Radiation Damage in Electron Cryomicroscopy

doi:10.1016/S0076-6879(10)81015-8

Methods in Enzymology

Volume 481, 2010, Pages 371-388

https://doi.org/10.1016/S0076-6879(10)81015-8 Get rights and content

Abstract

In an electron microscope, the electron beam used to determine the structures of biological tissues, cells, and molecules destroys the specimen as the image is acquired. This destruction occurs before a statistically well-defined image can be obtained and is consequently the fundamental limit to resolution in biological electron cryomicroscopy (cryo-EM). Damage from the destructive interaction of electrons with frozen-hydrated specimens occurs in three stages: primary damage, as electrons ionize the sample, break bonds, and produce secondary electrons and free radicals; secondary damage, as the secondary electrons and free radicals migrate through the specimen and cause further chemical reactions; and tertiary damage, as hydrogen gas is evolved within the sample, causing gross morphological changes to the specimen. The deleterious effects of radiation are minimized in cryo-EM by limiting the exposure of the specimen to incident electrons and cooling the sample to reduce secondary damage. This review emphasizes practical considerations for minimizing radiation damage, including measurement of electron exposure, estimation of absorbed doses of energy, selection of microscope voltage and specimen temperature, and selection of electron exposure to optimize images.

Section snippets

INTRODUCTION

In an electron microscope, electrons may interact with a specimen in one of two ways: electrons that scatter from the sample but retain their incident energy leave the structure unchanged; electrons that deposit some of their energy into the sample cause radiation damage and consequent structural changes. Contrast in bright field phase contrast electron microscopy (EM) comes primarily from the electrons that do not deposit energy into the specimen. If this type of scattering was the only type

MEASURING ELECTRON EXPOSURE

While discussing EM, many authors have used the words dose and exposure interchangeably. However, in more precise terminology, dose refers to the energy absorbed by the specimen while exposure indicates the amount of radiation incident on the specimen. Clearly, there is a relationship between the exposure of electrons to which a sample is subjected and the dose of energy absorbed. An experimentalist can perform an approximate conversion between these two quantities if the linear energy transfer

ENERGY ABSORBED BY SPECIMENS

The exposure of the sample to a flux of electrons is conveniently expressed in terms of electrons per Å² of specimen surface area (e⁻/Å²). The energy of the electrons is usually between 100 and 300 keV (1 eV = 1.602 × 10^− 19 J), although some microscopes employ accelerating voltages in the MV range. To convert from electron exposure to the dose of energy absorbed by the specimen, one must know the LET of incident electrons of a specific energy with a specific specimen. Glaeser has quoted values for the

RADIATION DAMAGE AND CHOICE OF ACCELERATING VOLTAGE

The probabilities of different types of interactions between an electron and a specimen are described by the cross-sections for each kind of interaction. Nondamaging interactions are always elastic events, where the kinetic energy of the incident electron is conserved. Only a small fraction of elastic events are destructive, causing the so-called “knock-on damage” that dislocates atoms from their chemical bonds (Glaeser et al., 2007). The vast majority of damaging interactions are inelastic

PRIMARY AND SECONDARY DAMAGE TO PROTEINS DURING IRRADIATION

At hundreds of keVs, the electron energies used for cryo-EM are significantly higher than the covalent bond energies in biological specimens, which are on the order of a few eVs. Most beam damage occurs due to electrons that lose between ~ 5 and ~ 100 eV during interaction with the specimen (on average ~ 20 eV) (Langmore and Smith, 1992). The deposited energy predominantly excites or ionizes the valence electrons that make up chemical bonds, breaking the bond and producing free radicals and causing

TERTIARY DAMAGE TO PROTEINS DURING IRRADIATION

In tertiary or global damage during irradiation, protein crystals and single particle EM or electron tomography samples become distorted, with bubbles becoming apparent in the extreme case. X-ray and EM analysis have shown that these distortions are coincident with the production of gas within the sample during irradiation. Bubbling of specimens in the electron beam (Dubochet et al., 1988) is due to the buildup of hydrogen gas in frozen-hydrated specimens (Leapman and Sun, 1995). Similarly,

CRYOPROTECTION AND OPTIMAL TEMPERATURES

Breaking of bonds by electrons occurs at any temperature. Therefore, cooling the specimen does not affect primary beam damage. However, it has long been known that cooling a specimen reduces the rate of fading of diffraction spots from 2D crystals (Hayward and Glaeser, 1979, Taylor and Glaeser, 1976). It is now thought that the mechanism of cryoprotection in both cryo-EM and X-ray crystallography is the mechanical restraint of molecular fragments by the ice matrix, preventing their movement so

QUANTIFICATION OF BEAM DAMAGE WITH INCREASING EXPOSURE

As has been recognized by many investigators, analysis of radiation damage in thin crystals offers a way of selecting optimal exposures for many types of samples by capturing the relevant effects of beam damage. Radiation damage of thin crystals results in a loss of diffraction intensity. This fading of the diffraction pattern does not automatically suggest destruction of the individual molecules that make up a crystal but instead could suggest that the crystallinity of the sample is destroyed.

OPTIMAL EXPOSURES FOR THIN CRYSTALS

For 2D crystals, the relationship between the exposure that maximizes the SNR at a resolution (i.e., the optimal exposure $N_{opt} (\vec{k})$ ) and the critical exposure, $N_{e} (\vec{k})$ , depends on whether data is collected by imaging or diffraction. In diffraction experiments, SNRs depend on the height of the intensity peak above the background noise. Therefore, the optimal exposure varies with the intensity of the spot. Spots with large ratios of peak height to background intensity have optimal exposures well

OPTIMAL EXPOSURES FOR SINGLE PARTICLE SAMPLES

For imaging of single particles, the SNR relationship derived by Hayward and Glaeser holds true. With these noncrystalline specimens, one must select the resolution at which the SNR is to be optimized in an image. While it may seem prudent to optimize the SNR at the highest resolution that the experiment aims to obtain, doing so may produce suboptimal SNRs at lower spatial frequencies needed to align particles so that images can be averaged coherently. Because critical exposures change rapidly

OPTIMAL EXPOSURES FOR TOMOGRAPHIC SAMPLES

For electron tomography, target resolutions are usually 20–60 Å, which is generally lower than for single particle EM and electron crystallography, and therefore higher electron exposures can be tolerated. The total electron exposure required for a given level of statistical significance at a given resolution in a 3D tomogram is the same as the exposure required for the same significance at the same frequency in a 2D image (Hegerl and Hoppe, 1976, McEwen et al., 1995). Therefore, the exposure

CONCLUDING COMMENTS

As described above, radiation damage imposes strict limitation on the electron exposure to which biological specimens can be subjected. Recent experiments have suggested that there is little advantage still to be gained from further optimization of specimen temperatures for the reduction of secondary damage. Radiation damage forces the experimentalist to work at conditions where every bit of extra signal in an image can improve a 3D model and every additional source of noise can reduce the

ACKNOWLEDGMENTS

We thank Robert Glaeser and Richard Henderson for many informative discussions and a critical reading of this chapter. L. A. B. was supported by a Vanier Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada and J. L. R. was supported by a New Investigator award from the Canadian Institutes of Health Research (CIHR). This work was funded by operating grant MOP 81294 from the CIHR.

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Citation Excerpt :
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Chapter Fifteen - Radiation Damage in Electron Cryomicroscopy

Abstract

Section snippets

INTRODUCTION

MEASURING ELECTRON EXPOSURE

ENERGY ABSORBED BY SPECIMENS

RADIATION DAMAGE AND CHOICE OF ACCELERATING VOLTAGE

PRIMARY AND SECONDARY DAMAGE TO PROTEINS DURING IRRADIATION

TERTIARY DAMAGE TO PROTEINS DURING IRRADIATION

CRYOPROTECTION AND OPTIMAL TEMPERATURES

QUANTIFICATION OF BEAM DAMAGE WITH INCREASING EXPOSURE

OPTIMAL EXPOSURES FOR THIN CRYSTALS

OPTIMAL EXPOSURES FOR SINGLE PARTICLE SAMPLES

OPTIMAL EXPOSURES FOR TOMOGRAPHIC SAMPLES

CONCLUDING COMMENTS

ACKNOWLEDGMENTS

J. Struct. Biol.

J. Struct. Biol.

J. Struct. Biol.

J. Struct. Biol.

J. Struct. Biol.

Ultramicroscopy

Curr. Opin. Struct. Biol.

J. Ultrastruct. Res.

Ultramicroscopy

Ultramicroscopy

Ultramicroscopy

Q. Rev. Biophys.

J. Struct. Biol.

Ultramicroscopy

Ultramicroscopy

Ultramicroscopy

Proc. Natl. Acad. Sci. USA