Making plant tissue accessible for cryo-electron tomography

  1. Matthias Pöge
  2. Marcel Dickmanns
  3. Peng Xu
  4. Meijing Li
  5. Oda H Schiøtz
  6. Christoph OJ Kaiser
  7. Jianfei Ma
  8. Anna Bieber
  9. Cristina Capitanio
  10. Johann Brenner
  11. Margot Riggi
  12. Sven Klumpe
  13. Manuel Miras
  14. Neda S Kazemein Jasemi
  15. Waltraud X Schulze
  16. Rüdiger Simon
  17. Wolf B Frommer
  18. Jürgen M Plitzko
  19. Wolfgang Baumeister

  • Curated by eLife

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    eLife Assessment

    In contrast to animal cells, mature plant cells contain large watery vacuoles commonly taking up more than 90% of the cell volume. Therefore, mature plant cells 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. This important study describes procedures for high-pressure-freezing, focused ion-beam milling, and cryo-electron tomography imaging of select plant tissue types that can be vitrified successfully. The paper provides solid evidence for the usefulness of these methods for those samples. Some concerns remain about the applicability of the methods to a wider range of plant tissue samples, including for example intact leaves of higher plants, for which vitrification remains difficult.

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Abstract

Cryo-electron tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues in situ. 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 light 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, Arabidopsis thaliana seed embryos and Limonium bicolor salt glands. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

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  1. eLife

    eLife Assessment

    In contrast to animal cells, mature plant cells contain large watery vacuoles commonly taking up more than 90% of the cell volume. Therefore, mature plant cells 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. This important study describes procedures for high-pressure-freezing, focused ion-beam milling, and cryo-electron tomography imaging of select plant tissue types that can be vitrified successfully. The paper provides solid evidence for the usefulness of these methods for those samples. Some concerns remain about the applicability of the methods to a wider range of plant tissue samples, including for example intact leaves of higher plants, for which vitrification remains difficult.

  2. eLife

    Reviewer #1 (Public review):

    [Editors' note: The reviewers discussed the revision. They agreed that the authors made a good faith effort to thoughtful response to the queries and concerns raised, and this manuscript should be published. Although some concerns about the applicability of the methods for a wider range of plant tissues remain. This has been expressed more clearly in the eLife Assessment above, and the authors may want to consider adding their own cautionary language to the manuscript, for example that they were not successful with intact plant leaves.]

    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.

    Comments on revised version:

    The authors made a good faith effort to thoughtful response to my queries and concerns, and this manuscript should be published. I really appreciate it. I suggest one final amendment, and it can simply be an insertion of a couple sentences. I'm still concerned by the more niche plant sample types being used, admittedly due to limitations in freezing of more mainstream plant species and/or tissues, and leaves in particular. The workflows described in the manuscript definitely could work IF freezing could be adequate. The authors go to lengths describing what makes it a challenge to vitrify, and they comment in rebuttal that they so far have been unsuccessful in intact leaves of higher plants. Yet, they do not explicitly make that statement in the manuscript itself. I want to be sure that a reader doesn't invest much time and money thinking this paper on "Making plant tissue accessible..." is actually not broadly accessible to any plant tissue. As described it is specific or workable in "select" plant tissue types or species due to freezing limitations and/or strategies employed. I am asking to place some simple cautionary guiding language in the discussion or elsewhere.

  3. eLife

    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.

    Comments on revised version:

    I am satisfied with the revision and have no more comments.

  4. eLife

    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.

    Comments on revised version:

    As I mentioned in my original review, there will be only a few plant cell types that can be vitrified and there is no solution for this in sight. This means that we will have to rely on "classical" electron microscopy for the foreseeable future for most experiments. Nevertheless, the publication shows great progress and there is a lot we can learn within those limitations.

  5. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    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.

    We agree that this would help the reader to keep track of the different workflow steps. Therefore, we prepared two additional supplementary figures:

    Figure 1 – figure supplement 1: Overview of the critical workflow steps for P. patens protenemata.

