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At least 1,621 records · Page 90Linked to original sources
PubMed1983

Demonstration of virus particles within immune complexes by electron microscopy.

A method has been developed to detect virus particles within immune complexes by electron microscopy (VICEM test). Radioiodinated or unlabeled adenovirus type 2 immune complexes (IC) in human serum were used to determine the optimal conditions. Best results were obtained when the immune complexes were precipitated with 5% polyethylene glycol 6000, adsorbed with 0.5% of protein A-containing Staphylococcus aureus (Cowan 1) and eluted, with brief sonication, in 2 M propionic acid. When the eluate was examined by the pseudoreplica method of electron microscopy, morphologically intact viruses, concentrated over five-fold and free from background serum proteins, were readily demonstrated. The method is applicable to virus IC with a wide-range of antigen-to-antibody ratios and requires a minimum of 10(4)--10(5) virus particles. This method should be useful for the characterization of IC in clinical specimens obtained from patients with viral infections and for the possible demonstration of viral agents in immune complex diseases of unknown etiology. A modification of the technique can also be used for the rapid serological identification of viruses by electron microscopy, as exemplified by studies using adeno- and herpes simplex viruses.

Adenoviruses, Human↗
PubMed1986

Demonstration of calicivirus in human faeces by immunosorbent and immunogold-labelling electron microscopy methods.

Immunosorbent electron microscopy (ISEM) and immuno-gold staining (IGS) electron microscopy methods have been applied to human faeces, shown by direct electron microscopy (EM) to contain calicivirus. Caliciviruses were successfully trapped on grids coated with positive rabbit or human antisera against calicivirus, but not with negative sera. Caliciviruses were specifically labelled with gold particles, when treated with positive rabbit or human antisera against calicivirus followed by protein A-gold and goat anti-rabbit or anti-human IgG gold conjugates in an indirect method in suspension. Goat anti-human IgM gold complexes did not react with the available antisera. Attempts to label caliciviruses trapped on the grids were unsuccessful. No cross-reactions were observed with Norwalk agent-like particles, hepatitis A virus or poliovirus type 3 by any of the methods. The results indicate that ISEM and IGS may be useful techniques for detection and identification of small viruses present in low concentrations in faeces.

Caliciviridae↗
PubMedDate not supplied

Evaluation of an immunogold electron microscopy technique for detecting bovine coronavirus.

A solid phase colloidal gold immunoelectron microscopy (IGEM) technique for detecting bovine coronavirus (BCV) was developed and shown to be specific. This test was compared with three other diagnostic tests using fifteen faecal samples. Bovine coronavirus was detected in 2 samples by direct electron microscopy (DEM), in 3 samples by immunosorbent electron microscopy, in 5 samples by haemadsorption-elution-haemagglutination and in 6 samples by IGEM. Ninety four faecal samples were tested by DEM and IGEM. Of 26 samples found to contain BCV by IGEM only 14 were positive by DEM. The IGEM technique is simple, efficient and less susceptible than others to non-specific reactions.

Animals↗
PubMed1989

Intravital microscopy: a new in vivo technique for visualizing and quantifying effects of regulatory peptides on choledochoduodenal junction motility.

Using intravital microscopy, we studied the in vivo effects of regulatory peptides on choledochoduodenal junction motility in guinea pigs. During basal and hormone-stimulated periods, intravital microscopy documented rhythmic, asymmetrical, "milking" contractions of the sphincter ductus choledochi (SDC) which occurred independent of sphincter ampullae (SA) contractions or were followed by SA contractions. Cholecystokinin octapeptide (CCK-8) (greater than or equal to 0.01 micrograms/kg) increased the frequency of SDC contractions and at higher doses (greater than or equal to 0.1 microgram/kg) increased the frequency of SA contractions. Pentagastrin (greater than or equal to 1.0 microgram/kg) and secretin (10 micrograms/kg) decreased the contraction frequencies of both sphincters. Biliary manometry demonstrated similar effects of these peptides on the frequency of the SDC and SA contractions, but also showed that CCK-8 (0.1 microgram/kg) increased the amplitude of SDC and SA contractions while pentagastrin (1 microgram/kg) decreased the amplitude of only SDC contractions. Tetrodotoxin and atropine did not affect hormone-induced changes in frequency, but tetrodotoxin reduced the increase in amplitude of contraction caused by CCK-8. We concluded that intravital microscopy provides a sensitive, in vivo technique to visualize and quantify the complex motility of a small structure like the choledochoduodenal junction.

Animals↗
PubMed1989

Native fibrin gel networks observed by 3D microscopy, permeation and turbidity.

