Making plant tissue accessible for cryo-electron tomography
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Curated by eLife
eLife Assessment
Thick multicellular plant samples provide unique challenges when it comes to cryo-preservation, which has resulted in limited successful examples for structural studies using in situ cryo-electron tomography. To address this deficiency, this important study describes procedures for high-pressure-freezing, focused ion-beam milling, and cryo-electron tomography imaging of certain plant types. The results described in the paper provide solid evidence for the usefulness of the methods described, although some reservations remain about the applicability of the methods to a wider range of plant cell types.
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Abstract
Abstract
Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.
Article activity feed
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eLife Assessment
Thick multicellular plant samples provide unique challenges when it comes to cryo-preservation, which has resulted in limited successful examples for structural studies using in situ cryo-electron tomography. To address this deficiency, this important study describes procedures for high-pressure-freezing, focused ion-beam milling, and cryo-electron tomography imaging of certain plant types. The results described in the paper provide solid evidence for the usefulness of the methods described, although some reservations remain about the applicability of the methods to a wider range of plant cell types.
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Reviewer #1 (Public review):
Summary:
This in situ cryo-ET workflow of selected plant structures provides several detailed strategies using plunge-freezing and the HPF waffle method and lift-out for notoriously difficult samples (compared to cell culture, yeast, and algae, which are far more prevalent in the literature).
Strengths:
A very difficult challenge whereby the authors demonstrate successful vitrification of selected plants/structures using waffle and lift-out approaches for cryoET. Because there are relatively few examples of multi-cellular plant cryo-ET in the literature, it is important for the scientific community to be motivated and have demonstrated strategies that it is achievable. This manuscript has a number of very helpful graphics and videos to help guide researchers who would be interested in undertaking that would …
Reviewer #1 (Public review):
Summary:
This in situ cryo-ET workflow of selected plant structures provides several detailed strategies using plunge-freezing and the HPF waffle method and lift-out for notoriously difficult samples (compared to cell culture, yeast, and algae, which are far more prevalent in the literature).
Strengths:
A very difficult challenge whereby the authors demonstrate successful vitrification of selected plants/structures using waffle and lift-out approaches for cryoET. Because there are relatively few examples of multi-cellular plant cryo-ET in the literature, it is important for the scientific community to be motivated and have demonstrated strategies that it is achievable. This manuscript has a number of very helpful graphics and videos to help guide researchers who would be interested in undertaking that would help shorten the learning curve of admittedly tedious and complex workflows. This is a slow and tedious process, but you have to start somewhere, and I applaud the authors for sharing their experiences with others, and I expect will help other early adopters to come up to speed sooner.
Weaknesses:
While important, the specific specimen and cell-types selected that were successful (perhaps other plant specimen and tissues tried were unsuccessful and thus not reported) in this approach did not demonstrate success to broadly applicable to other much more prevalent and interesting and intensive areas plant biology and plant structures (some mentioned in more detail below).
This manuscript is essentially a protocol paper and in its paragraph form, and even with great graphics, will definitely be difficult to follow and reproduce for a non-expert. Also considering the use of 3 different FIB-SEM platforms and 2 different cryo-FLM platforms, I wonder if a master graphic of the full workflow(s) could be prepared as a supplementary document that walks through the major steps and points to the individual figures at the critical steps to make it more accessible to the broader readership.
Multiple times in the manuscript, important workflow details seemed to point to and be dependent on two "unpublished" manuscripts:
(1) Line 583, 755, 790, 847-848, (Poge et al., will soon be published as a protocol).
(2) Lines 140, 695, 716 (Capitanio et al., will soon be described in a manuscript).
It is not clear if/when these would be publicly available. It may be important to wait until these papers can be included in published form.
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Reviewer #2 (Public review):
Summary:
Poge et al. present a workflow for studying plant tissue by combining high-pressure freezing, cryo-fluorescence microscopy, FIB milling, and cryo-electron tomography (cryo-ET). They tested various plant tissues, including Physcomitrium patens, Arabidopsis thaliana, and Limonium bicolor. The authors successfully produce thin lamellae suitable for cryo-ET studies. Using sub-tomogram averaging, they determined the Rubisco structure at subnanometer resolution, demonstrating the potential of this workflow for plant tissue studies.
Strengths:
This manuscript is likely the first to systematically apply FIB milling and cryo-ET to plant tissue samples. It provides a detailed methodological description, which is not only valuable for plant tissue studies but also adaptable to a broader range of biological …
Reviewer #2 (Public review):
Summary:
Poge et al. present a workflow for studying plant tissue by combining high-pressure freezing, cryo-fluorescence microscopy, FIB milling, and cryo-electron tomography (cryo-ET). They tested various plant tissues, including Physcomitrium patens, Arabidopsis thaliana, and Limonium bicolor. The authors successfully produce thin lamellae suitable for cryo-ET studies. Using sub-tomogram averaging, they determined the Rubisco structure at subnanometer resolution, demonstrating the potential of this workflow for plant tissue studies.
Strengths:
This manuscript is likely the first to systematically apply FIB milling and cryo-ET to plant tissue samples. It provides a detailed methodological description, which is not only valuable for plant tissue studies but also adaptable to a broader range of biological tissue samples. The study compares the plunge freezing method with a high-pressure freezing method, demonstrating that high-pressure freezing can vitrify thick tissues while preserving their native state. Additionally, the authors explore two methods for plant tissue sample preparation, the "waffle" method and in-carrier high-pressure freezing combined with the "lift-out" approach. The "waffle" method is suitable for samples less than 25um, while the in-carrier high-pressure freezing method can process samples up to 100um.
Weaknesses:
The described workflow is very complicated and requires special expertise. The success rate of this workflow is not very high, particularly for high-pressure freezing and life-out technology. Further improvements are needed for automation and increasing throughput.
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Reviewer #3 (Public review):
Summary:
The authors aimed to improve cryo-TEM workflows for plant cells. The authors present details on high-pressure-freezing protocols to vitrify, ion-mill, and image certain plant cell types.
Strengths:
Clear step-by-step outline on how to preserve and image cryo samples derived from plants.
Weaknesses:
A general current weakness of cryo-TEM is the problem of vitrifying cells that are embedded in tissues. The vast majority of cells in the plant body are currently not accessible to this technology. This is not a weakness of this specific manuscript but a general problem.
The manuscript is well organized and well written, and the discussion covers practically all questions I had while reading the results section. I only have a few comments, all of which I consider minor.
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