Journal of electron microscopy technique最新文献

Electron microscopic studies of membrane structure have been facilitated by the recent development of the composite replica technique in which the membrane is freeze-fractured, then inverted and the surface deep-etched and replicated. Examination in stereo of this composite preparation of two replicas with interposed half-membrane and associated surface elements reveals the physical relationship between structures on the surface and within the membrane. Composite replicas of the toad urinary bladder surface demonstrated connections of filamentous glycocalyx elements to intramembrane particles (IMPs). Using a bidirectional shadowing technique, many membrane surface particles also are shown to be associated with underlying IMPs, suggesting that these membrane surface particles are projections of the IMPs above the surface of the membrane. There is evidence that elements whose attachment sites relate to the half-membrane fractured away can be displaced from the membrane surface and lost. Labelling studies using colloidal gold-labelled antibodies were carried out to assess loss of surface mesh from fractured membrane. Gold distributions and amounts were similar in labelled surface replicas, label-fracture specimens, and labelled composite replicas, yet the amount of mesh detected in the composite replicas was less than in the surface replicas. This suggests that while some unlabelled or lightly labelled surface elements can be lost from fractured membranes, ligands stabilize elements and reduce their loss apparently by cross-linking them.

The quick-freeze, deep-etch, rotary-shadow technique provides a powerful tool to study the structural dynamics of extracellular matrices. Using this technique, we show that the extracellular investments of the Xenopus laevis egg are multilayered and securely anchored to the egg surface. The cortical cytoskeleton within the egg contains embedded cortical granules with surrounding endoplasmic reticulum and is capped by a thin reticular sheet that contacts the inner surface of the plasma membrane. The extracellular matrix undergoes three distinct changes at fertilization: a) formation of a "smooth" layer below the vitelline envelope (VE), b) transformation of the VE itself to an altered VE composed of concentric fibrous sheets, and c) formation of a dense, "briar-patch"-like fertilization layer at the upper surface of the VE.

The two main advantages of cryofixation over chemical fixation methods are the simultaneous stabilization of all cellular components and the much faster rate of fixation. The main drawback pertains to the limited depth (less than 20 microns surface layer) to which samples can be well frozen when freezing is carried out under atmospheric conditions. High-pressure freezing increases the depth close to 0.6 mm to which samples can be frozen without the formation of structurally distorting ice crystals. This review discusses the theory of high-pressure freezing, the design of the first commercial high-pressure freezing apparatus (the Balzers HPM 010), the operation of this instrument, the quality of freezing, and novel structural observations made on high-pressure-frozen cells and tissues.

"Freeze-fracture cytochemistry" encompasses a diversity of recently developed techniques in which freeze-fracture and cytochemistry are combined. Cytochemical labeling may, in principle, be integrated into one of three basic points in the standard freeze-fracture procedure; 1) before the specimen is frozen, 2) after it has been fractured, or 3) after it has been platinum shadowed and/or carbon coated. Visualization of the labeled cellular structures can be achieved by a variety of different methodologies. For example, the markers (usually colloidal gold particles) may be viewed embedded within a replica, or attached to it via fragments of membrane (or other cellular components). Sectioning is a central strategy in a number of techniques, either in combination with or in place of replication. The different combinations of methods that have been devised are not, for the most part, alternative ways of arriving at the same result; each provides quite distinct information about specific classes of membrane component or other structure in the cell. The purpose of this review is to present, within a single article, a systematic survey of the full range of techniques currently available in freeze-fracture cytochemistry. Emphasis is placed on explaining the principles underlying the methods and on illustrating their applications. With the success recently achieved, freeze-fracture cytochemistry has moved from the phase of experimental development to a position in which it may be expected increasingly to make significant contributions across a wide spectrum of problems in cell and membrane biology.

Molecular imaging by freeze-drying of molecules adsorbed to a mica substrate often provides better images of molecules than those attainable with other methods; the images are easier to interpret than those obtained with frozen thin film or negative staining, and the 3-dimensional information content is greater. The complete procedure for the production and examination of platinum-carbon replicas of molecules is described. Topics include production of a mica flake suspension, chemical pretreatment of the flakes to enhance adsorption, quick-freezing of the samples on mica, optimal operation of the freeze-fracture equipment, and orientation of replica topography. The production of stereo micrographs is analyzed in detail, with emphasis on the photographic procedures necessary for interpretation and on the identification of correct micrograph orientation. Guidelines are provided for the extrapolation from observed molecular size in platinum replicas to expected molecular weight.