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PubMed2001

Structural diversity of ex vivo amyloid fibrils studied by cryo-electron microscopy.

Cryo-electron microscopy studies are presented on amyloid fibrils isolated from amyloidotic organs of two patients with different forms of hereditary non-neuropathic systemic amyloidosis, caused, respectively, by Leu60Arg apolipoprotein AI and Asp67His lysozyme. Although ex vivo amyloid fibrils were thought to be more uniform in structure than those assembled in vitro, our findings show that these fibrils are also quite variable in structure. Structural disorder and variability of the fibrils have precluded three-dimensional reconstruction, but averaged cryo-electron microscopy images suggest models for protofilament packing in the lysozyme fibrils. We conclude that ex vivo amyloid fibrils, although variable, assemble as characteristic structures according to the identity of the precursor protein.

Amino Acid Substitution↗
PubMed1991

Development of the glycogen body of the Japanese quail, Coturnix japonica: II. Observations of electron microscopy.

Transmission electron microscopic observations of the relationships of the cells of the glycogen body and those of nervous tissue in the lumbosacral spinal cord show that one day after hatching, glycogen cells at the lateral margins of the glycogen body lie in close association with elements of the neuropil in the adjacent spinal cord. Glycogen cells and their processes appear to extend into the neuropil, where they contact other glycogen cells, blood vessels, neurons, and neuroglia. Junctional complexes and synapses occur among glycogen cells, astrocytes, and oligodendrocytes. Other indications of specialized activities were surmised by the presence of annulate lamellae in continuity with extensive arrays of smooth endoplasmic reticulum in several glycogen cells. These observations enhance our earlier views that cells of the avian glycogen body are metabolically active in the synthesis and degradation of glycogen for neuronal support and myelination in the central nervous system.

Animals↗
PubMed1990

Correlative light, transmission, and high resolution (SE-I) scanning electron microscopy studies of rhesus adrenocortical vascular morphology.

A detailed correlative morphologic description using light microscopy (LM), transmission electron microscopy (TEM) and high resolution SE-I scanning electron microscopy (SEM) was conducted on the capillary endothelium of the zona-fasciculata (Z-F) in juvenile male rhesus monkeys. The glucocorticoid synthesis and release phenomena, associated with stress stimulated release of adrenocorticotropic hormone (ACTH) via the hypothalamic-pituitary axis, intimately involves capillaries of the Z-F. A comprehensive study of all the ultrastructural features implicated in the transendothelial uptake of steroidogenic precursors and release of glucocorticoids in perfused rhesus adrenals has not previously been made. This report presents correlative images of transendothelial openings that include previously described single diaphragmed fenestrae and plasmalemma vesicles, and double diaphragmed transendothelial channels. New observations of endothelial cell pockets, tight junctional complexes and membrane filled ghost sacs were recorded from perfused rhesus adrenal. Membranous ghosts associated with adrenocortical endothelium were reported in a previous TEM study of perfused rat, however the potential argument existed that ghosts were artifactual. Their role as steroid hormone releasing structures remains an open question, yet their structural characteristics appear justified based on imaging of identical profiles observed in perfused rhesus adrenocortical specimens. These structural features are considered for the potential of gating and sorting of metabolites, and release of glucocorticoids in response to ACTH stimulated stress events.

Adrenal Cortex↗
PubMed1975

An experimental mouse testicular teratoma as a model for neuroepithelial neoplasia and differentiation. II. Electron microscopy.

The electron microscopic features of the stages of divergent neuroepithelial differentiation in the solid implants of a transplantable mouse testicular teratoma (OTT-6050) are presented and compared to the sequential stages of cytogenesis that have been described in the developing avian and mammalian central nervous system. Primitive neuroepithelial tumor cells showed the features of undifferentiated multipotential matrix (or ventricular) cells of the neural tube. They formed primitive medullary rosettes, from which various transitions were traced to more differentiated, cilia-containing ependymoblastomatous rosettes; the transitional features included increased granular endoplasmic reticulum and microvilli formation. Glial differentiation was characterized by the presence of mature ependymal rosettes and of astrocytes containing glial filaments. Neuronal differentiation included the development of synapses and the presence of dense-core vesicles in nerve cell processes. No intermediate cell forms were found that suggested multiple lines of differentiation occurring within a single cell.

Animals↗
PubMed2006

Cytoskeletal elements in bacteria Mycoplasma pneumoniae, Thermoanaerobacterium sp., and Escherichia coli as revealed by electron microscopy.

Recently, electron microscopic studies on the eubacteria Mycoplasma pneumoniae, Thermoanaerobacterium sp., and Escherichia coli have revealed the existence of cytoskeletal elements so far unknown in prokaryotes. The wall-less bacterium M. pneumoniae contains, in close vicinity to the inner face of the cytoplasmic membrane, a helically organized lining composed of protein elements that form a regular network of meshes that encloses the entire cytoplasm. Numerous regularly spaced pin-like structural elements, the stalks with terminal knobs, connect the lining with the cytoplasmic membrane. In this bacterium, a specific rod-like structural element is located in the tip region. Occasionally, it is bent or twisted. It consists of two matching blade-like sub-elements. A number of parallel linkers, extending from the edges of the rod, make contact with the lining. The proximal end of the rod is attached to a wheel-like complex. Fibrils originating from the wheel cross the cytoplasm and make contact with the lining. E. coli contains a similar helically organized lining close to the inner face of the cytoplasmic membrane. Groups of ribosomes (polysomes) were seen to be attached to the helical elements of the lining. A feature that is common to both bacteria and to Thermoanaerobacterium sp. appears to be that the lining and the fibrils crossing the cytoplasm contain a high number of copies of the bacterial elongation factor Tu (EF-Tu). This indicates that this protein may play an important role as a structural element in bacterial cytoskeletons. This notion was supported by experiments in which the cytoskeleton in E. coli was destabilized by induced expression of truncated EF-Tu, with the consequence of cell lysis, and by the finding that in vitro polymerization of monomeric EF-Tu into protofilaments was hindered in a mixture of full-size EF-Tu and truncated EF-Tu consisting of domain 3 only. Current research and developmental efforts are aimed at the design of a new class of antibacterial drugs, acting by destabilization of the EF-Tu-containing bacterial cytoskeleton, and of an innovative mode of inducible lysis of recombinant bacteria by controlled destabilization of the EF-Tu-containing cytoskeleton.

Cell Membrane↗
PubMed2006

Growth hormone cell phagocytosis in adenohypophysis of mosaic mice: morphological and immunocytochemical electron microscopy study.

An electron microscopy immunocytochemical study was performed to determine the expression pattern of growth hormone (GH) in mosaic mutant mice adenohypophysis. In normal condition GH was restricted to the secretory granules of all growth hormone cells. Mosaic mice adenohypophysis contained growth hormone cells which have distinctive GH labeled secretory granules at the level seen in control animals. Ultrastructurally, some GH cells of mosaic mice presented abnormalities, but labeling intensity of secretory granules in these cells was always comparable to the basal condition. The striking findings presence of two forms (simple and activated) of folliculo-stellate cells (FS) in close association trough gap or tight junction with GH cells localized especially near the perivascular space. Frequently, in cytoplasm of FS cells, large clusters containing fragments of GH labeled cell were present. Additionally, the existence of large intracellular, electron-lucent spaces, with remnant cellular material in parenchyma of mosaic mutant mice adenohypophysis could suggest intensive process of GH-cell destruction. Our electron microscopy immunocytochemical results provide evidence for loss of GH cells in mosaic mice by phagocytosis. We suppose that impaired body growth observed in mosaic mutant male rats may be, at least partially, a consequence of an alteration in somatotropic axis activity. Loss of GH cells in mosaic mice by phagocytosis supported by FS cells may contribute to this effect.

