Montage electron tomography of vitrified specimens

Abstract

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 15 Å were observed in the montaged tilt-series, and diverse cellular features were evident in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

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

“…This departure in image shift values introduced by global tile rotation was much larger than the applied translation alone at lower tilts and became particularly irregular as the stage was tilted to degrees greater than ±30°. As a result, the inconsistent overlap between tiles caused problematic stitching, consistent with previous reports 15,18 .…”

“…Compared to resin-embedded samples where the image contrast is strong 18 , reliable tile overlaps at a majority of the tilts are essential in vitrified unstained samples to achieve gap-free stitching 15 . 4) Sorting of individual tile tilt series from the complete montage collection for efficient data processing and sub-tomogram averaging is applicable only when each tile frame contains enough fringe-unimpacted FOV and retains the ROI throughout the tilt series.…”

“…The development of montage cryo-ET has been limited. To our knowledge, only one method 15 has been reported. As noted by the authors, major challenges included seamlessly joining cryo-EM montage tiles with improved automation 15 , processing large volume stitched tomograms 16 , and sub-tomogram averaging (STA).…”

Quantitative Cryo-Electron Tomography

“…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 .…”

“…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 ).…”