    Figure 1 – figure supplement 2: Overview of the used lift-out procedures.

    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.

    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.

    We thank the reviewer for the fair and detailed assessment of our manuscript.

    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.

    We thank the reviewer for the fair and detailed assessment of our manuscript.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    (1) I would like to see more part numbers of reagents and supplies used. They were largely absent.

    (2) The authors seem to refer to two other unpublished manuscripts, which have important details for this workflow, and are unavailable at the time of this submission.

    In point (1) and (2), the reviewer addresses crucial points. By the time we submit the revised version, the manuscript describing the FIB-view method in more detail is still in preparation. The step-by-step protocol was accepted for publication in Bio-Protocol but cannot yet be cited. We stated this as placeholders for the citations in the text which will be added in the version of record of this manuscript.

    Here, we wanted to give the reader an outline of the concepts which are required to image plant tissues with cryo-ET. Therefore, we omitted the full description of chemicals and supplies which will be listed in the step-by-step protocol.

    (3) A number of figure legends lack the actual specimen in the figure. Please add.

    We thank the reviewer for this comment and added specimen names and tissue type to the caption of Figures 2-4 and the figure supplements.

    (4) There are a few mixed orientations and missing figure panel labeling. Sometimes, in the same figure, the reader must compare horizontal and vertical sequences in the same panel. This makes it more difficult to figure out, especially when many panels are unlabeled (one example, Extended Data Figure 6). Every panel in the figure should have a letter. Consider using C1, C2, C3, C4 if they are a related series.

    We agree that some of the figures are difficult to understand due to different directions of image sequences. Instead of labeling every image of an image sequence with a unique letter, we decided to frame images which belong to an image sequence. These adjustments were consistently implemented for all main figures, supplementary figures and figures in the supplementary material.

    (5) Line 22. Insert "in situ" cryo-ET since cryo-FIB is part of the workflow.

    (6) Line 31. Remove redundant second "tissue" in the same sentence.

    (7) Line 31-32. Specify for At "seed embryos and Lb "salt glands".

    The suggested changes in (5)-(7) were implemented.

    (8) Line 53; add. air spaces in leaves. Why do you suggest vacuoles often contain solute "depleted" vacuoles? See: https://doi.org/10.1016/S0065-2296(08)60152-4. It's variable for sure, but I see that as an exception rather than as written, it seems to imply this is more likely than not, which I disagree with. Please modify. The primary driver for poor freezing is the very large vacuole itself and air spaces (at least in leaves).

    We changed the first two sentences starting from line 51: The most critical step in cryo-ET is the vitrification. For plant tissues, this process is complicated by the cell size (diameter typically 10 – 100 µm) which is larger than for most yeast, algae or mammalian cells, often contain water-filled vacuoles occupying up to 90 % of their cellular volume and, in the case of leaves, the tissue comprises air-filled spaces.

    (9) Lines 66-68. This oversimplification should be modified. Many freeze-substitution protocols include a fixative (typically osmium tetroxide) and 1-5% water in solvent, so technically not dehydrated (at least until resin steps).

    This line was changed to “samples are metal-impregnated and dehydrated upon embedding in a resin”.

    (10) Lines 66-69. I suggest that more recent papers that actually include resin electron-tomography (and cryo-ET) in plants with HPF data should be cited: https://doi.org/10.1093/jmicro/dfy133. There are plenty of organellar/sub-organellar studies of merit with FS-HPF (albeit I agree, not appropriate for molecular structure). The value and importance of your cryo-ET work are good, but it is very difficult and has limited accessibility. The merits of an in situ cryo-ET of plants paper will have more credibility to plant scientists who do the prior when properly framed. Please adjust accordingly.

    (12) I would have at least expected a brief review (yes, it's very limited) of plant-based cryo-EM studies for readers. There is not a lot, but it would add value and demonstrate the work of others who did cryo-ET in photosynthetic organisms that preceded this effort.

    Following the suggestions in (10) and (12), we elaborated on certain points in the introduction.

    We briefly review pioneering cryo-ET work on plant and algal system in one sentence added in line 64:

    “Previous cryo-ET work focused on Protoplasts [24], i.e. isolated cells without cell wall, isolated organelles [25], cyanobacteria [26], red algae [27] and single celled green algae [28,29].” Citing:

    (24) Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography., Sanchez Carrillo et al, 2023

    (25) Molecular architecture of thylakoid membranes within intact spinach chloroplasts, Wietrzynski et al, 2025

    (26) Structure and Function of a Bacterial Gap Junction Analog, Weiss et al, 2019

    (27) In situ cryo-ET structure of phycobilisome–photosystem II supercomplex from red alga, Li et al, 2021

    (28) Charting the native architecture of Chlamydomonas thylakoid membranes with single-molecule precision, Wietrzynski et al, 2020

    (29) Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii, Kelley et al, 2024

    To highlight the importance HPF-FS for EM of plant tissues, we now cite the suggested review and added a sentence in line 68 referencing two more reviews for the interested reader :

    “Currently, this is the most accessible and reliable approach to image plant tissues by electron microscopy [7,31].”

    (7) Electron tomography of plant organelles and the outlook for correlative microscopic approaches, Wang et al, 2019

    (31) Electron microscopy for imaging organelles in plants and algae, Weiner et al, 2022

    (11) Lines 74-75. While I understand the temptation to use "one-dimensional" and "two-dimensional", the chains are not straight lines, and certainly they are 3D structures. I suggest chains and sheets is sufficient here.

    The terms "one-dimensional" and "two-dimensional" were removed.

    (13) The Intro is pretty minimalist and ends rather abruptly, only mentioning one plant species used in the study. While it was the primary system used, please include the other plant targets and rationale.... Indeed, the title "Making plant tissue accessible for cryo-electron tomography" felt a bit misleading. I was expecting cells from non-embryonic roots, stem, or leaf tissue,e which would be the dominant targets for most plant scientists. Yes, A. thaliana embryos and salt glands from L. bicolor are useful plant targets, but certainly don't represent typical types of structures and potential for biotic and abiotic stress and developmental studies of multicellular plants. They certainly would be low-hanging fruit, but limited use cases for the workflow.

    (14) Please consider a title more appropriate based on the realities/constraints and limitations of what you were able to demonstrate in broadly relevant plant tissues. For example, "Making select plant tissue accessible for cryo-electron tomography" would more accurately reflect the scope of the achieved project, at least what was included in the manuscript.

    Regarding (13) and (14), we are aware that most research is done on root tips or leaf epidermis. We had no success with leaves. The air-filled space in the spongy mesophyll is very problematic for HPF and we did not manage to dissect the tissue to fit into HPF carriers without severely damaging the cells. We can imagine that other labs with access to cryo-ET setups similar to ours but more expertise in high-pressure freezing plant tissues may still be successful. On the other hand, we have initial results, indicating that Arabidopsis thaliana root tips can be vitrified using the described methods. Furthermore, we discuss the limitations of the method extensively. Therefore, we think that the title is still appropriate.

    We specified the names of the tested tissues in line 78 of the introduction.

    (15) Lines 91 and 92. I am a bit surprised that such high concentrations of sucrose (and glycerol) were used as cryo-protectants. This certainly was a hypertonic solution and would have caused plasmolysis and cell collapse (not dehydration as characterized). Good results (or at least freezing quality improvement) in cells equilibrated in much lower concentrations of sucrose (closer to those used in proline). I'm not saying you need to address it in the manuscript, but I am not surprised. Those would not have worked well as tested.

    We thank the reviewer for this advice. It may be worth screening more cryo-protectants at various concentrations for plunge freezing. We still think that most plant cells will not vitrify with this method.

