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At least 991 records · Page 55Linked to original sources
PubMed1995

Quantitative analysis of chondroitin sulphate retention by tannic acid during preparation of specimens for electron microscopy.

The ability of tannic acid to enhance binding of glycosaminoglycans to purified collagen was analysed in an in vitro system using amino sugar analysis on an amino acid analyser, transmission electron microscopy, and scanning electron microscopy. Collagen was purified by digestion with trypsin, papain, and hyaluronidase. Purified collagen was incubated with hyaluronic acid or with chondroitin sulphate glycosaminoglycan and then treated with tannic acid. Tannic acid was found to enhance retention during preparation for electron microscopy of either of the glycosaminoglycans onto collagen fibres. The ability of tannic acid to enhance binding of collagen and glycosaminoglycans might explain, at least in part, its structural reinforcement effect on resected synovial joint-apposing surfaces during preparation for scanning electron microscopy.

Amino Sugars↗
PubMed1995

Confocal imaging and immunogold electron microscopy of changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L.

Using anti-myosin antibodies, standard immunocytochemical techniques in conjunction with confocal scanning laser microscopy and colloidal gold immunoelectron microscopy we compare changes in the distribution patterns of myosin during the early stages of pollen hydration, germination, tube growth, and myosin associated with isolated vegetative nucleus and the generative cell in Nicotiana tabacum L. Furthermore, on the Western blots of pollen tube proteins, the antimyosin antibodies crossreact only with one polypeptide of approximately 174 kDa. Confocal immunofluorescence microscopy reveals that in hydrated pollen, myosin is discretely associated with the cytoplasmic organelles and numerous punctate structures present in the center of the pollen. Within 30 min following transfer of pollen into the germination medium, that is, with the onset of germination, the centrally located punctate structures are displaced, and we find accumulation of myosin-associated organelles towards one of the germinal apertures from which the pollen tube would emerge. Subsequently, after 45 min of germination with the emergence of germination structure, few punctate structures are detected in the vegetative cytoplasm while intense immunostain is detected just below the plasma membrane of the emerging pollen tube tip. In the older parts of both short and long pollen tubes after 90 to 120 min of pollen germination, few fluorescent structures were found in the pollen tubes, however, numerous punctate fluorescent spots were concentrated in the tip region over a distance of 2 to 3 microns below the plasma membrane of the tube tip. This is further substantiated by colloidal gold immunoelectron microscopy wherein clusters of gold particles are associated with vesicle-like structures in the tip region of the pollen tubes.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗
PubMed1994

[History of microscopy in Spain].

Nowadays, many Spanish research centers have excellent electronic microscopy services. The current situation, however, should not allow us to forget that the initial steps of microscopy in Spain were very difficult. The construction of excellent optical microscopies in the late XIX century, and their almost immediate introduction in Spain, coincides with a period of thriving scientific activity in our country. Both micrography and histology saw the highlights of their development in Spain, with scientists such as Ramón y Cajal, Río Hortega, Ferrán, Simarro, among others, all of them widely known at present. This article evokes briefly the vicissitudes of Spanish microscopy, from its very beginning in 1843, when the Allgemeine Anatomie by Jacob Henle was translated into Spanish, to present. Scientific historical facts in this article are often accompanied with anecdotes, which show the human aspect of those great scientists. The persevering task carried out by researchers whose names have been recorded in the history of Spanish science and technology, have established the grounds in which our current development is based.

History, 19th Century↗
PubMed1994

Proteoglycan ultracytochemistry and conventional and high resolution scanning electron microscopy of vertebrate cerebellar parallel fiber presynaptic endings.

