Plasma FIB milling for the determination of structures <i>in situ</i>

Berger, Casper; Dumoux, Maud; Glen, Thomas; Yee, Neville B.-y.; Mitchels, John M.; Patáková, Zuzana; Naismith, J.H.; Grange, Michael

doi:10.1101/2022.08.01.502333

Cited by 10 publications

(15 citation statements)

References 52 publications

(68 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For most tiles, the apparent thickness of LSU detections was at least 70 nm less than the estimated sample thickness, indicating that no detections were being made within 35 nm of the lamella edge. It is possible that this is caused by sample damage during FIB-milling as similar values for damaged areas in FIB-milled lamellae have been estimated from sub-tomogram averaging ( Berger et al, 2022 ). This means that LSU detections across the lamellae can be skewed by a change in thickness and sample damage.…”

Section: Resultsmentioning

confidence: 83%

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections

Elferich

Schiroli

Scadden

et al. 2022

eLife

View full text Add to dashboard Cite

A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study we use a new data-acquisition scheme called 'Defocus-Corrected Large-Area cryo-EM' (DeCo-LACE) to collect high-resolution images of entire sections (100 - 250 nm thick lamellae) of neutrophil-like mouse cells, representing 1-2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide 'maps' of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.

show abstract

Section: Resultsmentioning

confidence: 83%

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections

Elferich

Schiroli

Scadden

et al. 2022

eLife

View full text Add to dashboard Cite

show abstract

“…These changes are difficult to quantify in terms of damage, and they could in part be caused by other mechanisms such as ice accumulation after milling (23). Argon plasma FIB-damage has been assessed by estimating B-factors of subtomogram averages, comparing averages of particles at less than and greater than 30 nm from the lamella surface, which lacks positional precision (24). The 2DTM SNR represents a more quantitative metric to assess sample integrity.…”

Section: Discussionmentioning

confidence: 99%

Quantification of gallium cryo-FIB milling damage in biological lamella

Lucas

Grigorieff

2023

Preprint

View full text Add to dashboard Cite

Cryogenic electron microscopy (cryo-EM) has the potential to reveal the molecular details of biological processes in their native, cellular environment at atomic resolution. However, few cells are sufficiently thin to permit imaging with cryo-EM. Thinning of frozen cells to <500 nm lamellae by cryogenic focused ion beam (FIB) milling has enabled visualization of cellular structures with cryo-EM. FIB-milling represents a significant advance over prior approaches because of its ease of use, scalability, and lack of large-scale sample distortions. However, the amount of damage caused by FIB-milling to the generated thin cell section has not yet been determined. We recently described a new approach for detecting and identifying single molecules in cryo-EM images of cells using 2D template matching (2DTM). 2DTM is sensitive to small differences between a molecular model (template) and the detected structure (target). Here we use 2DTM to demonstrate that under the standard conditions used for machining lamellae of biological samples, FIB-milling introduces a layer of variable damage that extends to a depth of 60 nm from each lamella surface. This thickness exceeds previous estimates and limits the recovery of information for in situ structural biology. We find that the mechanism of FIB-milling damage is distinct from radiation damage during cryo-EM imaging. By accounting for both electron scattering and FIB-milling damage, we find that FIB-milling damage will negate the potential improvements from lamella thinning beyond 90 nm.

show abstract

“…Further investigation will be required. SEM imaging will damage the sample, the highest dose we employed data acquisition with the small pixel size was under 10 -2 e - /Å, lower than the one used during cryo-ET imaging (Berger et al 2022). Though damage increases at the acceleration voltage of SEM due to the increased scattering cross-section, the damage would be expected to be confined to the interaction layer, a depth less than 50 nm.…”

Section: Discussionmentioning

confidence: 99%

“…The use of pFIB to fabricate lamella has been demonstrated with plunge frozen vitrified materials as part of a high-resolution high throughput cryo-ET workflow (Berger et al, 2022). The cryo-ET workflow study indicated that milling with Ar plasma (30kV, 60 pA) beam adversely affected the resolution of sub-tomographically averaged ribosomes to a depth of around 30 nm.…”

Section: Discussionmentioning

confidence: 99%

Cryo-plasma FIB/SEM volume imaging of biological specimens

Dumoux

Glen

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 μm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free tool is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 μm) to produce lamella for cryogenic electron tomography.

show abstract

Plasma FIB milling for the determination of structures in situ

Cited by 10 publications

References 52 publications

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections

Quantification of gallium cryo-FIB milling damage in biological lamella

Cryo-plasma FIB/SEM volume imaging of biological specimens

Contact Info

Product

Resources

About