    (16) The first introduction of cryo "FLM" (Leica solution) was line 62, and "int-FLM" (Delmic solution) without reference to cryo was line 111. Thermo has an integrated system referred to as iFLM. Please consider that it may cause confusion to a novice, and they should probably be differentiated together at first introduction. I assume the use of int-FLM is meant to distinguish from Thermo iFLM.

    In order to clarify this and to name the different cryo-FLM modalities already in the introduction, we added a sentence in line 62:

    “For correlative light and electron microscopy (CLEM) approach it is possible to use FLMs equipped with cryo-compatible stages (e.g. Leica SP8/Stellaris or Linkam CMS196 stage) or FLMs integrated into the FIB chamber (int-FLM, e.g. Delmic METEOR or Thermo Fisher Scientific iFLM).”

    (17) Line 124-128. Figure 1 and the corresponding text. Sure, large/severe crystals in this low-resolution TEM image are visible, but this is insufficient to confirm that much smaller ice crystals (e.g., 5-10nm) that would not be visible from just this data. Please note that other evidence to confirm vitrification would require a closer inspection.

    We agree. That is an aspect we do not address. The tilt-series recorded on our HPF samples do not show the large patterns introduced by crystalline ice. Still, the best evidence for full vitrification would be a diffraction pattern without the characteristic diffraction spots of crystalline ice. Unfortunately, we did not record diffraction patterns. And because the pixel size of our raw tilt images is too large, there are no diffraction spots in the power spectra of tilt images even when they clearly contain crystalline ice. After cryo-ET imaging, we discarded the samples. Therefore, we cannot record diffraction patterns for the lamellae shown in this work.

    (18) Line 340. I think most cell types have sufficient contrast with certain platforms, such as Hydra Helios5 pFIB or ZEISS CrossBeam. This is likely a limitation of the platform used, Scios, Aquilos, and Aquilos 2 columns and detectors. In the alternative platforms, other cellular features can definitely be resolved and contrasted that are non-membranous. This is why I expect this valuable approach would have benefited this particular work and reduced the dependency and resolution challenges for cryo-FLM, as it was essentially not available/utilized in this workflow. This should be noted accordingly in discussion or elsewhere, as it is a limitation of the platforms used, not the potential for this kind of work in general, as implied.

    We present SEM imaging and integrated fluorescence light microscopy as possibilities for targeting. In line 340 where we discuss axial targeting. The point we want to make is that SEM and int-FLM imaging are complementary methods with different limitations. Irrespective of the SEM system, for example liquid-liquid phase-separated compartments may not generate high enough contrast in SEM images but can potentially be labelled with fluorophores and therefore reliably targeted with int-FLMs. During our work for this project we milled lamellae on protonema cell files that were oriented parallel to the surface of the trenches and no cellular material was exposed at the surface of the trench. In this case, SEM block face imaging could not be used for axial targeting and we had to rely on the int-FLM.

    (19) Line 621-622 and elsewhere. The fluorescence in the images from the Leica SP8 is of chlorophyll, which would dominate with an emission filter of 500-750nm. Chlorophyll is definitely not "green autofluorescence". It emits above 650nm quite strongly. However, in lines 644-645, using the Delmic platform, the text simply states an "emission wavelength = 525nm". First, while that may be the center point of this filter or the name in the software, the manuscript should include the full spectral bandwidth of this and any other filters reported in that platform. Based on the specific exclusion of chloroplast signals in the images (dark ovoid voids), I fully expect it blocks the chlorophyll signal. Thus, interpretability and relationship would simply show locations of cells, but not exactly match if using cryo-FLM and int-FLM at the same cell location in this use case. I interpret the green fluorescence signal in int-FLM to be most likely vacuole autofluorescence rather than general cytoplasmic autofluorescence. The readers should be informed of the importance of matching emission filters if this is critical for targeting their work and matching an integrated and non-integrated FLM image.

