Focused Ion Beam Electron Microscopy Research Articles

Dynamics of CLIMP-63 S-acylation control ER morphology

The complex architecture of the endoplasmic reticulum (ER) comprises distinct dynamic features, many at the nanoscale, that enable the coexistence of the nuclear envelope, regions of dense sheets and a branched tubular network that spans the cytoplasm. A key player in the formation of ER sheets is cytoskeleton-linking membrane protein 63 (CLIMP-63). The mechanisms by which CLIMP-63 coordinates ER structure remain elusive. Here, we address the impact of S-acylation, a reversible post-translational lipid modification, on CLIMP-63 cellular distribution and function. Combining native mass-spectrometry, with kinetic analysis of acylation and deacylation, and data-driven mathematical modelling, we obtain in-depth understanding of the CLIMP-63 life cycle. In the ER, it assembles into trimeric units. These occasionally exit the ER to reach the plasma membrane. However, the majority undergoes S-acylation by ZDHHC6 in the ER where they further assemble into highly stable super-complexes. Using super-resolution microscopy and focused ion beam electron microscopy, we show that CLIMP-63 acylation-deacylation controls the abundance and fenestration of ER sheets. Overall, this study uncovers a dynamic lipid post-translational regulation of ER architecture.

Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy

Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM) for the identification of synaptic contacts in neural tissue. Peroxidases, in particular, can efficiently metabolize 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in a multiplexed manner in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) that are favored for the large-scale ultrastructural assessment of synaptic architecture However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) can efficienty deposit EM-visible DAB products in thick tissue without detergent treatment opening the possibility for the multiplex labeling of genetically defined cell-types in combination with volume EM methods. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.

Metabolic design in a mammalian model of extreme metabolism, the North American least shrew (Cryptotis parva).

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused ion beam electron microscopy imaging and tandem mass tag mass spectrometry proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart, permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogeneous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism. KEY POINTS: Least shrews were studied to explore the relationship between metabolic function, mitochondrial morphology and protein content in different tissues. Liver and kidney mitochondrial content and enzymatic activity approaches that of the heart, indicating similar metabolic demand among tissues that contribute to basal and maximum metabolism. This allows an examination of mitochondrial structure and composition in tissues with similar maximum metabolic demands. Mitochondrial networks only occur in striated muscle. In contrast, the liver and kidney maintain individual mitochondria with limited reticulation. Muscle mitochondrial reticulation is the result of dense ATPase activity and cell-spanning myofibrils which require networking for adequate metabolic support. In contrast, liver and kidney ATPase activity is localized to the endoplasmic reticulum and basolateral membrane, respectively, generating a locally balanced energy conversion and utilization. Mitochondrial morphology is not driven by maximum metabolic demand, but by the cytosolic distribution of energy-utilizing systems set by the functions of the tissue.

The Three-Dimensional Ultrastructure of the Human Alveolar Epithelium Revealed by Focused Ion Beam Electron Microscopy.

Thin type 1 alveolar epithelial (AE1) and surfactant producing type 2 alveolar epithelial (AE2) cells line the alveoli in the lung and are essential for normal lung function. Function is intimately interrelated to structure, so that detailed knowledge of the epithelial ultrastructure can significantly enhance our understanding of its function. The basolateral surface of the cells or the epithelial contact sites are of special interest, because they play an important role in intercellular communication or stabilizing the epithelium. The latter is in particular important for the lung with its variable volume. The aim of the present study was to investigate the three-dimensional (3D) ultrastructure of the human alveolar epithelium focusing on contact sites and the basolateral cell membrane of AE2 cells using focused ion beam electron microscopy and subsequent 3D reconstructions. The study provides detailed surface reconstructions of two AE1 cell domains and two AE2 cells, showing AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. Furthermore, we show reconstructions of a surfactant secretion pore, enlargements of the apical AE1 cell surface and long folds bordering grooves on the basal AE1 cell surface. The functional implications of our findings are discussed. These findings may lay the structural basis for further molecular investigations.

Effects of annealing on the temperature coefficient of resistance of nickel film deposited on polyimide substrate

This study investigates the effects of annealing temperature as well as time on the temperature coefficient of resistance (TCR) of nickel (Ni) films deposited on flexible polyimide substrate by direct current magnetron sputtering. The morphological and microstructural characteristics of as-deposited and annealed Ni films were analyzed to understand the mechanisms of TCR variation. The crystalline phase, grain size change tendency and residual stress of films were evaluated by X-ray Diffractometer (XRD) technique. (111) is the preferred orientation of Ni and the grains have a growth after annealing. The residual stress in as-deposited films is about −446.3 MPa and reduced to −208.8 MPa after annealing at 400 °C for 6 h. Focused Ion Beam Electron Microscope (FIBEM) and Energy Dispersive X-ray Spectroscopy (EDS) investigations showed the microstructure of annealed films has a relative improvement and the oxygen content was little increased with increasing annealing temperature and time. Mean surface roughness determined by Atomic Force Microscope (AFM) is decreased from 4.5 nm to 1.25 nm with increasing annealing temperature. The TCR value increases with the increasing annealing temperature and time. When the annealing time exceeds 6 h, the TCR almost has no change or even begin to decline.

