摘要:

AbstractLowicryl K4M‐embedded Gram‐positive and Gram‐negative bacteria have a tendency to separate between the cell surface and the resin. This often leads to distortion of bacteria and more especially of mycobacteria. We describe attempts made to overcome this technical problem. Different assays were made onBacillus subtilis, Escherichia coli, andMycobacterium avium:(1) Modification of the bacterial surface by coating of bacteria with proteinic compounds; (2) treatment of bacteria with metallic salts known to modify cell wall polysaccharides; and (3) comparison between Lowicryl K4M and HM20.Conditions have been found in which the separation of all bacterial species from the resin is abolished. The most important factor appeared to be the treatment of bacteria before dehydration, with 0.5% uranyl acetate for 30 min. The second most important factor, especially forM. aviumand to a lower extent for Gram‐negative bacteria, was the use of Lowicryl HM20. Pre‐embedding in gelatin instead of agar improved sectioning ofM. avium, but had no effects on the other bacterial species. These conditions applied to macrophages infected withShigella dysenteriaeorM. aviumalso gave excellent results.In addition to sectioning improvement of bacteria, uranyl acetate improved the ultrastructure of bacteria and macrophages. All organelles were more clearly delineated and, hence, more easily identified. Finally, it was shown that UA treatment did not affect immunogold labeling of a variety of

ISSN:0741-0581    

DOI:10.1002/jemt.1060140402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractFor many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25°C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long‐term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin‐2 receptor (IL‐2R) on leukocyte surface membranes was imaged using colloidal gold‐labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important ad

ISSN:0741-0581    

DOI:10.1002/jemt.1060140403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractDuring the microstructural examination of ceramic thermal barrier coatings by transmission electron microscopy (TEM), initial efforts for the preparation of cross‐sectional thin foils from interface regions by conventional means were mostly failures. Delamination of the Y2O3‐stabilized ZrO2ceramic coating from the nickel‐base alloy substrate sometimes occurred during fine polishing at around 80 μm thickness but mostly occurred during dimpling. Because of this sensitivity, special techniques for mechanical handling were developed so that ion milling could give thin enough regions of the metal‐ceramic interface. TEM showed convincingly that the highly fragile nature of the coatings is in fact due to the extensive porosity at the interface developed as a result of heat t

ISSN:0741-0581    

DOI:10.1002/jemt.1060140404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractFab‐colloidal gold labelling in conjunction with negative staining and high‐resolution electron microscopy was used for targeting single protein units in regular arrays. These were bacteriophage T4 polyheads with Fab‐Au2.5, and a specific antibody binding site on the haemagglutinin polypeptide of influenza virus with Fab‐Au3, Fab‐Au2.5, and Fab‐Au1‐2. For the latter, IgG‐Au3was also used. Experimental details are summarized to provide generally applicable methods for the preparation of small gold colloids Fab‐Au and of labelling.The putative mechanism of protein‐gold complex formation and adsorption to preferred sites on Fab and IgG, most probably to sulphur‐rich regions, is discussed. The influence of pH during complex formation was found to be of minor importance in the samples investigated. Reported experimental details and our own experiences suggest that the importance of a protein's pI relative to its optimum gold complexing pH critically depends on the nature of the protein in question rather than being of general importance for protein

ISSN:0741-0581    

DOI:10.1002/jemt.1060140405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThis paper presents a reliable technique for the preparation of radioautographic specimens for study by high‐voltage electron microscopy. Tissues are fixed by ventricular perfusion of aldehydes, sectioned, collected on slides, and coated with nuclear track emulsion using a coating machine. Sections are developed after 17 weeks' exposure in either undiluted Microdol‐X or diluted D‐19 developer, fixed in 25% sodium thiosulfate, and stained through the emulsion with uranyl acetate in absolute methanol and aqueous lead citrate. The technique produces sections of even thickness and staining density with negligible numbers of background grains and non‐specific labeling. In this report, the efficacy of this technique for the study of the periosteal surface of demineralized alveolar bone specimens is demon

ISSN:0741-0581    

DOI:10.1002/jemt.1060140406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe microstructure and growth of ZnTe films deposited onto glass and freshly cleaved NaCl substrates are carefully studied by a TEM. Effect of different stimulator on the grain growth is also described.

ISSN:0741-0581    

DOI:10.1002/jemt.1060140407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractA method is described for electron microscopic observation of two‐dimensional paracrystals on unsupported lipid monolayers. The method uses a hydrophobic holey C‐coated grid placed on a monolayer made positively charged by the inclusion of stearylamine (SA) and has been used to align scallop thin filaments and reconstituted actin/tropomyosin filaments to form paracrystals. The use of unsupported monolayers allows the paracrystals to be viewed in either negative stain or with cryoelectron microscopy. Those paracrystals in frozen hydrated specimens have better order than those with negative stain.It was found that varying the lipid composition between the less fluid distearolyphosphotidylcholine/SA and the more fluid egg yolk phosphotidylcholine/SA alters the size and order of the paracrystals, the more fluid system having smaller, more ordered paracrystalline domains. The advantage of the technique for studying actin/thin filaments is the ability to form large two‐dimensional paracrystals under physiological conditions of [Mg2+] a

ISSN:0741-0581    

DOI:10.1002/jemt.1060140408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractWe have made several technical improvements for quick‐freeze, deep‐etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat andXenopuscultured on glass coverslips, and erythrocytes attached to coverslips coated with poly‐L‐lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick‐freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (<10 μm). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table–one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage

ISSN:0741-0581    

DOI:10.1002/jemt.1060140409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractConditions for cryofixation and freeze‐substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze‐substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the “classic” cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and–with certain reservations–dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze‐substitution yielded better ultrastructural preservation of the embryos with fe

ISSN:0741-0581    

DOI:10.1002/jemt.1060140410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe influence of fixation on membrane permeability has been examined in the isolated rat heart using lanthanum as a permeability probe. Normal and ischaemic hearts were probed at various stages during a conventional fixation programme with either ionic or colloidal lanthanum and compared with lanthanum saline administered prior to fixation. Fixation of the myocardium coincident with or followed by lanthanum probing resulted in an influx of the probe into most myocytes in normal tissue. Alterations in permeability after ischaemic episodes could not be distinguished from this artefact. However, lanthanum saline prior to fixation showed exclusion of the probe from normal tissue, while the increased permeability demonstrated after ischaemia was associated with declining myocardial performance during subsequent reperfusion. These results illustrate the need for caution in the application and evaluation of methods determining permeability in fixed tissue. Probes of differing size and charge permeated fixed tissue to varying degrees thereby implicating the formation of specific lesions during chemical fixation.

ISSN:0741-0581    

DOI:10.1002/jemt.1060140411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY