Low Temperature Electron Microscopy

Dubochet, Jacques; Booy, Frank P.; Freeman, Robert R.; Jones, and A V; Walter, C.A.

doi:10.1146/annurev.bb.10.060181.001025

Cited by 55 publications

(34 citation statements)

References 33 publications

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“…Their ultimate success has been described in detail in two very important review articles (Dubochet et al, 1981;Dubochet et al, 1988). Probably the article with the most impact, however, is the one that appeared in Nature (Adrian et al, 1984), which paved the way for the explosion of icosahedral virus structures obtained by cryoEM.…”

Section: Vitrified Frozen Hydrated Specimensmentioning

confidence: 99%

Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Taylor

Glaeser

2008

Journal of Structural Biology

154

119

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Methods for preserving specimen hydration in protein crystals were pursued in the early 1970s as a prerequisite for protein crystallography using an electron microscope. Three laboratories approached this question from very different directions. One built a differentially pumped hydration chamber that could maintain the crystal in a liquid water environment, a second maintained hydration by rapidly freezing the protein crystal and examining it in a cold stage, and the third replaced the water of hydration by using glucose in the same way as one had previously used "negative stains". Each of these early efforts succeeded in preserving the structures of protein crystals at high resolution within the vacuum of the electron microscope, as demonstrated by electron diffraction patterns. The next breakthrough came in the early 1980s when a technique was devised to preserve noncrystalline specimens by freezing them within vitreous ice. Since then, with the development of high stability cold stages and transfer mechanisms compatible with many instrument platforms, and by using commercially provided low-dose imaging techniques to avoid radiation damage, there has been an explosion of applications. These now include single particles, helical filaments, 2-D arrays and even whole cells, where the most exciting recent applications involve cryoelectron tomography. These achievements and possibilities generate a new set of research opportunities associated with increasing the reliability and throughput with which specimens can be studied by cryoEM.

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Section: Vitrified Frozen Hydrated Specimensmentioning

confidence: 99%

Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Taylor

Glaeser

2008

Journal of Structural Biology

154

119

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“…An effective way of decreasing radiation damage is to cool samples to temperatures around 100 K (15)(16)(17)(18)(19)(20), which is routinely done in x-ray crystallography and electron microscopy. At these temperatures, samples can tolerate a significantly higher dose of ionizing radiation, but eventually they show comparable signs of damage as observed at room temperature.…”

mentioning

confidence: 99%

Origin and temperature dependence of radiation damage in biological samples at cryogenic temperatures

Meents

Gutmann

Wagner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

184

153

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Radiation damage is the major impediment for obtaining structural information from biological samples by using ionizing radiation such as x-rays or electrons. The knowledge of underlying processes especially at cryogenic temperatures is still fragmentary, and a consistent mechanism has not been found yet. By using a combination of single-crystal x-ray diffraction, small-angle scattering, and qualitative and quantitative radiolysis experiments, we show that hydrogen gas, formed inside the sample during irradiation, rather than intramolecular bond cleavage between non-hydrogen atoms, is mainly responsible for the loss of high-resolution information and contrast in diffraction experiments and microscopy. The experiments that are presented in this paper cover a temperature range between 5 and 160 K and reveal that the commonly used temperature in x-ray crystallography of 100 K is not optimal in terms of minimizing radiation damage and thereby increasing the structural information obtainable in a single experiment. At 50 K, specific radiation damage to disulfide bridges is reduced by a factor of 4 compared to 100 K, and samples can tolerate a factor of 2.6 and 3.9 higher dose, as judged by the increase of R free values of elastase and cubic insulin crystals, respectively.

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“…Après sa thèse en Biophysique effectuée à Genève avec Eduard Kellenberger et soutenue en 1973, il dépose en 1978 un projet pour devenir responsable d'un groupe de recherche, au Laboratoire européen de biologie moléculaire (EMBL), à Heidelberg. Ce projet visait à utiliser le froid afin de pouvoir appliquer la microscopie électronique structurale au vivant et ainsi contourner le problème de l'observation d'objets hydratés dans le [6] 1990 : Apparition des microscopes FEG (Field Emission Gun) [31] 2008 : Apparition de caméras nouvelle génération [32,33] 1976 : Congélation d'échantillons hydratés [28] 1981 : Naissance de la cryo-microscopie [12,29,30] …”

Section: Jacques Dubochetunclassified

Prix Nobel de Chimie 2017 : Jacques Dubochet, Joachim Frank et Richard Henderson

et al. 2017

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Cette méthode pouvait paraître comme complètement impensable il y a quelques décennies. En effet, comment un échantillon biologique peut-il être observé, sous vide et sous un faisceau d'électrons, sans être instantanément détruit ? Pour trouver une explication et des solutions, revenons à l'origine de la microscopie. Les premiers microscopes furent des microscopes optiques, inventés en Hollande au XVII e siècle. Ils évoluèrent sans cesse au cours des siècles, permettant d'apporter de nombreuses connaissances dans divers domaines, allant de la physique à la biologie. Le pouvoir de réso-lution d'un microscope, ou sa capacité à discerner deux points adjacents, est lié à la longueur d'onde de la source de lumière (la résolution étant d'environ /2, c'est-à-dire longueur d'onde divisée par 2). Pour un microscope optique « classique », sa résolution est limitée à 0,2 μm. Afin d'étudier des objets de taille inférieure, il est donc nécessaire d'utiliser des ondes qui peuvent atteindre des longueurs d'onde suffisamment faibles pour pouvoir observer des objets de la taille des atomes, comme par exemple les électrons ( ~ 10 -3 nm). Ce n'est qu'au début des années 1930, qu'Ernst Ruska et Max Knoll [5] ont pu mettre au point un microscope « électronique ». Ils ont ainsi été à l'origine des premières images ayant une résolution de quelques dizaines de nanomètres. Différents types d'instruments ont ensuite été développés permettant d'observer, par exemple, les virus jusqu'alors invisibles : des microscopes électroniques à balayage en réflexion, qui permettent l'analyse de la surface d'échantillons épais, et des microscopes à transmission capables (en particulier) d'analyses à très haute résolution d'échan-tillons suffisamment minces pour que les électrons transmis puissent les traverser. L'observation par un microscope électronique repose sur une double contrainte : l'échantillon doit supporter le vide présent au sein de la colonne de transmission, et qui est nécessaire pour éviter

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Low Temperature Electron Microscopy

Cited by 55 publications

References 33 publications

Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Origin and temperature dependence of radiation damage in biological samples at cryogenic temperatures

Prix Nobel de Chimie 2017 : Jacques Dubochet, Joachim Frank et Richard Henderson

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