A stable iron-haematoxylin solution for staining the chromatin of cell nuclei

By using ferrous sulphate as mordant, sodium iodate as oxidant and aluminium chloride as stabilizer, it proved possible to formulate a stable single-solution iron-hæmatoxylin. This provides a resistant nuclear stain, sufficiently acid-fast to be suitable for counterstaining with van Gieson or the trichrome methods. This solution, ferrous hæmatein, can replace the celestin blue-hæmalum sequence.     

A new approach to the study of the E. coli nucleoid

E. coli were examined by the freeze-fracture thaw-fix technique, embedded in thin fibrin gels. After glutaraldehyde fixation the bacterial nucleoid was found spread out over the surrounding fibrin. Addition of calcium and uranyl acetate to the fixative preserved the nucleoid in compact form. The spread nucleoid was then examined against a smooth mica background after freeze-thaw and osmotic lysis. These spreads were critical-point dried, rotary shadowed with platinum–carbon and viewed as stereo-pair micrographs. Structures seen are tentatively interpreted as clusters of polyribosomes, extended DNA, and supercoiled DNA complexed with proteins or polyamines. After osmotic lysis, glutaraldehyde alone preserves the nucleoid in compact form. Only where strands are broken, in freeze-fracture or freeze-thaw lysis, must uranyl acetate be added to the fixative to preserve a compact structure.     

Uranyl sulphate: A new negative stain for electron microscopy

Uranyl sulphate is a negative stain of high quality for electron microscopy of macromolecules below their isoelectric point. The stain results in good contrast and high resolution as demonstrated by optical diffraction of periodic structure. Analysis of scanning transmission electron microscopic data reveals that uranyl sulphate is lower in background noise level than uranyl acetate and is dramatically more resistant toward granularization upon continued exposure to electron irradiation. Electron microscopic images of most macromolecules contrasted with uranyl sulphate were indistinguishable from those obtained with uranyl acetate. However, electron microscopic images of Reo virus contrasted with uranyl sulphate are always readily distinguished from those obtained with uranyl acetate.     

Electron microscopy of tRNA crystals: I. Thin crystals negatively stained with uranyl acetate

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNA^phe. The thickness of the thinnest was estimated at 130 Å corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.     

The correlation averaging of a regularly arranged bacterial cell envelope protein

An adaptation of the ‘correlation averaging’ method is described which allows reliable and almost fully automatic image averaging in the case of near-periodic structures notwithstanding the presence of substantial crystal imperfections; methods for assessing resolution and symmetry without reliance on crystallinity are also discussed. Electron micrographs of negatively stained and rotary shadowed preparations of the HPI-layer protein from the cell envelope of Micrococcus radiodurans have been averaged using the method, and the projected structure is described to a resolution of about 1·9 nm.     

Electron microscopic study of 5SrRNA crystals from Thermus thermophilus HB8

Several types of crystals were grown from 5SrRNA, which was purified from the highly thermophilic bacterium, Thermus thermophilus HB8. Crystal lattice parameters were determined by X-ray and electron diffraction. One type of crystal was suitable for electron microscopy after staining with uranyl acetate. Two projections derived from micrographs taken at different tilt angles were processed for image analysis. This result enabled us to deduce the arrangement of molecules within the crystal lattice.     

Electron microscopy of tRNA crystals. I. Thin crystals negatively stained with uranyl acetate

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNAphe. The thickness of the thinnest was estimated at 130 A corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.     

