Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate

Krishnan, M.; Nejadasl, Fatemeh Karimi; Vulović, Miloš; Koster, Abraham J.; Ravelli, Raimond B. G.

doi:10.1107/s090904951100820x

Cited by 91 publications

(76 citation statements)

References 95 publications

(122 reference statements)

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“…But this deposited energy suffices to break ionic and covalent bonds (radiolysis), since their binding energy is in the mentioned range, and also to heat the sample, if the dose rate exceeds its heat dissipation. Radiolysis of especially organic compounds leads ultimately to the formation of hydrogen-gas filled cavities (Leapman and Sun, 1995) that appear as "bubbling" effect in the TEM and locally destroy and deform the sample (Karuppasamy et al, 2011;Meents et al, 2010). Heating of the sample due to a high electron dose rate (~50 e Å -2 s -1 ) can cause ice crystallization (Karuppasamy et al, 2011).…”

Section: Dose-limitationmentioning

confidence: 99%

In situ architecture and cellular interactions of PolyQ inclusions

Bäuerlein¹

2018

Preprint

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Inclusions of misfolded proteins are found in the brain of patients of many neurodegenerative diseases, but the contribution of these aggregates to cytotoxicity remains poorly understood. In this PhD thesis, cryo-electron tomography is employed to analyze at unprecedented resolution the architecture of neuronal polyQ inclusions within their cellular environment. Cytoplasmic polyQ inclusions contain fibrils that strongly interact with cellular endomembranes, particularly those of the ER. These interactions alter ER organization and dynamics, and may even lead to ER membrane rupture. This preview image shows how the polyQ fibrils (cyan) of an inclusion body at the vicinity of the nucleus (grey) interact with the ER membranes (red). Vesicles (white) are abundant both within the inclusion and at its periphery.

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Section: Dose-limitationmentioning

confidence: 99%

In situ architecture and cellular interactions of PolyQ inclusions

Bäuerlein¹

2018

Preprint

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“…In addition, while the angle before PSS inflow between the two obtuse steps (

[\bar{4} 41]

and

[48 \bar{1}]

) had a value of ≈103° (vs theoretical value of 101.9°, see ref. ), after its introduction, rounding of the obtuse steps led to a progressive increase in this angle to produce pseudo‐[001] steps, again resulting in protrusions toward the facet boundaries.…”

Section: Resultsmentioning

confidence: 96%

A Mesocrystal‐Like Morphology Formed by Classical Polymer‐Mediated Crystal Growth

Smeets

Cho

Sommerdijk

et al. 2017

Adv Funct Materials

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Growth by oriented assembly of nanoparticles is a widely reported phenomenon for many crystal systems. While often deduced through morphological analyses, direct evidence for this assembly behavior is limited and, in the calcium carbonate (CaCO 3 ) system, has recently been disputed. However, in the absence of a particle-based pathway, the mechanism responsible for the creation of the striking morphologies that appear to consist of subparticles is unclear. Therefore, in situ atomic force microscopy is used to investigate the growth of calcite crystals in solutions containing a polymer additive known for its ability to generate crystal morphologies associated with mesocrystal formation. It is shown that classical growth processes that begin with impurity pinning of atomic steps, leading to stabilization of new step directions, creation of pseudo-facets, and extreme surface roughening, can produce a microscale morphology previously attributed to nonclassical processes of crystal growth by particle assembly.

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“…The role of electron beam damage has been well characterized for cryo-EM, where a relationship between cumulative electron flux and loss of structure has been described in detail (28)(29)(30)(31). Despite this knowledge it remains unknown how a loss of structure impacts the functionality of biomolecules, whether the catalytic mechanism of an enzyme, the protein coding function of an mRNA, or the transport, recognition, and binding of a transcription factor.…”

Section: Comparison Of Damage Between Cryo-em and Liquid-emmentioning

confidence: 99%

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

Moser

Shokuhfar

Evans

2018

Preprint

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Transmission electron microscopy of whole cells is hindered by the inherently large thickness and low atomic contrast intrinsic of cellular material. Liquid cell transmission electron microscopy allows samples to remain in their native hydrated state and may permit visualizing cellular dynamics in-situ. However, imaging biological cells with this approach remains challenging and identifying an optimal imaging regime using empirical data would help foster new advancements in the field. Recent questions about the role of the electron beam inducing morphological changes or damaging cellular structure and function necessitates further investigation of electron beam-cell interactions, but is complicated by variability in imaging techniques used across various studies currently present in literature. The necessity for using low electron fluxes for imaging biological samples requires finding an imaging strategy which produces the strongest contrast and signal to noise ratio for the electron flux used. Here, we experimentally measure and evaluate signal to noise ratios and damage mechanisms between liquid and cryogenic samples for cells using multiple electron imaging modalities all on the same instrument and with equivalent beam parameters to standardize the comparison. We also discuss considerations for optimal electron microscopy imaging conditions for future studies on whole cells within liquid environments.

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Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate

Abstract: The effects of dose and dose-rate were investigated for single-particle cryo-electron microscopy using stroboscopic data collection. A dose-rate effect was observed favoring lower flux densities.

Cited by 91 publications

References 95 publications

In situ architecture and cellular interactions of PolyQ inclusions

In situ architecture and cellular interactions of PolyQ inclusions

A Mesocrystal‐Like Morphology Formed by Classical Polymer‐Mediated Crystal Growth

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

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