Animals↗
PubMed2005

Characterization of Alzheimer paired helical filaments by electron microscopy.

We show how electron microscopy can be used to answer several critical issues in neurodegenerative disorders that course with the formation of aberrant filamentous structures. Thus, electron microscopy is a useful technique to study in vitro assembly of pathogenic proteins, to map the regions involved in filament formation, as well as to detect by immunoelectron microscopy which proteins bind to the filaments. Furthermore, electron microscopy is the main technique used to discover if an animal model develops fibrillar pathology and if those filaments are similar to those found in human patients. This review focuses on Alzheimer's disease and related tauopathies, although similar studies have been done with other neurodegenerative disorders as, for example, Huntington's disease.

Alzheimer Disease↗
PubMed2005

Introduction to 3D reconstruction of macromolecules using single particle electron microscopy.

Single-particle electron microscopy has now reached maturity, becoming a commonly used method in the examination of macromolecular structure. Using a small amount of purified protein, isolated molecules are observed under the electron microscope and the data collected can be averaged into a 3D reconstruction. Single-particle electron microscopy is an appropriate tool for the analysis of proteins that can only be obtained in modest quantities, like many of the large complexes currently of interest in biomedicine. Whilst the use of electron microscopy expands, new methods are being developed and improved to deal with further challenges, such as reaching higher resolutions and the combination of information at different levels of structural detail. More importantly, present methodology is still not robust enough when studying certain tricky proteins like those displaying extensive conformational flexibility and a great deal of user expertise is required, posing a threat to the consistency of the final structure. This mini review describes a brief outline of the methods currently used in the 3D analysis of macromolecules using single-particle electron microscopy, intended for those first approaching this field. A summary of methods, techniques, software, and some recent work is presented. The spectacular improvements to the technique in recent years, its advantages and limitations compared to other structural methods, and its future developments are discussed.

Cryoelectron Microscopy↗
PubMed1983

Scanning electron microscopy and X-ray microanalysis of foreign bodies associated with Silastic implants in humans.

Polydimethylsiloxane (Silastic) capsules containing megestrol acetate have been implanted in subcutaneous tissue as a method of long-term contraception. Histological studies revealed a granulomatous foreign body reaction around these capsules with birefringent crystals in multinucleated giant cells. Although several authors had interpreted these crystals as steroids, this seemed unlikely since the tissue had been processed with organic solvents. Analysis of these crystals by polarization microscopy, scanning electron microscopy, and X-ray microanalysis demonstrated that the material was talc. The talc was probably introduced as a contaminant from gloves during the implantation. Further analysis showed that capsule fragmentation could not have produced the material since Silastic particles could not be detected.

Contraceptive Agents↗
PubMed1987

Correlative light and electron microscopy of platelet adhesion and fibrinogen receptor expression using colloidal-gold labeling.

Differences in the shape change responses of platelets to various polymers may determine the thrombotic potential of these materials. Substrate-dependent variation in the expression and motility of the platelet fibrinogen receptor may underlie these differences due to this ligand's essential role in platelet aggregation. In this study we examine platelet activation on polyetherurethaneureas (PEUUs) presently being evaluated for vascular prosthetic applications. These polymers are prepared as 50-100nm thin films suitable as substrates for consecutive light microscopy, high voltage electron microscopy (HVEM), and SEM. 18nm colloidal gold coupled to fibrinogen permits visualization of that receptor's motility in living cells by video-enhanced light microscopy. Subsequent HVEM and SEM of identified cells provides correlative ultrastructure and surface morphology. The use of these novel support films coupled with the multiple modes of microscopy and colloidal gold labeled ligands permits in depth study of the molecular biology of cell adhesion to materials with varied, and known, surface properties. The motility of the platelet fibrinogen receptor was related to the extent of cytoskeletal reorganization, which, in turn, was influenced by polymer surface energetics. Platelets adherent to more hydrophobic PEUUs had greater receptor mobility and receptor redistribution than platelets adherent to more hydrophilic PEUUs. The most extensive receptor motility and redistribution was observed on Formvar, a non-PEUU with low surface-water energy, suggesting that additional surface properties are of importance in determining platelet spreading and fibrinogen receptor motility.