    We thank the reviewer for making us aware of this incomplete description. We included the spectral band widths of the emission filters and replaced the term “green-autofluorescence” with “cellular autofluorescence” as a more general term. Furthermore, we added a sentence to the caption of Figure 2 and Figure 2 - figure supplement 1 (previously Extended Data Fig 3):

    “Note that the excitation wavelengths and emission filters used for FLM imaging are different from those used for int-FLM imaging. Therefore, the fluorescence signals appear different. Ideally, for most reliable targeting, excitation and emission should be the same for all FLM imaging modalities.”

    And to methods section “Lateral targeting and trench milling” the following sentence:

    “If both FLM and int-FLM imaging are employed for targeting, the used excitation wavelengths and emission filters should ideally be the same for both imaging modalities. This assures most reliable targeting. That is not the case here. Therefore, the fluorescence signals of FLM and int-FLM images (Figure 2, Figure 2 - figure supplement 1) appear different.”

    (20) Please review the paper more closely, a number of typos and grammatical errors were noted.. such as but not limited to:

    - Line 319 swift

    - Line 725 patterns

    - Line 1002 autofluorescence

    - Line 1064 Luliia Lermak.

    We thank the reviewer for carefully reading our manuscript and uncovering these mistakes. The name “Iuliia Iermak”, however, both starting with capital “i” is spelled correctly. This is the official transliteration of Ukrainian names to Latin alphabet.

    Reviewer #2 (Recommendations for the authors):

    Minor comments:

    (1) The authors tested several cryoprotectants for the plunge freezing method, which were ineffective for thick samples. For high-pressure freezing, they used either 20% (m/v) Ficoll 400 or 40% (m/v) Ficoll 400. Did they test other cryoprotectants, and does the high concentration of Ficoll 400 affect the cells?

    We thank the reviewer for the overall positive assessment of our work. In fact, we only used 20% (m/V) Ficoll 400 for high-pressure freezing. The 40% (m/v) Ficoll 400 solution was our stock solution and mixed 1:1 with growth medium. For HPF, we only tested dextrans and in contrast to Ficoll 400 we often observed plasmolysis. This indicates that dextrans put P. patens under osmotic pressure. We did not test, however, what impact a high concentration of Ficoll 400 has on growth, development and physiology of P. Patens or the other tested samples. But we suspect the impact to be minimal because tissue samples were immersed in a Ficoll-containing buffer only for 10-60 s right before freezing.

    (2) Page 15, line 277. There is no Figure 6.

    (3) Page 27, line 550. Switched off?

    (4) Page 50, line 992, 4 ℃?

    All typos in (2)-(4) were corrected.

    Reviewer #3 (Recommendations for the authors):

    (1) I appreciate that the authors highlight some of the drawbacks of their methods in the discussion and mention potential improvements that need to be accomplished to make cryo-TEM a more standard method for plant cell biology. For tissues that are more deeply embedded in the plant body (practically all sub-epidermal layers in higher plants that are not embryonic or meristematic), there is no potential method on the horizon that would allow vitrification. This means that some 99% of the plant body can currently not be vitrified, and this will likely be the major hurdle in the coming years. I find the text line 326 "can still be a major challenge for some samples" an understatement. Most samples will show vitrification problems.

    This fact does not reduce the importance of their current study, but a sentence on this would inform non-experts that cryo- and classical TEM will have to run side by side for quite some time. Clarification may help, as reviewers constantly ask for the application of cryo-TEM to investigate samples that currently cannot be vitrified.

    We agree with the reviewer that the mentioned sentence is an understatement and changed it into “is still a mayor challenge.” Furthermore, we added a new sentence at the end of the paragraph starting in line 335 “Currently, most fully intact tissues remain only accessible by conventional electron microscopy employing chemical fixation, if the dissection to fit the sample into the small volume of HPF carriers is omitted.” to emphasize that conventional TEM is currently indispensable for many if not most projects.

    (2) As the authors correctly mention, the waffle method leads to injury of neighboring cells. Cell-walled plant cells often withstand pressures of 5-10 MPa and more. Squeezing to make them burst will likely lead to a significant pressure wave running through the tissue with potential artifact formation. I was wondering if the authors considered using young cultures where the protonema are just a few cells long and may completely fit into a grid hole?

