The biomass dynamics of cyanobacteria in an annual cycle determined by confocal laser scanning microscopy

We recently published a method for estimating cyanobacteria biomass in delta microbial mats from the Ebro river using confocal laser scanning microscopy (CLSM). The present paper uses this method for identifying different groups of cyanobacteria and for determining their biomass dynamics. Microcoleus chthonoplastes and the Lyngbya-Oscillatoria were the most important contributors to the cyanobacterial biomass throughout the study. Biomass values ranged from 1.29 to 6.55 mgC/cm2 sediment for Microcoleus chthonoplastes, and from 128 microgC/cm2 to 3.16 mgC/cm2 sediment for Lyngbya-Oscillatoria. This technique is useful for determining biomass and for studying filamentous cyanobacteria as well as ramified eukaryotic cells. Confocal serial sections through the samples can be obtained. Two-dimensional images from the samples can be used to calculate the biomass of individual cells.     

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.     

Environmental scanning electron microscopy investigations of biodeterioration

Case studies will be presented in which environmental scanning electron microscopy (ESEM) has been used to provide unique insight into the role of microorganisms in deterioration processes. ESEM is an excellent tool for demonstrating spatial relationships between microorganisms and substrata because hydrated, nonconducting samples can be viewed with a minimum of manipulation. Copper and iron-rich deposits associated with bacteria were detected within corrosion layers on copper and steel surfaces, respectively. Fungal mycelia growing on wooden storage spools were shown to penetrate protective grease on carbon steel wire rope in contact with the spool and to cause localized corrosion. Large numbers of marine bacteria were documented within paint blisters and disbonded regions of fiber-reinforced polymeric composites. In both cases, it appears that microbial gas production resulted in mechanical damage to the substrata.     

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.     

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.     

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.     

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.     

Evaluating sharpness functions for automated scanning electron microscopy

Fast and reliable autofocus techniques are an important topic for automated scanning electron microscopy. In this paper, different autofocus techniques are discussed and applied to a variety of experimental through-focus series of scanning electron microscopy images with different geometries. The procedure of quality evaluation is described, and for a variety of scanning electron microscope samples it is demonstrated that techniques based on image derivatives and Fourier transforms are in general better than statistical, intensity and histogram-based techniques. Further, it is shown that varying of an extra parameter can dramatically increase quality of an autofocus technique.     

Dinosaur eggshell study using scanning electron microscopy

Visualization and analysis of structural features in fossil dinosaur eggs by scanning electron microscopy augment information from traditional petrographic light microscopy. Comparison of characteristics in fossil and modern eggshells allows inferences to be made regarding dinosaur reproductive biology, physiology, and evolutionary relationships. Assessment of diagenetic alteration of primary eggshell calcite structure that occurs during fossilization provides important information necessary for taxonomic identification and paleoenvironmental interpretations.     

A method for imaging single clay platelets by scanning electron microscopy

A method for preparing and observing clay platelets for size and shape analysis using scanning electron microscopy (SEM) was developed. Samples of the clay platelets were prepared by polyelectrolyte-assisted adsorption onto a pyrolytic graphite surface. The use of graphite as a substrate was advantageous because of the low number of secondary electrons emitted from it during imaging by SEM. The resulting low background noise allowed the emission from the approximately 1 nm thick clay sheets to be clearly visualized. Images of centrifuged montmorillonite showed large exfoliated platelets with lateral dimensions between 200 and 600 nm. In contrast, uncentrifuged montmorillonite appeared to contain a large amount of unexfoliated clusters. Although it was not possible to obtain high-quality images of the smaller sheets of Laponite RD, the images of this material did contain size features comparable to the approximately 30 nm2 size reported previously using light scattering, as well as transmission electron and atomic force microscopies.     

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.     

Three‐dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscpy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze‐fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4–64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing‐over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high‐resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

Environmental scanning electron microscopy of marine aggregates

Marine aggregates were examined for the first time in the hydrated state using an environmental scanning electron microscope (ESEM). Sample preparation consisted of fixation followed by rinsing with distilled water to remove excess salts and fixative. Aggregates were continuously observed at resolutions comparable to conventional scanning electron microscopy through stages of hydration, from completely immersed to desiccated. Because no metallic coating is required, energy-dispersive X-ray spectroscopy (EDXS) can be used to analyse rapidly constituent elements occurring at low concentrations with no spectral interference. Subtle differences in mineral particles were seen in both EDXS spectra and in direct observation of relative hydration, reflecting apparent differences in mineralogy. ESEM enabled examination of effects of desiccation and rehydration on individual particles composed primarily of hydrated polymer and eliminated dehydration artefacts in delicate organisms.     

Three-dimensional reconstruction of biological macromolecular complexes from in-lens scanning electron micrographs

Two helical samples: F-actin and the bacteriophage T4 tail sheath were reconstructed in three dimensions from contrast enhanced (rotational shadowing and negatively stained) in-lens cryo-field emission scanning electron micrographs, using the iterative real-space helical reconstruction method. The F-actin – and bacteriophage T4 reconstructions compare favourably to an atomic model refined against fibre diffraction data and a cryo-electron microscopy reconstruction, respectively. These results show that single-particle methods, developed for macromolecules imaged in the transmission electron microscope can be applied to cryo-field emission scanning electron micrographs data with appropriate symmetry.     

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.     

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.     

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.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

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.