Assessing the veracity of Precambrian ‘sponge’ fossils using in situ nanoscale analytical techniques

Paleontological inferences, molecular clocks, and biomarker fossils indicate sponges evolved in the Cryogenian, but Precambrian sponge fossils are rare, poorly substantiated, and controversial. Spicule-like microstructures (SLMs) hosted in phosphatized fossils from the Ediacaran Doushantuo Formation (∼635–551Ma) at Weng’an of South China have been interpreted as cylindrical siliceous monaxons, and their hosting fossils as the oldest demosponges in the fossil record. In order to assess their veracity as the oldest spiculate demosponges, we utilize a suite of in situ nanoscale analytical techniques—including scanning electron microscopy, synchrotron X-ray fluorescence mapping, X-ray absorption near edge structure (XANES) spectroscopy, focused ion beam electron microscopy, and transmission electron microscopy—to evaluate the ultrastructures and elemental, chemical, and mineralogical compositions of the SLMs. Our data decisively shows that the SLMs are carbonaceous in composition and rectangular in transverse sections, and therefore, are not cylindrical siliceous spicules. Instead, the SLMs may be microbial strands, axial filaments of early hexactinellids, or acicular crystals molded by organic matter. Regardless, our new data invalidate the oldest and only Precambrian demosponges with mineralized spicules. These results indicate that interpretations of Precambrian sponge fossils should be scrutinized with compositional, mineralogical, and ultrastructural data collected using in situ analytical techniques. In addition, our conclusions affirm that no unequivocal biomineralizing sponges occur below the Ediacaran–Cambrian boundary. If hexactinellids and demosponges did diverge in the Cryogenian as suggested by molecular clocks and biomarkers, they either evolved biomineralization long after their divergence or their biomineralized spicules were never preserved until after the Ediacaran–Cambrian boundary. In either case, the dearth of biomineralizing sponge fossils in the Precambrian and their abundance in the early Cambrian must reflect a geobiologically significant aspect of the Precambrian–Phanerozoic transition.

Microbial biofilms and the preservation of the Ediacara biota

Laflamme, M., Schiffbauer, J.D., Narbonne, G.M., & Briggs, D.E.G. 2011: Microbial biofilms and the preservation of the Ediacara biota. Lethaia, Vol. 44, pp. 203–213. The terminal Neoproterozoic Ediacaran Period is typified by the Ediacara biota (ca. 579–542 Ma), which includes the first morphologically complex macroscopic organisms. Both the taphonomic setting that promoted the preservation of the soft-bodied Ediacara biota in coarse-grained sediments, and the influence of associated microbial coatings on this process, have generated debate. Specimens of Ediacaran discs (Aspidella) from the Fermeuse Formation of Newfoundland, Canada, were analysed using environmental scanning electron microscopy (ESEM) and focused ion beam electron microscopy (FIB-EM) to determine the relationship between the fossil specimens and the surrounding sediment. The presence of chemically distinct (Al–Mg–Fe–K- and to a lesser extent S-rich), finer-grained sediment (with organized iron sulphides) surrounding the upper and lower margins of the Ediacaran fossils is consistent with elemental analyses of well preserved bacterial biofilms from other localities. ESEM analyses reveal a contrast in the composition of the sediment bound within the discs, which contains a higher concentration of Al, Ca and K, and the purer Si-rich sediment that forms the surrounding matrix. This suggests that the coarse grained sediment was incorporated into the organism during life. Ediacaran discs were likely surrounded by a bacterial biofilm or thin microbial mat composed primarily of extracellular polymeric substances (or exopolysaccharide) during life, which added structural stability to these frond holdfasts, and facilitated their fossilization. Microbially mediated preservation in Fermeuse-style Ediacaran taphonomy provides an explanation for the dominance of Aspidella holdfasts in these settings, and suggests that preservation of Ediacaran fossils in the round may be much more prevalent than previously recognized. We suggest that the overwhelming dominance of circular to bulbous forms such as Aspidella in Ediacaran biotas around the world is a direct result of the interplay between microbial ecology and microbially mediated taphonomy. □Aspidella, Ediacaran preservation, environmental scanning electron microscopy, focused ion beam electron microscopy, palaeoecology, taphonomic bias.

Novel application of focused ion beam electron microscopy (FIB-EM) in preparation and analysis of microfossil ultrastructures: A new view of complexity in early Eukaryotic organisms

Coupled dual-beam focused ion beam electron microscopy (FIB-EM) has gained popularity across multiple disciplines over the past decade. Widely utilized as a stand-alone instrument for micromachining and metal or insulator deposition in numerous industries, the submicron-scale ion milling and cutting capabilities of FIB-EM systems have been well documented in the materials science literature. These capacities make FIB-EM a powerful tool for in situ, site-specific transmission electron microscopy (TEM) ultrathin foil preparation. Recent advancements in the field-emission guns (FEGs) of FIB-EM systems have provided spatial resolution comparable to that of many high-end scanning electron microscopes (SEM), thus providing enhanced imaging capacities with material deposition and material removal capabilities. More recently, FIB-EM preparation techniques have been applied to geological samples to characterize mineral inclusions, grain boundaries, and microfossils. Here, we demonstrate a novel method for analyzing three-dimensional (3D) ultrastructures of microfossils using FIB-EM. Our method, FIB-EM nanotomography, consists of sequential ion milling, or cross sectioning, and concurrent SEM imaging. This technique with coupled dual-beam systems allows for real-time, 3-D ultrastructural analysis and compositional mapping with precise site selectivity and may provide new insights in fossil ultrastructures. Using the FIB-EM nanotomography method, we investigated herkomorphic and acanthomorphic acritarchs (organicwalled microfossils) extracted from the $999 Ma Mesoproterozoic Ruyang Group of North China. The 3-D characteristics of such important but controversial acritarch features as processes and vesicularly enclosed central bodies are described. Through these case studies, we demonstrate that FIB-EM nanotomography is a powerful and useful tool for investigating the three-dimensionality of microfossil ultra- and nanostructures.