Electron microscopy reviews最新文献

The nicotinic acetylcholine receptor is a glycoprotein occurring in the electric tissue of the electric ray Torpedo sp. and the electric eel Electrophorus electricus in postsynaptic membranes in high densities. Since these membranes can be easily prepared they have been, since their discovery, a favourable object for electron microscopists. The receptor protein appears in negatively stained membranes as a ring with a diameter of about 75 Å. With improved techniques of preparing membranes which contain the receptor molecules in two-dimensional crystalline arrays and especially with computer-aided image processing, the ring appeared as an arrangement of five maxima (representing probably the five receptor subunits) with a five-fold axis of pseudosymmetry perpendicular to the membrane plane. At present the resolution obtained is better than 20 Å, enough to depict the receptor's overall shape and dimensions but not enough to resolve functional moieties, as for example the selectivity filter and the gating device of the ion channel, which is an integral part of the receptor complex.

The receptor-rich membranes turned out to be models for developing and comparing image processing methods. In this article some of these methods, especially the Circular Harmonic Averaging method, are critically reviewed.

This review aims to provide a comprehensive and in-depth survey of a cell organelle, the annulate lamellae, that is widely distributed and especially prevalent in both female and male sex cells as well as tumor and cancer cells. The organelle is also present in many somatic cells and plant cells. Emphasis is placed on the contributions that electron microscopy and associated experimental approaches have made in providing information about the distribution, ultrastructure, morphogenesis and relationships of annulate lamellae to other cellular organelles, especially the nuclear envelope and endoplasmic reticulum, as well as cell product. An increasing number of experimental manipulations have recently been shown to alter, either increase or decrease, the amount of annulate lamellae and these studies are explored in depth. Information about the origin and morphogenesis of annulate lamellae in different cells is summarized and extensive coverage is given to several hypotheses about possible annulate lamellae function. A detailed bibliography provides a thorough compilation of research dealing with annulate lamellae. A major goal of this extensive review is to generate increased awareness of, and interest in, this cell organelle for students and investigators of the cell who, by bringing current techniques in cell and molecular biology to bear, might focus and intensity studies on the function of an organelle whose precise role in the cell is presently enigmatic.

Melamine resins are derived from the heterocyclic compound triaminotriazine, C₃H₆N₆. Similar to proteins in structure and reactivity, water-soluble melamine resins can be used as water-embedding media for electron microscopy (Bachhuber and Frösch, 1983). The idea behind this approach was to study some of the artefacts of traditional embedding techniques and to work out conditions to eliminate as far as possible denaturing of proteins and extraction of lipids. Sectioned cells and tissues processed in the melamine resin Nanoplast show remarkable preservation of ultrastructure. Because they can be sectioned extremely thinly, melamine resins are particularly suitable for dark-field and electron spectroscopic imaging of unstained molecular suspensions providing in this way an unusually clear reproduction of ultrastructural detail such as the helical structure of isolated unstained double-strand DNA molecules (Frösch et al., 1987b).

In 1988, the melamine resin Nanostrat was introduced as an EM-compatible prolific substrate foil for cell culture (Westphal et al., 1988). Cells or bacteria cultivated on this material can be processed for various kinds of follow-up techniques like TEM, SEM, vertical sectioning and immunocytochemistry.

The key carboxylating enzyme of the reductive pentose phosphate cycle, D-ribulose-1,5-bisphosphate carboxylase/oxygenase [RuBisCo] isolated from the chemolithoautotrophic, H₂-oxidizing bacterium Alcaligenes eutrophus H16 has been analyzed by several different techniques that allow conclusions about structure and function-dependent structural changes. The techniques include a novel approach in which the enzyme was induced to form 2D-crystals suitable for electron microscopy in each of its three stable functional states: as active enzyme [E_a] (in the presence of Mg²⁺ and HCO^-₃); as inactivated enzyme [E_ia] (in the absence of Mg²⁺ and HCO^-₃) and as enzyme locked in an in vitro transition state [CABP-E] (E_a fully saturated with the transition state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate [CABP]). In conjunction with X-ray crystallography, X-ray small angle scattering and other biophysical and biochemical data, the results obtained by electron microscopy support the idea that drastic configurational changes occur. Upon transition from E_a to the CABP-E the upper and lower L4S4 halves of the molecule consisting of eight large and eight small subunits (L8S8; MW = 536,000 Da) are assumed to be laterally shifted by as much as 3.6 nm relative to one another while the location of the small subunits on top of the large subunits, and relative to them, remains the same. For the E_ia a similar sliding-layer configurational change in the range of 2–2.5 nm is proposed and in addition it is suggested that other configurational/ conformational changes take place. The proposed structural changes are discussed with respect to the current model for the tobacco enzyme and correlated with data obtained for various other plant and (cyano)bacterial L8S8 RuBisCOs leading to speculations about structure-function relationships.

Over the past decade new methods have been developed to visualize both the external and the protoplasmic surfaces of cultured cells in the electron microscope. In this review the emphasis is on cell monolayers, though some of the techniques are also applicable to cells in suspension.

There is no universal method which would satisfy all our requirements i.e. the preservation of native structure and antigenicity and the visualization of the whole cell surface at high resolution. While surface replicas of freeze-dried or critical point-dried cells are eminently suited for high resolution studies including gold immunolabelling, scanning electron microscopy provides a view of the whole cell and a large sample for ‘statistical’ evaluation. Whole mount preparations of cleaved cells prove useful in studies of plasma membrane associated structures such as the cytoskeleton.

A series of new procedures have been developed for studies of cytoskeleton/membrane interactions, identification of intramembrane particles and their contacts with the glycocalyx, to mention some of the biological problems. Although the lysis-squirting technique appears most suitable for the visualization and immunolabelling of protoplasmic surfaces of ventral membranes, dry- or wet-cleaving represent a useful alternative for studies of the protoplasmic surfaces of dorsal membranes and of the ventral membrane associated cytoplasmic domains.

An assessment of the methods is given though this should only serve as guidance and it is up to the experimentor to choose the most useful technique for the project under study. Briefly the aim of the project determines the choice of the method. A multi-methodical approach is recommended when one method does not provide satisfactory results.

An exoglucanase (exo-1,4-β-glucanase), purified from a cellulase produced by the fungus Trichoderma harzianum, was tagged with colloidal gold particles and applied on plant and fungal tissue sections for localizing β-(1 → 4)-linked glucans. The present review reports the conditions required for the preparation of this enzyme-gold complex and for its application in transmission electron microscopy. The exoglucanase-gold complex was found to be a powerful probe for labelling accurately cellulosic glucans in tissues processed under conventional procedures. This approach represents a promising alternative to previous methods such as autoradiography or subtractive cytochemistry in the study of wall topochemistry. It will undoubtedly acquire increasing applicability and relevance in various fields of plant science including physiology and pathology.