Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology

Berger, Casper; Premaraj, Navya; Ravelli, Raimond B. G.; Knoops, Kèvin; López‐Iglesias, Carmen; Peters, Peter J.

doi:10.1038/s41592-023-01783-5

Cited by 56 publications

(23 citation statements)

References 154 publications

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“…The potential of cryo-ET in studying intricate biological systems is further enhanced when combined with synergistic advancements such as focused ion beam (FIB) milling and correlative light and electron microscopy (CLEM). , FIB milling facilitates thinning of dense biological samples like cells or tissue slices, thereby preserving their near-native state for detailed visualization using cryo-ET. On the other hand, CLEM represents the convergence of live-cell imaging capabilities of light microscopy with the high-resolution structural analysis of EM.…”

Section: Cryo-electron Microscopy (Cryo-em)-based Technologymentioning

confidence: 99%

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

Bai,

Zhang,

Dong

et al. 2023

Chem. Rev.

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Protein misfolding and aggregation, a key contributor to the progression of numerous neurodegenerative diseases, results in functional deficiencies and the creation of harmful intermediates. Detailed visualization of this misfolding process is of paramount importance for improving our understanding of disease mechanisms and for the development of potential therapeutic strategies. While in vitro studies using purified proteins have been instrumental in delivering significant insights into protein misfolding, the behavior of these proteins in the complex milieu of living cells often diverges significantly from such simplified environments. Biomedical imaging performed in cell provides cellular-level information with high physiological and pathological relevance, often surpassing the depth of information attainable through in vitro methods. This review highlights a variety of methodologies used to scrutinize protein misfolding within biological systems. This includes optical-based methods, strategies leaning on mass spectrometry, in-cell nuclear magnetic resonance, and cryo-electron microscopy. Recent advancements in these techniques have notably deepened our understanding of protein misfolding processes and the features of the resulting misfolded species within living cells. The progression in these fields promises to catalyze further breakthroughs in our comprehension of neurodegenerative disease mechanisms and potential therapeutic interventions.

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Section: Cryo-electron Microscopy (Cryo-em)-based Technologymentioning

confidence: 99%

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

Bai,

Zhang,

Dong

et al. 2023

Chem. Rev.

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“…During his time at Maastricht University, Raimond elucidated the structure of beta-amyloid fibrils (Gremer et al, 2017), and was part of the team who showed that SARS-CoV-2 can productively infect human gut enterocytes (Lamers et al, 2020), both studies being published in Science. Raimond also pursued tomographic methods (Berger et al, 2023), putting to use his earlier experience on ID14-4 to image crystals (Brockhauser et al, 2008), and he continued his radiation damage investigations particularly on the effects of dose in single-particle cryo-EM (Zhang et al, 2021).…”

Section: Obituarymentioning

confidence: 99%

Raimond B. G. Ravelli (25 March 1968–30 June 2023)

Garman

2023

Acta Cryst Sect D Struct Biol

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in a beautiful and full chapel in Maastricht, the Netherlands, we remembered and bade farewell to a much-loved colleague and friend, Raimond Ravelli, who died on 30 June after a sudden unexpected 10-week illness. Raimond was a highly creative structural biologist who contributed hugely to the field. The bare facts of Raimond's far too short 55-year life give no indication of the profound impact of his innovations in X-ray crystallography and electron microscopy methodology, and make it easy to underestimate his own seminal contributions in answering numerous scientific questions in structural biology. More importantly though, facts cannot tell of the profound inspiration he gave to those fortunate to be his colleagues, collaborators and mentees.Raimond was born in De Bilt, near Utrecht, the youngest of three children. His nickname was 'Kikvors' ('frog') because he was always ready to jump and to surprise. He loved to read and build with LEGO (a fascination he retained all his life), but at school it soon became clear how intelligent he was. His scientific talent and passion surfaced as a teenager and at the age of 18 he participated in the finals of the Netherlands National Chemistry Olympiad. He performed best under pressure and in fact he later seemed to seek that pressure in his research life too. He loved to perform experiments, so it was no surprise to his family when he decided to study chemistry at the University of Utrecht, graduating in 1992 despite his extensive commitment to his rowing crew. Raimond stayed in Utrecht for his PhD research under the supervision of Henk Krabbendam and Jan Kroon, working on X-ray data-collection techniques, particularly Laue methodology

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“…Although many biological samples are thin enough to be imaged directly (10)(11)(12), other samples, most notably eukaryotic cells, need to be thinned prior to imaging (13). Currently, cryo-focused-ion beam milling (cryo-FIB milling) is the thinning method of choice, as it lacks several severe artifacts associated with cryo-sectioning (14,15). During cryo-FIB milling, material from the sample is ablated by focused ions, until a thin (up to ~300 nm) slice remains, which can subsequently be subjected to tomographic acquisition (16).…”

Section: Introductionmentioning

confidence: 99%

Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging

Tuijtel,

Cruz-León,

Kreysing

et al. 2024

Sci. Adv.

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Cryo–electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo–focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.

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Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology

Cited by 56 publications

References 154 publications

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

Raimond B. G. Ravelli (25 March 1968–30 June 2023)

Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging

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