Transmission Electron Microscopy Of Replicas Research Articles

Analyzing the cellular plasma membrane by fast and efficient correlative STED and platinum replica EM

The plasma membrane of mammalian cells links transmembrane receptors, various structural components, and membrane-binding proteins to subcellular processes, allowing inter- and intracellular communication. Therefore, membrane-binding proteins, together with structural components such as actin filaments, modulate the cell membrane in their flexibility, stiffness, and curvature. Investigating membrane components and curvature in cells remains challenging due to the diffraction limit in light microscopy. Preparation of 5–15-nm-thin plasma membrane sheets and subsequent inspection by metal replica transmission electron microscopy (TEM) reveal detailed information about the cellular membrane topology, including the structure and curvature. However, electron microscopy cannot identify proteins associated with specific plasma membrane domains. Here, we describe a novel adaptation of correlative super-resolution light microscopy and platinum replica TEM (CLEM-PREM), allowing the analysis of plasma membrane sheets with respect to their structural details, curvature, and associated protein composition. We suggest a number of shortcuts and troubleshooting solutions to contemporary PREM protocols. Thus, implementation of super-resolution stimulated emission depletion (STED) microscopy offers significant reduction in sample preparation time and reduced technical challenges for imaging and analysis. Additionally, highly technical challenges associated with replica preparation and transfer on a TEM grid can be overcome by scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides the highest spatial resolution of plasma membrane proteins and their underlying membrane and is, therefore, a suitable method to study cellular events like endocytosis, membrane trafficking, or membrane tension adaptations.

A conformational switch in clathrin light chain regulates lattice structure and endocytosis at the plasma membrane of mammalian cells.

The nanoscale localizations, interactions, and conformations of endocytic proteins are key regulators of clathrin-mediated endocytosis. Although super-resolution microscopy has revealed the detailed organization of endocytic proteins, the resolution of super-resolution imaging cannot assess molecular interaction and conformational changes. Fluorescence resonance energy transfer (FRET) occurs when dyes are separated by less than 10 nm for most fluorescent proteins pairs. Thus, it can be used to map close-range molecular complexes and dynamics. Yet, to understand how endocytosis works, measurements from FRET must be correlated to the distinct stages of endocytosis. To accomplish this, we developed a new correlative lifetime-based FRET (FLIM-FRET) and platinum replica transmission electron microscopy (PREM) method, named FRET-CLEM. Here, FRET-based atomic distances can be mapped directly to individual cellular structures visualized in EM at the plasma membrane. We used this method to measure the conformational changes in clathrin light chain (CLC), a component of the clathrin triskelion and assembled clathrin lattice. CLC conformational changes have been proposed to regulate the assembly of clathrin in solution. However, CLC conformational changes and their effects on clathrin lattice growth, curvature, and endocytosis at the membrane of living cells are unknown. Using FRET-CLEM, we discovered that CLC undergoes a conformational switch as clathrin lattices curve. Preventing this conformational switch with acute chemical tools increased clathrin lattice sizes and inhibited endocytosis. Therefore, a specific conformational switch in CLC regulates lattice curvature and endocytosis in mammalian cells. These new correlative light and EM data will help develop a complete mechanistic model of endocytosis. More generally, FRET-CLEM can map molecular interactions and conformational changes at targeted membrane-associated proteins at identified cellular compartments including exocytic sites and neuronal synapses.

The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo.

Canonical Wnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

To Pinpoint the Location and the Orientation of Proteins Associated with Dense-Core Vesicles (DCVs) using CLEM