Native fully hydrated fibrin gels formed at different fibrinogen and thrombin concentrations and at different ionic strengths were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels were found to be composed of straight rod-like fiber elements that often came together at denser nodes. In gels formed at high fibrinogen concentrations, or with high amounts of thrombin, the spaces between the fibers decreased, indicating a decrease of gel porosity. The fiber strands were also shorter. Gel porosity decreased dramatically in gels formed at the high ionic strengths. Shorter fibers were observed and fiber swelling occurred at ionic strengths above 0.24. Quantitative parameters for gel porosity, fiber mass/length ratio and diameter were also derived by liquid permeation and turbidometric analyses of the gels. Permeation analysis showed that gel porosity (measured as Ks) decreased in gels formed at higher fibrin and thrombin concentrations in agreement with the porosity observed by microscopy. The turbidometric analysis showed good agreement with the permeation data for gels formed at various thrombin concentrations, but supported the permeation data more poorly in gels formed at different fibrinogen concentrations, especially above 2.5 mg/ml. Turbidometric analysis showed that the fiber mass/length ratio and diameter decreased in gels formed at ionic strength up to 0.24, as was seen in the permeation study. However, at higher ionic strengths swelling of the fibers was suggested from the gel turbidity data and this was also indicated by microscopy. These findings are discussed in relation to previous hydrodynamic and electron microscopic studies of fibrin gels.

Fibrinogen↗
PubMed1992

SIMS microscopy: a tool to measure the intracellular concentration of carbon 14-labelled molecules.

Monolayer cultures of human fibroblasts were incubated for 24 h with 14C-arginine and observed by means of SIMS microscopy (ion microscopy). Carbon 14 imaging showed the intracellular distribution of labelled arginine which featured high nuclear incorporation. The local concentration of this amino acid in different cells and intracellular structures was assessed through local isotopic 14C/12C ratio measurement. This relates the signal intensity of the labelling isotope carbon 14 to that of the corresponding natural isotope (carbon 12) of known tissular concentration. Using this method we were able to measure minor variations in the molecular concentration of arginine (expressed as mumol/g of tissue) between different fibroblasts. Results of this study indicate that SIMS microscopy is well adapted to carbon 14 detection and can provide quantitative maps of the cellular and subcellular distribution of 14C-labelled molecules.

Adult↗
PubMed1990

Scanning tunneling microscopy of the enzymes of muscle glycogenolysis.

Scanning tunneling microscopy (STM) has been used to examine the structures of the skeletal muscle enzymes phosphorylase and phosphorylase kinase. The interaction of these two proteins represents the last step in the process of signal transduction which results in muscle glycogen being converted into metabolic energy for use in muscle contraction. Phosphorylase b has a molecular weight of 97,000 and the dimer is seen by STM to have dimensions of 11 X 5.7 nm. Phosphorylase b has a tendency to form linear arrays of dimers on the graphite surface used as the support for STM imaging. Phosphorylase kinase is imaged as a butterfly-like object with lateral dimensions of 36 X 27 nm. The molecular thicknesses given by scanning tunneling microscopy for these two non-conducting molecules is significantly less than expected. The height measurement in STM is dependent not only on the surface topology of the object being imaged, but also on the electronic work function of the object compared to that of the graphite surface on which it lies. In addition to the individual proteins, a complex between phosphorylase and phosphorylase kinase has been observed by scanning tunneling microscopy.

Animals↗
PubMed1986

Hypothalamic neuroendocrine correlates of cutaneous burn injury in the rat: I. Scanning electron microscopy.

Rats were given a standard scald burn on 60% of the body surface or only a sham burn and were sacrificed at intervals from 6 hr to 14 days later. Serum thyroxine (T4), free thyroxine index (FT4I) and triiodothyronine (T3) were depressed compared to values in respective shams as early as 6 hr post-burn. T4 and FT4I were less depressed on post-burn days (PBD) 2-3 than on PBD 1 and then exhibited a further fall. T3 remained depressed through PBD 14. Pineal melatonin content was elevated at 6 hr and fell to the normal daytime range in subsequent samples. The ventral portion of the diencephalon was prepared for scanning electron microscopy. Only in the burned rats and beginning on PBD 2, large numbers of supraependymal neurons (SEN) appeared in the ventricular space attached to the inferior walls and floor of the third cerebral ventricle. Transmission electron microscopy was used to confirm the neuronal nature of the SEN. Viewed by scanning electron microscopy, these persisted through PBD 14. SEN were interconnected by cables of their neurites exhibiting varicosities on individual neurites as they passed over perikarya of other SEN. Some SEN were seen to be only partially emerged from the underlying tissue and others were seen to send a thick process into the hypothalamic tissue. These observations indicate that after peripheral injury there is marked plasticity of the brain in an area thought to control the endocrine systems that show abnormalities after such a peripheral injury. The timing, location and nature of these anatomic changes indicate the possibility that at least some aspects of central nervous orchestration of the endocrine metabolic response to injury may be related to the emergence of a neuronal system receiving or sending messages through the cerebrospinal fluid and/or through new neurite circuits along the surface of the third ventricular wall. These structures may appear in response to initial primary hormonal changes and/or may play a role in maintaining the post-injury hormonal milieu manifested in part by a subsequent second fall in serum T4.

Animals↗
PubMedDate not supplied

Fluorescence microscopy in the detection of early myocardial infarction.

Fluorescent microscopy is amongst the many techniques devised for the post-mortem detection of early myocardial infarction. The method has the advantage of sensitivity, speed and simplicity, compared with more complex techniques such as enzyme histochemistry and electron microscopy. Disadvantages include the difficulty of permanent preservation of the sections and lack of sharp differentiation between normal and very early infarction--though the latter problem is common to most other methods, except enzyme histochemistry. There are two main techniques in fluorescent microscopy: The examination of sections specifically stained by fluorochrome dyes; The re-examination of haematoxylin-eosin sections for 'autofluorescence'.

Acridine Orange↗
PubMed1985

Differentiation of Pseudomonas aeruginosa and Enterobacteriaceae in direct smears based on measurements by scanning electron microscopy.

We believe that experienced observers can often distinguish between Enterobacteriaceae and Pseudomonas aeruginosa, by differences in dimensions in Gram-stained direct smears. Many microbiologists question whether this should be attempted because of overlap in dimensions. We have found that culture results confirm our observations about 80% of the time and that such reporting is helpful in diagnosis and treatment. We decided to try to verify the differences in dimensions objectively. Because of the limitations of making measurements by light microscopy, exudate from clinical specimens and from experimental mouse infections were examined by scanning electron microscopy. Discriminant function analysis was applied to the dimensions of length and width, and this showed that 83% of the organisms in both groups could be correctly classified on the basis of the dimensions. This supports our premise that experienced observers should be able to differentiate between these organisms in Gram-stained direct smears, using light microscopy with sufficient confidence to provide clinically useful information.

Animals↗
PubMedDate not supplied

Confocal microscopy in chemical neuroanatomy.

The application of computer-assisted confocal laser scanning microscopy in chemical neuroanatomy is briefly reviewed and illustrated with examples taken mainly from morphological studies of neurons in the spinal cord of the lamprey, a lower vertebrate. The principle of operation of the confocal microscopy system used, with emphasis on the computer-controlled image data acquisition and further processing of the large image volumes, is described. The imaging characteristics of confocal laser scanning microscopy in combination with fluorescence labelling, in particular the capacity to perform three-dimensional reconstructions of high optical resolution, are also described and illustrated with projection images from different viewing angles and with stereo pair reconstructions. Different possibilities for image processing, e.g. surface shading and surface extraction, are also described and illustrated.

Animals↗
PubMed1989

Human enamel structure studied by high resolution electron microscopy.

Human enamel structural features are characterized by high resolution electron microscopy. The human enamel consists of polycrystals with a structure similar to Ca10(PO4)6(OH)2. This article describes the structural features of human enamel crystal at atomic and nanometer level. Besides the structural description, a great number of high resolution images are included. Research into the carious process in human enamel is very important for human beings. This article firstly describes the initiation of caries in enamel crystal at atomic and unit-cell level and secondly describes the further steps of caries with structural and chemical demineralization. The demineralization in fact, is the origin of caries in human enamel. The remineralization of carious areas in human enamel has drawn more and more attention as its potential application is realized. This process has been revealed by high resolution electron microscopy in detail in this article. On the other hand, the radiation effects on the structure of human enamel are also characterized by high resolution electron microscopy. In order to reveal this phenomenon clearly, a great number of electron micrographs have been shown, and a physical mechanism is proposed.

Dental Caries↗
PubMed1992

Cryo-electron microscopy of vitrified muscle samples.

A great deal of information on the 3-dimensional structure of the protein assemblies involved in muscle contraction has been obtained using conventional transmission electron microscopy. In recent years, developments in cryo-electron microscopy have facilitated work with fully hydrated, non-chemically fixed specimens. It is shown how this technique can be used to visualize muscle sarcomere filaments in quasi-native conditions, to access hitherto inaccessible states of the crossbridge cycle, and to obtain new high resolution structural information on their 3-dimensional protein structure. A short introduction to the crossbridge cycle and its biochemically accessible states illustrates the problems amenable to studies using the electron microscope, as well as the possibilities offered by cryo-microscopy on vitrified samples. Work on vitrified cryo-sections and myosin filament suspensions demonstrates the accessibility of crossbridge states and gives implications on the gross structural features of myosin filaments. Recent studies on actin filaments and myosin (S1) decorated actin filaments provide the first high resolution data on vitrified samples. The use of photolabile nucleotide precursors allows the trapping of short lived states in the millisecond time range, thereby visualizing intermediate states of the crossbridge cycle.

