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Electron microscopy findings on an intraocular lens in the uveitis, glaucoma, hyphaema syndrome.

PURPOSE: To report the electron microscopic findings on an explanted intraocular lens in a patient with the uveitis, glaucoma, hyphaema syndrome. METHODS: Scanning and transmission electron microscopy were undertaken on a coccoon of cellular material from the tip of the intraocular lens haptic. RESULTS: Scanning electron micrographs showed densely packed coccoid-like structures on the haptic surface. By transmission electron microscopy these structures proved to be melanosomes. CONCLUSIONS: The scanning electron micrographs described in this report are similar to those reported in patients with chronic post-operative uveitis, but to our knowledge have not been shown before in association with the uveitis, glaucoma, hyphaema syndrome. Transmission electron microscopy determined that the coccoid-like structures were melanosomes. The melanosomes are probably derived from damaged pigment epithelial cells or iris stromal melanocytes secondary to recurrent chafing of the haptic against the posterior surface of the iris.

Aged↗

Scanning electron microscopy of acellular glomeruli in nephrotic syndrome.

All cellular elements were extracted from portions of 13 renal biopsies performed for nephrotic syndrome and the acellular glomerular basement membrane (GBM) examined by scanning electron microscopy (SEM). Five biopsy specimens did not contain subepithelial deposits by light microscopy (LM), immunofluorescence (IF), and transmission electron microscopy (TEM). SEM of the acellular GBM showed a uniform slightly granular surface identical to that previously reported for normal human GBM. Eight biopsy specimens contained subepithelial deposits by LM, IF, and TEM. SEM showed complete extraction of deposits and a spectrum of clearly visualized diffuse GBM alterations in response to subepithelial deposits. Shallow pits or indentations of the GBM were noted in two cases stage I MGN which correlated with numerous "pinholes" visualized by LM in tangential sections of the GBM on silver stain. A complex reticular pattern of GBM elaboration was observed in cases of stage II MGN corresponding to "spikes" visualized by LM on silver stain. SEM of glomeruli following cell extraction permits the first direct three-dimensional visualization of the GBM alterations occurring in various stages of subepithelial immune complex formation.

Adult↗

Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy.

Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm(2), 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (>80%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.

Albumins↗

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro.

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparations techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference. AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (-15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).

Animals↗

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001.

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars--possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924-930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.

Carbonates↗

Iso-butanol saturated water: a simple procedure for increasing staining intensity of resin sections for light and electron microscopy.

The addition of the alcohol iso-butanol (2-methylpropan-1-ol) to water was found to improve the post-staining procedures for semi-thin and ultrathin resin sections, for both light and electron microscopy. Stain penetration was enhanced with samples embedded in both acrylic and epoxy resins and provided structural information not previously seen. These improvements were found with general (non-specific) stains and a fluorescence stain for light microscopy, as well as for a range of heavy metal stains for electron microscopy. The use of this water/solvent medium also gave improved results when used in a variety of immunogold labelling procedures, resulting in a greater specific label density without affecting background gold levels. The use of this solvent/water medium may have wider applications for other types of staining.

Bacteria↗

Photonic nanopatterns of gold nanostructures indicate the excitation of surface plasmon modes of a wavelength of 50-100 nm by scanning near-field optical microscopy.

Scanning near-field optical microscopy images of metal nanostructures taken with the tetrahedral tip (T-tip) show a distribution of dark and bright spots at distances in the order of 25-50 nm. The images are interpreted as photonic nanopatterns defined as calculated scanning near-field optical microscopy images using a dipole serving as a light-emitting scanning near-field optical microscopy probe. Changing from a positive to a negative value of the dielectric function of a sample leads to the partition of one spot into several spots in the photonic nanopatterns, indicating the excitation of surface plasmons of a wavelength in the order of 50-100 nm in metal nanostructures.

Gold↗

Scanning electron microscopy of the interior of cells in Hürthle cell tumors.

Four cases of Hürthle cell tumor were examined by scanning electron microscopy after being macerated to remove all soluble components. By all morphological criteria, Hürthle cells are oncocytes with their usual augmented complement of mitochondria. The Hürthle cell mitochondria either are ovate with central stacks of cristae or elliptical or rod-like with cristae that often are finger-like. As in salivary gland oncocytes, the shelf-like cristae are anchored to the inner boundary membrane by tubular necks. In some Hürthle cells, all of the mitochondria exhibit reticulate cristae. A few mitochondria harbor a globular inclusion in their inner compartment. The Golgi apparatuses are relatively simple, consisting of imbricated saccules that are edged by small, bud-like structures. The rare lumina in the midst of clusters of Hürthle cells are lined by numerous microvilli. Thus, scanning electron microscopy of macerated Hürthle cell tumors has revealed a number of features, especially of their mitochondria, that have escaped detection by transmission electron microscopy.

Adenoma, Oxyphilic↗

Surface characterization by atomic force microscopy of sterilized PLGA microspheres.

