Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

Yang, Jie; Larson, Matthew R.; Sibert, Bryan; Kim, Joseph Y.; Parrell, Daniel; Sanchez, Juan; Pappas, Victoria; Kumar, Anil; Cai, Kai; Thompson, Keith; Wright, Elizabeth

doi:10.1101/2021.12.31.474669

Cited by 16 publications

(14 citation statements)

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“…This may affect excessively thick lamellae (>300 nm). Unlike recently published montage tomography workflows (Peck et al, 2021;Yang et al, 2022), PACE-tomo prioritises radiation damage avoidance, data quality, and throughput. Together with the automation of lamella cryoFIB milling (Buckley et al, 2020;Tacke et al, 2021;Zachs et al, 2020), automated tilt series alignment and tomogram reconstruction (areTomo (Zheng et al, 2022), emClarity (Ni et al, 2022), dynamo (Burt et al, 2021)), PACE-tomo could play an important role in streamlining in situ cryoET workflows and determining high-resolution structures of macromolecular complexes in their native cellular environment.…”

Section: Discussionmentioning

confidence: 99%

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Parallel cryo electron tomography on in situ lamellae

Eisenstein

Yanagisawa

Kashihara

et al. 2022

Preprint

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In situ cryo electron tomography of cryo focused ion beam milled samples emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. The lamella-shaped samples, however, have a limited area and are created with a necessary pretilt. This severely limits the possibilities for recording tomographic tilt series in a high-throughput manner. Here, we utilise a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining sample areas conventionally used for tracking of specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows the collection of sample areas that were previously unreachable, thus maximising the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high-throughput and high-quality of PACE-tomo.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Parallel cryo electron tomography on in situ lamellae

Eisenstein

Yanagisawa

Kashihara

et al. 2022

Preprint

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“…Camera size improvements can impact the resolution achieved but not the number of extracted particles ( Kudryashev et al, 2012 ). To overcome such a trade-off between resolution and FOV, montage data collection schemes are being explored for cryo-ET ( Peck et al, 2021 ; Yang et al, 2022 ). These approaches are especially useful for lamellae where, after a timely workflow to obtain them, the maximum information can be extracted by imaging the lamella as much as possible .…”

Section: Resolution Vs Field Of Viewmentioning

confidence: 99%

“…Relevant ongoing developments that help expand cryo-ET are pointed out by a black arrow at their corresponding length scale area. Montage ET allows image acquisition of larger field of views (FOV) without compromising resolution at the cost of elongated acquisition times ( Peck et al, 2021 ; Yang et al, 2022 ). While microfluidic cryofixation of larger specimens during live-cell microscopy imaging is a promising tool to explore the opportunities for correlative light and electron microscopy studies ( Fuest et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Cryo-Electron Tomography

Navarro

2022

Front. Mol. Biosci.

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The three-dimensional organization of biomolecules important for the functioning of all living systems can be determined by cryo-electron tomography imaging under native biological contexts. Cryo-electron tomography is continually expanding and evolving, and the development of new methods that use the latest technology for sample thinning is enabling the visualization of ever larger and more complex biological systems, allowing imaging across scales. Quantitative cryo-electron tomography possesses the capability of visualizing the impact of molecular and environmental perturbations in subcellular structure and function to understand fundamental biological processes. This review provides an overview of current hardware and software developments that allow quantitative cryo-electron tomography studies and their limitations and how overcoming them may allow us to unleash the full power of cryo-electron tomography.

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“…Such actin bundles were not observed in control neurons, being organized to either be more disperse or 'swirl' with curvature (Fig 2). Future work will include implementing montage tilt series collection to have a wider field of view [9], biophysical measurements of the actin filaments in CIP4-transfected cells vs. non-transfected cells, live-cell imaging of cells cultured on grids to understand CIP4 dynamics, pharmacological experiments, and transfecting neurons with other constructs in the CIP4 family. Such studies will provide ultrastructural insights on the process and control of neuritogenesis [10].…”

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confidence: 99%