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At least 1,333 records · Page 74Linked to original sources
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↗
PubMed1976

Studies on the biological effects of deuteriated organic compounds. II. Morphological changes of Epidermophyton floccosum exposed to perdeuteriated n-hendecanoic acid studied with interference contrast and scanning electron microscopy.

In order to visualise the morphological changes of Epidermophyton floccosum associated with exposure to perdeuteriated n-hendecanoic acid, the architecture of the dermatophyte was investigated by means of interference contrast and scanning electron microscopy. The morphology of mycelia grown on substrate containing perdeuteriated n-hendecanoic acid, or the unlabelled analogue, was compared. The perdeuteriated n-hendecanoic acid produced a characteristic undulant effect of the hyphae. The characteristic wave-like appearance of the mycelia looked similar to the curling effect occuring after treatment of dermatophytes with griseofulvin, but was not so pronounced. Perdeuteriated n-hendecanoic acid, unlike the unlabelled analogue, also seems to cause a reduction of the number of chlamydospores perforations of the macroconidia. The changes in the morphological structure of Epidermophyton floccosum exposed to pereuteriated n-hendecanoic acid have been investigated. Morphological examination of mycelia exposed to this substance by interference contrast microscopy demonstrated a picture of defect hyphae and macroconidia. By the aid of scanning electron microscopy we have attempted to obtain a better visualization of these changes at ultrastructural level.

Chemical Phenomena↗
PubMed1993

Scanning electron microscopy of synaptonemal complexes.

The novel application of scanning electron microscopy to study whole-mount surface-spread synaptonemal complex complements of rye (Secale cereale) and rat (Rattus norvegicus) is described. Scanning electron microscopy is able to resolve the third dimension in such preparations and improve the tracing of the continuity of lateral elements without losing information that could be obtained by conventional transmission electron microscopy. This improvement is likely to benefit detailed studies of chromosome synapsis and karyology, and may provide a means of circumventing technical obstacles inhibiting the use of surface-spreads as substrates for in situ hybridization under the electron microscope.

Animals↗
PubMed2000

Three-dimensional electron cryo-microscopy as a powerful structural tool in molecular medicine.

Electron cryo-microscopy has established itself as a valuable method for the structure determination of protein molecules, protein complexes, and cell organelles. This contribution presents an introduction to the various aspects of three-dimensional electron cryomicroscopy. This includes the need for sample preservation in the microscope vacuum, strategies for minimizing radiation damage, methods of improving the poor signal-to-noise ratio in electron micrographs of unstained specimens, and the various methods of three-dimensional image reconstruction from projections. The various specimen types (e.g., flat and tubular two-dimensional crystals, protein filaments, individual protein molecules, and large complexes) require different means of three-dimensional reconstruction, and we review the five major reconstruction techniques (electron crystallography, helical reconstruction, icosahedral reconstruction, single-particle reconstruction, and electron tomography), with an emphasis on electron crystallography. Several medically relevant three-dimensional protein structures are chosen to illustrate the potential of electron cryo-microscopy and image reconstruction techniques. Among the structural methods, electron cryo-microscopy is the only tool for studying objects that range in size from small proteins over macromolecular complexes to cell organelles or even cells.

Cryoelectron Microscopy↗
PubMed2003

Identification of caveolae-like structures on the surface of intact cells using scanning force microscopy.

Caveolae are small, functionally important membrane invaginations found on the surface of many different cell types. Using electron microscopy, caveolae can be unequivocally identified in cell membranes by virtue of their size and the presence of caveolin/VIP22 proteins in the caveolar coat. In this study we have applied for the first time scanning force microscopy (SFM), to visualize caveolae on the surface of living and fixed cells. By scanning the membranes of Chinese hamster ovary cells (CHO), using the tapping mode of the SFM in fluid, we could visualize small membrane pits on the cell membranes of living and fixed cells. Two populations of pits with mean diameters of around 100 nm and 200 nm were present. In addition, the location of many pits visualized with the SFM was coincident with membrane spots fluorescently labeled with a green fluorescent protein-caveolin-1 fusion protein. Scanning force microscopy on cells treated with methyl-beta-cyclodextrin, an agent that sequesters cholesterol and disrupts caveolae, abolished pits with a measured diameter of 100 nm but left pits of around 200 nm diameter intact. Thus, the smallest membrane pits measured with the SFM in CHO cells were indeed very likely to be identical to caveolae. These experiments show for the first time that SFM can be used to visualize caveolae in intact cells.

Animals↗
PubMed1997

[Imaging of laser thermokeratoplasty lesions by optical low coherence tomography and polarization microscopy after Sirius Red staining].