Atomic force microscopy (AFM) is recognized a suitable and powerful technique for surface and morphological analysis. Even if until now this technique has not been frequently used in the pharmaceutical field, it can contribute to an accurate morphologic characterization of microspheres and nanospheres. In this work, atomic force microscopy has been used to perform the surface characterization of sterilized microspheres. The aim is to investigate the morphologic modifications induced by gamma irradiation on poly(lactide-co-glycolide) microspheres loaded with ovalbumin and to compare the results obtained by AFM to those obtained by scanning electron microscopy (SEM). The results obtained show that, with respect to SEM, AFM can give some additional information regarding the modifications induced by gamma-irradiation on microspheres surface morphology. The significant changes in surface roughness after irradiation are indicative of damage due to gamma-irradiation. The unchanged surface roughness values calculated for microspheres containing PEG in their matrix, suggest that this polymer exerts a protective effect towards gamma-irradiation.

Drug Carriers↗

Measurement of thickness within sections by quantitative electron microscopy.

To apply the method of quantitative electron microscopy to the measurement of mass in thin sections, the thickness of the section at or very near the structure to be studied must be known. Dowex anion exchange resin AG 1 x 2, stained with phosphotungstic acid (PTA) at pH 6.4, was used as a thickness standard which could be embedded and sectioned. The sectioned PTA-Dowex appeared uniformly stained and exhibited suitable electron opacity. The stoichiometry of the reaction between PTA and the Dowex resin was measured by three independent methods based on gravimetric, colorimetric, and nitrogen determinations whose results showed close agreement. From the PTA uptake, the density of the stained spheres was calculated. Mass of a defined area of PTA-Dowex was measured by quantitative electron microscopy, and from this mass and density, the volume and then the thickness were calculated. The values for thickness were compared to those obtained by interference microscopy on the embedding medium alone in the same sections.

Colorimetry↗

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell.

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

Animals↗

Nonlinear optical microscopy at wavelengths exceeding 1.4 microm using a synchronously pumped femtosecond-pulsed optical parametric oscillator.

A singly resonant femtosecond-pulsed synchronously pumped optical parametric oscillator (SPOPO) is reported that provides a wavelength-tunable alternative to the Cr:forsterite source for nonlinear microscopy applications. Periodically poled lithium niobate within an external resonator was synchronously pumped by a commercial fs-pulsed Nd:YLF laser source to provide fs-pulsed continuously tunable radiation from 1.45 to 1.63 microm. Third harmonic generation (THG) microscopy and three-photon laser scanning fluorescence microscopy (3PLSM) of a range of samples were subsequently performed using this long-wavelength SPOPO source. The ability to tune the excitation wavelength delivered to the sample significantly improved the efficiency of both the 3PLSM and THG imaging experiments.

Animals↗

Light, conventional and environmental scanning electron microscopy of the trichomes of Cucurbita pepo subsp. pepo var. styriaca and histochemistry of glandular secretory products.

BACKGROUND AND AIMS: In the present study, the differences between glandular and non-glandular trichomes, the secretory process and the method of secretion were studied. Previous studies on leaves of Styrian oil pumpkin (Cucurbia pepo var. styriaca) plants have shown that four morphologically and ontogenetically independent glandular and non-glandular trichome types and one bristle hair type can be distinguished. The four types of trichomes can be categorized into three glandular trichome types: type I, a short-stalked trichome with four head cells including a 'middle-cell', two stalk cells and one basal cell; type II, a long-stalked trichome with two head cells, a 'neck-cell' region and a long stalk area; type IV, a 'stipitate-capitate' trichome with a mesophyll cell basement, a short stalk and a multicellular head; type III, a non-glandular 'columnar-digit' trichome, which consists of two head cells continuous with three-celled stalk, and the basal cell. METHODS: The histochemical studies (the main classes of metabolite in secreted material of glandular trichomes) were conducted in fresh and fixed hand sections, using the following tests: Sudan black B, Nile blue A, osmium tetroxide, neutral red, Naturstoffreagent A, FSA (fuchsin-safranin-astra blue), NADI (naphthol + dimethylparaphenylenediamine) and ruthenium red. Each suggested differences in the intercalations during the ontogenetical development of each trichome during the development stage. KEY RESULTS: The histochemical reactions revealed the main components of the materials secreted by all types of trichomes, which include lipids, flavones and terpenes and the different cell wall compositions. Glandular secretions were observed during environmental scanning electron microscopy (ESEM) and the trichomes compared with those seen by conventional scanning electron microscopy (CSEM). CONCLUSIONS: Scanning electron microscopy and histochemical analysis demonstrated that each of the trichomes studied produced and released secretory products in a characteristic way.

Cucurbita↗

Analysis of calcitonin gene-related peptide (CGRP)-containing nerve fibres in the rat spinal cord using light and electron microscopy.

Calcitonin gene-related peptide (CGRP) content in areas surrounding the central canals of cervical, thoracic, lumbar and sacral spinal cords of rats were investigated by light microscopy, conventional transmission electron microscopy (CTEM) (100-200 kV) and high-voltage transmission electron microscopy (HVTEM) (1000 kV) using immunocytochemistry. Tissues were examined using either the peroxidase-antiperoxidase technique or an immuno-cryoultramicrotomy technique. Light microscopically, more intense CGRP localization was observed ventral to the central canals in the lumbar and sacral cord compared with the dorsal area in the same regions. HVTEM revealed that high levels of CGRP labelling were found adjacent to the basal side of ependymal cells ventral to the central canal. Analysis using CTEM operated at 200 kV demonstrated that the CGRP immunoreactivity was present within unmyelinated nerve fibres in the vicinity of the basal side of ependymal cells ventral to the central canal. Immuno-cryoultramicrotomy revealed that immuno-gold particles indicating CGRP labelling were localized on vesicle-like electron-dense bodies in unmyelinated and some fine caliber myelinated nerve fibres. Existence of CGRP as a primary afferent marker in the area surrounding the central canal suggest that there may be an intimate relationship between the modulation of nociceptive information and the area surrounding the central canal in rat spinal cord.

Animals↗

In-situ etching observation of human teeth in acid agent by atomic force microscopy.

Atomic force microscopy was applied to the in-situ observation of the etching process of human teeth by acid agents. The change of surface morphology was observed consecutively before and during etching for the same area in the same specimen. The course of the etching process in enamel from dissolution of smear layer just after injection of acid agent, appearance of enamel prisms and progress of demineralization were quantitatively analysed for three fundamental acid agents of 2% phosphoric acid, 10% citric acid and 10% polyacrylic acid. Then the depth profile, etching amount, etching rate and thickness of smear layer were evaluated. Observation by scanning electron microscopy was also done and compared with the results by atomic force microscopy.

Acid Etching, Dental↗

Utility of electron microscopy in the assessment of transthoracic needle lung biopsy specimens.

RATIONALE AND OBJECTIVES: The authors determine the usefulness of electron microscopy (EM) in the workup of patients with certain intrathoracic masses undergoing transthoracic needle biopsy (TNB). METHODS: Over a 4-year period, 1603 patients underwent TNB at our institution. Of these, 79 had EM examination of the aspirated material. The study is a retrospective review of this latter group. Previous use of EM for TNB had suggested that it may he helpful in those with pleural, chest wall, and mediastinal lesions. The 40 men and 39 women had pulmonary (n = 49), mediastinal (n = 17), pleural (n = 10), and chest wall (n = 3) lesions. RESULTS: The adequate specimen rate was 59% (47 of 79) for light microscopy (LM) and 37% (29 of 79) for EM. Of the 28 patients with satisfactory specimens for both LM and EM, the correct diagnosis was obtained by LM 79% (22 of 28) and EM 96% (27 of 28) of the time. Electron microscopy was most helpful in patients with mediastinal (6 of 6 correct versus 3 of 6 for LM) and pleural (3 of 3 versus 1 of 3) lesions. CONCLUSIONS: In specific circumstances, EM can be a very useful adjunct to LM in patients undergoing TNB. Problems with sample adequacy must be addressed.

Adult↗

Electron microscopy of human immunodeficiency virus.

Two cell lines of human T lymphocytes (H9 and CEM) chronically infected with isolates of human or simian immunodeficiency viruses were examined by electron microscopy. Scanning electron microscopy of H9 cells showed characteristic morphological changes in the cells after infection with human T cell lymphotropic virus type III (HTLV-IIIB); these included loss of cell microvilli and their replacement by rounded surface protrusions. Virus particles were present over the whole cell surface, but were more numerous between, rather than on, surface protrusions. In contrast, CEM cells infected with three other isolates of the virus showed little change in morphology compared with uninfected cells; they typically had a single large aggregate of virus particles at the posterior end of the cell. Transmission electron microscopy of sections of infected cells showed budding virus in only a small proportion of cells although many had mature virus particles on their surface. The immature particles released by budding had a ring-like morphology in contrast to the mature virus with its characteristic elongated nucleoid. Surface spikes were rarely seen on sectioned virus particles, but were more obvious on the simian immunodeficiency virus than on human isolates. Negative staining of purified virus preparations showed that the peripheral dense material of immature particles had a striated structure. Surface spikes were not seen by negative staining on either immature or mature virus particles. A third type of smaller particle with surface spikes was found in all negatively stained preparations.

Animals↗

X-ray microscopy.

The subject of X-ray microscopy (high-resolution X-ray imaging of general nonperiodic structures), an area in which much progress has been made in recent years, is reviewed. The main techniques are briefly described. Achievable performance levels, which for many years were highly speculative, can now be understood with fair accuracy in terms of basic X-ray and specimen properties, and techniques have progressed to the point where actual results are nearing those levels. In terms of specimen size and imaging resolution, X-ray microscopies lie between electron and light microscopy, and are thus suited to imaging extremely large and complex structures; in addition, they demand little or no specimen preparation, and can be used to observe local composition and chemical state as well as structure. Thus X-rays, which have played the leading role in imaging crystallizable materials, may also prove to be highly valuable in the imaging of very large non-crystalline structures. Throughout the treatment, attention is paid to the relationships connecting the subject with X-ray crystallography.

Crystallography, X-Ray↗