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PubMed2003

Characterisation and quantification of angiogenesis in beta-tricalcium phosphate implants by immunohistochemistry and transmission electron microscopy.

The aim of this study is the histological characterisation of angiogenesis in a macroporous biomaterial with quantification techniques used in oncology. Porous tricalcium phosphate implants were seated in the tibias of 12 rabbits. This work allows (1) morphological study with photonic microscopy, transmission electron microscopic and immunohistochemistry labelling for (2) quantification of vascularisation using anti-CD31 monoclonal antibody (3) quantification of proliferation using anti-PCNA polyclonal antibody (4) study of two angiogenic growth factors: VEGF and FGF-2. Quantification of angiogenesis revealed an outbreak kinetic with early vascular growth in first several days and a second growth phase after 4 weeks. This study reveals in macropores many isolated cells without adjacent vascular lumen, with endothelial phenotype. Expression of angiogenic growth factors reveals that all endothelial cells were VEGF-negative throughout the test period. FGF-2 expression by endothelial cells began 2 weeks post-implantation. Osteoblasts strongly expressed two markers throughout the test period. Furthermore, the procedure described here can be used to compare angiogenesis in different biomaterials or in the same biomaterial with the influence of macroporosities.

Animals↗
PubMed2002

The use of field emission scanning electron microscopy to assess recombinant adenovirus stability.

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

Adenoviruses, Human↗
PubMed1993

Progress in scanning electron microscopy of frozen-hydrated biological specimens.

Modern scanning electron microscopy yields structural information down to 2 to 5 nm from thin, beam transparent biological specimens. This paper examines the possibilities of garnering this level of structural information from bulk, frozen-hydrated samples. Freeze-fractured, frozen-hydrated yeast cells, frequently taken as a yardstick to monitor progress in low-temperature scanning electron microscopy, have been used to optimize both metal shadowing methods and observation parameters (e.g. accelerating voltage, electron beam irradiation of the specimen). Uncoated frozen-hydrated yeast cells do not change electrically at an accelerating voltage of 30 kV. Increasing charging effects are however observed with decreasing accelerating voltages. Very thin metal films are therefore used for specimen coating to localize and enhance the specific secondary electron signal. Planar-magnetron sputtering of a 1 nm metal layer provides high resolution secondary electron images, at 30 kV, of freeze-fractured, frozen-hydrated yeast cells in an in-lens field-emission scanning electron microscope. Structural information comparable to that of transmission electron microscopy of freeze-fractures is attained. Planar-magnetron sputtering of either chromium, tungsten or platinum results in essentially the same information density (smallest visible significant structural detail). Frozen-hydrated samples are very beam sensitive and have to be observed under minimum dose conditions.

Cryopreservation↗
PubMed1985

Animal model. Light and electron microscopy of hepatocytes of cats with Chediak-Higashi syndrome.

Cats with a condition resembling human Chediak-Higashi syndrome (CHS) are the most recently described of five species of animals with similar syndromes. In this study, hepatocytes of cats with CHS were examined by light microscopy, histochemistry, and transmission electron microscopy. Enlarged cytoplasmic granules, morphologically consistent with lysosomes, were present in many of the CHS cat hepatocytes. The enlarged lysosomes were generally larger and more numerous in centrilobular hepatocytes and were generally larger in older cats. The lesions were similar to those reported in other species with CHS suggesting that CHS cats are a valid animal model of human CHS.

Animals↗
PubMed1990

Amorphisation of MgO single crystal specimens prepared by ion milling for transmission electron microscopy studies.

Single crystal MgO specimens having low load Vickers indentations were thinned in an ion milling machine employing a single ion gun, and their characteristics were investigated with optical microscopy and high voltage electron microscopy (HVEM). It was found that the state of cleanliness of the specimen chamber of the ion milling machine had a very marked influence on the quality of the thinned specimens. If the specimen chamber was not well cleaned before ion milling a fresh specimen, the latter tended to show amorphisation due to the deposition on the specimen of the debris left in the chamber from the previously ion-milled specimens. Such observations were made from MgO specimens ion milled in several different types of commercial ion milling machine employing a single gun. It is proposed that to obtain good-quality ion milled TEM specimens, it is important to clean the specimen chamber thoroughly prior to milling.

Magnesium Oxide↗
PubMed2001

Nervous system-derived chondroitin sulfate proteoglycans regulate growth cone morphology and inhibit neurite outgrowth: a light, epifluorescence, and electron microscopy study.