BACKGROUND: Information on the extent and degree of the thermal effect produced is of great importance for control of the laser dosage in laser thermokeratoplasty (LTK) and for postoperative follow-up. We investigated on acute LTK effects which information images obtained by optical low coherence tomography (OCT) offer compared to those obtained by polarization microscopy. METHODS: Porcine eyes were irradiated through a 400 microns quartz fiber using light from a laser diode emitting up to 300 mW at a wavelength of 1.86 microns. Thermal lesions of varying strength were scanned using an experimental OCT device with about 25 microns lateral and 20 microns axial resolution. Histologic evaluation of the scanned areas was done by polarization microscopy after Sirlus-Red staining, and similar lesions were also analyzed by TEM. RESULTS: Both methods differentiated three damage zones a transition zone, a zone of moderate coagulation, and a central zone of strong coagulation. In the transition zone, increased birefringence was seen in polarization microscopy, which correlated with increased light scattering seen in the OCT images. In the moderately coagulated zone, a decrease in birefringence was associated with an even stronger increase of the OCT signal. In the central zone, a loss of the fibrillar tissue structure was observed, which led to a complete loss of birefringence and a strong reduction of the OCT signal. CONCLUSIONS: Although OCT does not provide the detailed information on thermal changes of tissue seen by the histologic method, it offers information on the extent and degree of tissue changes without preparation artifacts and provides a non-invasive method of immediate and follow-up control of LTK lesions. A quantitative analysis of changes in corneal thickness and curvature is much simpler than by a slit lamp. Time-resolved measurements of corneal light scattering may be used for on-line control of the laser-light dosage during LTK.

Animals↗
PubMed2001

Non destructive analysis of the wax layer of apple (Malus domestica Borkh.) by means of confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was used to non-destructively analyse the changes in the structure and thickness of the cuticle during storage of apples (Malus domestica Borkh.). Interpretation of the confocal images was performed by comparison with scanning electron microscopy and environmental scanning electron microscopy images. The natural reflectance of the wax and the auto-fluorescence of the underlying cells made it possible with CLSM to distinguish the wax from the underlying layers without any pretreatment of the fruit. The thickness of the consecutive layers (wax, cutin, cells) could be estimated from measurements of the reflection and fluorescence intensities as a function of the number of pixels. The mean wax-layer thickness measured in this way amounted to 2.58 microm, 3.41 microm or 4.14 microm for the cultivars Jonagold, Jonagored and Elstar, respectively. Changes in the wax structure and cells of the same important Belgian apple cultivars as mentioned above were monitored during nine months of storage in ultra low oxygen and after exposure to ambient conditions. The changes in the wax ultrastructure and cell morphology are likely related to water losses and specific protection of the apple cultivars against water losses during storage and shelf life.

Fruit↗
PubMed2000

The role of chlamydia in the pathogenesis of pulmonary emphysema. Electron microscopy and immunofluorescence reveal corresponding findings as in atherosclerosis.

Chlamydia pneumoniae has been detected in atherosclerotic plaques by various means. Chlamydiae are able to cause persistent infections. Serologically elevated antibody titers are found in severe chronic obstructive pulmonary disease. In atherosclerosis and pulmonary emphysema, inflammatory reactions can be seen by means of light microscopy. Specimens from patients with obliterative arteriosclerosis undergoing thrombendarteriectomy and with advanced emphysema undergoing lung volume reduction surgery were examined using scanning (SEM) and transmission (TEM) electron microscopy, and using immunofluorescence with monoclonal antibodies and antiserum against chlamydiae. SEM shows spherical bodies (SBs) with a diameter from 0.3 microm to 0.6 microm on the surface of the alveoli and bronchioles, as well as in atherosclerotic plaques. In atherosclerosis and emphysema, SBs reveal a double membrane, adherence to collagen fibers, tissue destruction, as well as intracellular and interstitial localization in TEM. They show in parts a densely packed central structure. SBs are seen both in alpha-1-antitrypsin deficiency emphysema and smoker's emphysema. Using immunofluorescence microscopy, spots are seen in corresponding distributions to the SBs. Morphological findings are typical for aberrant chlamydiae seen in persistent infections. Chronic infection and bacterial colonization associated with progressive disease seems to be relevant not only in atherosclerosis but also in pulmonary emphysema.

Arteriosclerosis↗
PubMed1997

Detection of sperm within Gyrodactylus (Platyhelminthes, Monogenea) tissues using fluorescence and transmission electron microscopy.

