Transmission Electron Microscope Preparation Research Articles

Scanning electron microscope cathodoluminescence imaging of subgrain boundaries, twins and planar deformation features in quartz

The study of deformation features has been of great importance to determine deformation mechanisms in quartz. Relevant microstructures in both growth and deformation processes include dislocations, subgrains, subgrain boundaries, Brazil and Dauphiné twins and planar deformation features (PDFs). Dislocations and twin boundaries are most commonly imaged using a transmission electron microscope (TEM), because these cannot directly be observed using light microscopy, in contrast to PDFs. Here, we show that red-filtered cathodoluminescence imaging in a scanning electron microscope (SEM) is a useful method to visualise subgrain boundaries, Brazil and Dauphiné twin boundaries. Because standard petrographic thin sections can be studied in the SEM, the observed structures can be directly and easily correlated to light microscopy studies. In contrast to TEM preparation methods, SEM techniques are non-destructive to the area of interest on a petrographic thin section.

Morphological characterization of Phaeocystis antarctica (Prymnesiophyceae)

Zingone A., Forlani G., Percopo I. and Montresor M. 2011. Morphological characterization of Phaeocystis antarctica (Prymnesiophyceae). Phycologia 50: 650–660. DOI: 10.2216/11-36.1Different stages of the life cycle of Phaeocystis antarctica are described by light and electron microscopy on the basis of cultured material isolated from the Atlantic Sector of the Antarctic Circumpolar Convergence, relatively close to the type locality. Species identity was confirmed molecularly on the basis of small-subunit ribosomal DNA. Colonies (up to 2000 µm in diameter) are surrounded by a rather thick membrane and are generally spherical, in some cases presenting a conical protrusion with a pointed end at one side. Colonial cells (4–6 µm) have a rounded or slightly quadrangular outline and possess two chloroplasts. In small and medium-sized colonies, cells may be densely packed under the peripheral colony skin; otherwise they are more or less evenly spaced. Nonmotile cells within or outside the colonies often show one to three short protrusions emerging from a depression between the chloroplast edges. These protrusions may develop into two flagella and one haptonema even within intact colonies. Flagellates of different size (3.5–6.5 µm) are found in the cultures. Their cell body is rounded, elliptical, or pear-shaped, and has a deep, saddle-shaped groove that is dorsoventrally oriented and flanked by the apical edges of the chloroplasts. Two markedly unequal flagella (< 12 and < 18 µm, respectively) and a straight and relatively long (< 4.5 µm) haptonema emerge from the apical groove. Flagellates with scales and five-filament structures were found only in one transmission electron microscope preparation. Scales are extremely thin and of two different sizes, with no clear pattern visible in the material observed. Overall, both flagellates and colonies of P. antarctica present some distinctive characteristics that are discussed in comparison with the other known species in the genus and in the frame of a hypothetical life cycle of the species.

Supramolecular structure of humic acids by TEM with improved sample preparation and staining

Understanding the role of the dissolved organic matter in the environment will benefit from the characterization of its components at the supramolecular scale. With its very high spatial resolution the transmission electron microscope (TEM) allows the determination of colloidal size, structure, and shape on aggregated substances as well as single particles. It also allows the determination of the chemical composition if coupled with an X-ray detector. However, TEM preparation artifacts may hamper the relevance of such observations. This study demonstrates the capacity of TEM for the size and shape analysis of humic substances. Three sample preparation techniques (ultramicrotomy sectioning after embedding into a hydrophilic resin, drop deposition, and absorption) were evaluated with the Fluka humic acid (HA). Additionally, several staining agents (cesium hydroxide, lead citrate, uranyl acetate, and ruthenium tetraoxide) were used to increase the humic acid contrast for TEM observations. An improved simple preparation method with selective staining of the HA is proposed which permits the imaging of the humic acid macromolecules. The results show that the Fluka humic acid is composed mainly of fibrils generated by the interconnection of basic molecules of about 20 nm diameter with some isolated larger macromolecules (30-200 nm).

Microfilament–Disrupting Agent Latrunculin A Induces and Increased Number of Fenestrae in Rat Liver Sinusoidal Endothelial Cells: Comparison With Cytochalasin B

This report describes the effect of the actin–disrupting marine toxin latrunculin A on the cytoskeleton and fenestrae of liver endothelial cells (LECs). Fluorescence microscopy and whole mount–transmission electron microscopic preparations of isolated, purified, and cultured LECs showed that latrunculin A, which sequesters actin monomers and depolymerizes actin filaments, caused profound changes in microfilament organization in LECs. Scanning electron microscopic preparations showed that latrunculin A almost doubles the number of fenestrae within 10 minutes, whereas the diameter is only slightly reduced. All new fenestrae possess the earlier described fenestrae–associated cytoskeleton ring. Cytochalasin B, which disrupts the network of actin filaments, principally by capping the fast growing end of actin filaments, produced comparable effects with regard to actin organization and the number and size of fenestrae. After 1 hour of treatment, an equal maximum number of fenestrae was observed for both agents. The effect of latrunculin A was obtained at concentrations about 100 times lower than cytochalasin B. Thus, two agents that alter the state of actin organization in LECs, albeit by different mechanisms, cause the doubling of the number of fenestrae within 10 to 30 minutes. This indicates that the state of assembly of the actin cytoskeleton is important in the numerical dynamics of LEC fenestrae and that the actin cytoskeleton of LECs is probably the main mechanical regulator for sieving between the sinusoidal blood and the parenchymal cells. Latrunculin A represents a new agent in the study of the de novo formation of fenestrae.

Subdomains of the rough endoplasmic reticulum in colon goblet cells of the rat: lectin-cytochemical characterization.

Using lectin binding, we characterized subdomains of the rough endoplasmic reticulum (rER) in goblet cells of the rat colon. In this cell type, special rER regions can be differentiated on the basis of their content of low electron density and dilated cisternal spaces in conventional transmission electron microscopic preparations. The fine fibrillar content of these cisternal regions demonstrated high-affinity binding with lectins from wheat germ, Helix pomatia, Griffonia simplicifolia I-A4 and -B4, and Ricinus communis I, although not with the sialic acid-specific Limax flavus lectin and the fucose-binding Ulex europaeus I lectin. Sugar-inhibitory experiments indicated that glycoconjugates packed within these regions bound the lectins with higher affinity than molecules present in the Golgi apparatus and secretory granules. Furthermore, the lectin binding patterns of the rER subdomains differed from those of the Golgi apparatus and mucin granules: the terminal sugar residues sialic acid and fucose were demonstrable in the Golgi apparatus and mucin granules and were absent from the rER, while galactose-recognizing lectins bound intensely at these rER regions, weakly to Golgi elements, and were almost absent from mucin granules.

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus.

Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunoreactive to anti-actin at about 60 h (12-14 degrees C). Initially, each myoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.

Effects of cytochalasin B on the fine structure of organized endodermal cells of the early chick embryo.

Fresh pullet eggs (White Leghorn) were incubated for 36 to 48 hours. The blastoderms were exposed to cytochalasin B (CB), 10 or 40 microgram/ml, for 2, 5, and 15 minutes prior to fixation by immersion in buffered chick Ringers solution containing CB, previously dissolved in dimethysulfoxide (DMSO), or by sub-blastodermic injection. Controls fixed in ovo possess relatively flat surfaces with bulges due to uptake of yolk. Numerous microappendages (blebs, microvilli and ruffles) are present, especially at cell margins. DMSO-controls present a similar cell surface except that small blebs are more prominent. The plasmalemmas of CB-treated endodermal cells possess numerous large blebs (2-10 micron in diameter), smaller blebs (0.2 micron) and microvilli. Cell dissociation occurs in selected areas resulting in rounded cells, devoid of microappendages, with peripheral processes. Transmission electron microscopic preparations of tissues similar to those used for scanning electron microscopy reveal that large blebs are filled with membranous material. Microfilaments are present but lack their normal subplasmalemmal arrangement. Microtubules and other cell organelles are apparently unaffected by CB. Evidence in this study supports the concept that cytochalasin B exerts its influence through alteration of the plasmalemma.