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• W4387376589 abstract "Cryo-EM imaging of vitreous samples is limited to a few hundred nanometers in thickness. Focused ion beams can mill windows into cells and tissues for imaging, but they damage biological samples. In this issue of Structure, Yang et al. (2023) quantitatively describe this damage and suggest ways to minimize it. Cryo-EM imaging of vitreous samples is limited to a few hundred nanometers in thickness. Focused ion beams can mill windows into cells and tissues for imaging, but they damage biological samples. In this issue of Structure, Yang et al. (2023) quantitatively describe this damage and suggest ways to minimize it. Cryo-electron tomography (cryo-ET) is able to provide unique insights into the molecular architecture of cells, reveal structures of macromolecules, and visualize their interactions in situ. However, due to the limited mean free path of electrons in vitreous ice (∼300 nm), most biological samples are too thick to be imaged by cryo-EM directly. Focused ion beam (FIB) milling tools have been developed in order to reduce the thickness of a wide range of samples to 100–200 nm,1Marko M. Hsieh C. Schalek R. Frank J. Mannella C. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy.Nat. Methods. 2007; 4: 215-217https://doi.org/10.1038/nmeth1014Crossref PubMed Scopus (364) Google Scholar,2Rigort A. Bäuerlein F.J.B. Leis A. Gruska M. Hoffmann C. Laugks T. Böhm U. Eibauer M. Gnaegi H. Baumeister W. Plitzko J.M. Micromachining tools and correlative approaches for cellular cryo-electron tomography.J. Struct. Biol. 2010; 172: 169-179https://doi.org/10.1016/j.jsb.2010.02.011Crossref PubMed Scopus (163) Google Scholar enabling recording of high-quality cryo-ET data. Tomograms from cryo-FIB-milled lamellae can reveal molecular details in cells3Mahamid J. Pfeffer S. Schaffer M. Villa E. Danev R. Cuellar L.K. Förster F. Hyman A.A. Plitzko J.M. Baumeister W. Visualizing the molecular sociology at the HeLa cell nuclear periphery.Science. 2016; 351: 969-972https://doi.org/10.1126/science.aad8857Crossref PubMed Scopus (340) Google Scholar and tissues,4Schaffer M. Pfeffer S. Mahamid J. Kleindiek S. Laugks T. Albert S. Engel B.D. Rummel A. Smith A.J. Baumeister W. Plitzko J.M. A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.Nat. Methods. 2019; 16: 757-762https://doi.org/10.1038/s41592-019-0497-5Crossref PubMed Scopus (117) Google Scholar potentially followed by structural analysis of macromolecules by subtomogram averaging. Cryo-FIB milling is performed by beams of high-energy charged particles such as Ga+ ions or, more recently, argon plasma.5Berger C. Dumoux M. Glen T. Yee N.B.-Y. Mitchels J.M. Patáková Z. Darrow M.C. Naismith J.H. Grange M. Plasma FIB milling for the determination of structures in situ.Nat. Commun. 2023; 14: 629https://doi.org/10.1038/s41467-023-36372-9Crossref PubMed Scopus (9) Google Scholar High-energy charged particles sputter atoms from vitreous biological samples but can damage the molecules in the remaining lamellae. In order to extract maximal structural information from biological samples, it is important to understand the extent of this damage and develop the tools to minimize it. In simulations, a 30 keV beam could implant Ga+ ions in vitreous ice to a depth of 30 nm. The implanted ions could displace or damage the molecule by ion-atom collisions and generate potentially damaging secondary electrons.6Fukuda Y. Leis A. Rigort A. Preparation of vitrified cells for TEM by cryo-FIB microscopy.in: Biological Field Emission Scanning Electron Microscopy. John Wiley & Sons, Ltd, 2019: 415-438https://doi.org/10.1002/9781118663233.ch19Crossref Scopus (2) Google Scholar A recent report by Berger and colleagues in 2023 experimentally demonstrated that ∼30-nm ribosomes close to the edges of the plasma-milled lamellae of cells are damaged, and subtomogram averaging of such ribosomes results in structures at lower resolution.5Berger C. Dumoux M. Glen T. Yee N.B.-Y. Mitchels J.M. Patáková Z. Darrow M.C. Naismith J.H. Grange M. Plasma FIB milling for the determination of structures in situ.Nat. Commun. 2023; 14: 629https://doi.org/10.1038/s41467-023-36372-9Crossref PubMed Scopus (9) Google Scholar Lucas and Grigorieff showed that ribosomes located close to the edge of Ga+-milled lamellae of cells have lower signal-to-noise ratio in 2D micrographs.7Lucas B.A. Grigorieff N. Quantification of gallium cryo-FIB milling damage in biological lamellae.Proc. Natl. Acad. Sci. USA. 2023; 120e2301852120https://doi.org/10.1073/pnas.2301852120Crossref Scopus (7) Google Scholar In both reports, the magnitude of the damage to the structures decreased with the depth in the lamellae and was reduced to a negligible amount 45–60 nm away from the edge of the lamellae (Figure 1). In this issue of Structure, Yang and colleagues8Yang Q. Wu C. Zhu D. Li J. Cheng J. Zhang X. The reduction of FIB damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysis.Structure. 2023; 31https://doi.org/10.1016/j.str.2023.07.002Abstract Full Text Full Text PDF Scopus (1) Google Scholar performed an independent quantification of Ga+-induced damage of ∼30-nm icosahedral parvovirus particles in a phosphate buffer solution. The authors produced structures of viruses in blocks of 10-nm thickness, reaching a resolution in the 3 Å range. Based on this analysis, standard ions with a particle energy of 30 keV damaged the macromolecules up to 50 nm inside the lamellae. This is in good agreement with the previous reports building up a consensus about the amount of damage and its depth. Analyzing the spectral properties of the sample damage, the authors decomposed the damage into two types: at depths of over 30 nm, the damage was similar to a pre-exposure by electrons, whereas at lower depths, additional damage was detected at low frequencies. These considerations and the measurements by Lucas and Grigorieff7Lucas B.A. Grigorieff N. Quantification of gallium cryo-FIB milling damage in biological lamellae.Proc. Natl. Acad. Sci. USA. 2023; 120e2301852120https://doi.org/10.1073/pnas.2301852120Crossref Scopus (7) Google Scholar suggest that the damage at a depth of less than 30 nm can be caused by a combination of direct Ga+ ion damage and the impact of secondary electrons. At depths of more than 30 nm, most of the damage can be attributed to secondary electrons. This conclusion is consistent with simulations indicating that 30 keV Ga+ ions reach up to 30 nm away from the surfaces.6Fukuda Y. Leis A. Rigort A. Preparation of vitrified cells for TEM by cryo-FIB microscopy.in: Biological Field Emission Scanning Electron Microscopy. John Wiley & Sons, Ltd, 2019: 415-438https://doi.org/10.1002/9781118663233.ch19Crossref Scopus (2) Google Scholar Yang and colleagues suggest two complementary approaches to improve the data quality. First, a depth-dependent weighting function can be applied to macromolecule images during image processing and averaging. The weighting function minimizes the high-frequency contribution for macromolecules located close to the edges of the lamellae, resulting in an increased high-resolution signal of the resulting reconstructions. Such weighting can be performed post-acquisition and could become a standard part of processing pipelines for tomograms from FIB-milled samples. The second approach tested by the authors is to reduce the energy of ions at the final stages of FIB milling. Structural analysis of viruses in lamellae milled with 8 keV ions demonstrated that the damage is detected at up to 30 nm, as opposed to 50 nm for 30 keV ions. Previous approaches included polishing of lamellae with ions of the same energy but at a lower current.9Lam V. Villa E. Practical approaches for cryo-FIB milling and applications for cellular cryo-electron tomography.Methods Mol. Biol. 2021; 2215: 49-82https://doi.org/10.1007/978-1-0716-0966-8_3Crossref PubMed Scopus (12) Google Scholar The reduction of energy for polishing has been suggested by Lucas and Grigorieff7Lucas B.A. Grigorieff N. Quantification of gallium cryo-FIB milling damage in biological lamellae.Proc. Natl. Acad. Sci. USA. 2023; 120e2301852120https://doi.org/10.1073/pnas.2301852120Crossref Scopus (7) Google Scholar and successfully implemented and described by Yang and colleagues. Reduction of ion energy comes at a price of slower etching speed; it is therefore practical to use it only at the last steps of milling. Automated routines for FIB milling can potentially perform such polishing with minimal user intervention. The depth of damage is important, as for high-resolution structural analysis by cryo-EM/ET, the sample should be as thin as possible. Thicker specimens introduce more inelastic scattered electrons that decrease the signal-to-noise ratio of the images. On the other hand, as the top and the bottom sides of the lamellae are damaged, particles in these areas will contribute less to the high-resolution structures. Furthermore, for high-resolution structural analysis, a large number of individual particles have to be identified and averaged; this consideration also favors the need for thicker lamellae. Therefore, the selection of an optimal lamellae thickness is non-trivial. For ribosomes in cells, a lamellae thickness of up to 180 nm was demonstrated to not limit the resolution in the 4–7 Å range.10Tuijtel M.W. Kreysing J.P. Welsch S. Hummer G. Beck M. Thinner is not always better: Optimising cryo lamellae for subtomogram averaging.Preprint at bioRxiv. 2023; https://doi.org/10.1101/2023.07.31.551274Crossref Scopus (0) Google Scholar However, for other macromolecules with lower molecular weights and/or abundance in cells, different data collection throughput, and different resolution targets, the optimal thickness may differ and should be considered as a part of the experimental design. The authors thank the Helmholtz Society for support. Mikhail Kudryashev is supported by the Heiselberg Award from the DFG (KU3221/3-1). The authors declare no conflicts of interest. The reduction of FIB damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysisYang et al.StructureJuly 31, 2023In BriefYang et al. developed a quantitative analysis method of cryo-FIB damage using high-concentrated virus sample. The damage depth of 30-keV gallium FIB was estimated as 50 nm. Cryo-lamellae quality can benefit from low energy milling and the resolution can be increased by a depth-dependent weighting function. Full-Text PDF" @default.
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