A rapid fluorescent staining method demonstrating spermatozoa within gyrodactylid monogeneans is described. Gyrodactylids fixed and stained in 2% acetic acid containing 1 microgram ml-1 bisBenzimide (Hoechst 33258) were viewed using epifluorescence microscopy. In addition to staining sperm in the testis, seminal vesicle and seminal receptacle, the technique highlighted sperm in the gut and cytoplasmic lining of the uterus, locations from which they had not previously been recorded. The technique was more rapid than transmission electron microscopy (TEM), which was used to confirm the observations. Fluorescence microscopy provided an overview of sperm orientation and distribution that could otherwise be obtained only from serial ultrathin sections. Using the fluorescence technique and/or TEM we have located migrating sperm in the tissues of Gyrodactylus turnbulli, G. bullatraudis and G. gasterostei, although these sperm do not appear to enter the tissues of embryos in utero. The technique can be used to study insemination patterns in gyrodactylid populations and in experimental studies of gyrodactylid reproduction.

Animals↗
PubMed1991

Morphologic changes in the human amnion epithelium that accompany labor as seen with scanning and transmission electron microscopy.

Scanning and transmission electron microscopy were used to assess the influence of normal, active labor on the ultrastructure of the human amnion epithelial membrane. Amnion membranes (reflected and placental portions) were obtained from patients either in active labor who were delivered vaginally or by cesarean section after 6 to 12 hours of labor or from patients who underwent elective cesarean section before clinical signs of overt labor. Scanning electron microscopy revealed that reflected amnion membranes that were obtained from patients who were not in labor consisted of a uniform single layer of epithelial cells with numerous microvilli on the apical surface and closely associated cellular borders. In contrast, amnion membranes that were obtained from patients who were in labor consisted of a single layer of epithelial cells, which was interrupted by wide intercellular gaps and extracellular extrusions. Transmission electron microscopy showed that intercellular junctions tended to be less complex in patients who were in labor versus patients who were not in labor. Although lipid droplets were prevalent in both patient groups, specimens that were obtained from patients who were in labor had more lipid droplets per cell than specimens from patients who were not in labor. These results support the theory that the complex biochemical events that culminate in parturition are accompanied and/or preceded by demonstrable morphologic changes in the amnion membrane.

Amnion↗
PubMed1987

Colloidal gold fluorescent microspheres: a new retrograde marker visualized by light and electron microscopy.

A new retrograde tracer, rhodamine latex microspheres, permits labeled neurons to be visualized with fluorescence light microscopy. However, their use has been limited to the light microscope. We now have developed colloidal gold fluorescent microspheres which identify retrogradely labeled neurons first by fluorescence microscopy and then by electron microscopy. This new fluorescent/EM tracer will find widespread use in the field of neuroscience to elucidate the ultrastructural integrity of neuronal networks.

Animals↗
PubMed1984

Transmission and scanning electron microscopy of experimental pulmonary simian varicella (Delta herpesvirus) infection in African green monkeys (Cercopithecus aethiops).

The lungs of African green monkeys infected with simian varicella virus (Delta herpesvirus) were examined by scanning and transmission electron microscopy. Endothelial cell shrinkage, oedema formation and necrosis characterized the scanning electron microscopy of the affected vasculature. Oedema of the interalveolar septa with swelling of the capillary endothelial cells was observed by transmission electron microscopy. Numerous viral particles were associated with a generalized area of necrosis of the alveolar wall.

Animals↗
PubMed1992

Epi-polarization and incident light microscopy readily resolve an autoradiographic or heavy metal label from an obscuring background or second label.

Difficulty encountered in resolving grains of exposed photographic emulsion in autoradiographs of the densely melanized retinal pigment epithelium was solved by using epi-polarized or incident light microscopy. The apparatus used included a metallurgical illuminator specifically designed for epi-polarization microscopy or, as a less expensive but only slightly less effective alternative, a modified fluorescence illuminator. The black melanin granules absorb incident light (as they do in vivo) while the silver grains reflect it producing a "darkfield-like" representation. Brightfield and darkfield-like images can be alternated easily and quickly, or both can be viewed simultaneously. Epi-polarization microscopy has wider application in resolving a reflective label over any opaque background staining or dark second label.

Animals↗
PubMed1985

Pathological changes during the development of the vestibular sensory and ganglion cells of the Bronx waltzer mouse. Scanning and transmission electron microscopy.

Vestibular receptors and ganglia of homozygous Bronx waltzer (bv/bv) mice were investigated by scanning and transmission electron microscopy at various stages between 3 days and 90 days after birth. Scanning electron microscopy revealed that there was already a considerable lack of hair bundles in the maculae utriculi, as well as in the cristae ampullares by the 3rd day after birth. During development, the growth of the remaining hair bundles was observed but the most of them exhibited morphological abnormalities. Transmission electron microscopy revealed early degeneration of sensory cells followed by delayed maturation of the remaining sensory cells. The sensory cells which seem unaffected displayed immature features in adult animals. In type I hair cells, the calyces were incomplete, contacts between the cell and the afferent calyces were immature and synaptic bodies persisted. In some type II hair cells, there was an abnormal overabundance of afferent nerve endings, which implies that these type II cells could be immature type I cells. Immature features were also observed in the vestibular ganglia, particularly the absence of the myelin sheath around the perikarya. We discuss the relationship between these vestibular morphogenetic abnormalities and those described in the cochlear system.