Proteoglycans influence aging and plasticity in the nervous system. Particularly prominent are the chondroitin sulfate proteoglycans (CSPGs), which are generally inhibitory to neurite outgrowth. During development, CSPGs facilitate normal guidance, but following nervous system injury and in diseases of aging (e.g., Alzheimer's disease), they block successful regeneration, and are associated with axon devoid regions and degenerating nerve cells. Whereas previous studies used non-nervous system sources of CSPGs, this study analyzed the morphology and behavior of sensory (dorsal root ganglia) neurons, and a human nerve cell model (SH-SY5Y neuroblastoma cells) as they contacted nervous system-derived CSPGs, using a variety of microscopy techniques. The results of these qualitative analyses show that growth cones of both nerve cell types contact CSPGs via actin-based filopodia, sample the CSPGs repeatedly without collapse, and alter their trajectory to avoid nervous system-derived CSPGs. Turning and branching are correlated with increased filopodial sampling, and are common to both neurons and Schwann cells. We show that CSPG expression by rat CNS astrocytes in culture is correlated with sensory neuron avoidance. Further, we show for the first time the ultrastructure of sensory growth cones at a CSPG-laminin border and reveal details of growth cone and neurite organization at this choice point. This type of detailed analysis of the response of growth cones to nervous system-derived CSPGs may lead to an understanding of CSPG function following injury and in diseases of aging, where CSPGs are likely to contribute to aberrant neurite outgrowth, failed or reduced synaptic connectivity, and/or ineffective plasticity.

Actins↗
PubMed2002

Investigating the ultrastructure of fibrous long spacing collagen by parallel atomic force and transmission electron microscopy.

The ultrastructure of fibrous long spacing (FLS) collagen fibrils has been investigated by performing both atomic force microscopy (AFM) and transmission electron microscopy (TEM) on exactly the same area of FLS collagen fibril samples. These FLS collagen fibrils were formed in vitro from type I collagen and alpha1-acid glycoprotein (AAG) solutions. On the basis of the correlated AFM and TEM images obtained before and after negative staining, the periodic dark bands observed in TEM images along the longitudinal axis of the FLS collagen fibril correspond directly to periodic protrusions seen by AFM. This observation is in agreement with the original surmise made by Gross, Highberger, and Schmitt (Gross J, Highberger JH, Schmitt FO, Proc Natl Acad Sci USA 1954;40:679-688) that the major repeating dark bands of FLS collagen fibrils observed under TEM are thick relative to the interband region. Although these results do not refute the idea of negative stain penetration into gap regions proposed by Hodge and Petruska (Petruska JA, Hodge AJ. Aspects of protein structure. Ramachandran GN, editor. New York: Academic Press; 1963. p. 289-300), there is no need to invoke the presence of gap regions to explain the periodic dark bands observed in TEM images of FLS collagen fibrils.

Animals↗
PubMed1995

Distribution of thrombospondin and integrin alpha V in DCIS, invasive ductal and lobular human breast carcinomas. Analysis by electron microscopy.

The ultrastructural distribution of thrombospondin (TSP) and its cell surface receptor, integrin alpha V, was studied in two cases of human breast carcinoma: one of ductal carcinoma in situ (DCIS) with an invasive component, and one of invasive lobular carcinoma. In DCIS, moderate immunolabelling for TSP and integrin alpha V was observed in the rough endoplasmic reticulum and at the plasma membrane of intraductal carcinoma cells. TSP was also associated with extracellular matrix collagen fibrils surrounding in situ carcinoma cells. In the invasive part of this ductal carcinoma, most of the malignant cells were negative for TSP, while integrin alpha V was moderately expressed in these cells. In sharp contrast, typical strands of invasive lobular carcinoma cells in "Indian file" showed moderate TSP immunostaining in the rough endoplasmic reticulum and strong immunoreactivity for TSP at the plasma membrane and in the extracellular matrix. Moderate to strong immunoreactivity for integrin alpha V was also observed in invasive lobular carcinoma cells. Because of the role of TSP during cancer cell invasion, the different expression patterns of TSP in invasive ductal versus lobular carcinoma may well reflect biological differences between these two types of breast carcinoma and could account for the peculiar diffuse invasive behaviour of breast lobular carcinoma cells.

Antigens, CD↗
PubMed1984

Catecholamine-containing pancreatic islet cells of the domestic fowl. Light, fluorescence and electron microscopy, and immunohistochemistry.

In an attempt to identify pancreatic islet cells emitting formaldehyde-induced fluorescence (FIF), the pancreatic islets of the domestic fowl were studied by combined fluorescence, ultrastructural, silver-impregnation and immunohistochemical methods in the same section or in consecutive semi-thin and ultra-thin sections. The results indicate that islet cells emitting intense FIF exhibit a strongly argyrophil reaction with the Grimelius' silver method and also immunohistochemical reaction with anti-glucagon serum, but not with anti-5-HT serum. Therefore, the fowl islet A cell, a peptide hormone-producing cell, stores simultaneously catecholamine as biogenic amine. The islet B and D cells did not display any FIF, any argyrophil reaction with the Grimelius' silver method, or any immunoreactivity with anti-glucagon or anti-5-HT sera. The fluorescent but non-argyrophil cells dispersed in the exocrine acinus may well be PP cells.

Animals↗