Electron microscopy reviews最新文献

A general description is given of the various techniques that may be used in ultrastructural studies of the cytoskeleton. Electron microscopy of the cytoskeleton of Dictyostelium discoideum serves as a source of examples illustrating the general effects of detergent treatment and fixation techniques. A concise review is given of the structure and function of the actin microfilament system and the cytoplasmic microtubules in Dictyostelium, based on electron microscopical, light microscopical and biochemical studies. Special attention is paid to their involvement in cell movement and chemotaxis. Conventional thin sectioning, fast freezing freeze substitution, whole mounts, freeze fracturing and freeze etching and negative staining techniques are discussed and their respective advantages and limitations are mentioned. A recently developed technique, wet-cleaving, is described which gives promising results in experiments in which the inside of the plasma membrane with the adhering cortical cytoskeleton is studied. This technique may turn out to be useful in high-resolution scanning electron microscopy. A description is given of protocols that proved to be successful in the author's and other laboratories, In a few cases the feasibility of immunogold labelling (illustrated by anti-tubulin labelling of cytoplasmic microtubules) is also dealt with.

The molecular and cell biologic data supporting the established model of the intracellular secretory (transport) pathway for glycoproteins in the yeast Saccharomyces cerevisiae have been reviewed and confronted with our electron-cytochemical findings. These in situ findings show a new class of constitutive intracellular conveyors—the coated globules—and also suggest substantial alternatives in the cellular mechanism of the vacuole biogenesis. The controversial question of the Golgi compartment identity in S. cerevisiae is revived.

This review includes details of recent macromolecular crystallizations using lipid monolayers. Crystallization conditions are discussed together with suggestions for improving resolution.

In the last few years the role of pollen and the pollen tube in the fertilization process in higher plants has received considerable attention.

By ultrastructural, biochemical and immunofluorescent investigations it has been shown that a cytoskeletal apparatus plays a central role in pollen tube growth. Microfilaments and microtubules, in which main components are, respectively, actin and tubulin, represent the most investigated cytoskeletal components. New information has been recently provided by the identification of myosin and also of a kinesin-like protein.

The pollen tube cytoskeleton consists of two different cytoskeletal systems: the vegetative cell cytoskeleton, namely the cytoskeleton of the pollen grain and pollen tube, and the gamete cytoskeleton (generative cell and sperm cell cytoskeleton). The vegetative cell cytoskeleton plays a fundamental role in assuring the cytoplasmic movement of organelles, vesicles and gametes from the pollen grain to the pollen tube apex and consists mainly of microtubules and microfilaments. Also myosin and the kinesin-like protein are involved in the process of organelle and vesicle movement. The gamete cytoskeleton has a central role in sperm cell formation and in the reshaping process during gamete movement inside the pollen tube. It consists mostly of microtubules and partially characterized microtubule-associated structures. Actin filaments have recently also been identified.

Light microscopy (LM) enables biological specimens to be examined without fixation or dehydration but the resolution is insufficient for studies of cell ultrastructure. Electron microscopy (EM) improves the resolution, but requires the specimen to be fixed or frozen, which may cause alterations in cell structure. Using soft X-rays to image specimens improves the resolution, relative to LM, and avoids tissue pretreatment. Staining is not required since within the ‘water window’ (2.3–4.4nm), carbon absorbs more strongly than oxygen. The lower attenuation of soft X-rays, relative to electrons, by biological material allows specimens several microns thick to be examined.

Several sources for generating water-window X-rays are briefly described and examples of images obtained with each are presented. The specimens imaged include both plant and animal material either in the fixed or natural state. Of the different systems currently used to collect images only contact imaging is considered in detail. By placing the specimen against photosensitive resist, which acts as the image recording medium, an absorption map of the specimen is produced. This latent image is then chemically developed and, after coating, the resist is examined by scanning EM, or, if a replica is produced, by transmission EM. Using laser-produced plasmas such images are produced within a very short exposure time, typically 1–10 nsec, thus avoiding any radiation-induced damage to the specimen which other X-ray imaging techniques may cause.

Negative staining, some closely related alternative preparation techniques and radiation stability are considered. An attempt is made to clarify the mechanism of action and ultimate resolution limit of negative staining. The results of electron diffraction investigation of thermitase micocrystals embedded in glucose and glucose + stains are presented. It is shown that at doses not exceeding 10 electrons/nm² electron diffraction from thermitase crystals demonstrate diffraction fields up to 0.2 nm. When adding heavy-atom salts to glucose or using negative staining, the relative intensities of reflections change and electron diffraction patterns for every type of heavy-atom additive (or negative stain) have their specific features. Such characteristic changes of reflection intensities indicate specific interaction of these additives (or stains) with the object. In the case of electron diffraction from the crystals stained using the routine negative staining technique the ordering was preserved down to 0.4–0.5 nm. Increasing the dose up to the normal value results in fading of distant reflections. Thus, negative staining with radiation doses less than the critical one could yield resolution down to 0.4 nm. Yet, the structure may change due to interaction with the stain. Nevertheless, the possibility that such resolution could be obtained for a limited number of objects should not be excluded. Some examples of the application of negative staining for investigation of quaternary and domain structure of proteins (nitrogenase, glutamine synthetase, mitochondrial ATP-synthase, membrane monooxygenase enzymes), tubular and two-dimensional protein crystals (catalase, phosphorylase, HWV protein, hydrogenase), as well as ribosomes and bacteriophages are given in the review.

We describe here a method for computing a three dimensional map of a periodic specimen from a single electron micrograph of an obliquely cut section. Neighbouring areas of such an image display successively the contents of the unit cell of the structure. The reconstruction procedure can be considered in two steps. The first step involves restacking of successive areas to produce an image akin to that produced by serial section reconstruction. The resolution normal to the section would, at this stage, be limited by the thickness of the section, since the micrograph represents a projection of the density in the section. However, because of the periodic nature of the specimen, the image contains redundant information, which can be used in an attempt to deconvolute the section thickness and thus produce improved resolution normal to the section. The computation can be carried out directly with the densities or more conveniently, particularly for three dimensional crystals, by using Fourier transforms. The approach, which is most powerful when the section is thin, is insensitive to the collapse of the section caused by electron irradiation. Striated muscle provides particularly suitable specimens for such analysis and we present, as examples, computed maps of the M-band of fish muscle and of insect flight muscle in rigor.

Whole mounts of intact virus-infected cells have been used for several decades to examine virus-cell relationships and virus structure. The general concept of studying virus structure in association with the host cell has recently been expanded to reveal interactions between viruses and the cytoskeleton. The procedure permits utilization of immuno-gold protocols using both the transmission and scanning electron microscopes. The grid-cell-culture technique is reviewed to explain how it can be exploited to provide valuable information about virus structure and replication in both diagnostic and research laboratories. The use of the technique at the research level is discussed using bluetongue virus as a model. The procedure can provide basic structural information about intact virions and additional data on the intracellular location of viruses and virus-specific structures and about the mode of virus release from infected cells. Application of immunoelectron microscopy reveals information on the protein composition of not only released virus particles but also cell surface and cytoskeletal-associated viruses and virus-specific structures. Collectively, this simple and physically gentle technique has provided information which would otherwise be difficult to obtain.