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PubMed2000

Scanning electron microscopy and fluorescent in situ hybridization of experimental Brachyspira (Serpulina) pilosicoli infection in growing pigs.

Two groups of six 8-week-old pigs were challenged with 1x10(9) cfu Brachyspira (Serpulina) pilosicoli or Serpulina intermedia daily for 3 consecutive days to study the pathology of porcine colonic spirochetosis by scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with oligonucleotide probes targeting ribosomal RNA specific for B. pilosicoli and the genus Brachyspira/Serpulina. Six pigs served as noninoculated controls. The animals were euthanatized successively between postinoculation days 14 and 24. B. pilosicoli was reisolated in feces from all of the inoculated pigs; however, only two pigs developed transient watery diarrhea. S. intermedia was reisolated from four of the inoculated pigs, but clinical signs were not observed. Gross examination of the B. pilosicoli-infected pigs revealed dilated large intestines with a hyperemic mucosa, whereas the large intestines of the S. intermedia-inoculated pigs and the control pigs appeared normal. SEM examination of B. pilosicoli-infected pigs revealed degenerated epithelial cells and spirochetal colonization of the colonic mucosa in four pigs. By FISH, B. pilosicoli cells were found colonizing and invading the surface epithelium and the crypts in all the pigs. Spirochetal crypt colonization markedly exceeded the occurrence of spirochetes on the mucosal surface. SEM examination of S. intermedia-inoculated pigs revealed no abnormalities, and Serpulina cells were detected only sporadically in the otherwise normal-appearing mucosa of four pigs by FISH. The results provide further evidence that B. pilosicoli is associated with colitis in pigs, although the gross lesions are mild. The spirochete is capable of colonizing the large intestine, inducing mucosal damage, invasion of the crypt and surface epithelium, and focal infiltration of the lamina propria. In addition, the study shows the applicability of FISH for specific identification of B. pilosicoli in formalin-fixed tissue.

Animals↗

PubMed1981

The application of scanning electron microscopy to the study of the cytoskeleton of cells in culture.

This paper reviews the methods available for studying the structural organization of the cytoskeleton of cells in culture. These are transmission electron microscopy of whole cells, immunofluorescence, and detergents extraction procedures. Published methods for detergent extraction are critically reviewed with special emphasis on those factors that are most relevant for the preparation of cytoskeletons for observation by scanning electron microscopy. These include the type of detergent, the use of chemical crosslinking reagents or specially formulated buffers to stabilize the cytoskeleton during extraction, the osmotic and salt composition of the extraction medium, the use of cool sputter coaters, and the application of minimal amounts of metal during coating. Finally, examples are given of the types of information about the three dimensional organization of the cytoskeleton that can be obtained by SEM.

Animals↗

PubMedDate not supplied

Scanning electron microscopy as a diagnostic procedure in giant cell arteritis.

Forty-five consecutive patients (32 women and 13 men) underwent biopsy of the temporal artery because of suspected giant cell arteritis. Their ages ranged from 38 to 84 years, mean 68.1 years. Five patients (11.1%) four of them women, were found to be affected by the disease. Their ages ranged from 54 to 80 years, mean 69 years. Clinical and laboratory findings included elevated erythrocyte sedimentation rate, prolonged fever, continuous headache, sudden onset of unilateral blindness, intermittent mandibular claudication, severe anemia and myalgia. None of these, whether present in isolation or in various combinations, were of significant diagnostic value. All biopsies were examined both by light microscopy and by scanning electron microscopy. The former examination took about 5-7 days to complete, and the latter about 3 hours. Light microscopy studies showed that 46.6% of the arterial biopsies were normal, 42.3% were arteriosclerotic and 11.1% (5 specimens) were characteristic of giant cell arteritis. Scanning electron microscopy revealed that the biopsies obtained from all five patients found to have temporal arteritis displayed the "occlusive" pattern: the three-laminar appearance of the artery was markedly distorted or lost, the internal elastic lamina was barely detectable, and the densely hypertrophied media and intima filled the arterial lumen, virtually obliterating it. We conclude that scanning electron microscopy is a quick and accurate procedure for diagnosis of temporal arteritis and that positive findings may be taken as an indication for immediate steroid treatment.

Adult↗

PubMed1988

[Periosteal bone resorption in the area of the metaphysis of growing bone as a precursor of epiphyseal injuries. A polarization optical and scanning electron microscopy study].

Macerated epiphyses of the growing infant bone exhibit an irregular surface which can already be observed at the macroscopic level. Polarizing microscopy demonstrates extended zones of bone resorption on the cortical surface, deductable from numerous lacunae of Howship. Ensuing experimentally induced epiphysiolyses, cortical bone fragments adhering to the inner surface of the periosteum are demonstrable by scanning electron microscopy. Having been torn out of the bone together with the periosteum, these cortical fragments leave corresponding defects on the bone surface. Due to the remodelling of the bone, involving the readjustment of the shape of the extremity, the attachment of the periosteum is relatively poor in the metaphyseal region of the growing bone. The influence of pathological forces can therefore easily cause a detaching of the periosteum in this region. The latter results in a significant weakening of the epiphyseal fastening in the zone between the epiphyseal plate and the metaphysis. The patterns of injury in the region of the growth plate are therefore essentially determined by the varying attachment of the periosteum to the metaphysis.

Adolescent↗

PubMedDate not supplied

Scanning electron microscopy of normal cells, dyskaryotic cells and malignant cells exfoliated from the uterine cervix.

The same exfoliated benign, dyskaryotic and malignant cervical cells were observed by light microscopy and scanning electron microscopy in order to investigate the reflection of the course of malignancy in the surface ultrastructure. The surface ultrastructural features (microridges and microvilli) of normal squamous cells and metaplastic cells showed regularities, with an approximately even thickness, length and distribution of microvilli. In dyskaryotic cells of the superficial type, the microridgelike structure was retained, and some regularity of the microvilli remained. However, in dyskaryotic cells of the deep-layer type, the microvillous surface ultrastructure had a pronounced irregularity and diversity. In malignant cells, there was unevenness in the projections and in the thickness of the cytoplasm, and these cells presented, as a whole, a bizarre appearance. The surface ultrastructure of malignant cells thus reflected a strong atypia of the cytoplasm.

Carcinoma in Situ↗

PubMed1992

Review and new case reports on scanning electron microscopy of pili annulati, Monilethrix and Trichothiodystrophy.

Pili annulati, Monilethrix and Trichothiodystrophy are uncommon conditions in which the hair shaft has a distinct appearance as seen by optical microscopy and scanning electron microscopy (SEM). We report several new cases and review the characteristic ultrastructural abnormalities investigated by SEM. Pili annulati: abnormal areas which show a longitudinal, "curtain-like" folding of the cuticular cells, alternating with normal areas. The latter present regularly non-systematized, superficial depressions. Hair specimens of the patient's mother show the same surface irregularities. Monilethrix: most hair shafts show variations in thickness giving a typical nodal appearance. In the isthmus area we noticed longitudinal ridging and cuticular scales that are extended lengthwise in a fish-scale-like pattern. In the nodus area a smooth surface due to a complete loss of cuticular cells, was observed. Trichothiodystrophy: the hair morphology observed by SEM is characterized by severe cuticular and secondary cortical degeneration along the entire length of the flattened hair shaft, with longitudinal ridging, cuticle loss, trichlorrhexis nodosa formation and trichoschisis. SEM observations show morphological abnormalities which are characteristic for each pathological condition described. This method may provide data that add some clarity in the surface changes of the different hair shaft anomalies.

Adult↗

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↗

PubMed2006

High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy.

Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component alpha-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.

Cell Cycle↗

PubMed1991

Quantitative light and scanning electron microscopy of ferret sperm.

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

Acrosome↗

PubMed1996

Advantages of backscatter electron imaging scanning electron microscopy for intracellular localization of cardiac analytes by gold conjugated antibody.

