrogen in zirconium alloy systems that are important for nuclear power applications.

Statistics for real-time deformability cytometry: Clustering, dimensionality reduction, and significance testing.

Real-time deformability (RT-DC) is a method for high-throughput mechanical and morphological phenotyping of cells in suspension. While analysis rates exceeding 1000 cells per second allow for a label-free characterization of complex biological samples, e.g., whole blood, data evaluation has so far been limited to a few geometrical and material parameters such as cell size, deformation, and elastic Young's modulus. But as a microscopy-based technology, RT-DC actually generates and yields multidimensional datasets that require automated and unbiased tools to obtain morphological and rheological cell information. Here, we present a statistical framework to shed light on this complex parameter space and to extract quantitative results under various experimental conditions. As model systems, we apply cell lines as well as primary cells and highlight more than 11 parameters that can be obtained from RT-DC data. These parameters are used to identify sub-populations in heterogeneous samples using Gaussian mixture models, to perform a dimensionality reduction using principal component analysis, and to quantify the statistical significance applying linear mixed models to datasets of multiple replicates.

Standardized microgel beads as elastic cell mechanical probes.

Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acrylamide (PAAm) microgel beads mimicking size and overall mechanics of biological cells. We produced mono-disperse beads at rates of 20-60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads' elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors.

Linear complexions: Confined chemical and structural states at dislocations.

For 5000 years, metals have been mankind's most essential materials owing to their ductility and strength. Linear defects called dislocations carry atomic shear steps, enabling their formability. We report chemical and structural states confined at dislocations. In a body-centered cubic Fe-9 atomic percent Mn alloy, we found Mn segregation at dislocation cores during heating, followed by formation of face-centered cubic regions but no further growth. The regions are in equilibrium with the matrix and remain confined to the dislocation cores with coherent interfaces. The phenomenon resembles interface-stabilized structural states called complexions. A cubic meter of strained alloy contains up to a light year of dislocation length, suggesting that linear complexions could provide opportunities to nanostructure alloys via segregation and confined structural states.

Combining structural and chemical information at the nanometer scale by correlative transmission electron microscopy and atom probe tomography.

In many cases, the three-dimensional reconstructions from atom probe tomography (APT) are not sufficiently accurate to resolve crystallographic features such as lattice planes, shear bands, stacking faults, dislocations or grain boundaries. Hence, correlative crystallographic characterization is required in addition to APT at the exact same location of the specimen. Also, for the site-specific preparation of APT tips containing regions of interest (e.g. grain boundaries) correlative electron microscopy is often inevitable. Here we present a versatile experimental setup that enables performing correlative focused ion beam milling, transmission electron microscopy (TEM), and APT under optimized characterization conditions. The setup was designed for high throughput, robustness and practicability. We demonstrate that atom probe tips can be characterized by TEM in the same way as a standard TEM sample. In particular, the use of scanning nanobeam diffraction provides valuable complementary crystallographic information when being performed on atom probe tips. This technique enables the measurement of orientation and phase maps as known from electron backscattering diffraction with a spatial resolution down to one nanometer.

Real-time deformability cytometry as a label-free indicator of cell function.

The mechanical properties of cells are known to be a label-free, inherent marker of biological function in health and disease. Wide-spread utilization has so far been impeded by the lack of a convenient measurement technique with sufficient throughput. To address this unmet need, we have recently introduced real-time deformability cytometry (RT-DC) for continuous mechanical single-cell classification of heterogeneous cell populations at rates of several hundred cells per second. Cells are driven through the constriction zone of a microfluidic chip leading to cell deformations due to hydrodynamic stresses only. Our custom-built image processing software performs image acquisition, image analysis and data storage on the fly. The ensuing deformations can be quantified and an analytical model enables the derivation of cell material properties. Performing RT-DC we highlight its potential to identify rare objects in heterogeneous suspensions and to track drug-induced changes in cells. In summary, RT-DC enables marker-free, quantitative phenotyping of heterogeneous cell populations with a throughput comparable to standard flow cytometry.

Atomic-scale quantification of grain boundary segregation in nanocrystalline material.

Grain boundary segregation leads to nanoscale chemical variations that can alter a material's performance by orders of magnitude (e.g., embrittlement). To understand this phenomenon, a large number of grain boundaries must be characterized in terms of both their five crystallographic interface parameters and their atomic-scale chemical composition. We demonstrate how this can be achieved using an approach that combines the accuracy of structural characterization in transmission electron microscopy with the 3D chemical sensitivity of atom probe tomography. We find a linear trend between carbon segregation and the misorientation angle ω for low-angle grain boundaries in ferrite, which indicates that ω is the most influential crystallographic parameter in this regime. However, there are significant deviations from this linear trend indicating an additional strong influence of other crystallographic parameters (grain boundary plane, rotation axis). For high-angle grain boundaries, no general trend between carbon excess and ω is observed; i.e., the grain boundary plane and rotation axis have an even higher influence on the segregation behavior in this regime. Slight deviations from special grain boundary configurations are shown to lead to unexpectedly high levels of segregation.

Three-dimensional grain mapping by x-ray diffraction contrast tomography and the use of Friedel pairs in diffraction data analysis.

X-ray diffraction contrast tomography (DCT) is a technique for mapping grain shape and orientation in plastically undeformed polycrystals. In this paper, we describe a modified DCT data acquisition strategy which permits the incorporation of an innovative Friedel pair method for analyzing diffraction data. Diffraction spots are acquired during a 360 degrees rotation of the sample and are analyzed in terms of the Friedel pairs ((hkl) and (hkl) reflections, observed 180 degrees apart in rotation). The resulting increase in the accuracy with which the diffraction vectors are determined allows the use of improved algorithms for grain indexing (assigning diffraction spots to the grains from which they arise) and reconstruction. The accuracy of the resulting grain maps is quantified with reference to synchrotron microtomography data for a specimen made from a beta titanium system in which a second phase can be precipitated at grain boundaries, thereby revealing the grain shapes. The simple changes introduced to the DCT methodology are equally applicable to other variants of grain mapping.