Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Elastofibroma dorsi. Cytologic, histologic, immunohistochemical and ultrastructural studies.

Cytologic, light and electron microscopic, and immunohistochemical studies were conducted on a case of elastofibroma. Aspiration cytology showed a characteristic "braidlike" or "fern leaf-like" structure. Immunohistochemically the accumulate was shown to be elastin. Transmission electron microscopy indicated electron-dense, granular aggregates surrounded by microfilaments and collagen, while scanning electron microscopy revealed balls with a ball-of-yarn-like structure consisting of small fibrils, probably of elastin. These structures are unique to this disease and useful for diagnosis.     

Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.     

Microscopies,spectroscopies and spectrometries applied to marine corrosion of copper

Used in combination, surface analytical techniques can resolve spatial relationships between bacteria and localized corrosion, determine specific corrosion mechanisms and differentiate between abiotic and biotic processes. Confocal laser scanning microscopy and scanning vibrating electrode microscopy were used to demonstrate that marine bacteria and anodic sites are co‐located. Environmental scanning electron microscopy coupled with energy dispersive X‐ray spectros‐copy was used to demonstrate dealloying of nickel from copper: nickel alloys. X‐ray absorption spectroscopy, and transmission electron microscopy equipped with electron energy loss spectrometry were used to determine the speciation of copper associated with corrosion products.     

The ups and downs of beam damage,contrast and noise in biological electron microscopy

A personal account of the early problems associated with contrast in images obtained by electron microscopy from biological specimens is presented, together with the effects of electron beam damage. The author's experiences with different types of electron microscope as well as problems of contrast enhancement is described. A short account is given of the physical effects occuring during specimen preparation and their relation to structural preservation when attempting to achieve atomic resolution. Recent developments in biological electron microscopy are also discussed with a view to future trends.     

Colloidal gold, a useful marker for transmission and scanning electron microscopy.

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.     

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.     

Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots.

Ultrastructural perturbations resulting from defects in polymerization of fibrinogen Dusart, a congenital dysfibrinogenemia with the amino acid substitution A alpha 554 arginine to cysteine, were investigated by a variety of electron microscope studies. Polymerization of this mutant fibrinogen on addition of thrombin is impaired, producing clots with decreased porosity and increased resistance to fibrinolysis, resulting in thrombotic complications in the family members with this dysfibrinogenemia. Electron microscopy of rotary-shadowed individual molecules revealed that, in contrast to control fibrinogen, most of the alpha C domains of fibrinogen or fibrin Dusart appeared to be free-swimming appendages that do not exhibit intra- or intermolecular interactions either with each other or with the central domains. The location of albumin on the alpha C domains was demonstrated by electron microscopy using anti-albumin antibodies. Electron microscopy of negatively contrasted fibrin Dusart fibers indicated that they were less ordered than control fibers and had additional mass visible. Electron microscopy of freeze-dried, unidirectionally shadowed fibers showed that they were twisted with a shorter pitch. Scanning electron microscopy revealed that intact clots were made up of thin fibers with many branch points and very small pore sizes. The viscoelastic properties of Dusart fibrin clots measured with a torsion pendulum indicated a marked increase in stiffness consistent with the structural observations.     

High-pressure freezing, cellular tomography, and structural cell biology

Structural cell biology, which we define as electron microscopic analysis of intact cells, suffered a loss of interest and activity following the advances in light microscopy beginning in the 1990s. Interestingly, it is the wealth of detailed observation in the light microscope that is one of the driving forces for the current renewed interest in electron microscopy (EM). A great many cellular details are simply beyond the resolving power of the light microscope. In this article, we describe how electron microscopists are responding to the demands for better preservation of cells and for ways to view cell ultrastructure in three dimensions at high resolution. We discuss how low temperature methods, especially high-pressure freezing and freeze substitution, reduce the artifacts of conventional EM specimen preparation. We also give a brief introduction to cellular electron tomography, a powerful analytical method that can give near-atomic resolution of cell ultrastructure in three-dimensional (3-D) models.     

Genomic,Proteomic, Morphological,and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia – Salmonella genera.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

A Method for the Examination of the Same Cell Using Light, Scanning and Transmission Electron Microscopy

A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis.     

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

Simplified preparation of mycoplasmas, an acholeplasma, and a spiroplasma for scanning electron microscopy.

A simple, effective procedure was developed for scanning electron microscopic examination of mycoplasmas and similar organisms. Cultivation of several mycoplasmal species, an acholeplasma, and a spiroplasma in broth media in Leighton tubes with cover slips resulted in attachment of the organisms to the cover slips. The attached cells were easily processed for either scanning electron microscopy or light microscopy. By eliminating the need for centrifugation, which was used in previously described techniques, physical stress on the cell is minimized. The effects of different preparative procedures on the morphology of Mycoplasma gallisepticum are described.     

Ultrastructural evidence for the presence of bacteria, viral-like particles, and mycoplasma-like organisms associated with Giardia spp

Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.     

Scanning electron microscopy of the eggs of Ascaris lumbricoides, A. suum, Toxocara canis, and T. mystax.

Eggs of Ascaris lumbricoides, A. suum, Toxocara canis, and T. mystax were examined by scanning electron microscopy (SEM). All species under study exhibited pronounced surface ridges. The ridges formed distinctive patterns in T. canis and T. mystax. In the Ascaris species, the ridges are similar except that they are more pronounced in the eggs of A. suum. Operculumlike structures were observed only in Ascaris. Correlation of data from SEM with previously reported transmission electron microscopy suggests that the surface ridges seen in Ascaris eggs are formed by the chitinous layer of the shell.     

Effect of the lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells

Abstract The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.     

The use of an inexpensive,general purpose microcomputer in quantitative cytochemistry

Summary The histochemical localization of alkaline phosphatase in sheep parathyroid gland was investigated using light and electron microscopy. The reaction products of enzyme activity were observed by light microscopy in pericytes. By electron microscopy they were limited to the intercellular spaces between the gland cells, being exclusively confined to the external surface of plasma membrane.     

Ultrastructure of the Secondary Egg Envelope of Cyprinodontidae of the Genus Epiplatys Gill, 1862 (Pisces,Teleostei)

Abstract The structure of the secondary egg envelope of seven species of Cyprinodontidae belonging to the genus Epiplatys was observed by transmission and scanning electron microscopy. The transverse structure of the secondary envelope examined by transmission electron microscopy is a constant feature in all the species studied and represents a good criterion for identification. In contrast, the surface structure of the secondary envelope when observed by scanning electron microscopy exhibits a great diversity of form. It is possible to distinguish each of the seven different species by the pattern of ornamentation, the width of the polygons and the size of the granules comprising them on the secondary egg envelope.     

The fine structure of the periphyton of Chara sp. and Potamogeton natans from Wytham Pond, Oxford, and its significance to the macrophyte-periphyton metabolic model of R. G. Wetzel and H. L. Allen

The fine structure of the periphyton of Chara sp. from Wytham pond, Oxford, is examined using transmission and stereoscan electron microscopy. As a result of this investigation the structural proximity of each periphyte component as required by Wetzel and Allen's metabolic model of macrophyte-periphyton interaction is amply confirmed. The application of stereoscan electron microscopy to a study of feeding in periphyton consumers is also examined.