Biocompatible Materials↗
PubMed2001

pH-Responsive Polymer Microspheres: Rapid Release of Encapsulated Material within the Range of Intracellular pH Financial support was provided by the NSF (Cooperative Agreement No. ECC9843342 to the MIT Biotechnology Process Engineering Center), the NIH (GM26698), and the Department of the Army (Cooperative Agreement DAMD 17-99-2-9-001 to the Center for Innovative Minimally Invasive Therapy). D.M.L. wishes to thank the NIH for a Postdoctoral Fellowship (NRSA Fellowship No. 1 F32 GM20227-01). Scanning electron microscopy and confocal microscopy images were acquired by William Fowle at the Northeastern University Center for Electron Microscopy. Dr. David Putnam, David Ting, and Tommy Thomas are thanked for helpful discussions.

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Journal Article↗
PubMed2004

The role of electron microscopy for the diagnosis of glomerulopathies.

CONTEXT: Electron microscopy has been used for the morphological diagnosis of glomerular diseases for more than three decades and its value has been widely emphasized. However, recent reports have analyzed the routine use of electron microscopy critically. Its use in other areas of diagnosis such as tumor diseases has declined considerably; in addition, in view of the unavoidable financial pressure for the reduction of costs due to investigations and diagnostic routines, the selection of cases for electron microscopy has been quite rigorous. OBJECTIVE: To identify the glomerular diseases that depend on electron microscopy for a final diagnosis, by means of reviewing renal biopsies performed over a 12-year period. DESIGN: Prospective SETTING: Hospital Ana Costa, Hospital Guilherme Alvaro and Serviço de Anatomia Patológica de Santos, Santos, São Paulo, Brazil. PARTICIPANTS: 200 consecutive renal biopsies obtained from private hospitals and the teaching hospital from 1979 to 1991 were studied. MAIN MEASUREMENTS: All cases were analyzed via light microscopy, immunofluorescence and electron microscopy. The diagnosis was first made via light microscopy plus immunofluorescence and then via electron microscopy. RESULTS: Electron microscopy was diagnostic or essential for diagnosis in 10.0% of the cases, corresponding to 3.4% of primary glomerulopathies and 100% of hereditary glomerulopathies. Electron microscopy was contributory (useful) to the diagnosis in 5.5% of the cases, confirming the preliminary diagnosis formulated on the basis of clinical and laboratory data and light microscopy plus immunofluorescence findings. We obtained a 7.5% rate of discordant immunofluorescence, which was considered as such when negative immunofluorescence findings were not confirmed by electron microscopy. The final diagnosis with the use of light microscopy plus immunofluorescence alone was 77.0%. CONCLUSIONS: It was possible to diagnose with certainty a great percentage of glomerulopathies (82.5-90% of the cases) based on the light microscopy and immunofluorescence findings alone. Electron microscopy was essential for the diagnosis of hereditary nephropathies.

Biopsy, Needle↗
PubMed2003

Pulsed and scanned carbon dioxide laser resurfacing 2 years after treatment: comparison by means of scanning electron microscopy.

Studies have reported short-term and long-term (1-year) findings for laser skin resurfacing. Two of the most popular systems used for this procedure, the continuous-wave Sharplan 40C SilkTouch system and the pulsed Coherent 5000C UltraPulse system with a computer pattern generator, were previously compared for a range of follow-up times up to 1 year, using light microscopy and transmission electron microscopy. This study analyzed the 2-year morphological differences using scanning electron microscopy. Tissue samples were obtained from 10 patients (age range, 50 to 72 years; skin types II and III) who had undergone laser resurfacing 2 years previously. One half of the face of each patient had been treated with the continuous-wave system and the other half with the pulsed system. The samples were subjected to scanning electron microscopy. On the continuous-wave-treated side, significantly better dermal collagen organization was observed at 2 years, with plump-appearing fibers that were closely knit to form a compact structure. On the side treated with the pulsed system, the collagen fibers in the papillary dermis were more loosely arranged and appeared drier. In both the continuous-wave-treated and pulsed-treated areas, the epidermis appeared healthy and exhibited some signs of age-related deterioration, with slightly flatter plaques and somewhat more flaking keratin on the pulsed-treated side. Probably because of the greater degree of residual thermal damage associated with the continuous-wave system, at 2 years after treatment there was more prolific synthesis and better orientation of collagen fibers, which were maintained for longer times, compared with the pulsed-treated specimens.

Aged↗
PubMed1989

Scanning electron microscopy of the small intestine mucosa in children with celiac disease after long-term dietary treatment.

Jejunal mucosal specimens from twenty children with celiac disease were studied by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) after one year of dietary treatment. An ultrastructural morphometric study was performed in five patients who had an intestinal permeability (IP) test. Seventeen patients were tested for serum antigliadin antibodies (AGA). In ten children, in whom LM showed partial villous atrophy, SEM and TEM examination confirmed the lesion. In the second group (10 children) with normal morphology at routine LM, SEM showed lesions of variable degree in 70% of cases. The morphological ultrastructural investigation showed good correlation with the immunological and functional data (IP test): ultrastructural damage of the jejunal mucosa after one year of a gluten-free diet was found in patients with positive serum AGA and an abnormal IP test. Furthermore, the morphometric study of the ultrastructural alterations allowed a quantitative, closer correlation between morphological and functional data. Our results suggest: 1) SEM and TEM investigations offer additional and more complete information on celiac patients, over LM alone. 2) The morphometric evaluation of the ultrastructural alterations highlights quantitative and reproducible correlations between morphological and clinical data, not strengthened by the subjective, qualitative study.

Adolescent↗
PubMed1996

Modified technique to recover microsporidian spores in sodium acetate-acetic acid-formalin-fixed fecal samples by light microscopy and correlation with transmission electron microscopy.

Microsporidia are an emerging cause of significant disease, particularly in the immunocompromised host. Until recently, the diagnosis of enteric infections has required invasive sampling, the use of expensive technology, and considerable technological expertise. The purpose of the present study was to examine three modifications to the processing of fecal specimens for light microscopy (LM) examination for microsporidian spores: the use of pretreatment with potassium hydroxide, modified centrifugation conditions, and a modified staining technique. A sodium acetate-acetic acid-formalin-fixed fecal sample containing numerous microsporidian spores confirmed to be positive by transmission electron microscopy (TEM) was used in all studies performed. A simulation of a heavy to lightly infected individual was used. The results of LM were correlated with those of TEM. Duplicate smears were stained with Weber's modified trichrome and Giemsa (GS) stains. The stained slides were randomized and examined blindly by LM at x 625 and x 1,250 magnifications. A portion of the dilutions after centrifugation were fixed for TEM. The Weber modified trichrome stain performance rating was higher than the Giemsa stain rating because of ease of interpretation, and material stained with Weber modified trichrome stain required less examination time at a lower magnification. The number of positive smears and the quantity of spores detected were significantly higher following pretreatment of the sample with KOH. TEM was positive only when numerous spores were present, but the quality of the photomicrographs was superior after pretreatment with KOH. Pretreatment of sodium acetate-acetic acid-formalin-fixed fecal samples with 10% KOH and then a 5-min centrifugation time and staining with Weber modified trichrome stain provide for the excellent recovery of microsporidia in the routine diagnostic parasitology laboratory.

AIDS-Related Opportunistic Infections↗