Enhanced effects of nonisotopic hafnium chloride in methanol as a substitute for uranyl acetate in TEM contrast of ultrastructure of fungal and plant cells

This ultrastructural study showed that nonisotopic methanolic hafnium chloride and aqueous lead solution was an excellent new electron stain for enhancing TEM contrasts of fungal and plant cell structures. The ultrastructural definition provided by the new stain was often superior to that provided by conventional staining with uranyl acetate and lead. Definition of fine ultrastructure was also supported by quantitative data on TEM contrast ratios of organelles and components in fungal and plant cells. In particular, polysaccharides, which were localized in cell walls, glycogen particles, starch grains, and plant Golgi vesicle components, were much more reactive to the new stain than to the conventional one. The new nonisotopic stain is useful for enhancing the contrast of ultrastructure in biological tissues and is a safer alternative to uranyl acetate. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Reconstruction of glutamine synthetase using computer averaging.

The axial projection of the glutamine synthetase molecule has been reconstructed from electron micrographs of a stained preparation by using a new method of correlation search and averaging. The average over 50 individual molecules appears as a radial pattern with sixfold symmetry. The handedness evident in the average is attributed to nonuniformity of the negative stain.     

Dimensions of active cytochrome c oxidase in reconstituted liposomes using a gold ball shadow width standard: a freeze-etch electron microscopy study

The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported. Freeze-etched cytochrome oxidase vesicles and gold ball calibration grids were simultaneously shadowed with platinum/carbon. The shadow width distribution of the cytochrome oxidase located in and spanning the membrane was measured. The membrane fracture face edge and cross-fractured bilayer membrane edge were also measured. Dimensions of the cytochrome oxidase were found to be 5·8 ± 0·3 nm in diameter parallel to the membrane and 8·2 ± 0·3 nm long across the membrane. The bilayer membrane dimensions were 3·0 ± 0·3 nm for the half bilayer and 5·8 ± 0·3 nm for the cross-fractured bilayer membrane edge thickness. The length of the cytochrome oxidase was observed to span the bilayer membrane. Previous X-ray diffraction measurements on similar hydrated liquid crystalline artificial membranes were found to be in good agreement with the freeze-etched results. Membrane widths from thin-sectioned cytochrome oxidase vesicles were measured and found to be 5·8–5·9 nm in non-post-stained sections. Post-staining with uranyl acetate and/or lead citrate was shown to increase this average thickness. The technique of freeze-etching electron microscopy in conjunction with the gold ball shadow width calibration experiment has been shown to provide accurate and precise measurements of membranes and a functional intramembrane protein in a hydrated non-crystalline sample.     

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: The utility of methylamine tungstate stain and butvar support film in the study of macromolecules by transmission electron microscopy

The structure of ornithine decarboxylase (M_r ≈? 1.04 × 10⁶) from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted ± 45°. The two edge views were related by a 30° rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-Å resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10° with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.     

An image reversal process for electron microscope film

An improved technique has been developed to produce a positive image directly on some standard electron microscope films (Kodak 4489, SO 163, and Ilford Electron Microscope Film). This technique has been useful for processing micrographs of freeze-etched and shadowed specimens, and eliminates the need for producing positive image transparencies before printing.     

Three-dimensional view of the chromatin in freeze-fractured chicken erythrocyte nuclei.

The freeze-fracture thaw-fix (FfTF) technique described in earlier papers is applied in the present work to more detailed study of the chicken erythrocyte, by transmission replicas and high resolution scanning electron microscopy (3 nm scan beam size). The three-dimensional structure of the chromatin, and possibly the non-histone protein matrix, of fractured nuclei is to a large extent retained in this method of preparation and seen in stereomicrographs. In these micrographs the helical sub-structure of the 25 nm chromatin strands can be seen at about the same resolution as that of previously published micrographs in which extracted chromatin is viewed by negative contrast or after metal shadowing. The useful resolution of the secondary electron micrographs, for a suitably mounted specimen, is shown to be as good as that of transmission micrographs of platinum-carbon replicas of the same material.     

Electron microscopy of the poly-l-lysine α-helix

Dark field electron micrographs of polylysine molecules in the α-helical form were analyzed by digital Fourier techniques to determine the extent to which the helix was still represented in the images. A total of 50 molecular images were selected following defined criteria, 13 from spreads of molecules from aqueous solution and 37 from methanol solution. As controls, similar shapes were chosen from the random structure seen in the carbon specimen support without polylysine. Computer analysis of the 50 molecular images indicated the presence of a helical structure with a pitch of 0.54±0.01 nm and a pitch angle of 25.6°±1.5° (standard error) in over 70% of the images. Moreover, 63% of the total α-helix measured by circular dichroism for the molecules in solution was still represented in the micrographs. The controls had no helical characteristics.     

A microcomputer program in “BASIC” for the accurate determination of the height of small particles

A formula is derived to enable the calculation of the true height of an object, such as a shadowed latex bead, from electron micrographs. Knowing only the angle of shadowing and the length of the evaporated shadow, and by substituting these values in the derived formula, a microcomputer may be programmed to carry out the necessary computations. An example of such a microcomputer program is given. The correct determination of the height of particles by electron microscopy using the shadowing technique is one of the most accurate methods available for the determination of small particle height.     

High resolution analysis of paired helical filaments in Alzheimer's disease

Results of this comparative study indicated that electron microscopy of supra-ultrathin sections provided the best resolution, revealing the subunit structures, globules (32 Å ± 4), and longitudinal bars (47 Å ± 6) of paired helical filaments (PHF). A three-dimensional model of PHF substructure is therefore presented. The image of brightfield electron micrographs of isolated PHF positively stained with 2% phosphotungstic acid (PTA) also provided high resolution and revealed the presence of the beaded structures of these heavily stained filaments in the crossing-over region of PHF. Noisy background of the negatively stained preparation of isolated PHF substantially reduced the resolution. Severe flattened morphology of this preparation further complicated the interpretation of image analysis. Darkfield electron micrographs offered high-image contrast with low noise in the background clearly demonstrating the ropelike twisting configuration of PHF, but did not reveal the substructures of PHF. The tilting (±45°) analysis of these PHF prepared by uranyl acetate staining and rotary-shadowing and unidirectional shadowing methods with platinum showed that these heavy metals were deposited on the top filaments (away from the carbon film on the grid) in the crossing-over region of PHF.     

Low cost internegatives from electron micrographs of metal shadowed objects

Some observers prefer a contrast reversal of electron micrographs of metal shadowed objects, so that the metal-free shadows appear dark on the print. There are several ways of obtaining contrast reversal, and the reasons for using 35 mm internegatives are given and a setup for re-photography of electron microscopy films is described together with a modified enlarger. Both are equipped with nearpoint illumination, which can be diffused in a simple manner with minimal loss of intensity. Other features are a particularly rigid camera stand combined with a numbering device, and a glass- and scratch-free negative holder for the enlarger. The choice of lens for the re-photography is discussed.     

Photoelectron microscopy of cell surfaces

Photoelectron micrographs of fixed, unstained, uncoated chicken embryo fibroblasts and absolute photoelectron quantum yields in the 180-230 nm wavelength band of L-fucose, D-galactose, D-glucose, N-acetyl-D-glucosamine, and the sucrose polymer Ficoll are reported. The quantum yields of the saccharides are low compared to the reference dye, phthalocyanine, and fall in the same range as those previously measured for amino acids and membrane phospholipids. Photoelectron micrographs of the unstained and uncoated cells inhibit considerable surface detail. The photoelectron quantum yield data and the micrographs indicate that surface relief is the dominant source of contrast.     

Electron microscopy of cellulose in entire tissue

Microfibrillar structures can be seen in ultrathin sections of cell walls which have been post-stained with uranyl acetate and lead citrate. This stain is removable by washing, suggesting that a physical mechanism is involved. It is suggested that these structures are the cellulose microfibrils of the wall; they appear to be 3.5 nm in diameter and not fasciated into larger units. Freeze-etching of untreated tissue supports this conclusion. These two techniques seem to have many advantages over methods previously used for the study of microfibril arrangement and structure.