Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

Transmission electron microscopy (TEM) is an important tool for studies in cell biology, and is essential to address research questions from bacteria to animals. Recent technological innovations have advanced the entire field of TEM, yet classical techniques still prevail for most present-day studies. Indeed, the majority of cell and developmental biology studies that use TEM do not require cutting-edge methodologies, but rather fast and efficient data generation. Although access to state-of-the-art equipment is frequently problematic, standard TEM microscopes are typically available, even in modest research facilities. However, a major unmet need in standard TEM is the ability to quickly prepare and orient a sample to identify a region of interest. Here, I provide a detailed step-by-step method for a positional correlative anatomy approach to flat-embedded samples. These modifications make the TEM preparation and analytic procedures faster and more straightforward, supporting a higher sampling rate. To illustrate the modified procedures, I provide numerous examples addressing research questions in Caenorhabditis elegans and Drosophila. This method can be equally applied to address questions of cell and developmental biology in other small multicellular model organisms.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

Atom probe tomography was utilized to measure directly the chemical compositions of Al3(Zr1−xTix) precipitates with a metastable L12 structure formed in Al-0.1Zr-0.1Ti (at.%) alloys upon aging at 375°C or 425°C. The alloys exhibit an inhomogeneous distribution of Al3(Zr1−xTix) precipitates, as a result of a nonuniform dendritic distribution of solute atoms after casting. At these aging temperatures, the Zr:Ti atomic ratio in the precipitates is about 10 and 5, respectively, indicating that Ti remains mainly in solid solution rather than partitioning to the Al3(Zr1−xTix) precipitates. This is interpreted as being due to the very small diffusivity of Ti in α-Al, consistent with prior studies on Al-Sc-Ti and Al-Sc-Zr alloys, where the slower diffusing Zr and Ti atoms make up a small fraction of the Al3(Sc1−xTix/Zrx) precipitates. Unlike those alloys, however, the present Al-Zr-Ti alloys exhibit no interfacial segregation of Ti at the matrix/precipitate heterophase interface, a result that may be affected by a significant disparity in the evaporation fields of the α-Al matrix and Al3(Zr1−xTix) precipitates and/or a lack of local thermodynamic equilibrium at the interface.

Calcite spherulites have been observed in many laboratory experiments with different bacteria, and spherulitic growth has received much interest in mineralogy research. However, the nucleation and growth mechanism, as well as geological significance of calcite spherulites in solution with bacteria is still unclear. Herein, spherulites composed of an amorphous core, a Mg-calcite body and an organic film were precipitated by the Curvibacter lanceolatus HJ-1 bacterial strain in a solution with a molar Mg/Ca ratio of 3. Based on the results, we provide a possible mechanism for the biomineralization of Mg-calcite spherulites. First, amorphous calcium carbonate particles are deposited and aggregated into a stable sphere-like core in combination with organic molecules. The core then acts as the nucleus of spherulitic radial growth. Finally, the organic film grows on the surface of Mg-calcite spherulites as a result of bacterial metabolism and calcification. These findings provide insight into the growth mode and crystallization of biogenic spherulites during biomineralization, and are of significance in the application of novel biological materials.

Scanning electron microscopy (SEM) is widely used in the science of materials and different parameters were developed to characterize the surface roughness. In a previous work, we studied the surface topography with fractal dimension at low scale and two parameters at high scale by using the variogram, that is, variance vs. step log–log graph, of a SEM image. Those studies were carried out with the FERImage program, previously developed by us. To verify the previously accepted hypothesis by working with only an image, it is indispensable to have reliable three-dimensional (3D) surface data. In this work, a new program (EZEImage) to characterize 3D surface topography in SEM has been developed. It uses fast cross correlation and dynamic programming to obtain reliable dense height maps in a few seconds which can be displayed as an image where each gray level represents a height value. This image can be used for the FERImage program or any other software to obtain surface topography characteristics. EZEImage also generates anaglyph images as well as characterizes 3D surface topography by means of a parameter set to describe amplitude properties and three functional indices for characterizing bearing and fluid properties.

