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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↗
PubMed1981

Hyperplastic lesions of the human breast: scanning electron microscopy and a review of current knowledge.

Although the etiology of human breast cancer is unknown, certain epidemiological parameters such as risk of breast cancer development. Concurrent, retrospective and prospective studies have shown that one particular component of benign breast disease -- epithelial hyperplasia, may be responsible for the increase in risk. Several classification have been devised for epithelial hyperplasia of the human breast using light microscopy. By scanning electron microscopy epithelial hyperplasia in ducts has several patterns: focal hyperplasia, simple columnar bridging, complex bridging and solid hyperplasia. Although these patterns may be seen in non-cancerous ducts in both malignant and non-malignant cases, alterations in microvillus structure and number have only been seen to date in malignant cases.

Breast↗
PubMed1999

Surface ultrastructure of pit organ, spectacle, and non pit organ epidermis of infrared imaging boid snakes: A scanning probe and scanning electron microscopy study.

Boid snakes possess unique infrared imaging pit organs. The ultrastructure of the surfaces of these organs scatter or reflect electromagnetic radiation of specific wavelengths. Pit organ epidermal surfaces of boid snakes are covered with arrays of pore-like structures called micropits. In order to determine the dimensions of this complicated surface structure, we have performed the first ultrastructural analysis on snake epidermis by high-resolution microscopy techniques. Using scanning probe microscopy and scanning electron microscopy, we found that the epidermis of pit organ, maxillary non pit organ, spectacle, and ventral scales contain arrays of micropits. These scale surfaces also contain major surface features of overlapping plate-like structures. Pit organ micropits averaged 319 nm in diameter and 46 nm in depth and were spaced an average of 808 nm from each other. These micropits were significantly deeper, of greater diameter, and spaced at greater distances apart than those of the other scales. Plate structures of the pit organs had a mean distance between plates of 3.5 microm and a mean plate step height of 151 nm. These differences serve to strengthen the argument that arrays of micropit and plate surface structures function as spectral filters or anti-reflective coatings with respect to incident electromagnetic radiation.

Animals↗
PubMed1994

Morphologic characteristics of equine endometrium classified as Kenney categories I, II, and III, using light and scanning electron microscopy.

Pathologic changes in the endometrium of mares may be rated according to Kenney's method of classification. Category I endometrium contains healthy tissue with no or few widely scattered pathologic changes. At the opposite end, severe widespread pathologic changes are associated with category III. Uterine biopsy specimens were collected aseptically from 16 mares during the estrous and diestrous stages of the cycle. Pathologic changes were evaluated, using light microscopy, and endometrium was classified as Kenney's category I, II, or III. Endometrial tissue of category I (n = 5 mares in estrus; n = 3 in diestrus); category II (n = 3 in estrus; n = 4 in diestrus), and category III (n = 4 in estrus; n = 4 in diestrus) were processed for scanning electron microscopy (SEM). All specimens were fixed immediately after biopsy because it was found that numerous bleb-like projections were formed when fixation was delayed. Category I endometrium had normal glands, and fibrotic tissue was not observed by light microscopy. Scanning electron microscopy revealed numerous hexagonally shaped cells that were covered with many microvilli. Ciliated cells also were observed, and they contained long healthy cilia. Category II endometrium had 2 to 4 nests surrounded by collagen fibers. Of the 4 specimens, 3 had moderate leukocyte infiltration (59 +/- 14.8 WBC/4 high-power fields [450 x]). Scanning electron microscopy revealed some inflammatory changes with slight swelling of the cell surface. Several cells in category II endometrium lacked microvilli, but they were interdispersed among many healthy hexagonal cells. Many nests were seen in category III tissue, and 2 specimens had severe infiltration of WBC (232,264 cells/4 fields).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

High-resolution scanning electron microscopy of human metaphase chromosomes.

Human metaphase chromosomes, prepared for light microscopy were examined by scanning electron microscopy. Use of an osmium impregnation technique eliminated the need for sputter-coating and allowed high-resolution visualization of uncoated specimens. Chromosomes were of three-dimensional cylindrical profile, with well-defined chromatids and centromeres. Prior to Giemsa-banding a smooth surface morphology was observed. Relaxation of chromosome integrity by Giemsa-banding pretreatment allowed resolution of several orders of chromosome structure not previously demonstrated by scanning electron microscopy. The observed organization of the chromatin fibres allowed parallels to be drawn with the radial loop model of chromosome construction as described by Marsden and Laemmli.

Chromosome Banding↗
PubMed1990

Analysis of glass ionomer cement with use of scanning electron microscopy.

In the study, scanning electronmicroscopy and x-ray microanalysis techniques were used to examine a silver-reinforced glass ionomer cement. Scanning electron microscopy was used to evaluate surface topography and microstructure. Microcracking and a bimodal pore distribution were observed. The cement consisted of unreacted glass particles surrounded by a silica gel "halo" and embeded in an organic salt matrix. Back-scattered electron microscopy and energy-dispersive spectroscopy detected discrete clusters of silver atoms within the material. Other elements, such as calcium and aluminum, were dispersed homogeneously. Wave-length dispersive spectroscopy was used to detect fluoride, which also appeared to be homogeneously distributed. Electron microscopy and x-ray microanalysis techniques could prove valuable in clarifying the microstructure and fluoride release mechanisms of glass ionomer cements. Refinement of some aspects, including sample preparation, will be necessary to obtain consistently reliable results.

Aluminum↗