© Nature Publishing Group1984
© Nature Publishing Group1984
© Nature Publishing Group1984
© Nature Publishing Group1984
© Nature Publishing Group1984
Cryo-electron microscopy of viruses
TL;DR: Cryo-electron microscopy of vitrified specimens offers possibilities for high resolution observations that compare favourably with any other electron microscopical method.
Abstract: Thin vitrified layers of unfixed, unstained and unsupported virus suspensions can be prepared for observation by cryo-electron microscopy in easily controlled conditions. The viral particles appear free from the kind of damage caused by dehydration, freezing or adsorption to a support that is encountered in preparing biological samples for conventional electron microscopy. Cryo-electron microscopy of vitrified specimens offers possibilities for high resolution observations that compare favourably with any other electron microscopical method.
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TL;DR: Water is the most abundant component of biological material, but it is systematically excluded from conventional electron microscopy, because water evaporates rapidly under the vacuum conditions of an electron microscope.
Abstract: Water is the most abundant component of biological material, but it is systematically excluded from conventional electron microscopy. This is because water evaporates rapidly under the vacuum conditions of an electron microscope. Cryoelectron microscopy has long been seen as a possible avenue to overcome this limitation, but until recently the direct observation of frozen-hydrated specimens was relatively unsuccessful because of a number of serious difficulties. These were, in particular, due to the absence of a good cryospecimen holder, the inherently low contrast of hydrated specimens and the structural damage due to ice crystals formed during freezing. As a consequence, the cryomethods which have flourished in electron microscopy during the last 20 years were not aimed at preserving the hydration of the specimen in the electron microscope. Freezing was only used as an aid to preparation. The objects ultimately observed in the electron microscope were dry and at room temperature. Such cryomethods have recently been reviewed in detail (Robards and Sleytr 1985).
2,137 citations
1,828 citations
TL;DR: The design, synthesis and characterization of a new class of organic nanotubes based on rationally designed cyclic polypeptides are reported, which may have possible applications in inclusion chemistry, catalysis, molecular electronics and molecular separation technology.
Abstract: Hollow tubular structures of molecular dimensions may offer a variety of applications in chemistry, biochemistry and materials science. Concentric carbon nanotubes have attracted a great deal of attention, while the three-dimensional tubular pore structures of molecular sieves have long been exploited industrially. Nanoscale tubes based on organic materials have also been reported previously. Here we report the design, synthesis and characterization of a new class of organic nanotubes based on rationally designed cyclic polypeptides. When protonated, these compounds crystallize into tubular structures hundreds of nanometres long, with internal diameters of 7-8 A. Support for the proposed tubular structures is provided by electron microscopy, electron diffraction, Fourier-transform infrared spectroscopy and molecular modelling. These tubes are open-ended, with uniform shape and internal diameter. We anticipate that they may have possible applications in inclusion chemistry, catalysis, molecular electronics and molecular separation technology.
1,508 citations
TL;DR: The theoretical behaviour of the FSC in conjunction with the various factors which influence it are discussed: the number of "voxels" in a given Fourier shell, the symmetry of theructure, and the size of the structure within the reconstruction volume.
764 citations
TL;DR: The recent advances in electron detection and image processing are reviewed and the exciting new opportunities that they offer to structural biology research are illustrated.
738 citations
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TL;DR: The structural and thermal properties of pure frozen water important for electron microscopy are summarized in an appendix as discussed by the authors, and a possible model for the mechanism of beam damage in aqueous solutions is proposed.
Abstract: SUMMARY
Thin layers of pure water or aqueous solutions are frozen in the vitreous state or with the water phase in the form of hexagonal or cubic crystals, either by using a spray-freezing method or by spreading the liquid on alkylamine treated films. The specimens are observed in a conventional and in a scanning transmission electron microscope at temperatures down to 25 K. In general, the formation of crystals and segregation of solutes during freezing, devitrification and evaporation upon warming, take place as foreseen by previous X-ray, thermal, optical and electron microscopical studies. Electron beam damage appears in three forms. The devitrification of vitreous ice. The slow loss of material for the specimen at a rate of about one molecule of pure water for every sixty electrons. The bubbling in solutions of organic material for doses in the range of thousands of e nm−2. We propose a possible model for the mechanism of beam damage in aqueous solutions.
The structural and thermal properties of pure frozen water important for electron microscopy are summarized in an appendix.
437 citations
TL;DR: High-resolution electron diffraction patterns have been obtained from frozen, hydrated catalase crystals to demonstrate the feasibility of using a frozen specimen hydration technique.
Abstract: High-resolution electron diffraction patterns have been obtained from frozen, hydrated catalase crystals to demonstrate the feasibility of using a frozen specimen hydration technique. The use of frozen specimens to maintain the hydration of complex biological structures has certain advantages over previously developed liquid hydration techniques.
400 citations
TL;DR: In this article, it was shown that macroscopic parts of samples of pure liquid water and of dilute aqueous solutions can be vitrified completely by jet-freezing of micrometre-sized aqous droplets distributed in n -heptane as an emulsion.
Abstract: Pure water can only be vitrified by the very slow condensation of vapour on a metal surface maintained at very low temperatures1,2. Attempts to form vitreous ice by rapid cooling of liquid water invariably lead to formation of ice Ih (ref. 3). (Pryde and Jones4 did report a heat capacity change of rapidly cooled water at 126 K which they attributed to a glass transition, but could not reproduce this result in subsequent experiments.) Dilute aqueous solutions in contrast to concentrated aqueous solutions5 behave similarity to water and separate during freezing, even with the highest cooling rates available, into pure ice and concentrated solute6. We report here that macroscopic parts of samples of pure liquid water and of dilute aqueous solutions can be vitrified completely by jet-freezing of micrometre-sized aqueous droplets distributed in n -heptane as an emulsion—the resulting supercooling effect of ∼40 K being essential for vitrification7.
366 citations
356 citations
TL;DR: In this paper, the Fourier transform of the image of a thin crystal of catalase, which has discrete diffraction maxima in the resolution range of 10 to 2.5 nm, as a function of defocusing, was determined by finding the relative contributions from phase and amplitude contrast.
Abstract: The effects of defocusing and spherical aberration in the electron microscope image are most simply and directly displayed in the Fourier transform of the image. We have investigated the process of image formation by determining the changes in the transform of the image of a thin crystal of catalase, which has discrete diffraction maxima in the resolution range of 10 to 2.5 nm, as a function of defocusing. The changes in amplitude and phase of these diffraction maxima have been measured and compared with the predictions of a first-order theory of image formation. The theory is generally confirmed, and the transfer function of the microscope is completely determined by finding the relative contributions from phase and amplitude contrast. A 'true' maximum contrast image of the catalase crystal, compensated for the effects of defocusing, is reconstructed from the set of micrographs in the focal series. The relation of this compensated image to individual underfocused micrographs, and the use of underfocus contrast enhancement in conventional electron microscopy, are discussed. This approach and the experimental methods can be extended to high resolution in order to compensate for spherical aberration as well as defocusing. In as much as spherical aberration is the factor presently limiting the resolution of electron lenses, this could provide a considerable extension of the resolution of the electron microscope.
339 citations