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PubMed1994

Proteoglycan ultracytochemistry and conventional and high resolution scanning electron microscopy of vertebrate cerebellar parallel fiber presynaptic endings.

The parallel fiber "en passant" synaptic endings of mouse cerebellar molecular layer have shown by means of transmission electron microscopy, the presence of an electron dense extravesicular material in samples perfused with Alcian blue. This alcianophilic material was digested in cerebellar tissue previously treated with testicular hyaluronidase, suggesting the presence of hyaluronic acid or chondroitin 4- or 6-sulphate. Freeze-fractured Rhesus monkey cerebellar cortex prepared for conventional scanning electron microscopy also revealed the presence in fractured synaptic varicosities of parallel fibers of a high mass density material, in which the synaptic vesicles are embedded. Examination of cryofractured primate cerebellar cortex coated with thin chromium films, 1-2 nm thick, in the high resolution field emission scanning electron microscope showed the SE-I topographic contrast of an extravesicular material deposited in axoplasmic matrix of fractured parallel synaptic endings. The precise localization of this material corresponds to that observed in transmission electron microscopy and conventional freeze-fracture scanning electron microscopy. These electron microscopic findings tend to agree with the omnipresence in several vertebrates of a presynaptic axoplasmic material, which seems to be proteoglycan in nature.

Alcian Blue↗
PubMed1988

Solid phase immune electron microscopy for diagnosis of transmissible gastroenteritis in pigs.

A serological trapping technique is described for detecting transmissible gastroenteritis (TGE) virus in faeces. The technique involves the coating of electron microscope grids with protein A and specific TGE virus antiserum. Optimal conditions for performing this solid phase immune electron microscopy technique were a concentration 250 micrograms ml-1 of protein A; 1:100 diluted rabbit anti-TGE virus hyperimmune serum for coating the grids and overnight incubation with virus samples. The possibility of detecting coronavirus in crude faeces was highly improved with solid phase immune electron microscopy, compared with conventional negative staining electron microscopy, by specific trapping of virus and prevention of adsorption of contaminants. The sensitivity of the method was evaluated by coded investigation of a dilution series of stock virus mixed with different pools of faeces. The improvement of virus detection in faeces by solid phase immune electron microscopy, compared with standard electron microscopy was at least 100-fold. Faecal shedding of coronavirus by pigs infected with virulent and attenuated strains of TGE virus was studied. Virus detection in faeces by a standard electron microscopy technique was not practical, since the virus was obscured by a large quantity of debris in the faeces. By using solid phase immune electron microscopy, however, the aspect of the specimens on the grids improved so much that, in addition to more common immunofluorescence, the technique might be useful as a diagnostic test for TGE. Virus was detected in daily faecal samples from one or two days after experimental infection with virulent TGE virus, until death in five out of eight animals. Unlike immunofluorescence, solid phase immune electron microscopy may be used for diagnosis in living animals.

Animals↗
PubMed2001

Sub-Angstrom high-resolution transmission electron microscopy at 300 keV.

Sub-Angstrom transmission electron microscopy has been achieved at the National Center for Electron Microscopy (NCEM) by a one-Angstrom microscope (OAM) project using software and enhanced hardware developed within a Brite-Euram project (Ultramicroscopy 64 (1996) 1). The NCEM OAM provides materials scientists with transmission electron microscopy at a resolution better than 1 A by using extensive image reconstruction to exploit the significantly higher information limit of an FEG-TEM over its Scherzer resolution limit. Reconstruction methods chosen used off-axis holograms and focal series of underfocused images. Measured values of coherence parameters predict an information limit of 0.78 A. Images from a [1 1 0] diamond test specimen show that sub-Angstrom resolution of 0.89 A has been achieved with the OAM using focal series reconstruction.

Journal Article↗
PubMed1995

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., 100,000 x) 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.

Carbon↗
PubMed1989

A new method of the immunohistochemical detection of cellular antigens for light and electron microscopy.

A new immunohistochemical method for light and electron microscopy of tissue- and cell-specific antigens by using ferric colloid-labeled antibody is presented. The antibodies labeled with the cationic cacodylate ferric colloid are stable and bind specifically to the target antigens to show clearly the site of antigens in tissue sections and on free cells by Prussian blue reaction for light microscopy and by the specific figure of electron opaque ferric colloid particles for electron microscopy. The staining procedure is very simple and it gives clear picture. So the method will be of beneficial for general laboratory use in immunohistochemical researches.

Animals↗
PubMed1983

Stereoscanning electron microscopy of argon laser excised laryngeal papilloma.

Laryngeal papillomas were removed from two patients with the argon laser and examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and stereoscanning electron microscopy (SSEM). Two techniques, lipid-carbohydrate retention for TEM specimens and sonication for SEM, were applied in studying the papillomas. Lipid-carbohydrate retention results were in agreement with previous work in papilloma ultrastructure. The char interface was observed with the sonication technique. Peripheral to the laser impact site, four zones were observed: 1. coagulation necrosis, 2. contracted epithelial cells, 3. minimally damaged epithelial cells, and 4. morphologically undamaged epithelial cells. In Zone 2, cells were violently popped-off as heat was conducted through the epithelium. The potential for spread of papillomas is discussed and suction of the vapor plume recommended when removing laryngeal papillomas with the argon laser.

Humans↗
PubMed2000

[Observation of biofilms inside tracheal tubes by electron microscopy and the relationship between biofilms and VAP].

OBJECTIVE: To observe the formation of biofilms inside tracheal tubes and to evaluate the effect of biofilm on the development of ventilator associated pneumonia (VAP). METHODS: Biofilms inside tracheal tubes from intubated patients were observed by scanning electron microscopy and transmission electron microscopy, meanwhile bacteria were detected in the specimens from the lower respiratory tract and the inside of tracheal tubes. RESULTS: 76% (19/25) of inner surface of tracheal tubes was coated with a confluent amorphous matrix by scanning electron microscopy. The average duration of trach-intubation in this group [(10.7 +/- 7.9) days] was longer than that in the other group [(2.1 +/- 0.8) days] in which tracheal tubes were not coated with the confluent amorphous matrix (P < 0.05). The presence of many bacteria(14/18) within these amorphous matrix was confirmed by transmission electron microscopy. Of the sixteen tracheal tubes, eleven tubes grew the same organisms that had been isolated from the secretions of the lower respiratory tract before extubation. In some VAP patients (7/8), organisms isolated from tracheal tubes were the same organisms which were pathogenic organisms of VAP. CONCLUSIONS: There was a close connection between the formation of bacterial BF and the long-term indwelling tracheal tubes. The presence of bacterial BF in the inner of tracheal tubes might be associated with the pathogenic organisms of VAP.

Adult↗
PubMed1979

Scanning electron microscopy of bronchiolar neuroepithelial bodies in neonatal mouse lungs.

Scanning electron microscopy of the bronchiolar neuroepithelial bodies (NEB's) in the neonatal mouse lungs was undertaken and correlated with the accompanying transmission electron microscopy. The NEB's appeared as isolated organoids along the entire length of the bronchioles, and often were located at the branching points. The boundary of the NEB's was outlined by the ciliated and Clara cells. Both granulated cells and modified Clara cells participated in the formation of the NEB. The modified Clara cells covered most of the surface of the NEB leaving only small oval areas for the exposed surfaces of the specialized cells which contained numerous cytoplasmic granules. Short and regular microvilli projected from the exposed surfaces of the granulated cells, while only sparse microvilli of irregular length were seen on the surfaces of the modified Clara cells. This scanning electron microscopy of the NEB's further established these organoids as separate morphological entities. In addition, the findings that the NEB's could be easily identified with the scanning electron microscope and subsequently dissected out for further studies would help future investigations of their functions which are not clearly known.

Animals↗
PubMed1982

[Modern electron microscopy methods in the study of ultrastructure and function of the terminal vascular system].

The present survey deals with the possibilities of electron microscopic methods, with the gain of information by its use and with its prospective significance in solving actual problems in the subject of microcirculation. The paper consists of the subsequent parts: 1. Fixation methods; 2. Topical methods for ultrastructural investigation of defined areas of the terminal vascular bed and the transmural ways of transport of molecules; 3. Freeze etching technics; 4. Electron microscopic autoradiography; 5. Transmission electron microscopy including methods for three-dimensional reconstruction of vascular segments; 6. Scanning electron microscopy; 7. X-ray microanalysis; 8. Demonstration of cell receptors with the aid of lectines; 9. Methods for quantitative analysis. In each chapter the possibilities and disadvantages of the methods and the most frequently occurring artefacts were dealt. The survey concludes with a consideration of the prospective development of electron microscopic technics and its use in the investigation of the terminal vascular bed. In the opinion of the authors the methodical development will take the subsequent direction: 1. Further completion of the preparative methods and its development to routine technics combined with the more general use of necessary tools; 2. Further completion and spreading of quantitative investigation methods; 3. More effective use of the synthetic analysis solving a problem, that means, the simultaneous application of different methods (for instance: transmission electron microscopy, scanning electron microscopy, experimental methods and so on).

Animals↗
PubMed1975

Primary neural induction as studied by scanning electron microscopy.

Normal primary neural induction has been studied by scanning electron microscopy and the results compared with those obtained by TEM. Mesoderm cells are usually in contact with several other cells, both mesodermal and endodermal in origin. By SEM the ectoderm layer has been shown to be in contact with the underlying mesoderm cells. Tufts of fibrous basement membrane are also present between the two cell types. TEM specimens also show an intermediate basement membrane.

Animals↗
PubMed1984

High-resolution electron microscopy of hydroxyapatite grown in dilute solutions.

High-resolution electron microscopy (HREM) was applied to the study of seeded crystal growth of hydroxyapatite (HA) in supersaturated solutions. The HA seed crystals were rod-shaped, elongated along the c-axis, and showed smooth contours. The seed crystals exhibited hexagonal prism facets, and one end was rhombohedrally terminated, whereas the opposite end was blunt. Growth experiments were carried out at 37 degrees C, and solution compositions were carefully selected to avoid the involvement of precursor phases during the HA growth. After five-hour growth, the total amount of precipitation was from 3 to 7% of the initial crystal mass added to the solutions; examination of the crystals by HREM disclosed the formation of projections on the end surfaces of the HA crystals. High-resolution TEM clearly showed lattice fringes with predictable spacings over the entire crystal specimen, including the formed projections. Analysis of selected regions by optical diffraction of the HRTEM fringe negatives showed that the nature and orientation of the projections were similar to those of the underlying seed crystal. High-resolution SEM of the HA crystal after five-hour growth disclosed step-like structures on the prism faces, as well as the projections localized on the ends. These results strongly suggest that more than one growth process may be involved, perhaps related to the distinct faces of the HA seed crystals.

Chemical Precipitation↗
PubMed1988

Clinical applications of electron microscopy in the analysis of collagenous biomaterials.

Scanning and transmission electron microscopy are of clinical value in assessing the interaction between biomaterials and ingrowing tissues. Ultrastructural information allows the clinician and biomaterials specialist to determine events occurring during wound healing and the biocompatibility of prosthetic devices. This paper reviews some of the experimental and clinical studies done in our laboratory on the use of natural and reconstituted collagen as replacements for connective tissues. Consideration is given to collagen flakes used for the treatment of dermal ulcers, a collagen fiber prosthesis used for tendon and ligament replacement, the effects of chemical preservatives on cartilage used for replacement of tissues during plastic surgery and the growth and orientation of nerve cells on reconstituted collagen fibers. Our results show that reconstituted collagen can be prepared into prosthetic devices which encourage cell attachment and orientation thereby facilitating healing of injured tissues. Furthermore chemical preservation of cartilagenous tissues kills chondrocytes resulting in eventual resorption by inflammatory cells.

Biocompatible Materials↗
PubMed1994

Conformation, order-disorder conformational transitions and gelation of non-crystalline polysaccharides studied using electron microscopy.

Direct imaging of polysaccharides using transmission electron microscopy (EM) is an important alternative to physical characterization of non-crystalline polysaccharides in solution. The polymer nature of stiff-chain polysaccharides is quite apparent from direct visualization of the electron micrographs, despite the fact that commonly employed preparation techniques reduce the resolution limit to about 1-2 nm. Electron microscopy has recently been used to study polysaccharides with emphasis both on quantitative properties like contour length, end-to-end distance and chain stiffness, and on qualitative structural features such as cyclization at the macromolecular level. The structural richness observed for polysaccharides of the beta-D0glucan family after a denaturation-renaturation treatment of the specimen, in particular, illustrates the unique potential of EM as a tool for obtaining conformational information about carbohydrate macromolecules. Examples of the latter also include the recent discoveries of cyclic beta-D-glucan and l-carrageenan structures. The EM technique provides information that is not only complementary to what can be obtained using other physical techniques, but also offers important insight otherwise masked by the averaging implicit in most physical techniques used to study aqueous polysaccharide solutions.

Gels↗
PubMed2006

Ultrastructure of spermatozoa of tench Tinca tinca observed by means of scanning and transmission electron microscopy.

Structure of tench (Tinca tinca L.) spermatozoa was investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spermatozoa of 26.1+/-3.8 microm total length possessed typical primitive simple structure, called "aqua sperm", without acrosomal head structures. It was probably the smallest spermatozoon described among cyprinid fishes. Heads were mostly composed of dense and slightly granular material, which appeared to be fairly homogeneous except for the occasional appearance of vacuoles. The midpiece remained separated from the flagellum by the cytoplasmic channel; it was cylindric/cone-shaped, 0.86+/-0.27 microm in length and 1.17+/-0.24 microm in width at proximal part. The proximal centriole was located in the "implantation fossa". The distal centriole appeared almost tangential to the nucleus and it functioned as a basal body for the flagellum. It had an orientation of 140 degrees with respect to the distal centriole. The sperm flagellum with 25.45+/-2.47 microm of total length had no any fin. The diameter of the flagellum perpendicular to the plane of the doublet of central microtubules was 173.67+/-20.45 nm and horizontal plane of the central microtubules was 200.71+/-20.45 nm. Peripheral doublets and the central doublet of microtubules measured 23.39+/-3.18 and 35.88+/-4.44 nm in width, respectively. The diameter of a microtubule was only 9.14+/-2.97 nm. A vesicle was attached to the most basal region of the flagellum and located just under plasma membrane of the flagellum.

Acrosome↗
PubMed1998

Electron microscopy in tumor diagnosis: indications for its use in the immunohistochemical era.

The electron microscope reveals much more information on tumor cell structure than can be obtained with the light microscope, and some of the data are useful in diagnostic studies. In this review, major contributions of electron microscopy in the main tumor categories are discussed. To select between immunocytochemistry and electron microscopy, the probable contributions of each in the context of the particular case must be assessed. Usually, electron microscopy will only be requested after a battery of immunostaining procedures has been performed and found to be insufficient, but there are occasions when ulstrastructural study is logically the first choice after routine light microscopy. It is worth taking tissue for possible electron microscopy from any tumor that is known or anticipated to be a diagnostic problem.

Biomarkers, Tumor↗
PubMed1992

Relative usefulness of electron microscopy and immunocytochemistry in tumour diagnosis: 10 years of retrospective analysis.

AIMS: To determine retrospectively the relative usefulness of electron microscopy and immunocytochemistry for tumour diagnosis; to monitor the influence of new antibodies and antisera on the use of these techniques in one laboratory. METHODS: During 1980 to 1989 inclusive, 726 tumours were examined by electron microscopy, 862 by immunocytochemistry, and 286 by both techniques. The choice of techniques and, for immunocytochemistry, the range of antibodies used were compared between each category of final diagnosis. RESULTS: During the study period there was a sharp fall in the use of electron microscopy and a corresponding rise in immunocytochemistry. These trends applied to all categories of final tumour diagnosis, but the use of electron microscopy was sustained longer for lesions suspected or eventually confirmed to be melanomas or amine precursor uptake decarboxylation cell carcinoma (APUDomas)--for example, carcinoid tumours. The immunocytochemistry:electron microscopy use ratios ranged from 2.07:1 to 0.44:1 for the categories in which lymphoma and APUDoma, respectively, were the final diagnoses. The abandonment of electron microscopy for suspected or confirmed lymphomas and carcinomas corresponded to the increasing availability of relevant antisera and antibodies. CONCLUSIONS: The wider application of immunocytochemistry for tumour diagnosis is endorsed, but electron microscopy should be retained for selected cases in which the results of immunocytochemistry might be predictably ambiguous or otherwise unhelpful.

Apudoma↗
PubMed1978

Localization of chitin in algal and fungal cell walls by light and electron microscopy.

Chitin was visualized in cell walls after hydrolysis with potassium hydroxide and subsequent postfixation of the deacetylated polysaccharide (chitosan) in OsO4. Areas of chitin deposition appeared dark borwn by light microscopy and electron dense in the electron microscope. With this method, the presence of chitin was demonstrated in the cell walls of the green alga Pithophora oedogonia (Montagne) Wittrock and two fungi, Ceratocystis ulmi Buism. (C. Moreau) and Blastocladiella emersonii Cantino and Hyatt. Most of the chitin in P. oedogonia ws found in the crosswall disk and small amounts occurred in the outer longitudinal walls. The septal disk of C. ulmi also contained chitin, but significant amounts were present in the inner and outer regions of longitudinal walls as well. Chitin was present throughout the walls of B. emersonii. Small amounts of chitin were not easily demonstrated by this technique, but removal of chitosan by exposure to dilute acetic acid before osmium fixation disrupted cell wall integrity, suggesting that small amounts of the structural polysaccharide had been removed.

Ascomycota↗
PubMedDate not supplied

Ultrastructure of normal epithelial cells in Papanicolaou-stained cervical smears. An application of a modified open-face embedding technique for transmission electron microscopy.

The results of an open-face embedding technique applied to Papanicolaou-stained uterine cervical smears are presented. By this technique, cells are smeared on a plastic sheet, fixed in 1.5% buffered glutaraldehyde and stained by the Papanicolaou method; areas of special interest are then selected by light microscopy for electron microscopic observations. Thus, a comparison is possible between the light microscopic characteristics of cells in smear preparations and their ultrastructural counterparts. The good ultrastructural preservation allowed detailed study of nuclei and cytoplasm. The endocervical cells had intact nuclei with nuclear envelope, Golgi apparatus, well-preserved mitochondria and two types of secretory material. The superficial and intermediate squamous cells showed loss of desmosomes; dense, fine cytoplasmic fibrils; small, fatty droplets; and degenerated nuclei with loss of the nuclear envelope. Compared with ultrastructural studies on cervical tissues, the intermediate cells in cervical smears were more degenerated, presumably because predominantly degenerated intermediate cells exfoliate. This study indicates that the light microscopic impression of the nuclear appearance is misleading: in the routine smear the nuclei of superficial and intermediate cells may look "intact" and "well preserved" whereas ultrastructurally the nuclei are degenerated.

Cytological Techniques↗