Cryo-high-resolution scanning electron microscopy was used to analyze the conformation of fibronectin fibrils formed in human skin fibroblast cultures or in a cell-free system by treating soluble plasma fibronectin with guanidine. Structurally, fibrils assembled in the cell-free system and in culture were similar. Assembly of both fibrillar networks involves interactions with the III1 and amino terminal repeats of fibronectin; their conformations consist of either smooth surfaces or patches of smooth surfaces and nodules randomly spaced along the fibril. The random distribution of these two conformations in fibrils indicates that fibronectin fibrils are capable of undergoing localized conformational changes. The nodules may be discrete domains of 3 to 4 type III repeats, as they can be labeled with the monoclonal antibody IST-2 to the III13-14 repeats in fibronectin and are found in 160 kDa and 85 kDa fragments of fibronectin that only contain type III repeats. In our study, smooth regions of fibrils were never recognized by the IST-2 antibody, suggesting that the epitope in the III13-14 repeats is masked in these regions. These results demonstrate that fibronectin fibrils are flexible and certain epitopes of fibronectin may be buried, or exposed, depending on the conformation of the fibril. They also show that fibrils assembled in cell-free conditions can be a powerful tool for studying fibril formation.

Accurately identifying and extracting clusters from atom probe tomography (APT) reconstructions is extremely challenging, yet critical to many applications. Currently, the most prevalent approach to detect clusters is the maximum separation method, a heuristic that relies heavily upon parameters manually chosen by the user. In this work, a new clustering algorithm, Gaussian mixture model Expectation Maximization Algorithm (GEMA), was developed. GEMA utilizes a Gaussian mixture model to probabilistically distinguish clusters from random fluctuations in the matrix. This machine learning approach maximizes the data likelihood via expectation maximization: given atomic positions, the algorithm learns the position, size, and width of each cluster. A key advantage of GEMA is that atoms are probabilistically assigned to clusters, thus reflecting scientifically meaningful uncertainty regarding atoms located near precipitate/matrix interfaces. GEMA outperforms the maximum separation method in cluster detection accuracy when applied to several realistically simulated data sets. Lastly, GEMA was successfully applied to real APT data.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Convergent Beam Electron Diffraction and Transmission Electron Microscopy Study of Interfacial Defects in Gallium Nitride Homoepitaxial Films

A promising novel type of electrostatic phase plate for transmission electron microscopy (TEM) is presented. The phase plate consists of a single microcoaxial cable-like rod with its electrode exposed to the undiffracted electrons. The emerging field is used to shift the phase of the undiffracted electrons with respect to diffracted electrons. The design overcomes the drawback of the spatial frequency-blocking ring electrode of the Boersch phase plate. First, experimental phase-contrast images are presented for PbSe and Pt nanoparticles with clearly varying phase contrast, which depends on the applied voltage and resulting phase shift of the unscattered electrons. With the new phase-plate design, we show for the first time the reconstruction of an object wave function based on a series of only three experimental phase-contrast TEM images obtained with an electrostatic phase plate.

The impact of electron probe microanalysis on the study of geological and planetary materials has been tremendous. Electron microprobes evolved into routine analytical instruments in geological research laboratories as instrument capabilities improved and applications to geologic/planetary materials expanded. The contributions of electron probe microanalysis to the characterization of minerals, both terrestrial and extraterrestrial, and to other significant geological research, such as light element analysis, trace element analysis, and element mapping, is described.

Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

Duplex stainless steels (DSS) may be defined as a category of steels with a two-phase ferritic–austenitic microstructure, which combines good mechanical and corrosion properties. However, these steels can undergo significant microstructural modification as a consequence of either thermo-mechanical treatments (ferrite decomposition, which causes σ- and χ-phase formation and nitride precipitation) or plastic deformation at room temperature [austenite transformation into strain-induced martensite (SIM)]. These secondary phases noticeably affect the properties of DSS, and therefore are of huge industrial interest. In the present work, SIM formation was investigated in a 2101 lean DSS. The material was subjected to cold rolling at various degrees of deformation (from 10 to 80% thickness reduction) and the microstructure developed after plastic deformation was investigated by electron backscattered diffraction, X-ray diffraction measurements, and hardness and magnetic tests. It was observed that SIM formed as a consequence of deformations higher than ~20% and residual austenite was still observed at 80% of thickness reduction. Furthermore, a direct relationship was found between microstructure and magnetic properties.

Nanoparticle tracking analysis (NTA) was first applied to biologically active nanocomplexes to obtain concurrent information on their size, state of aggregation, concentration, and antigenic specificity in liquid. The subject of the NTA was an immunogenic complex (a candidate nanovaccine) comprised of spherical particles (SPs) generated by thermal remodeling of the tobacco mosaic virus and Rubella virus tetraepitopes exposed on the surface of SP.

The scanning electron microscope provides a platform for subnanometer resolution characterization of material morphology with excellent topographic and chemical contrast dependent on the used detectors. For imaging applications, the predominantly utilized signals are secondary electrons (SEs) and backscattered electrons (BSEs) that are emitted from the sample surface. Recent advances in detector technology beyond the traditional Everhart–Thornley geometry have enabled the simultaneous acquisition and discrimination of SE and BSE signals. This study demonstrates the imaging capabilities of a recently introduced new detector system that consists of the combination of two in-lens (I-L) detectors and one in-column (I-C) detector. Coupled with biasing the sample stage to reduce electron–specimen interaction volumes, this trinity of detector geometry allows simultaneous acquisition of signals to distinguish chemical contrast from topographical changes of the sample, including the identification of surface contamination. The I-C detector provides 4× improved topography, whereas the I-L detector closest to the sample offers excellent simultaneous chemical contrast imaging while not limiting the minimization of working distance to obtain optimal lateral resolution. Imaging capabilities and contrast mechanisms for all three detectors are discussed quantitatively in direct comparison to each other and the conventional Everhart–Thornley detector.

Errors in quantitative electron microprobe analysis arise from many sources including those associated with sampling, specimen preparation, instrument operation, data collection, and analysis. The relative magnitudes of some of these factors are assessed for a sample of NiAl used to demonstrate important concerns in the analysis of even a relatively simple system measured under standard operating conditions. The results presented are intended to serve more as a guideline for developing an analytical strategy than as a detailed error propagation model that includes all possible sources of variability and inaccuracy. The use of a variety of tools to assess errors is demonstrated. It is also shown that, as sample characteristics depart from those under which many of the quantitative methods were developed, errors can increase significantly.

The cusp epithelium is a specialized branch of the superior epithelium that surrounds the developing teeth of chitons and is responsible for delivering the elements required for the formation of biominerals within the major lateral teeth. These biominerals are deposited within specific regions of the tooth in sequence, making it possible to conduct a row by row examination of cell development in the cusp epithelium as the teeth progress from the unmineralized to the mineralized state. Cusp epithelium from the chiton Acanthopleura hirtosa was prepared using conventional chemical and microwave assisted tissue processing, for observation by light microscopy, conventional transmission electron microscopy (TEM) and energy filtered TEM. The onset of iron mineralization within the teeth, initiated at row 13, is associated with a number of dramatic changes in the ultrastructure of the apical cusp cell epithelium. Specifically, the presence of ferritin containing siderosomes, the position and number of mitochondria, and the structure of the cell microvilli are each linked to aspects of the mineralization process. These changes in tissue development are discussed in context with their influence over the physiological conditions within both the cells and extracellular compartment of the tooth at the onset of iron mineralization.