ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04263.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYIn high‐resolution image analysis, it is often desirable to return to a chosen cell after it has been restained or subjected to histochemical procedures. The reading of the vernier on the microscope stage is too coarse for relocating of non‐distinct single cells, because the accuracy, determined by visual interpolation, is limited, at best, to 1/20th of a millimetre, or 50 μm. When one works with haematologic samples, e.g. lymphocytes, the precision of relocating a cell has to be better than 50 μm; i.e. the cell should reappear near the centre of the visual field of a x 100 oil‐immersion objective.We describe a simple device by means of which distinct marks can be made on a slide with specimen (but before coverslipping) and will provide suitable origins for a coordinate system that will cover the entire prep

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04264.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThe study of distribution and quantitation of a fluorescent probe in living epithelia with the aid of an inverted microscope requires that individual cells can be analysed without optical interference from adjacent cells. This report describes the application of fluorescence microscopy and fluorometry to a recently developedin vitroculture system of renal epithelial cells.Epithelial cells derived from the mammalian renal cortical collecting tubule (CT) and the thick ascending loop of Henle (TAL) are cultivated as continuous monolayers in serum‐free, hormone‐supplemented media. A specific mitochondrial marker (DASPMI) is added to the medium and incorporated into the cytoplasm. The microscopic image reveals that the mitochondrial fluorescence distribution differs between CT and TAL cultures. The fluorometric quantitation shows a normally distributed histogram of medium‐range intensity in TAL cell cultures while CT cultures exhibit a two‐peak pattern of mitochondrial fluorescence distribution among epithelia

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04265.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYAn improved method for reinforcing freeze‐fracture replicas, which is especially suitable for tough tissues, is described. For this purpose, polystyrene dissolved in chloroform is used to produce a solid protecting layer of plastic on top of the replica, enabling it to withstand the stresses associated with the thawing and digestion of the tissue with strong acids. The method results in production of large clean replicas from tissues such as skin or peripheral nerve which are difficult to process. The method can also be used profitably for reinforcing other softer and more homogeneous tissue

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04266.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYGold, platinum and tungsten films were deposited by low energy input (7 mA, 450 V), or high deposition rate (80 mA, 1500 V), diode sputter coating and by ion beam sputter coating. Film structures on Formvar coated grids and on the surface of coated erythrocytes, resin embedded, sectioned, and recorded at high magnification in a TEM were compared using computer‐assisted measurements and analysis of film thickness and grain size.The average grain size of the thinnest gold and platinum films was relatively independent of the mode or rate of deposition but as the film thickness increased, significant differences in grain size and film structure were observed.Thick platinum or gold films deposited by low energy input sputter coating contained large grain size and electron transparent cracks; however, more even films with narrower cracks but larger grain size were produced at high deposition rates. Ion beam sputter coated gold had relatively large grain size in 10 nm thick films, but beyond this thickness the grains coalesced to form a continuous film. Platinum films deposited by ion beam sputter coating were even and free of electron transparent cracks and had a very small grain size (1–2 nm), which was relatively independent of the film thickness.Tungsten deposition either by low energy input or ion beam sputter coating resulted in fine grained even films which were free of electron transparent cracks. Such films remained granular in substructure and had a grain size of about 1 nm which was relatively independent of film thickness. Tungsten films produced at high deposition rates were of poorer quality.We conclude that thick diode sputter coated platinum and gold films are best deposited at high deposition rates provided the specimens are not heat sensitive, the improvement in film structure being more significant than the slight increase in grain size. Thick diode or ion beam sputter coated gold films should be suitable for low resolution SEM, and thin discontinuous gold films for medium resolution SEM. Diode sputter coated platinum should be suitable for medium resolution SEM and ion beam sputter coated platinum for medium and some high resolution SEM. 1–5 nm thick tungsten films, deposited by low energy input or ion beam sputter coating should be suitable for high resolution SEM, particularly where contrast is of less importance than resol

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04267.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYA new method is described for preparing thin sections for transmission electron microscopy of ciliated respiratory epithelial cells cultivatedin vitroon Teflon membranes. Hamster tracheal explant cultures were treated with collagenase to promote epithelial migration. Patches of monolayer growth occurred in the vicinity of the explants when the latter were incubated on FEP‐etched Teflon membranes in Chamber/Dishes. Cells in the monolayer maintained a high degree of differentiation as evidenced by active ciliary motion. The monolayer was fixed and processedin situ.During the embedding process the chamber was supported on a solid Teflon cylinder to keep the membrane taut as it was heated to polymerize the resin. The membrane was removed and the monolayered cells were re‐embedded and sectioned. Electron microscopic examination of cultures revealed clear cell profiles on a flat, even basal plane. This method provides for a normal spatial configuration of cells in a monolayer format, and should prove useful for electron microscopy studies of any cells cultured on flexible fi

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04268.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYBidirectional shadowing in freeze‐etching may be achieved by firing an electron‐beam shadowing source, rotating the specimen stage through a desired angle, and re‐firing the shadowing source. It is demonstrated that portrait shadow‐casting, which permits information to be drawn from much of the specimen region lying within primary shadows, can be readily achieved using a 90° specimen rotation. With 180° specimen rotation, particle‐size analysis is feasible. Particle‐height analysis is demonstrated using membrane‐associated particles as an example. Data from suitable sets of micrographs can also be used for the estimation of particle‐width exaggeration due to the accumulation of the shadowing‐metal cap. Fibre‐width analysis, using the linear regression method, is demonstrated by a study of native cellulosic microfibrils. Mean microfibril widths were f

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04269.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYChanges in the surface morphology of mature urediospores ofUromyces viciae‐fabaehave been studied with respect to different preparative procedures for the scanning electron microscope. Specimens were examined in the critical point‐dried, freeze‐dried and frozen‐hydrated states. Dried urediospores were significantly different in appearance from those which were hydrated. Shrinkage probably accounted for some of the surface features of dried spores, particularly amongst those which had been critical point‐dried. No significant effects on spore surface texture resulted from chemical immersion fixation, chemical vapour fixation, dehydration or the mode of sputter‐coating. It is suggested that the presence or absence of unfreezable water, involved in maintaining the structural stability of polymers which provide the specimen surface with its characteristic morphology, may have an important influence on the gross texture of cell surfaces as viewed with the scanning electron

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04270.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThe technique of delaying fixation until after freeze‐fracture and thawing, described in an earlier paper (Haggis&Bond, 1979), has been developed further for study of cells in culture, principally mouse lymphocytes stimulated by concanavalin A. Using a thin layer of cells, a cryoprotectant concentration of either 10% glycerol or dimethylsulphoxide, is sufficient to give good structural preservation after rapid freezing and thawing. Nuclear matrices and Triton‐permeabilized cells have been prepared from stimulated lymphocytes for comparative study. Polylysine‐coated fibrin support films have been found to provide a convenient means of handling cells and subcellular preparations during freeze fracture, critical point drying and mounting for high‐resolution scanning electron mic

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04271.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThe VCD/HXSA ultra‐low viscosity medium is characterized by a viscosity of only 20 cps at 298 K. This is extremely useful for rapid embedding schedules and facilitates cutting large sections even from difficult materials. The suitability of this medium for ultrastructural studies is tested with plant specimens ranging from soft, highly vacuolated parenchymatous tissue to hard thick‐walled cells impregnated with a variety of substances or covered with mucilage. The results, when compared with those from similar materials infiltrated with Spurr's and Epon embedding media, show that the general preservation of the cellular structure is comparable for all the three media tested. In addition, embeddment in VCA/HXSA medium results in better preservation of some vacuolar features and in the reduction of plasmoly

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04272.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY