High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils

High-resolution cryo-scanning electron microscopy was used to examine fibronectin fibrils formed in culture by human skin fibroblasts and in a cell-free system by denaturing soluble plasma fibronectin with guanidine. These studies indicate that the conformation of fibrils formed in culture and in a cell-free system appeared to be similar and that fibronectin fibrils have at least two distinct structural conformations. Fibronectin fibrils can be very straight structures with smooth surfaces or highly nodular structures. The average diameter of the nodules in these fibrils is 12 nm. Both conformations can be seen within an individual fibril indicating that they are not different types of fibronectin fibrils but rather different conformational states. Immunolabeling studies with a monoclonal antibody, IST-2, to the heparin II binding domain of fibronectin revealed that the epitope was buried in highly smooth fibrils, but it was readily exposed in nodular fibrils. We propose, therefore, that fibronectin fibrils are highly flexible structures and, depending on the conformation of the fibril, certain epitopes on the surface may be buried or exposed.     

Correlative light and backscattered electron microscopy of bone--Part I: Specimen preparation methods

A method for preparing nondecalcified bone and tooth specimens for imaging by both light microscopy (LM) and backscattered electron microscopy in the scanning electron microscope (BSE-SEM) is presented. Bone blocks are embedded in a polymethylmethacrylate (PMMA) mixture and mounted on glass slides using components of a light-cured dental adhesive system. This method of slide preparation allows correlative studies to be carried out between different microscopy modes, using the same histologic section. It also represents a large time savings relative to other mounting methods whose media require long cure times.     

Scanning electron microscopy and transmission electron microscopy study of hot-deformed γ-TiAl-based alloy microstructure

The aim of this work was to assess the changes in the microstructure of hot‐deformed specimens made of alloys containing 46–50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 °C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

Correlative fluorescence and electron microscopy in tissues: immunocytochemistry

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

Human chromatid ultrastructure: new observations with scanning and transmission electron microscopy

Two new observations have been made on human chromatid/chromosome ultrastructure using both scanning and transmission electron microscopy (SEM, TEM). A bipartite, apparently half-chromatid-like structure was observed in whole human chromosomes studied with SEM and in longitudinally sectioned chromosomes analyzed with TEM. In addition, we also observed a zipper-like configuration as the parallel sister chromatids separated likely due to the supercoiled structure of the chromosome and chromatid. It is possible that either or both of these new observations resulted from our (improved) method of preparing the chromosomes for SEM and TEM.     

Cathodoluminescence in transmission electron microscopy

We describe a cathodoluminescence spectrometer that is attached to an analytical transmission electron microscope. After a brief consideration of the set‐up and the peculiarities of recording spectra and of mapping defect distributions in panchromatic and monochromatic cathodoluminescence, we discuss two examples of applications. Emphasis is placed on the potential for obtaining novel information about materials and processes on a microscopic and a nanoscopic scale by combining cathodoluminescence with the structural and chemical information for the same site of the specimen. We select an example concerning the role of In distribution in light emission from InGa/GaN quantum wells and a second one concerning the analysis of the initial electron radiation damage of Cu(In,Ga)Se₂ photovoltaic films.     

Scanning electron microscopy studies of protein-functionalized atomic force microscopy cantilever tips

Protein-functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand-receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein-functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein-coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA-coated surface did not significantly alter the surface morphology. However, we found that avidin-functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand-receptor binding force experiments.     

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer‐based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.     

A novel sample holder allowing atomic force microscopy on transmission electron microscopy specimen grids: repetitive, direct correlation between AFM and TEM images

A novel sample holder that allows atomic force microscopy (AFM) to be performed on transmission electron microscope (TEM) grids is described. Consequently, AFM and TEM images were repeatedly obtained on exactly the same sample area. For both techniques, a thin carbon film was used as the imaging substrate. Although these techniques have been previously used in conjunction, AFM and TEM images on exactly the same area have not been repeatedly obtained for any system. Correlation of AFM and TEM images is useful for work where the three‐dimensional topographical information provided by the AFM could be used to better interpret the two‐dimensional images provided by the TEM and vice versa. To demonstrate the applicability of such correlation, new results pertaining to a fibrillar collagen system are summarized.     

Atomic force and scanning electron microscopy of atmospheric particles

Atomic force microscopy (AFM) and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) have been used for both morphological and elemental mass analysis study of atmospheric particles. As part of the geometrical particle analysis, and in addition to the traditional height profile measurement of individual particles, AFM was used to measure the volume relative to the projection area for each particle separately, providing a particle shape model. The element identification was done by the EDS analysis, and the element mass content was calculated based on laboratory calibration with particles of known composition. The SEM-EDS mass measurements from two samples collected at 150 and 500 m above the surface of the Mediterranean Sea were found to be similar to mass calculations derived from the AFM volume measurements. The AFM results show that the volume of most of the aerosols that were identified as soluble marine sulfate and nitrate aerosol particles can be better estimated using cylindrical shapes than spherical or conical geometry.     

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.     

Cross-sectional transmission electron microscopy of silicon LSI circuits and josephson junction devices

Cross-sectional transmission electron microscopy (XTEM) has been used to diagnose silicon LSI circuits and Josephson junction devices. For LSI circuits, some typical failure problems have been presented. For Nb-Si-Nb Josephson junction, microholes in the thin silicon layer have observed, and they are responsible for the short circuiting of these devices.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Identification of weak interfaces in composites using transmission electron microscopy

A new experimental technique was developed to identify crack paths with a resolution of nanometres in fibre-reinforced composites. Cracks were introduced through Vickers indentations on one side of the sample prior to starting the thinning process. Indentations were placed close to the fibres in order to get enough cracks at the fibre/matrix interface in the electron-transparent region of the thinned sample. The technique was used in a Nicalon-fibre Al₂O₃ matrix composite prior to and after a heat treatment at 1200 °C for 1 h. The analysis of the crack paths allowed the identification of the weakest interface in each condition.     

Processing and characterization of canine mixed mammary tumor using transmission electron microscopy

Canine mammary gland tumors represent the second most frequent type of neoplasm in dogs, being an important problem within veterinary medical field. Canine mixed mammary tumors are the most common; the use of a transmission electron microscope (TEM) can contribute as a tool in its diagnosis by determining the characteristics of cellular components from numerous neoplasms. The aim of this study was to characterize cytologically canine mammary mixed tumor by the use of the TEM. A biopsy collected from an 11 years old bitch Shih‐Tzu and analyzed by histopathology was used for ultrastructural analysis. Specimens obtained were double stained using uranyl acetate and lead citrate prior to observation in the TEM. The protocol established to transmission electron microscopy observation allowed the identification of main cellular characteristics of canine mixed mammary tumors; however, it was not possible a detailed visualization of the organelles due to the preservation of the biopsy in formaldehyde.     

Investigation of cholesterol,bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallstones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue—cholesterol, red, orange—bilirubin, yellow, green—protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultra-thin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.