The parallel fiber "en passant" synaptic endings of mouse cerebellar molecular layer have shown by means of transmission electron microscopy, the presence of an electron dense extravesicular material in samples perfused with Alcian blue. This alcianophilic material was digested in cerebellar tissue previously treated with testicular hyaluronidase, suggesting the presence of hyaluronic acid or chondroitin 4- or 6-sulphate. Freeze-fractured Rhesus monkey cerebellar cortex prepared for conventional scanning electron microscopy also revealed the presence in fractured synaptic varicosities of parallel fibers of a high mass density material, in which the synaptic vesicles are embedded. Examination of cryofractured primate cerebellar cortex coated with thin chromium films, 1-2 nm thick, in the high resolution field emission scanning electron microscope showed the SE-I topographic contrast of an extravesicular material deposited in axoplasmic matrix of fractured parallel synaptic endings. The precise localization of this material corresponds to that observed in transmission electron microscopy and conventional freeze-fracture scanning electron microscopy. These electron microscopic findings tend to agree with the omnipresence in several vertebrates of a presynaptic axoplasmic material, which seems to be proteoglycan in nature.

Alcian Blue↗
PubMed1993

Phase-shifting interference microscopy applied to the analysis of cell behaviour.

The theory of phase-shifting interferometry is not new but it is only recently, with the advent of solid-state detector arrays and fast image processors, that it has become a practical imaging technique. In conjunction with transmission interference microscopy, phase-shifting presents a new way of introducing contrast into the images of transparent microscopic objects such as cultured cells. An earlier paper from our laboratory has emphasised the advantages of transmission interference microscopy over phase contrast or differential interference contrast microscopy for the computerised analysis of cell behaviour. Phase-shifting greatly improves the accuracy, long-term stability and range of application of this technique but it has not previously, to our knowledge, been combined with transmission interference microscopy for the study of cultured cells. The resulting image is especially well suited to quantitative analysis by computer since it is a direct representation of the distribution of non-aqueous cellular material in the specimen. The image is not degraded by uneven illumination; by heterogeneous sensitivity of the detector array; or by differential absorption of light in the optics or specimen. Our main purpose in developing the method is to obtain sequences of images of the motile behaviour of cells in culture for analysis by computer. This type of analysis is potentially a powerful tool for studying the motile responses of cells and the operation and control of their locomotory machinery. Not only can the method be used for studying cell translocation and the dynamics of intracellular movement of non-aqueous material, but it is now possible to study in detail the time course of growth in individual cultured cells.

Animals↗
PubMed1993

Conventional and high resolution field emission scanning electron microscopy of vertebrate cerebellar parallel fiber-Purkinje spine synapses.

Purkinje dendrite-parallel fiber spine synapses of human, teleost fishes, Rhesus monkey and mouse cerebellar cortex have been studied by means of conventional scanning electron microscopy (SEM) using ethanol-cryofracturing technique and by high resolution field emission scanning electron microscopy (HRFESEM) for studying the outer and inner surface morphology of pre- and postsynaptic endings. Transmission electron microscopy of mouse cerebellar cortex either by means of ultrathin sections and freeze-etching replicas have been complementarily used for proper identification and comparative observations. Normal teleost fishes showed short neck and neckless Purkinje spines with exhibited spread or extended postsynaptic densities. In pathological human cerebellum, the ethanol cryofracturing technique exposed the outer surface of edematous flat and invaginated Purkinje spine synapses. In fractured presynaptic endings HRFESEM showed in Rhesus monkey cerebellar cortex a homogeneous extravesicular material binded to the synaptic vesicles and joining them to the presynaptic membrane. HRFESEM partially resolved the synaptic cleft as currently observed in high magnification transmission electron microscopy. Round subunits, 25-35 nm in diameter, were observed associated to postsynaptic membrane, apparently corresponding to the localization and distribution of E face postsynaptic intramembrane particles, which suggest that such subunits correspond to the domains of neurotransmitter postsynaptic receptors.

Animals↗
PubMed1993

[Detection of porcine epidemic diarrhea virus using electron microscopy in the Czech Republic].

Coronavirus-induced porcine epidemic diarrhoea (PED) was diagnosed in two swine herds. The causal agent was demonstrated in intestinal contents by electron microscopy and identified by immunoelectron microscopy using specific immune serum to the reference strain PED-CV77. Experimental transmission to hysterectomy-derived, colostrum-deprived piglets with an intestinal contents filtrate was successful. The virus was demonstrable by electron microscopy in the intestinal contents between 12th hour and 4th day, and in small intestinal epithelial cells 18 hours after infection. Scanning electron microscopy revealed shortening and fusion of villi of small intestinal mucosa.

Animals↗
PubMed1993

[Diagnostic value of electron microscopy on epithelial tumors of the ovary].

The study on transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in 52 cases of ovarian epithelial tumors (OET) was presented. The characteristic features of ultrastructure and cell surface structure of different types of OET, also the diagnostic features of cancer cells were determined and discussed. It has a great value in differentiation of histologic types of OET and in early diagnosis of cancer. In 3 cases of borderline malignant tumor, some malignant cells, which were impossible to distinguish by light microscopy, were discovered by TEM and SEM. The diagnostic value of electron microscopy in early malignancy of tumors is more important.

Adenocarcinoma, Mucinous↗
PubMed2000

Microscopy investigations. Techniques, results, limitations.

This paper critically reviews current microscopy techniques that are used to image resin-dentin interfaces and more in particular to investigate the process of hybridization and resin-tag formation. A short description of the four most commonly used microscopy techniques with their necessary specimen-preparation methodology is provided along with a discussion of their possibilities, limitations and potential artifact formation. With this critical appraisal on microscopy techniques available to study adhesive interfaces, it is hoped to encourage the use of high-resolution analytical tools to further elucidate the mechanisms of bonding at the ultra-structural level. Besides ultra-morphologic characterization, more research should be devoted to study the chemical and physical properties of resin-dentin bonds. The final objective of such fundamental research should be to establish a more durable and reliable adhesive restorative technique.

Artifacts↗
PubMedDate not supplied

Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy.

BACKGROUND: Keloids and hypertrophic scars result from aberrant wound healing and remain a potential complication of any surgical procedure or trauma. Investigation of aberrant wound healing has been limited to the study of growth factors, collagen precursors, and DNA synthesis in simple in vitro systems, which necessitate removal or destruction of cells or factors in the growth environment of cell cultures. Multiphoton microscopy (MPM) can use endogenous chromophores such as collagen and nicotinamide adenine dinucleotide hydrogenase to produce thin optical sections of thick living tissues without the use of dyes or stains. Endogenous second-harmonic-generation (SHG) signals in collagen can be collected to form an MPM image. OBJECTIVE: To present a novel wound-healing model used to investigate keloid-derived fibroblast activity and collagen production in the same intact tissue-engineered construct over time. METHODS: Artificial tissue constructs called RAFTs (produced by suspension of keloid or normal dermal fibroblasts in type I collagen gel with an overlying keratinocyte layer) were cultured at air-fluid interface. Multiphoton microscopy SHG images of collagen in the intact tissue constructs consisting of normal or keloid-derived fibroblasts were obtained. The constructs were then incised with a scalpel. Serial MPM and phase-contrast microscopy images were obtained to monitor changes in the extracellular matrix in response to wounding of the artificial skin construct over 8 days. RESULTS: The tissue-engineered constructs formed a bilayer resembling the dermis and epidermis of human skin. Phase-contrast microscopy revealed migration of keratinocytes into the defect created by scalpel wounding. The constructs were found to contract with time after wounding. The MPM SHG images showed collagen deposition in the tissue constructs after wounding. Tissue constructs with keloid-derived fibroblasts were found to deposit collagen at a higher rate than those with normal fibroblasts. CONCLUSIONS: The MPM model described herein permits serial observation of the same intact specimens without the need for fixation or cytotoxic stains. Furthermore, it demonstrates the biologic activity of RAFT artificial tissue constructs.

Cells, Cultured↗
PubMed2004

In vivo confocal microscopy in patients with central cloudy dystrophy of François.

