Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples

McDowall, Alasdair W.; Chang, Jianhong; Freeman, Robert R.; Lepault, Jean; Walter, C.A.; Dubochet, Jacques

doi:10.1111/j.1365-2818.1983.tb04225.x

Cited by 270 publications

(138 citation statements)

References 29 publications

(20 reference statements)

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“…Cryoultramicrotomy (8)(9)(10) is currently the method of choice for producing thin (50-200 nm) frozen-hydrated sections of cells and tissues. In spite of considerable efforts to develop cryosectioning into a routine technique, it remains a challenge to its practitioners to prepare such sections and to deposit them on EM grids with good thermal and electrical contact.…”

mentioning

confidence: 99%

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

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

389

306

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Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

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mentioning

confidence: 99%

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

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

389

306

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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.

182

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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“…Samples are cryo-immobilized by rapid freezing, which vitrifies the solution and avoids the formation of ice crystals that can damage the biological structure (McDowall et al, 1983;Dubochet et al, 1988). However, specimens that cannot be snap-frozen directly to an electron transparent thickness, such as whole cells, must be thinned before analysis in the cryo-TEM.…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Preparation of Intact Solid–Liquid Interfaces by Label-FreeIn SituLocalization and Cryo-Focused Ion Beam Lift-Out

et al. 2016

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Scanning transmission electron microscopy (STEM) allows atomic scale characterization of solid-solid interfaces, but has seen limited applications to solid-liquid interfaces due to the volatility of liquids in the microscope vacuum. Although cryo-electron microscopy is routinely used to characterize hydrated samples stabilized by rapid freezing, sample thinning is required to access the internal interfaces of thicker specimens. Here, we adapt cryo-focused ion beam (FIB) "lift-out," a technique recently developed for biological specimens, to prepare intact internal solid-liquid interfaces for high-resolution structural and chemical analysis by cryo-STEM. To guide the milling process we introduce a label-free in situ method of localizing subsurface structures in suitable materials by energy dispersive X-ray spectroscopy (EDX). Monte Carlo simulations are performed to evaluate the depth-probing capability of the technique, and show good qualitative agreement with experiment. We also detail procedures to produce homogeneously thin lamellae, which enable nanoscale structural, elemental, and chemical analysis of intact solid-liquid interfaces by analytical cryo-STEM. This work demonstrates the potential of cryo-FIB lift-out and cryo-STEM for understanding physical and chemical processes at solid-liquid interfaces.

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Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples

Cited by 270 publications

References 29 publications

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

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

Site-Specific Preparation of Intact Solid–Liquid Interfaces by Label-FreeIn SituLocalization and Cryo-Focused Ion Beam Lift-Out

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