ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03069.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYA VG Microscopes HB501 field‐emission high‐resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark‐field images were obtained from frozen‐hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze‐dried in the STEM and low‐dose (≅ 103e/nm2) dark‐field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel‐detection electron energy‐loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di‐decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm2. For freeze‐dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy‐dispersive X‐ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100‐s analysis time is sufficient to quantify the calcium content of 400‐nm2regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03070.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYIt is generally agreed that surface‐tension forces and the direct interaction between the specimen and either the air‐water interface or the water‐substrate interface can influence significantly the preparation of biological materials for electron microscopy.Even so, there is relatively little systematic information available that would make it possible to control surface‐tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1°, and perhaps less than 0·2°, over areas as large as 1 μm2or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose‐embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air‐water interface rather than adsorption of the specimen to the substrate. Surface‐tension forces at the air‐water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air‐water interface can be variously manipulated in the search to find desirable conditions of

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03071.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYBiological objects may be arrested in defined stages of their activity by fast freezing and may then be structurally examined. If the time between the start of activity and freezing is controlled, structural rearrangements due to biological function can be determined. Cryo‐electron microscopy shows great potential for the study of such time‐dependent phenomena. This study examines the actin polymerization process using cryo‐electron microscopy of vitrified specimens. Actin filaments are shown to undergo a structural change during polymerization. In the early stages of the polymerization process (t<2 min), filaments exhibit a pronounced structural variation and frequently show a central low‐density area. In the later stages of the polymerization, F‐actin‐ADP filaments have a more uniform appearance and rarely display a central low‐density area. These findings, analysed on the basis of a previously proposed polymerization model, suggest that polymerization intermediates (F‐actin‐ATP and more probably F‐actin‐ADP‐Pi) and filaments at steady state (F‐actin‐ADP) have different structures. To investigate the physiological relevance of these results at the cellular level, the potential of cryo‐substitution in preserving the structure of muscular fibre was assessed. Optical diffraction patterns of relaxed and contracted frog cutaneous muscle are similar to the corresponding X‐ray diffraction patterns. The resolution of the images extends to about 7 nm. These results show that dynamic study of muscle contraction is p

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03072.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYA review of low‐temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high‐resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient‐temperature SEM. This is because frozen‐hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen‐hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high‐resolution LTSEM. The advantages, problems and requirements for this approach

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03073.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYA dedicated cryopreparation system, the SCU 020 (Balzers), is introduced and described in detail for use in low‐temperature scanning electron microscopy (LTSEM). The basic unit consists of two parts: (i) a high‐vacuum preparation chamber equipped with a cold‐stage, motor‐driven fracturing microtome, planar magnetron (PM) sputter source, quartz‐crystal thin‐film monitor, Meissner cold trap, and turbo molecular pump stand; and (ii) a second part (separated from the first by a sliding, high‐vacuum valve) residing in the SEM chamber. This is equipped with an anti‐contamination cold trap, a fully movable goniometer cold stage (having motor drives forx, y, and rotation) and replaces the SEM's original stage (Raith). The SCU 020 is entirely self contained allowing independence from, and synchroneity with, the SEM of choice.LTSEM micrographs of specimen (that are fully frozen hydrated or partially freeze‐dried) surfaces or fracture faces, without or with various metal coatings, can be examined over a broad temperature range (‐150 to +50°C). This is made possible by the combined application of the two, independently controlled, cold stages and the on‐line, high‐vacuum, specimen cryo transfer between them.In‐situetching is simple and straightforward. Intramembranous particles and membrane fracture steps, typically imaged in transmission electron microscopy (TEM), are resolved by PM sputtering with platinum at low specimen temperature and high‐resolution LTSEM in

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03074.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYComparison of different harvesting and preparation pathways showed that low‐temperature SEM is an adequate method to conserve the stomatal aperture for SEM. Both critical point drying and freeze drying cause considerable artefacts.Exposure to site‐relevant concentrations of ozone led to reduced width of the stomatal aperture. Moreover, unetchable droplet‐like exudates were found on the outer face of mesophyll cells of leaves where the trees had been exposed to ozone. These exudates were later followed by collapsed mesophyll cells and ended in necrotic zones before premature leaf

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03075.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThe preparation of cells for electron microscopy, in particular the fixation and embedding routine, influences the antigenicity, often resulting in a markedly reduced labelling intensity. To overcome the difficulties associated with fixation‐induced changes in antigenicity, we produced antibodies against pre‐fixed human apolipoprotein (apo) A‐I. Purified apo A‐I was fixed with 4% formaldehyde and was used to raise polyclonal antibodies in rabbits. The antiserum was purified by protein‐A‐Sepharose followed by affinity chromatography with the fixed antigen coupled to vinylsulphone‐activated agarose. The specificity of the antibodies was ascertained by enzyme‐linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed lipoproteins. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, indicating that the antibodies were directed against epitopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells. When HepG2 cells were exposed to anti‐apo A‐I antibodies followed by a secondary FITC‐labelled antibody, fluorescence was found intracellularly in distinct regions. Electron microscopy revealed that the endoplasmic reticulum, and in particular the trans elements of the Golgi complexes, were the main compartments stained for apo A‐I both in HepG2 cells, as shown by the immunoperoxidase technique, and in human hepatocytes, as shown by the protein‐A‐gold technique on ultrathin cryosections. The findings demonstrate the potential of using antibodies to fixed antigens as a strategy to overcome impaired localization due to fixation in cytochemistry at the light microscopic and

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03076.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYA wide variety of methods by which cytochemistry and freeze‐fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze‐fracture, but differ inhowthis is achieved, in preciselywhattype of information is obtained, and inwhichtypes of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze‐fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze‐fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face‐on, or for examining the fracture path through the sample (without replica). Recent developments have centred on the use of replicas to stabilize half‐membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen— specifically, turning it over during processing—so that additional structural information can be obtained.This article aims to explain how modern freeze‐fracture cytochemisty works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze‐fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane b

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03077.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYDuring the past years, the methods of ultrastructural visualization of intracellular and cell‐surface proteins have been improved considerably, mainly as the result of the development of low‐temperature preservation in combination with immunocyto‐chemical labelling procedures using poly‐ or monoclonal antibodies. In this contribution we will discuss the combination of immunogold labelling with cryoultramicrotomy and two replica methods, i.e. freeze‐etching and label‐fracture. The main advantage of cryoultramicrotomy is that it enables post‐sectioning labelling, thus providing complete accessibility of all cellular antigens, located both intracellularly and on the cell surface. Important parameters that influence the labelling (i.e. label‐efficiency), including penetration of the label and antibodies in the section, effects of fixatives on antigenicity, and steric hindrance, will be discussed in detail. The replica methods have the advantage of enabling an analysis of the lateral distribution of antigens located at the cell surface. The label efficiency is of particular importance in these studies and in this context several parameters will be discussed, including accessibility and effe

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03078.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY