High resolution dark‐field electron microscopy

Dubochet, Jacques

doi:10.1111/j.1365-2818.1973.tb03837.x

Cited by 18 publications

(6 citation statements)

References 18 publications

Supporting

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“…While standard plastic supports (-300 A) already approgch or exceed this figure, drop films ( -60 A ) leave a considerable margin for specimen dimensions before the resolution limiting thickness is reached. Dark-field electron microscopy has advantages in high resolution since contrast is improved and phase effects are eliminated (Dubochet, 1975). However, this imaging mode is especially sensitive to mass thickness.…”

Section: Volume Of Drop X Concentration Of Solution = Mass Of Formvarmentioning

confidence: 99%

Ultrathin formvar support films for transmission electron microscopy

Davison

Colquhoun

1985

J. Elec. Microsc. Tech.

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We have found that ultrathin Formvar films are easily and reliably made at an air-water interface by the drop method. By varying the concentration of Formvar in the drop, films of different characteristics can be obtained. Concentrations of 0.25-0.4% in ethylene dichloride produce extremely flat, ultrathin, and stable films that are especially suited for shadowed and negatively stained preparations. Low concentrations ( < 0.1%) produce nets consisting of many tiny holes which, after carbon stabilization, are ideal for supporting high-resolution samples. Above 0.5%, films made by the drop method develop bubbles, and this bubble defect makes them unsuitable for section support.For section support, Formvar films made by the stripping method off mica are far superior to those made off glass. The films are more uniform in surface contour and thickness. They are less readily attacked by alcohols. Consequently, they are more resistant to staining procedures involving organic solvents and continue to be strong and uniform for section support.

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Section: Volume Of Drop X Concentration Of Solution = Mass Of Formvarmentioning

confidence: 99%

Ultrathin formvar support films for transmission electron microscopy

Davison

Colquhoun

1985

J. Elec. Microsc. Tech.

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“…Lattice distortions were recognized by examination of the micrographs or their optical diffraction patterns. Measurements of cell dimensions on 29 specimens all recorded at x 45,000, which were judged to be ] Others have shown that a weak beam somehow stabilizes specimens (1,7,17). Nevertheless, the stain distribution is still sensitive to the effects of further irradiation (3).…”

Section: Hexagonal Lattice Symmetrymentioning

confidence: 99%

Gap junction structures. IV. Asymmetric features revealed by low-irradiation microscopy.

Baker¹,

Caspar²,

Hollingshead³

et al. 1983

The Journal of Cell Biology

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Micrographs of mouse liver gap junctions, isolated with detergents, and negatively stained with uranyl acetate, have been recorded by low-irradiation methods. Our Fourieraveraged micrographs of the hexagonal junction lattice show skewed, hexameric connexons with less stain at the threefold axis than at the six indentations between the lobes of the connexon image. These substructural features, not clearly observed previously, are acutely sensitive to irradiation. After an electron dose less than that normally used in microscopy, the image is converted to the familiar doughnut shape, with a darkly stained center and a smooth hexagonal outline, oriented with mirror symmetry in the lattice. Differences in appearance among 25 reconstructed images from our low-irradiation micrographs illustrate variation in staining of the connexon channel and the space between connexons. Consistently observed stain concentration at six symmetrically related sites ~34 tk from the connexon center, 8 ° to the right or left of the (1, 1) lattice vector may reveal an intrinsic asymmetric feature of the junction structure. The unexpected skewing of the six-lobed connexon image suggests that the pair of hexagonal membrane arrays that form the junction may not be structurally identical. Because the projected image of the connexon pair itself appears mirror symmetric, each pair may consist of two identical connexon hexamers related by local (noncrystallographic) twofold axes in the junctional plane at the middle of the gap. All connexons may be chemically identical, but their packing in the hexagonal arrays on the two sides of the junction appears to be nonequivalent.Gap junctions in liver (13, 28) and many other tissues (5, 10) are built of connexon units (6) hexagonally arrayed with crystaUike regularity in the pair of connected cell membranes. Makowski et al. (21) proposed a model for the junction structure based on data from x-ray diffraction, electron microscopy and chemical analysis in which the connexon units were pictured as symmetric hexamers of identical connexin molecules that are equivalently paired across the gap with dihedral symmetry in the two-sided hexagonal plane group (p622; see reference 16). This model predicts that, as normally viewed in electron micrographs of untilted, negatively stained specimens, the junction lattice should appear mirror-symmetric in projection (with p6m plane group symmetry).Conventional high-irradiation micrographs of negatively stained specimens show doughnut-shaped connexons that appear nearly circularly symmetric in the Fourier-averaged images, and, therefore, the hexagonal lattice array, appear to have mirror symmetry (6). Higher resolution, hexagonally averaged reconstructions calculated by Zampighi and Unwin (33) from minimal irradiation micrographs of two forms of gap junctions, negatively stained with uranyl acetate, showed hexagonal shaped connexons arrayed with approximate mirror symmetry in the projected lattice images. However, the hexagonally shaped connexon images occured in ...

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“…Small structures might have been lost due to masking effects of the heavy metal. The section might have collapsed during drying although a surrounding contrasting medium is generally supposed to act as a sustaining medium (Dubochet, 1973). Further, effects such as compression during sectioning and shrinkage during drying might have influenced the appearance of the structures in the section.…”

Section: Discussionmentioning

confidence: 99%

The myofibrillar M‐band in the cryo‐section—analysis of section thickness

Thornell

Sjöstróm

1975

Journal of Microscopy

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SUMMARY Information regarding the formation and the thickness of cryo‐sections is of importance for an adequate interpretation of cryo‐sectioned biological material. In this study we have taken advantage of the regular arrangement of filaments in myofibrils in an analysis of these matters. It is concluded that the sections are formed partly by fracturing in a way similar to that visualized in replicas made by the freeze‐fracturing and ‐etching procedure.

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High resolution dark‐field electron microscopy

Cited by 18 publications

References 18 publications

Ultrathin formvar support films for transmission electron microscopy

Ultrathin formvar support films for transmission electron microscopy

Gap junction structures. IV. Asymmetric features revealed by low-irradiation microscopy.

The myofibrillar M‐band in the cryo‐section—analysis of section thickness

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