New approach in physical failure analysis based on 3D reconstruction

In this work we present a new approach in physical failure analysis. Fault isolation can be done using volume diagnosis techniques. But when studying the identified defect sites by Focused Ion Beam (FIB) cross-sectioning, correct interpretation of the cross-sectional views strongly relies on choosing an appropriate cutting strategy. However, volume diagnosis techniques do not provide any information on which cutting directions and settings to choose to avoid misinterpretation of the spatial evolution of the defects. The proposed approach is to acquire FIB-SEM tomographic datasets at the defect sites to unequivocally characterize the defects in three-dimensional visualizations, independent of particular cross-sectioning strategies. In this specific case we have applied the methodology at a microcontroller for automotive applications on which a couple of floating VIAS were found. Thanks to the complete information obtained with the tomography, the defect could be assigned to a specific class of etching tools, and the root cause thus be solved

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently labeled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods, such as synchrotron x-ray computed tomography with propagation-based phase contrast or serial blockface scanning electron microscopy, might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here, we showcase the utility of a femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

Electron backscatter diffraction analysis of non-conductive samples using in-situ charge compensation

Electron backscatter diffraction (EBSD) is a powerful technique for structural characterization of micro- or nanocrystalline samples in the scanning electron microscope (SEM). Reliable EBSD analysis of non-conductive samples has so far required the use of conductive coatings or variable pressure (VP) SEM. An in-situ charge compensation system provides improved EBSD analysis of non-conductive samples without the drawbacks of these conventional methods. Similar to VP-SEM, charge neutralisation is achieved by gas ionization, but at negligible loss of resolution due to a significantly reduced interaction volume of beam and backscattered electrons with gas particles.Angel Larrea and Sonia Serrano-Zabaleta acknowledge funding from project MAT2012-30763 and the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza.Peer Reviewe

Quantification of total calcium in terminal cisternae of skinned muscle fibers by imaging electron energy-loss spectroscopy

Skinned muscle fibers are ideal model preparations for the investigation of Ca-regulatory mechanisms. Their internal ionic milieu can be easily controlled and distinct physiological states are well defined. We have measured the total Ca content in the terminal cisternae of such preparations using imaging electron energy-loss spectroscopy (Image-EELS) as a new approach for quantification of sub-cellular element distributions. Murine muscle fibers submitted to a standardized calcium-loading procedure were cryo-fixed with a combined solution exchanger/plunge freezing device. Energy-filtered image series were recorded from ultrathin freeze-dried cryosections of samples immobilized in either relaxed or caffeine-contracted state. From these image series, electron energy-loss spectra were extracted by digital image-processing and quantitatively processed by multiple-least-squares-fitting with reference spectra. The calculated fit coefficients were converted to Ca-concentrations by a calibration obtained from Ca-standards. Total Ca-contents in the terminal cisternae of skinned skeletal muscle fibers decreased upon caffeine-induced Ca-release from 123 ± 159 (±11) to 73 ± 102 (±8) mmol/kg d.w. (weighted mean ± SD (±SEM))