Animals↗
PubMed1995

Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy.

To elucidate the protein composition of the nemaline bodies present in the muscle fibres of patients with congenital nemaline myopathy (CNM), we studied muscle biopsies with monoclonal antibodies against alpha-actinin and desmin in combination with a modified Gomori trichrome method. Electron microscopy of immunolabelled resin embedded sections was used for cytochemical localisation of alpha-actinin and desmin. Light microscopy of sections immunolabelled for alpha-actinin showed a cross-striation of the muscle fibres corresponding to the Z band pattern, focal thickening of the Z bands and additional reactivity with a granular pattern corresponding to the presence of nemaline bodies. Labelling of desmin did not show a similar pattern. Electron microscopy confirmed the presence of alpha-actinin in the nemaline bodies and Z bands, whereas desmin was only found in intermediate filaments around the Z bands. Western blots showed single, sharp alpha-actinin bands indistinguishable from normal. Our results provide direct evidence for the presence of alpha-actinin in nemaline bodies and a lack of quantitative or qualitative differences between the alpha-actinin of normal and CNM muscle.

Actinin↗
PubMedDate not supplied

Configuration of interdomain linkers in pyruvate dehydrogenase complex of Escherichia coli as determined by cryoelectron microscopy.

The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains). In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC. From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions. To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin. In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core. Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro. Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core. The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3. These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations.

Bacterial Proteins↗
PubMed2004

Characterization of ionizing radiation-induced ring chromosomes by atomic force microscopy.

We applied atomic force microscopy (AFM) to the structural analysis of radiation-induced ring chromosomes. Constrictions observed on the metaphase ring chromosome were found to correspond to the centromere regions of the ring chromosome in comparison with the AFM image of the centromere of rod chromosomes and with the fluorescence in situ hybridization (FISH) technique. Section analysis by AFM revealed that some ring-like chromosome fragments and ring-like chromatid fragments were thicker than standard chromosomes or chromatids, suggesting that they were ring chromosomes viewed edge on. Topographic analysis by AFM makes it possible to distinguish a ring viewed edge on that is difficult to recognize as a ring by light microscopy and to discriminate between a centric ring chromosome and an acentric ring chromosome using the same slides prepared for light microscopy.

Centromere↗
PubMed2005

Intravital imaging of tumour vascular networks using multi-photon fluorescence microscopy.

The blood supply of solid tumours affects the outcome of treatment via its influence on the microenvironment of tumour cells and drug delivery. In addition, tumour blood vessels are an important target for cancer therapy. Intravital microscopy of tumours growing in 'window chambers' in animal models provides a means of directly investigating tumour angiogenesis and vascular response to treatment, in terms of both the morphology of blood vessel networks and the function of individual vessels. These techniques allow repeated measurements of the same tumour. Recently, multi-photon fluorescence microscopy techniques have been applied to these model systems to obtain 3D images of the tumour vasculature, whilst simultaneously avoiding some of the problems associated with the use of conventional fluorescence microscopy in living tissues. Here, we review the current status of this work and provide some examples of its use for studying the dynamics of tumour angiogenesis and vascular function.

Animals↗
PubMed2004

Corneal thickness measurement by confocal microscopy, ultrasound, and scanning slit methods.

PURPOSE: To measure corneal thickness by using a calibrated confocal microscope and to compare this measurement to thickness determined by ultrasonic and noncontact scanning slit pachymetry. DESIGN: Comparison of corneal thickness measured by using four instruments in normal subjects. METHODS: Thickness measured by a clinical confocal microscope (Tandem Scanning) was calibrated from measurements of polymethylmethacrylate contact lenses with known thickness. Corneal thickness was measured in one eye of 24 normal subjects by using this instrument, two ultrasonic pachymeters (DHG-1000 and Sonogage), and a noncontact optical scanning slit pachymeter (Orbscan II). RESULTS: Mean corneal thickness measured by confocal microscopy was 516 +/- 30 microm (+/-SD). This was less than the mean thickness measured by both ultrasonic pachymeters, 554 +/- 28 microm by the DGH, and 555 +/- 28 microm by the Sonogage (P <.001). Thickness measured by the Orbscan II pachymeter was 540 +/- 35 microm (P <.001, compared with either confocal or ultrasound) after applying an "acoustic factor" of 0.92, a default correction of the software. CONCLUSION: Corneal thickness measured by calibrated confocal microscopy is approximately 39 microm (7.0%) less than thickness measured by two commonly used ultrasonic pachymeters and approximately 24 microm (4.4%) less than thickness measured by the corrected Orbscan II pachymeter. These differences are important for planning and measuring the effects of refractive and other surgical procedures. The precision of confocal microscopy is limited by corneal motion in an anterior-posterior direction. The difference between instruments suggests that verification of clinical ultrasonic pachymeters should be revisited.

Adult↗