Exocytosis of catecholamine (adrenaline and noradrenaline) from adrenal chromaffin cells is important for stress responses and insulin release from pancreatic beta cells is critical in regulating blood glucose levels. These exocytic processes are highly regulated by a dozens of key proteins in eukaryotic cells. While the origin, transport, and delivery of cargo-loaded vesicles (dense-core vesicles) and the proteins involved is well studied, how these proteins are organized with respect to dense-core vesicles (DCVs) at the nanoscale remains to be explored. Here, we directly image several proteins that are key regulators of the transport, docking, and priming of DCVs at the plasma membrane. Specifically, correlative super-resolution (dSTORM) light and platinum replica transmission electron microscopy (CLEM) imaging was performed on PC12 cells (a rat adrenal chromaffin-derived neuroendocrine cell line) that were transfected with proteins of interest. We aligned super-resolution dSTORM images of Rab proteins (Rab27a and Rab3a) and Rab effector proteins (granuphilin and Rabphilin) with corresponding TEM images of the plasma membrane and observed the precise correlation between proteins and vesicles at the plasma membrane. The nano-scale molecular architecture of DCVs generated from this correlative imaging will help us understand how key proteins guide the docking, priming, and fusion of DCVs in endocrine cells.

High resolution imaging of the cortex isolated from sea urchin eggs and embryos.

The cellular cortex-consisting of the plasma membrane and the adjacent outer few microns of the cytoplasm-is a critically important, dynamic and complex region in the sea urchin egg and embryo. Some 40 years ago it was discovered that isolated cortices could be obtained from eggs adhered to glass coverslips and since that time this preparation has been used in a wide range of studies, including seminal research on fertilization, exocytosis, the cytoskeleton, and cytokinesis. In this chapter, we discuss methods for isolating cortices from eggs and embryos, including those undergoing cell division. We also provide protocols for analyzing cortical architecture and dynamics using specific localization methods combined with super-resolution Structured Illumination and Stimulated Emission Depletion light microscopy and platinum replica transmission electron microscopy.

Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation.

We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation.

Assessment of the platinum deposition behaviour on stainless steel surfaces in a boiling water reactor plant

ABSTRACTWith the aim of mitigating stress corrosion cracking (SCC) in reactor internals and recirculation pipes, the boiling water reactor plant Leibstadt (KKL) changed in 2008 from normal water chemistry to hydrogen water chemistry in combination with online noble metal chemical addition (OLNC). For this purpose, a Na2Pt(OH)6 solution is injected into the reactor feed water, where platinum (Pt) particles are formed and deposited on water-wetted surfaces. If a stoichiometric excess of hydrogen and a uniform surface coverage with extremely fine Pt particles exist simultaneously at critical locations, a low electrochemical corrosion potential, and therefore a low SCC susceptibility can be expected. To study the Pt deposition behaviour and to assess the effectiveness of the OLNC technology under real plant conditions, stainless steel coupon specimens were exposed to the reactor water for different durations at two locations in KKL. The specimens were then analysed at PSI for their Pt surface loading. In addition, a replica technique and transmission electron microscopy were used to determine the Pt particle size and distribution on the active specimens. The investigations indicated that the pre- and post-OLNC exposure times play an important role for the Pt deposition and retention on specimens and that a remobilisation of Pt in the plant takes place. Unfortunately, most of the released Pt does not redeposit on other steel surfaces in the reactor system. The flow velocity of the Pt containing reactor water across the specimens also has an impact on the Pt surface loading. The average Pt particle size is in the expected range, but the distribution of the Pt particles on plant specimens seems to be rather erratic.

The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis.

Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.

Nanocrystallization in Oxyfluoride Glasses Controlled by Amorphous Phase Separation.

Transparent bulk glass-ceramics containing ZnF2, K2SiF6, and KZnF3 nanocrystals are successfully obtained from xKF-xZnF2-(100 - 2x)SiO2 oxyfluoride glasses for the first time to the best of our knowledge. The glass transition temperatures of heat-treated samples increase with time and approach values that resemble the temperatures chosen for thermal treatment. During nucleation and crystal growth, the residual glass around the crystals is depleted in fluoride which as glass component usually leads to a decrease in viscosity. The crystallization behavior notably depends on the glass composition and changes within a small range from x = 20 to 22.5 mol %. The occurrence of liquid/liquid phase separation in dependence of the composition is responsible for the physicochemical changes. Two different microstructures of droplet and interpenetrating phase separation and their compositional evolution are observed by replica transmission electron microscopy technique in the multicomponent glassy system. This study suggests that the size and crystal phase of precipitated crystallites can be controlled by the initial phase separation.

Two classes of gap junction channels mediate soma-germline interactions essential for germline proliferation and gametogenesis in Caenorhabditis elegans.

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.

Direct observation of liquid crystals using cryo-TEM: specimen preparation and low-dose imaging.

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset enabling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present comparative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understanding of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies.

Correlative super-resolution fluorescence and metal-replica transmission electron microscopy.

Super-resolution localization microscopy is combined with a complementary imaging technique, transmission electron microscopy of metal replicas, to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. Robust correlation on the scale of 20 nm is validated by imaging endogenous clathrin (with 2D and 3D PALM/TEM) and the method is further used to find the previously unknown 3D position of epsin on clathrin coated structures.

Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi

Background Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility.Methodology/Principal FindingsHere, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea.Conclusions/SignificanceGraphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating.

高分子材料の摩擦・摩耗面の分析と解析

Friction and wear properties of polymeric materials are reviewed with special reference to morphologies analyzed by transmission electron microscopy (TEM) of carbon replica films obtained from the worn surfaces. Tribological properties of polyethylene as functions of molecular weight and lubricants are related to mechanism for the formation of fine wear powders as revealed by TEM, including mechanism of self-lubrication of ultrahigh molecular weight polyethylene in water. Tribologies of isotactic polypropylene, polyacetal-based polymer alloy and other polymers are also reported based on the carbon replica TEM method. It is concluded that TEM analysis using carbon replica film obtained from the worn polymer surfaces is a powerful method for clarifying polymer wear mechanisms.

Comparative Study of Nanoscale Surface Structures of Calcite Microcrystals Using FE-SEM, AFM, and TEM

The bulk morphology and surface features that developed upon precipitation on micrometer-size calcite powders and millimeter-size cleavage fragments were imaged by three different microscopic techniques: field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) of Pt-C replicas, and atomic force microscopy (AFM). Each technique can resolve some nanoscale surface features, but they offer different ranges of magnification and dimensional resolutions. Because sample preparation and imaging is not constrained by crystal orientation, FE-SEM and TEM of Pt-C replicas are best suited to image the overall morphology of microcrystals. However, owing to the decoration effect of Pt-C on the crystal faces, TEM of Pt-C replicas is superior at resolving nanoscale surface structures, including the development of new faces and the different microtopography among nonequivalent faces in microcrystals, which cannot be revealed by FE-SEM. In conjunction with SEM, Pt-C replica provides the evidence that crystals grow in diverse and face-specific modes. The TEM imaging of Pt-C replicas has nanoscale resolution comparable to AFM. AFM yielded quantitative information (e.g., crystallographic orientation and height of steps) of microtopographic features. In contrast to Pt-C replicas and SEM providing three-dimensional images of the crystals, AFM can only image one individual cleavage or flat surface at a time.

A substructure mixtures model for the cyclic plasticity of single slip oriented nickel single crystal at low plastic strain amplitudes

The cyclic plasticity behavior of nickel single crystals oriented for single slip is characterized by uniaxial, symmetric, tension–compression, strain controlled tests carried out at constant plastic strain amplitudes ranging from 5(10 −5) to 1(10 −3). Annealed single crystals are cycled in this manner to post-cyclic saturation and microstructural characterizations, including transmission electron microscopy and optical micrographs of specimen surface replicas are used to verify and evaluate dislocation substructures. Stress–strain and microstructure data are used to construct a mixtures model that couples cyclic plasticity models for three substructures as well as a model for reverse magnetostriction (Villari effect) that is a significant component of inelastic strain at the lower plastic strain amplitudes. The model is used to correlate the stress–plastic strain hysteresis loop responses over the range of plastic strain amplitudes and from cumulative plastic strains from 0.3 to post-cyclic saturation. Complex evolution of substructure plastic strain amplitudes toward their so-called intrinsic values upon the formation of persistent slip bands is modeled. Additionally, bulk Young’s modulus is found to vary significantly with plastic strain amplitude and cumulative plastic strain. A correlation of this behavior is included.

