Macromolecular substructure in nuclear pore complexes by in-lens field-emission scanning electron microscopy

Scanning electron microscopy (SEM) has produced a wealth of novel images that have significantly complemented our perception of biological structure and function, derived initially from transmission electron microscopy (TEM) information. SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by reduced resolution in comparison with TEM. Recently, SEM resolution has been considerably improved by the advent of high-brightness sources used in field-emission instruments (FEISEM) which have produced resolution of around 1 nm, virtually equivalent to TEM “working resolution.” Here we review our findings in the use of FEISEM in the imaging of nuclear envelopes and their associated structures, such as nuclear pore complexes, and the relationships of structure and function. FEISEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding its functional significance.     

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.     

Double-layer coating for high-resolution low-temperature scanning electron microscopy

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in low-temperature scanning electron microscopy of bulk specimens. At high primary magnifications (e.g. 100 000x) a hydrated sample is usually severely damaged after one slow scan (about 3000 e^— nm^—2). The consequences of this beam damage are significantly reduced by coating the frozen-hydrated sample with a 5–10-nm-thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g. 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field-emission SEM and a sensitive backscattered electron detector, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.     

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.     

A novel application of microsphere perfusion and scanning electron microscopy to the identification of pulmonary arterioles in guinea-pig and rabbit lungs

In arterioles of the lung the intravascular blood pressures are lower than in comparable vessels in the systemic circulation and the arteriole walls are thinner. Therefore, it is very difficult to distinguish between arterioles and venules of the same size using scanning electron microscopy. This study describes a novel application of latex microsphere perfusion and scanning electron microscopy which distinguishes between pulmonary arterioles and venules on the basis of endothelial cell morphology. Microspheres, 90 and 45 μm in diameter, were perfused into the arterial side of the pulmonary circulation of guinea-pig and rabbit lungs. Scanning electron microscopy of the arterioles on both sides of the lodged microspheres indicated that the endothelial cells are spindle shaped. In contrast, the endothelial cells of equal diameter venules are polygonal. Furthermore, the nuclei of the arteriolar endothelial cells were significantly (P = 0·019) narrower than those of endothelial cells in venules of equal diameter. Finally, it was observed that the differences between arteriole and venule endothelial cells persisted distally to the capillaries.     

High-resolution field-emission scanning electron microscopy of nuclear pore complex

The nuclear pore complex (NPC) is a large macro-molecular assembly inserted into the nuclear envelope (NE). It controls the traffic of proteins, RNA, and RNA proteins between nucleus and cytoplasm. Its chemical composition and function are now intensively investigated in many organisms. To understand this unique membrane transport system, we must know the supramolecular organization of the NPC. In recent years, high-resolution field-emission scanning electron microscopy has made important contributions to our knowledge of NPC structure. It provided the first images of the complex and beautiful fish trap-like structure of its intranuclear surface, documented in this review. It also has provided the first images of a new intranuclear structure, a system of branching hollow cables connecting the nuclear interior with the NPCs at the nuclear surface. Most likely this is an intranuclear transport system, assuring efficient exchange between the nuclear interior and the NE, especially in large nuclei.     

Effects of brefeldin-A on Golgi morphology in human cultured fibroblasts observed in three-dimensional stereo scanning electron microscopy

Brefeldin A (BFA) has been reported to cause disassembly of the Golgi. We have used three-dimensional (3-D) high-resolution scanning electron microscopy (HRSEM) to investigate these effects in human skin fibroblast cells. The spontaneous reassembly during prolonged exposure to BFA and some effects of forskolin were observed. A BFA concentration of 5μg/ml caused Golgi complexes to become vesicular, resulting in a progressive decrease in the size of the Golgi. Morphologic changes were visible within 2 min of BFA incubation, and by 30 min no identifiable Golgi could be found. Spontaneous reassembly of the Golgi apparatus upon the removal of the BFA or with continued long-term exposure with BFA could not be confirmed. Preliminary experiments with forskolin were not effective in reversing or inhibiting the effects of BFA in human fibroblast cells grown in culture. This inability for spontaneous reassembly and nonreversal by forskolin may reflect a differential effect of BFA in various cell types. HRSEM has proven to be useful for observing 3-D morphologic effects of BFA in Golgi.     

Rapid diagnosis of fungal infection of intravascular catheters in newborns by scanning electron microscopy.

Intravascular catheters carry a significant risk of becoming colonized with bacteria and fungi and are important risk factors of septicemia in premature neonates. The study was undertaken to evaluate whether scanning electron microscopy (SEM) examination of removed catheters can be useful in early diagnosis of plastic infection by Candida, providing information useful for initiation of an eventual therapy. The evolution of biofilms in 28 catheters (umbilical or central) implanted in 24 newborns for prematurity was studied by SEM and transmission electron microscopy (TEM). In 4 of 24 patients, SEM examination revealed the presence of Candida in form of yeast or hyphae. In one of these patients, TEM confirmed the presence of organisms. In each case, hemoculture and culture of the catheter itself confirmed the diagnosis. The study demonstrates that SEM can identify fungi in the biomaterials covering the catheter surface in a few hours, allowing an early diagnosis of plastic infection.     

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.     

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.     

Field-emission scanning electron microscopy of the internal cellular organization of fungi

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.     

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.     

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.
AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).     

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.     

A scanning and transmission electron microscopy study of the parabronchial unit in quail (Coturnix coturnix) and town pigeons (Columba livia).

A combined scanning electron (SEM) and transmission electron microscopy (TEM) investigation was undertaken to gain insight into the complex structural pattern of the atrial compartment and the gas exchange tissue of parabronchial units in quail and town pigeons. The aim was also to depict the changes taking place in the parabronchial unit in the late prehatching and early posthatching periods in quail. The standard SEM and TEM investigation was carried out in 13 mature quail and 8 town pigeons. The developmental study involved embryonic quail (Days 15, 16, 17), newly hatched quail, quail 24 h after hatching, and quail aged 2, 10, 19, and 25 days (3 individuals per group). The luminal relief of the parabronchus is formed by anastomosing interatrial septa delineating the atrial pits, which are thinner and shallower in pigeons. The atrial bottom opens in mature individuals into 3-6 infundibula. The extracellular material represented by trilaminar substance, which does not appear until hatching, veils the surface relief of the parabronchial epithelium, which is consequently hardly accessible to three-dimensional visualization. Only in town pigeons with fewer discontinuous layers of extracellular material was it possible to visualize the surface of the atrial epithelium, that is, of the granular and squamous atrial cells. The SEM analysis has convincingly shown the intricate spatial organization of atria, infundibula, and air and blood capillaries of the gas exchange tissue. The retinacula, that is, parallelly arranged processes of squamous respiratory cells bridging the air-capillary lumina, were evidenced by SEM and TEM. The complex structure of the avian parabronchus has been successfully demonstrated in the present SEM and TEM study.     

Junctional complex revisited by high-resolution scanning electron microscopy

The present study correlates the ultrastructural morphology of junctional complexes as revealed by transmission electron microscopy (TEM) with that observed by high-resolution scanning electron microscopy (HRSEM), thanks to a new modification of the osmium tetroxide maceration technique. The removal of all cytoplasmic organelles by this technique allows the inspection of the inner side of the plasmalemma. With this treatment, a continuous band of tightly packed particles is observed at the most apical portion of lateral membranes. Just below this band, irregular clusters of apparently identical particles are placed all around the cellular contour. The topographical correspondence among these clusters and spot desmosomes seen by TEM identifies them as desmosomes. The continuous band seems to represent the combination of both zonulae, occludens and adherens. Regarding the nature of the particles, we suppose that they probably consist of peripheral membrane proteins clustered at the cytoplasmic surface of intercellular junctions and involved in the linkage between cytoskeleton and plasmalemma.     

New system for secondary electron detection in variable-pressure scanning electron microscopy

A novel secondary electron detection system combining a two‐stage detector head and a differential pumping system is presented. The detector head consisted of a scintillation Everhart‐Thornley detector and a microsphere plate, separating it from the lower vacuum in the intermediate chamber (below 0.1 mbar). The system was arranged asymmetrically, which should contribute to a lower gas leakage through the plate and a longer life span of the plate. The system offered all the advantages of the scintillator detector in a wide range of gas pressures, from high vacuum to those of the order of 10 mbar, typical of high‐pressure scanning electron microscopy.     

A rapid and simple technique for correlating light microscopy,transmission and scanning electron microscopy of fixed tissues in Epon blocks

A simplified and standardized technique for close correlation between light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) is described. Perfusion and immersion fixed tissue specimens were embedded in Epon 812 and cut for conventional LM and TEM. The Epon blocks with remaining tissue were thereafter treated with epoxy solvent (ethanol-NaOH solution) for partial epoxy resin removal only (dissolving rate approx 33μm/h). The blocks with partially blotted tissue specimens were then critically point dried and gold coated for SEM. This method, in an easy way, allows repeated observations with LM, TEM and SEM with preserved fine structure and exact correlation. Since the technique is so simple and there is no need for special equipment the method can easily be adopted in all laboratories with basic SEM standards.     

Manipulating biological samples for environmental scanning electron microscopy observation

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.