Animals↗
PubMed1995

Differentiation of filoviruses by electron microscopy.

Cultured monolayers of MA-104, Vero 76, SW-13, and DBS-FRhL-2 cells were infected with Marburg (MBG), Ebola-Sudan (EBO-S), Ebola-Zaire (EBO-Z), and Ebola-Reston (EBO-R) viruses (Filoviridae, Filovirus) and examined by electron microscopy to provide ultrastructural details of morphology and morphogenesis of these potential human pathogens. Replication of each filovirus was seen in all cell systems employed. Filoviral particles appeared to enter host cells by endocytosis. Filoviruses showed a similar progression of morphogenic events, from the appearance of nascent intracytoplasmic viral inclusions to formation of mature virions budded through plasma membranes, regardless of serotype or host cell. However, ultrastructural differences were demonstrated between MBG and other filoviruses. MBG virions recovered from culture fluids were uniformly shorter in mean unit length than EBO-S, EBO-Z, or EBO-R particles. Examination of filovirus-infected cells revealed that intermediate MBG inclusions were morphologically distinct from EBO-S, EBO-Z, and EBO-R inclusions. No structural difference of viral inclusion material was observed among EBO-S, EBO-Z, and EBO-R. Immunoelectron microscopy showed that the filoviral matrix protein (VP40) and nucleoprotein (NP) accumulated in EBO-Z inclusions, and were closely associated during viral morphogenesis. These details facilitate the efficient and definitive diagnosis of filoviral infections by electron microscopy.

Animals↗
PubMed1995

Immobilizing and imaging microtubules by atomic force microscopy.

Microtubules isolated from pig brains have been immobilized on an inorganic substrate for use in AFM studies. The method employs 4-aminobutyldimethylmethoxysilane and glutaraldehyde to activate a silicon wafer for binding the biopolymer. The covalent bond ensures the positional stability of the tubules on the substrate, and allows reproducible scanning probe experiments. Microtubules have been imaged both by atomic force and scanning tunneling microscopy, yielding results very similar to electron microscopy. The average apparent height of the tubules is smaller than observed with transmission electron microscopy (25 nm) and is smaller in buffer solution (10 nm) than in air (15 nm). The biopolymer surface is softer under buffer than in air. The highest resolution was obtained with the tapping mode where surface features as small as 10 nm in X and Y have been resolved. Gold-coated tubules bound on silicon have been successfully imaged by STM, while images of uncertain origin were generated for tubules deposited on graphite. It is shown that artefacts imaged on a blank graphite surface can easily be confounded with collapsed tubules.

Animals↗
PubMed2006

Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches.

The aim of this article is to review the basic principles of two-photon excitation fluorescence (2PEF) microscopy and to compare the advantages and disadvantages of 2PEF imaging to other microscopy methodologies. 2PEF imaging is a nonlinear approach that generates images of optical sections and that is particularly well suited for deep-tissue and in vivo imaging of live animals. The nonlinear excitation used for 2PEF offers the advantage, too, of being able to generate contrast from second or third harmonic generation as well as coherent anti-Stokes Raman scattering. We also review the recent use of nonlinear excitation to provide image resolution beyond the diffraction limit and discuss the progress in non-scanning (planar) 2PEF microscopy, an approach that holds great potential for large-scale quantitative imaging and plate reading, e.g., in screening applications.

Animals↗
PubMed2006

Intravital fluorescence microscopy in pulmonary research.

Over the last several years, microscopy as a scientific tool has reinvented itself evolving from a group of principally descriptive methodologies to encompass a wide range of primary tools and techniques to investigate the molecular organization of organs, tissues and cells. Advances in microscope and camera design, fluorescent dye technology, the development of fluorescent proteins as well as the advent of inexpensive powerful computers, has led to the feasibility of simultaneous sub micron resolution and quantitation of multiple concurrent molecular markers for both protein and DNA. Confocal microscopy has allowed optical sectioning and reconstruction of tissues in three dimensions. Finally, the development of multiphoton methodologies as an extension of optical sectioning microscopy has further improved the potential utility of this technology when examining living or light scattering tissues such as the lung. In order to illustrate the utility of two-photon methods in pulmonary biology, we present the application of this approach to the study of cellular trafficking in situ and to the study of pulmonary vasoregulation in an ex vivo rodent model.

Animals↗