ISSN:1059-910X    

DOI:10.1002/jemt.1070240502

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

年代:1993

数据来源: WILEY

摘要:

AbstractEarly chick embryos (stages 11–29), or parts thereof, were frozen by the high pressure technique and subjected to a variety of freeze substitution protocols. In addition to hexadecene, unincubated albumen was used as a cryoprotectant to surround the specimens during the freezing event. The quality of freeze ranged from poor to very good in these highly aqueous specimens; very good freeze quality was observed minimally to depths of 30–40 μ. Freeze substitution was carried out with a custom‐made aluminum chamber, automatic freeze substitution machine, or a combination of the two, in acetone containing (1) uranyl acetate; (2) acrolein and/or tannic acid; or (3) a sequence of (2), osmium, and glutaraldehyde. Excellent quality of morphology was cryopreserved with all solutions, given optimally cryoimmobilized tissue. Mitotic and pericentriolar microtubules and cytoskeletal elements were cryopreserved with all methods; membranous organelles were better preserved with osmium. Outstanding preservation of morphology of the ground substance of very hydrated embryonic cells, though limited in depth, was accomplished by high pressure cryoimmobilization and cryopreservation, demonstrating that the technique can be successfully applied to highly aqueous tissues. © 1993 Wiley‐

ISSN:1059-910X    

DOI:10.1002/jemt.1070240503

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

年代:1993

数据来源: WILEY

摘要:

AbstractIn order to test the ability of freeze substitution to accurately preserve the ultra‐structure of the actin component of the cytoskeleton, the structure of rotary shadowed actin filaments was compared following preparation by glutaraldehyde fixation and freeze etch or freeze substitution. Freeze substituted actin filaments were further processed by either etching away frozen organic solvent or critical‐point‐drying before rotary shadowing. Comparison of filament diameters showed no significant difference between actin filaments that were directly etched and those that were freeze substituted and then etched. However, freeze substituted and then critical‐point‐dried filaments were significantly larger in diameter than filaments that were directly etched in water. The long pitch (right‐handed) two start helix was not affected by the different methods of preparation. However, the left‐handed “genetic” helical repeat that was prominent in actin filaments prepared by freeze etch was more difficult to detect in freeze substituted specimen, especially following critical‐point‐drying. Although the organization and distribution of actin filaments in extracted cells was similar in both freeze substituted and freeze etched specimens, there were some detectable differences. In cells that were freeze substituted and then critical‐point‐dried, filaments appeared to intersect at greater angles and seemed more “taut.” These results suggest that freeze substitution can preserve the overall morphology of actin filaments, but some chemical or physical modification of macromolecular surface structure may occur during the substitution process and these changes may be further exaggerated by subsequent processing

ISSN:1059-910X    

DOI:10.1002/jemt.1070240504

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe determination of ion concentrations within cells and sub‐cellular compartments remains a difficult procedure, as the volumes to be analyzed are rather small. X‐ray microanalysis is sufficiently sensitive, and has adequate resolution, to measure these concentrations. The major difficulties are related to the preparation of material for analysis. We have compared the measurement of sodium, potassium, and chloride contents in a salt tolerant unicellular alga,Dunaliella parva, following either freeze‐substitution (using two different resins) or molecular distillation drying. All three procedures gave similar results: after freeze substitution, ion contents were marginally (but not significantly) higher following embedding in Nanoplast MUV 116 resin than in Spurr resin. Since the Nanoplast can be polymerised at low temperatures, it has advantages over the Spurr resin. © 1993 Wiley‐L

ISSN:1059-910X    

DOI:10.1002/jemt.1070240505

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

年代:1993

数据来源: WILEY

摘要:

AbstractConsidering the increasing necessity for improved preparation techniques in biological electron microscopy as a basis for the identification and localization of cellular substances within the compartments of the cell, this review is focussed on the method of freeze substitution as an important link between the cryofixation (ultrarapid freezing) and resin embedding of biological specimens. The theory and practice of freeze substitution is summarized with particular interest in the physical and thermodynamic as well as in the chemical basis of this technique. A survey of practical aspects of the technical process of freeze substitution concerning the equipment and various protocols successfully applied in biological systems is also given. The main advantage of freeze substitution versus conventional chemical fixation is seen in the maintenance of the hydration shell of molecules and macromolecular structures. This results in an improved fine structural preservation, superior retention of the antigenicity of proteins and decreased loss of unbound, diffusible cellular components. Examples of excellent visualization of the ultrastructure of macro‐molecular complexes (nucleic acids, extracellular material, membranes etc.), small organisms (bacteria, algae, cyanobacteria and fungi) and large biological samples such as plant and animal tissue as well as the plant‐pathogen (fungus) interface and infection structures are presented. Recent data on the molecular characterization of freeze‐substituted biological tissue are exemplified with special emphasis on the subcellular detection of soluble components (elements, lipids, proteins and drugs) and the inter‐/intracellular localization of proteins including foreign proteins in transgenic plants. The molecular analysis of freeze‐substituted specimens is achieved by the combination of low temperature preparation techniques in biological electron microscopy with various detection methods such as X‐ray microanalysis, immunocytochemistry and high resolution autoradiography. © 1993 Wil

ISSN:1059-910X    

DOI:10.1002/jemt.1070240506

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe 3‐dimensional distribution of transverse (T) tubules at myotendinous junctions (MTJs) was studied by intermediate voltage (400 kV) electron microscopy of thick sections of rat vastus intermedius and chicken pectoralis muscles stained with lanthanum nitrate. Transversely oriented T tubules were seen to run at the level of A‐I junctions (the rat vastus intermedius) and Z bands (the chicken pectoralis), but were absent from such levels adjacent to the end of MTJ processes. These tubules opened to the lateral wall of sarcolemmal infoldings of MTJs and to the lateral cell surface. Longitudinally running T tubules were seen to connect with the transverse T tubules and to open at the bottom of junctional folds. The lack of T tubules at the final sarcomeric regions seems to indicate that the terminal sarcomeric half in close proximity to MTJs may be activated from the sarcoplasmic reticulum which forms couplings with the MTJ sarcolemma and/or longitudinal tubules in the MTJ processes. © 1993 Wiley‐Lis

ISSN:1059-910X    

DOI:10.1002/jemt.1070240507

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

年代:1993

数据来源: WILEY

摘要:

AbstractA scanning electron microscope equipped with a low vacuum specimen chamber and a Robinson's backscattered electron detector was employed to observe the natural surfaces of human buccal enamel before and after 30 percent phosphoric acid etching sequentially up to 90 sec at the same sites with no coatings. Furthermore, successive etching patterns were compared between deciduous and permanent teeth. On the imbrication lines of young permanent teeth, prismend pits surrounded with a “prismless” structure occasionally disappeared after acid etching and became a prismless enamel. Sequential etching caused the prismless areas and the areas of a type 1 etching pattern to decrease, and a cone‐shaped prism structure and a complex type of the type 1 and type 2 etching pattern (type 1–2) to appear. The former was a transitional type between the prismless enamel and type 2 prisms. These etched surfaces show type 2 prisms after deeper etching. Small dome‐shaped structures, slightly elevated on the attrited enamel surfaces, were found only in deciduous teeth. After acid etching, such areas which retained the prismless enamel rose to the underlying surfaces of cone‐shaped prisms. © 1993 Wil

ISSN:1059-910X    

DOI:10.1002/jemt.1070240508

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe present recent developments of a widefield computer/microscope system and image reconstruction algorithm for producing three‐dimensional (3D) increased depth of field images in the form of brightfield stereo pairs of thick specimens. The theoretical principle of this image reconstruction technique is based on Weiner‐type inverse filtering. A number of extensions and refinements to our previous work have included further testing of the system with a broader class of specimens and the implementation of several pragmatic refinements important for future 3D microscopy systems. These refinements include histogram modification routines for improving visualization, a preprocessing routine to eliminate edge artifacts due to circular convolution and other effects, stereo viewing angle optimization, a rule of thumb estimate for the axial sampling rate, and incorporation of a variation of the Fast Fourier Transform and filtering operations that significantly reduce computational time. Images of spyrogyra, neonatal rat hippocampal neurons, and cervical/vaginal cell smears are presented to show the utility of these methods for 3D visualization. The primary advantages of these methods are that they operate with an ordinary transmitted light microscope and are inexpensively implemented on a personal computer with reasonable computation time. © 1993 Wiley‐Lis

ISSN:1059-910X    

DOI:10.1002/jemt.1070240509

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

年代:1993

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070240510

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

年代:1993

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070240511

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

年代:1993

数据来源: WILEY