    We specifically needed two-week-old protonemata for a project we are working on with collaborators. Otherwise, this is a great suggestion. Using tissue for HPF that is small enough to fit into the cavities formed by grid bars and support film is the best way to avoid tissue damage.

    (3) It would be helpful if the authors would briefly explain why the cryo pictures show open-ended membranes, e.g., in Figure 3, ER in e, Golgi in d, or relatively sharp corners (cristae in e). Are these reconstruction artifacts or potential preparation artifacts (e.g., by the described bending of lamellae or similar due to milling)?

    This is an important point to raise. The open-ended Golgi and ER membranes are caused by the missing wedge which is the most prominent but unavoidable imaging artifact in cryo-ET. To clarify this for readers, we added a sentence in line 198:

    “The seemingly discontinuous membranes in Fig. 3d, e are caused by the missing wedge, a known imaging artifact in cryo-ET.”. Citing the paper “On geometric artifacts in cryo-electron tomography” by Turoňová et al., 2016.

    Regarding the sharp membrane corners of cristae, that is hard to say for certain. It is not the missing wedge in this case. For all our P. Patens preparations, cristae always looked like that which is different from cristae in other organisms we investigated with cryo-ET. It could be a characteristic feature of cristae in P. patens. But it could also be an artifact of the preparation which we can never fully exclude. It is not ion beam damage, however, because it is confined to a few tens of nanometers from the surface of the lamella and cristae have this appearance throughout the whole thickness of the lamella (150 to 350 nm).

    (4) Please explain the nature of the dark spots in e.g., Figure 5e.

    We marked this ice contamination introduced during transfer between microscopes as we did it in Figure 1 and added the description to the figure caption.

    (5) Line 105 "recording the green cellular autofluorescence". The autofluorescence of chlorophyll is actually red. The software seems to render it green. I would suggest deleting "green".

    We followed the suggestion and removed “green” on multiple occasions in the methods section.

  6. Version published to 10.7554/elife.106455.2 on eLife
  7. Version published to 10.7554/elife.106455 on eLife
  8. Version published to 10.1101/2025.02.14.638237v2 on bioRxiv
  9. 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.

  10. eLife

    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.

  11. eLife

    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.

  12. eLife

    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.

  13. Version published to 10.7554/elife.106455.1 on eLife
  14. Mentioned by @somssich.bsky.social on Bluesky

    📜 Making plant tissue accessible for cryo-electron tomography 🧑‍🔬 Matthias Pöge1, @madic.bsky.social, Wolfgang Baumeister, et al. 📔 @biorxiv-plants.bsky.social 🔗 www.biorxiv.org/content/10.1... #️⃣ #PlantScience #PlantMicroscopy #PlantImaging #CryoET

  15. Mentioned by @prepub-singlecell.bsky.social on Bluesky

    Making plant tissue accessible for cryo-electron tomography #SingleCell 🧪🧬🖥️ https://www.biorxiv.org/content/10.1101/2025.02.14.638237v1

  16. Mentioned by @madic.bsky.social on Bluesky

    Our methods paper made it to bioRxiv: doi.org/10.1101/2025... Full #cryoET workflows for #plant tissues. Plus some general tricks and tweaks on serial Lift-Out and cryo-FLM. 🧪🧵1/5 #teamtomo #PlantScience #Physcomitrium #Arabidopsis #cryoEM #CLEM

  17. Mentioned by @biorxivpreprint.bsky.social on Bluesky

    Making plant tissue accessible for cryo-electron tomography https://www.biorxiv.org/content/10.1101/2025.02.14.638237v1

  18. Mentioned by @biorxiv-plants.bsky.social on Bluesky

    Making plant tissue accessible for cryo-electron tomography https://www.biorxiv.org/content/10.1101/2025.02.14.638237v1

  19. Version published to 10.1101/2025.02.14.638237v1 on bioRxiv