OBJECTIVE: To report in vivo corneal confocal microscopic findings of patients with central cloudy dystrophy of François. METHODS: Two unrelated patients, a 78-year-old man and a 75-year-old woman, with central cloudy dystrophy of François were examined using routine slitlamp biomicroscopy and confocal microscopy. RESULTS: In both cases, slitlamp biomicroscopy showed bilateral polygonal opacities separated by clear spaces. The corneal opacities were most prominent centrally and were located in the deeper stromal layer immediately anterior to the Descemet membrane. By confocal microscopy, normal superficial and basal epithelial layers, midstromal layers, and endothelial layers were noted in both cases. However, small highly refractile granules and deposits were observed in the anterior stromal layer in both cases. Also, multiple dark striae among the extracellular matrix with increased intensities were observed in the posterior stroma adjacent to the corneal endothelial layer in both cases. CONCLUSIONS: Abnormal stromal deposits and multiple dark striae were observed in central cloudy dystrophy of François using in vivo corneal confocal microscopy. Use of confocal microscopy to investigate these abnormal stromal opacities may be helpful in differentiating various corneal stromal pathologic features.

Aged↗
PubMed1997

Scanning slit confocal microscopy of fungal keratitis.

In vivo scanning slit confocal microscopy was performed in a patient with Fusarium solani keratitis. Morphologically distinctive abundant filamentous structures were observed intrastromally. Confocal microscopy of the culture plate growing F solani from the patient's corneal scraping revealed filaments morphologically similar to the filaments observed in vivo. After 1 week of medical therapy, subsequent confocal microscopy showed an increased load of filaments, supporting the decision to perform a penetrating keratoplasty. Confocal microscopy confirmed that all of the fungus was eradicated. This aided in the decision to administer corticosteroids and quickly discontinue antifungal agents.

Adolescent↗
PubMed1997

Displacement of gold marker in immunoelectron microscopy of human respiratory cilia.

Preembedding immunogold electron microscopy was performed to evaluate the position of outer arm dynein heavy chains in normal human respiratory cilia. Anti-dynein antibody (AD2), which is specific for sea urchin sperm flagellar dynein heavy chains, was used as primary antibody. Direct cross-sections of cilia were selected, and the distance between the center of a cilium and the center of a colloidal gold particle attached to the cilium (X) was measured. The distance between the center of a cilium and the farthest edge of an outer dynein arm of the cilium was measured by ordinary electron microscopy (Yo) and by immunoelectron microscopy (Yi). X was significantly longer than Yo and Yi. If it is assumed that the structure of respiratory cilia is dense and that antibodies are located at the outer side of the actual position of the heavy chains, then the average distance difference of approximately 90-120 A may represent the length of two conjugated antibodies. This length should be kept in mind when performing immunoelectron microscopy. The data suggest that AD2 recognizes the outer arm dynein heavy chains of normal human respiratory cilia.

Adult↗
PubMed1999

Investigation of protein partnerships using atomic force microscopy.

The origin of contrast in atomic force microscopy (AFM) lies in the probe's response to forces between itself and the sample. These forces most commonly result from changes in height as the tip is scanned over the surface, but can also originate in properties inherent in the sample. These have been exploited as further means of contrast and have spawned an array of similar imaging techniques, such as chemical force microscopy, magnetic force microscopy, and frictional force microscopy. All of these techniques use AFM as an extremely sensitive gauge to map forces at discrete sites on the surface. A natural extension of this approach is to map forces in an array, in order to create a force map. AFM can be used in aqueous or fluid environments, thus allowing the exploration of forces in biological systems under physiologically relevant conditions. By immobilizing one half of an interacting pair of proteins onto the tip and the other half onto the substrate, it is possible to investigate the electrostatic and hydrophobic interactions between them. We employed these techniques to examine the interaction between a pair of proteins of known affinity that are involved in exocytosis (NSF and alpha-SNAP) and separately to demonstrate how two-dimensional force mapping can be applied to the nuclear envelope to identify nuclear pore complexes.

Animals↗
PubMed1999

Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum.

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography. The autoradiographs were visualized by bright-field microscopy (BFM), reflection contrast microscopy (RCM), and transmission electron microscopy to evaluate the potentials of RCM visualization in high-resolution LM autoradiography. RCM visualization of ultrathin vs. semithin resin sections showed an improved stratum corneum morphology. Histological staining was superfluous. The localization of (3)H-E2 in human stratum corneum using high-resolution LM autoradiography and RCM was as accurate as with high-resolution EM autoradiography.