Cryo-SEM and subsequent TEM examinations of identical neural tissue specimen

Low temperature scanning electron microscopy of frozen-fractured specimens under cryo-protecting, non-dehydrating, and non-etching “wet” conditions, that is, direct cryo-SEM, was followed by transmission electron microscopy (TEM) with the same neural tissue specimens. In comparison to replica TEM, direct cryo-SEM can obtain images with a smooth gradation of contrast. The major advantage of direct cryo-SEM combined with TEM was that time was saved in SEM preparation. It had a high potentiality at a wide-range survey of multi-dimensional specimen structures with less-artifacts. Because the specimens were prepared as quickly as possible under “wet” conditions, the target structures could be examined under lower through higher magnifications. In the present study, neuronal and glial elements, such as plasma membranes and cell organelles that include the synaptic vesicles, were localized on the fractured surface. In subsequent TEM examination, it was confirmed that the underlying internal structures could be further characterized from cytological as well as molecular biological aspects. In addition, direct cryo-SEM distinctively demonstrated small intra-membrane particles (ca. 10 nm in diameter). However, due to electron lucency, they could not be confirmed in the re-processed TEM specimens. Applying the present protocol, stereological and internal architectural examinations of the neural tissues have been simultaneously conducted at ultra-fine levels.

Gap Junction Formation and Connexin Distribution in Pig Trophoblast before Implantation

This study describes the gap junctions in extraembryonic cell layers of the preimplantation pig embryo (trophectoderm and endoderm constituting the trophoblast). Using specific antibodies against connexins 31, 32 and 43, we found these connexins in embryos by immunodetection using Western blot and immunofluorescence analysis. By immunofluorescence, the first foci of connexin 31 were detected in the four-cell stage blastomeres, and the first diffuse gap junctions appeared at the eight-cell stage. Intercellular communication was observed with Lucifer yellow transfer to start also at the eight-cell stage around the onset of compaction. Typical gap junctions developed in the trophectoderm of blastocysts, as observed by transmission electron microscopy of thin sections and freeze-fracture replicas. Connexin proteins were differently expressed in time and space: connexin 31 was continuously present in trophectoderm, connexin 32 was essentially found in endoderm during elongation; connexin 43 was distributed in both trophectoderm and endoderm during blastulation and expansion. Connexin 43 was also found in two isoforms, phosphorylated or not, at day 14. Such developmentally regulated connexin expression may be essentially useful to control the exponential growth of trophoblast in preimplantation pig blastocysts.

Cuticular Architecture of Hassalstrongylus epsilon (Nematoda: Trichostrongyloidea).

Hassalstrongylus epsilon is a small nematode, whose adult forms are found among the intestinal microvilli of the water rat Nectomys squamipes, Brants 1827 (Rodentia: Muridae). The external appearance of the cuticle, which presents transversal striations and longitudinal ridges, is described using scanning electron microscopy. Transmission electron microscopy of thin sections and replicas of quick-frozen, freeze-fractured, deep-etched and rotatory shadowed samples showed the presence in the cuticle of struts that arise from the fluid median layer, extending outward to the epicuticle. The cuticle showed the presence of five layers: epicuticle, cortical, fibril-rich, fluid median and fibrous. The cuticle layers were made of an assemblage of fibers that create compartments, which were larger in the fluid region than in the fibril-rich median layer.

Mechanisms of brittle intergranular fracture in Al-Li alloys and comparison with other alloys

Abstract Evidence that brittle intergranular fracture in Al-Li alloys is associated with segregation of lithium, and maybe the formation of two-dimensional grain-boundary phases, is briefly reviewed. Particular attention is drawn to similarities between embrittlement of Al-Li alloys and segregation-induced embrittlement in other materials such as steels. High-resolution fractographic studies of Al-Li alloys to elucidate the atomic mechanisms of brittle intergranular fracture are then described. Brittle intergranular fracture surfaces were often featureless when examined by scanning electron microscopy, but transmission electron microscopy of replicas often revealed very shallow dimples, indicating that crack growth occurred by a very localized microvoid-coalescence process. The observations are explained in terms of decreased cohesion across grain boundaries due to lithium enrichment and grain-boundary structural changes. Low cohesion may facilitate void nucleation by decohesion (in the absence of grain-b...