A study of staining for electron microscopy using collagen as a model system—VIII. Simulation of the negative staining pattern

Simulated negative staining patterns of collagen fibrils were prepared for visual display by a graphical procedure in which amino acid side-chains along the staggered molecules were weighted according to their stain-excluding capacity. The simulated patterns were then compared directly with electron-optical images of collagen fibrils negatively stained with sodium phosphotungstate or lithium tungstate. These visual comparisons confirm previous observations that satisfactory matching occurs when side-chains are weighted according to their ‘bulkiness’ (average cross-sectional area or ‘plumpness’). Optimal matching at the edges of the overlap zones occurred when a hairpin-like conformation was assumed for the N-terminal telopeptides and a condensed conformation for the hydrophobic part of the C-terminal telopeptides. The negative staining pattern is known to include some element of positive staining; visual matching suggests that this additional uptake of positive staining ions occurs predominantly in the more accessible gap zone in a fibril D-period. A slight mismatching between observed and simulated patterns can be understood if the gap zone suffers greater axial shrinkage than the overlap zone when specimens are prepared for electron microscopy.     

A study of staining for electron microscopy using collagen as a model system—VI. Uranyl nitrate negative staining patterns and their correlation with sequence data

Collagen is used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) were compared with chemical data by a computer-aided correlation procedure. The stain used was uranyl nitrate, pH 3.2 and 4.9. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some contribution of positive staining can also be demonstrated by the analysis described here.     

Diffraction contrast in reflection electron microscopy—I. Screw dislocation

The diffraction contrast theory has been used to anslyze the reflection electron microscopy (REM) image features of a normally emerging screw dislocation and the dependence of these features on the incident beam conditions. The reflected beam rocking curve has been assumed to be a Gaussian-like function but with different half-widths on the two sides of a specific Bragg angle. The calculated images show some fine structures around the dislocation core which depend on the incident beam conditions. This dependence suggests a method of measuring the rotation field from surface defects. Experimentally obtained REM images show features which are in good agreement with the diffraction contrst theory.     

A study of staining for electron microscopy using collagen as a model system—IV. Phosphotungstate/tungstate negative staining patterns and their correlation with sequence data

The type I collagen fibril (for which the axial distribution of amino acid residues is known) has been used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils were compared with chemical data by a computer-aided correlation procedure. Stains used were: phosphotungstic acid, pH 3.2 and 7.0, lithium tungstate, pH 7.2, and methylamine tungstate, pH 6.6. In all cases, the results of the correlation analyses point to the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains as the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some preferred uptake of heavy metal ions on charged side chains (a positive staining contribution) can be demonstrated by partial correlation analysis but, under the staining conditions used here, the effect is largely masked by the much greater negative staining component.     

A study of staining for electron microscopy using collagen as a model system—V. The effect of suberimidate fixation on negative staining patterns

The effects of the fixative dimethylsuberimidate (DMS) on negative staining patterns were studied using reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) as a model system and comparing electron-optical data and chemical data by a computer-aided correlation procedure. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant factor in determining the stain-excluding property of a DMS-fixed negatively stained collagen fibril as it is in unfixed collagen. Some contribution of positive staining can also be demonstrated after DMS-fixation by partial correlation analysis. Other evidence suggests that (unlike glutaraldehyde) DMS does not produce any morphological alterations to the negative staining pattern.     

Nuclear lamina—like filaments and nuclear matrix in allium cepa as revealed by scanning electron microscopy

In this study,freeze-fractured specimens of allium cepa root tip meristems were examined under the scanning electron microscope(SEM),This technique permitted the visualization of the outer membrane of the nuclear envelope with nuclear pore complexes and polyribosomes.Some of the cell nuclei prepared with this procedure had fissures of various widths on their nuclear envelopes through which the nuclear lamina-like filaments(LLF) undernearth the nucleoplasmic side of the envelopes were clearly visible.The diameters of these filaments veried between 25 and 125nm.Many of the LLFs showed granular thickenings at places,and were attached to the inner surface of nuclear envelope in some regions .Similar LLFs were also seen at the peripheries of the freeze-fractured faces of nuclei.Meanwhile,the spatial relation between the nuclear matrix filaments(NMF) and other nuclear structures(nucleoli,chromation and peripheral lamina-like filaments) was revealed in these fractured preparations.In addition,the methods and techniques in studying the nuclear lamina morphology and the roles played by NMFs in activities of various nuclear sturctures were discessed in brief.     