Autoradiography↗
PubMed1997

Improving the diagnostic accuracy of cytologic cerebrospinal fluid examinations in acute lymphoblastic leukemia using high-power microscopy and terminal deoxynucleotidyl transferase determinations.

In patients with acute lymphoblastic leukemia (ALL), cytologic examination of cerebrospinal fluid (CSF) is becoming increasingly important for clinical management. In order to enhance the diagnostic accuracy of CSF cytology results, the value of using terminal deoxynucleotidyl transferase (Tdt) and high-power (1,000x) light microscopy, together with conventional cytologic examination was assessed. In 33 CSF samples from ten multiply examined Tdt-positive ALL patients, original cytologic interpretations were compared to Tdt results. Cytology samples were reviewed by two pathologists (one with hematopathologic expertise). The cases in which cytologic interpretation did not correlate with Tdt result were first reviewed via 1,000x microscopy without knowledge of Tdt result, then re-reviewed with knowledge of Tdt result. Conventional cytology alone diagnosed 64% of cases accurately (Tdt representing the comparative standard). High-power microscopy increased the correlation to 82%. Use of high-power microscopy and knowledge of Tdt result together produced a total of 85% of cases with correlation of results. High-power microscopic examination therefore contributes significantly to the accurate diagnosis of ALL, and knowledge of the Tdt result at the time of cytologic examination produces an additional advantage in providing an objective measure for CSF involvement by leukemia. Using all three methods in conjunction is recommended in order to increase the overall accuracy of CSF examination for the detection of leukemic involvement in ALL patients.

Adolescent↗
PubMed2001

High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells.

High-resolution, fluorescence deconvolution (DV) microscopy was implemented to obtain a detailed view of the organization and structural composition of gap junctions assembled from one or two different connexin isotypes in live and fixed cells. To visualize gap junctions, the structural protein components of gap junction channels, the connexin polypeptides alpha1(Cx43), beta1(Cx32), and beta2(Cx26), were tagged on their C-termini with the autofluorescent tracers green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants. Tagged connexins were expressed in transiently transfected HeLa cells. Comprehensive analysis including dye-transfer analysis demonstrated that the tagged connexins trafficked, assembled, and packed normally into functional gap junction channel plaques. Such gap junction plaques were examined by single, dual, and triple-color DV microscopy. High-resolution images and three-dimensional volume reconstructions of gap junction plaques were obtained by this technique, which revealed several new aspects of gap junction structure. Specifically, the studies demonstrated that the mode of channel distribution strictly depends on the connexin isotypes. Here we present such images, and volume reconstructions in context with images obtained by other light, and electron microscopic techniques, such as laser scanning confocal, conventional wide-field fluorescence, thin section, and freeze-fracture electron microscopy. In addition, we give a simple description of the principal mechanisms of DV microscopy, name advantages and disadvantages, and discuss issues such as dual-color imaging using CFP and YFP, spatial resolution, colocalization, and avoiding imaging artifacts.

Connexin 26↗
PubMed1982

Intraoperative microscopy of bile--is it useful?

Direct microscopy of the bile was performed during cholecystectomy in 111 patients in an attempt to identify those with a high risk of wound infection. Bacteria were identified in 23 patients, 11 of 83 undergoing cholecystectomy alone and 12 of 28 undergoing exploration of the common bile duct (P less than 0.01). These 23 patients were randomly allocated to an antibiotic group or a control group; there was one wound infection in the antibiotic group and two in the control group. A total of 14 patients developed wound sepsis. Infection was more likely if the common bile duct was explored (6 of 28) rather than cholecystectomy alone (8 of 83). There was a poor correlation between microscopy and culture of the bile for bacteria and there was no increase in sepsis when bacteria were observed on microscopy. We were not able to identify a high risk group of patients by intraoperative microscopy of bile.

Adult↗