Myoglobin and myosin light chain 1 (MLC1) are intracellular human cardiac marker proteins which are released as a consequence of ischemia. Human cardiomyocytes were isolated from fresh biopsies and also maintained for several passages in cell culture. The cardiomyocytes were fixed in 100% methanol at -20 degrees C, and labeled. The immunolocalization of intracellular antigen by fluorescence conjugated imaging was compared with scanning electron microscopy (SEM) backscatter electron (BSE) imaging of gold conjugated antibody. Ultra-violet light microscopy showed the intracellular distribution of both proteins to be mainly in the nuclear envelope, the cytoplasm immediately surrounding the nucleus and along portions of the cell membrane. To confirm this observed distribution of myoglobin and MLC1, labeling was repeated with antimyoglobin and anti-MLC1 monoclonal antibodies conjugated to colloidal gold particles. The advantage of colloidal gold labeling is that the intracellular antigen-antibody complexes may be more precisely located because of the significant improvement in resolution provided by BSE imaging in the SEM. BSE imaging confirmed the presence and subsarcolemma localization of myoglobin in cardiomyocytes directly isolated from fresh biopsies. The distribution of colloidal gold-conjugated antibodies did not coincide with the intracellular distribution of the two proteins in the cardiomyocytes grown in cell culture as indicated by immunofluorescence. A relatively random, intracellular gold particle distribution was confirmed by x-ray microanalysis. BSE imaging resulted in consistent auto-backscatter labeling patterns very similar to the labeling patterns obtained with immunofluorescent labeling. X-ray microanalysis confirmed that these auto-backscatter labeling patterns were formed by concentrations of intracellular phosphate. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting indicated that myoglobin and MLC1 were no longer present in detectable quantities in these cells after several passages. Polymerase chain reaction (PCR) amplification of mRNA for human myoglobin and cardiac MLC1 confirmed the absence of their transcripts. Electrophoretic analysis of proteins in cardiomyocytes grown in cell culture confirmed an increasing presence of alkaline phosphatase. Staining of this enzyme with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium showed that alkaline phosphatase was distributed in the same intracellular pattern as the fluorescence conjugated anti-body and the phosphatase auto-backscatter. These results indicate that high-resolution backscatter SEM imaging may be used as necessary control to confirm fluorescence light microscope intracellular labeling of antigens.

Alkaline Phosphatase↗

PubMed1976

[Certain structural details of developing in vitro human embryonal brain explants studied with the aid of fluorescence and scanning electron microscopy].

The bundles growing away from the explants were mainly formed by neuroblast processes. It has been shown that the degree of differentiation of neuroblasts depends on "the age" of embryonal brain. The bundles of neuroblasts are accompained by slightly differentiated cells, having their own processes descending to the surface of the lining. These processes attached to the surface of a bundle cover and have no connection with the inner structures of the bundle. The use of a scanning electron microscopy allows to detect some new details of the behaviour of nerve cells in vitro.

Brain↗

PubMed2002

Diurnal difference in the amount of immunogold-labeled glucomannans detected with field emission scanning electron microscopy at the innermost surface of developing secondary walls of differentiating conifer tracheids.

The differences between cell wall formation at night, when the tangential strain used as an index of the volumetric changes in differentiating cells is high, and in the day, when the tangential strain is low, were investigated in Cryptomeria japonica D. Don. Samples containing differentiating xylem were collected at 0500 hours and 1400 hours. The innermost surface of developing secondary walls in differentiating tracheids was observed by field emission scanning electron microscopy. In the specimens collected at 0500 hours, an amorphous material was observed covering the cellulose microfibrils. The cell wall surface was immunogold-labeled with an anti-glucomannan antiserum. After chlorite treatment, the amorphous material disappeared, and immunogold labeling was rarely observed. In the specimens collected at 1400 hours, cellulose microfibrils were clearly evident, and amorphous material and immunogold labeling were rarely observed. We thus confirmed that much amorphous material containing glucomannans is observed at night, when differentiating tracheids are turgid due to the increase in their volume, while the amorphous material was rarely observed during the day when cellulose microfibrils are clearly observed.

Cell Wall↗

PubMed1986

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy.

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.

Animals↗

PubMed2001

Immunogold labelling of fibroblast focal adhesion sites visualised in fixed material using scanning electron microscopy, and living, using internal reflection microscopy.

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell-substrate interface was removed with detergent. The antigen blocking procedure, size of label (5 nm) and duration of silver-enhancement (6 min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided 'sectional' views through the cell. This enabled visualisation of vinculin exclusively within the cell-substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.

Acrylic Resins↗

PubMed2006

Variation in articular cartilage in rats between 3 and 32 months old. A histomorphometric and scanning electron microscopy study.

In this study we assess the thickness, the cellular density, the cell sizes and the collagen of the three superficial cartilage zones and the morphology of the articular surface of the femoral trochlea in rats with 3, 12 and 32 months of age. The cartilage was studied using light microscopy and scanning electron microscopy. The quantitative results are expressed as means +/- SEM. The data were compared statistically (P < 0.05). Both the thickness and the cellular density significantly diminish with age, in the three cartilage zones studied. The reduction of cellular density is more pronounced in the superficial and intermediate zones of the cartilage (zones I and II, respectively). In zone III (deep zone), the cellular density declines only as from 12 months of age. The area of the chondrocytes diminishes in the superficial and deep zones, but only as from 12 months old. In the intermediate zone, there is no chondrocyte hypotrophy with age. The types of collagen in the zones of the cartilage change with age. In the superficial zone, the collagen type I predominates at 3 months of age while the collagen type II predominates at 12 and 32 months of age. In the intermediate and deep zones, the collagen type I that predominates at 3 months of age is substituted by the collagen type III at 12 and 32 months of age. The articular surface in the 3-month-old rats is relatively smooth, presenting few undulations. In 12-month-old animal cartilages, few fissures and craters are found. In the 32-month-old animals, it was observed a higher number of this kind of degenerative changes and with a more severe look.

Aging↗

PubMedDate not supplied

A study of primary dental enamel from preterm and full-term children using light and scanning electron microscopy.

PURPOSE: The aim of this study was to examine the enamel thickness of the maxillary primary incisors of preterm children with very low birth weight (< 1,500 g) compared to full-term children with normal birth weight. METHODS: A total of 90 exfoliated maxillary primary central incisors were investigated using light microscopy and scanning electron microscopy (SEM). Three serial buccolingual ground sections of each tooth were examined under light microscopy, and maximum dimensions of the prenatally and postnatally formed enamel were measured. RESULTS: The enamel of preterm teeth was approximately 20% thinner than that for full-term teeth. Most of the reduction was observed in the prenatally formed enamel. This was 5 to 13 times thinner than that for full-term children (P<.001). The "catch-up" thickness of postnatally formed enamel did not compensate fully for the decrease in prenatal enamel (P<.001). Although none of the teeth used in this study had enamel defects visible to the naked eye, 52% of preterm teeth showed enamel hypoplasia under SEM, compared with only 16% found on full-term teeth (P<.001). These defects were present as pits or irregular, shallow areas of missing enamel. CONCLUSIONS: Preterm primary dental enamel is abnormal in surface quality, and is significantly thinner compared to full-term enamel. The thinner enamel is due mainly to reduced prenatal growth and results in smaller dimensions of the primary dentition.

Child, Preschool↗

PubMedDate not supplied

An improved scanning method based on characteristics of the human visual system for scanning electron microscopy.

An improved scanning method for the scanning electron microscope (SEM) is proposed. Here, quincuncial scanning (sampling) instead of a conventional (raster) scanning is used. This scanning method is very effective for quality improvement of an SEM image obtained under undersampling conditions (rough sampling). The present study focuses on characteristics of the human visual system, specifically the low response of eyes in diagonal directions. When using this method coupled with a high-precision interpolation, the number of pixels necessarily doubles. It is not surprising that it is advantageous for printing. A more important advantage is the fact that SEM images can be acquired with a shorter recording time. Hence, this type of scanning will be helpful for quick and frequent recordings in a "snapshot" mode, which up to now has not been achieved successfully by SEM.

Humans↗

PubMed2002

Scanning electron microscopy of Spinochordodes tellinii (Camerano, 1888), (Gordiacea, Nematomorpha).

There are many species of Nematomorpha which are deficiently described and therefore pose doubts about their actual taxonomic position. This is the case with Spinochordodes tellinii (Comerano, 1888), which was transferred to four different genera and has been recently considered as species incertae sedis. A female of Spinochordodes tellinii is redescribed in this work under light microscopy and Scanning Electron Microscopy. Cuticle details, shapes and areolar distribution and the features as well as the location of spiniform structures are analysed. The systematic position is discussed.

Animals↗