Imaging the boundaries—innovative tools for microscopy of living cells and real-time imaging

Recently, light microscopy moved back into the spotlight, which is mainly due to the development of revolutionary technologies for imaging real-time events in living cells. It is truly fascinating to see enzymes “at work” and optically acquired images certainly help us to understand biological processes better than any abstract measurements. This review aims to point out elegant examples of recent cell-biological imaging applications that have been developed with a chemical approach. The discussed technologies include nanoscale fluorescence microscopy, imaging of model membranes, automated high-throughput microscopy control and analysis, and fluorescent probes with a special focus on visualizing enzyme activity, free radicals, and protein–protein interaction designed for use in living cells.     

M?ssbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins

M?ssbauer spectroscopy has indicated significant differences in the iron-containing cores of various haemosiderins. In the present study, haemosiderin was isolated from a number of animal species including man. In addition, haemosiderin was isolated from patients with primary idiopathic haemochromatosis or with secondary (transfusional) iron-overload. The iron cores of the animal and normal human haemosiderin appear to be very similar by M?ssbauer spectroscopy, and the electron diffraction data indicate a ferrihydrite structure similar to that of ferritin cores. The haemosiderin isolated from secondary iron-overload shows anomalous behaviour in its temperature-dependent M?ssbauer spectra. This can be understood in terms of the microcrystalline goethite structure of the cores as indicated by electron diffraction. The haemosiderin cores obtained in the case of primary haemochromatosis have an amorphous Fe(III) oxide structure and show M?ssbauer spectra characteristic of a magnetically disordered material, which only orders at very low temperatures.     

Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants

Environmental scanning electron microscopy (ESEM) enables the investigation of hydrated and uncoated plant samples and the in situ observation of dynamic processes. Water vapor in the microscope chamber takes part in secondary electron detection and charge prevention. Two ESEM modes are available and offer a broad spectrum of applications. The environmental or wet mode prevents sample dehydration by the combination of sample cooling (5°C) and a vapor pressure of 4–6 Torr. In the low vacuum mode, the maximum chamber pressure is limited to 1 Torr (corresponding to about 5% relative humidity in the chamber) and allows the simultaneous use of a backscattered electron detector for imaging material contrast. A selection of characteristic plant samples and various applications are presented as a guide to ESEM for plant scientists. Leaf surfaces, trichomes, epicuticular waxes, and inorganic surface layers represent samples being comparatively resistant to dehydration, whereas callus cells and stigmatic tissue are examples for dehydration- and beam-sensitive samples. The potential of investigating dynamic processes in situ is demonstrated by studying anther opening, by tensile testing of leaves, and by performing hydration/dehydration experiments by changing the vapor pressure. Additionally, automated block-face imaging and serial sectioning using in situ ultramicrotomy is presented. The strengths and weaknesses of ESEM are discussed and it is shown that ESEM is a versatile tool in plant science.     

A study of staining for electron microscopy using collagen as a model system—VII. The effect of formaldehyde fixation

Exposure to formaldehyde brings about small but readily detectable changes in the staining behaviour of collagen fibrils. These changes can be interpreted in chemical terms by comparing fibril staining patterns with artificial patterns computer-generated from sequence data. Positive staining with phosphotung-state (where heavy metal is confined to anions), shows that most of the lysyl and hydroxylysyl side-chains lose their charge character as a result of formaldehyde treatment and cease to take up staining ions. The charge character of arginyl (and probably histidyl) residues is unaltered and these residues continue to react with stain. Acidic residues are also unaffected. These results accord with biochemical evidence that the initial reaction between proteins and formaldehyde leading to subsequent cross-linking involves modification of ε-amino (and α-amino) groups. They show too that the secondary condensation producing the actual cross-link does not alter the charge character of the second group, at least when it is on an arginyl (or histidyl) side-chain.Formaldehyde-induced changes in stain deposition can also be detected after negative staining, although they are slight compared with those brought about by glutaraldehyde. Unlike glutaraldehyde, formaldehyde introduces no bulky polymeric adducts into the fibril structure, and the conspicuous stain-excluding bands seen in negative staining patterns following glutaraldehyde fixation are absent after exposure to formaldehyde. For this reason, where chemical fixation is used to stabilize macromolecules and supramolecular aggregates prior to negative staining and high resolution electron optical imaging, formaldehyde would seem to be preferable to glutaraldehyde. Data from fibril staining patterns and from thermal stability measurements (made on collagen gels) show that formaldehyde fixation does not preclude a subsequent reaction with glutaraldehyde.As with other fixatives, there is reduced accessibility to stain after formaldehyde treatment. Accessibility is least in the overlap zone where the denser packing of collagen molecules provides greater opportunities for intermolecular cross-linking. Gel electrophoresis confirms that formaldehyde-induced cross-links in fibrils are predominantly intermolecular.     

Pyrogallol red-vanadium complex—a new stain for electron microscopy

 We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (λ_max=520 nm) to violet (λ_max=539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultrathin sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to proteinaceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level. Accepted: 14 June 1996     

A study of positive staining for electron microscopy using collagen as a model system—III. The effect of suberimidate fixation

Collagen is used as a model system to study the structural effects of the fixative dimethylsuberimidate (DMS) on a protein. Using reconstituted fibrils of type I calf skin collagen(of known amino acid sequence), electron-optical data from positive staining patterns were compared with chemical data by a computer-aided correlation procedure. The results show that, under the conditions used, the reaction between DMS and collagen is specific for the ϵ-amino groups of lysyl and hydroxylysyl residues and that no reaction takes place between DMS and arginyl residues. Other evidence suggests that (unlike glutaraldehyde) no polymerization of the DMS occurs and that a cross-link between the two ϵ-amino groups can only form when they are sufficiently close to be bridged by the DMS monomer.     

Correlative video-light–electron microscopy: development,impact and perspectives

Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the ‘history,’ or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light–electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular ‘context’ to the fluorescent dynamic structures of interest. CLEM ‘multiplies’ the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging.     

MASDET—A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy

Electron microscopy has been used to measure the mass of biological nanoparticles since the early 60s, and is the only way to obtain the mass of large structures or parameters such as the mass-per-length of filaments. The ability of this method to sort heterogeneous samples both in terms of mass and shape promises to make it a key tool for proteomics down to the single cell level. A new multiplatform software package, MASDET, that can be run under MATLAB or as a standalone program is described. Based on a user-friendly graphical interface MASDET streamlines mass evaluation and greatly increases the speed of required optimisation procedures. Importantly, the immediate application of Monte-Carlo simulations to describe multiple scattering is possible, allowing the mass analysis of thicker samples and the generation of mass thickness maps.     

Breaking bad news in genetic counseling—problems and communication tools

Breaking bad news is a common problem for clinical geneticists in their daily work. Just like doctors of other specialties, e.g., oncologists, they can use proven communication tools instead of relying only on professional sense. The latter is, of course, always the most important for experienced doctors, but the use of protocols such as SPIKES and EMPATHY facilitates both the delineation of difficult information and the process of its transmission. The article gives an overview of the best tools of this type available to medical professionals dealing with genetic counseling.     

“Chromosome exo-skeleton” in plant cells visualized by scanning electron microscopy

Summary Cytoskeletons surrounding the chromosomes of the root tip cells ofPisum sativum and the generative cells ofAllamanda schottii were visualized using Triton X-100 extraction and scanning electron microscopy. The cytoskeleton surrounding the chromosome consisted of a reticulate network of fibres. This is the first report showing the existence of a chromosome exo-skeleton in plant cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid)