PONE-D-23-34295Sample preparation and data collection for Serial Block Face Scanning Electron Microscopy of Mammalian Cell MonolayersPLOS ONE

Dear Dr. Bhabha,

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preparation of EM samples. We thank Ari Davydov for critical reading and feedback on 

the manuscript and all members of the Bhabha / Ekiert labs for helpful discussions. We 

gratefully acknowledge the following funding sources: SSP-2018-2737 (Searle Scholars 

Program, to G.B.), R01AI147131 (NIAID, to G.B.), Irma T. Hirschl Career Scientist Award 

(to G.B.). G.B. is a Pew Scholar in the Biomedical Sciences, supported by The Pew 

Charitable Trusts (PEW-00033055). The NYU Microscopy Core is partially supported by 

NYU Cancer Center Support Grant NIH/NCI P30CA016087, and Zeiss Gemini 300 SEM 

with 3View was purchased with support of NIH S10 ODO019974"

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"We gratefully acknowledge the following funding sources: SSP-2018-2737 (Searle Scholars Program, to G.B.), R01AI147131 (NIAID, to G.B.), Irma T. Hirschl Career Scientist Award (to G.B.). G.B. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts (PEW-00033055). The NYU Microscopy Core is partially supported by NYU Cancer Center Support Grant NIH/NCI P30CA016087, and Zeiss Gemini 300 SEM with 3View was purchased with support of NIH S10 ODO019974."

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Partly

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: Yes

Reviewer #4: N/A

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5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Antao and co-authors entitled “Sample preparation and data collection for Serial Block Face Scanning Electron Microscopy of Mammalian Cell Monolayers” is a lab protocol. It intends to orient a novice user in the volumetric microscopy methods and to help such a user to make an informed decision on the exact protocol that best addresses their research objectives. Although the intent is commendable, the manuscript content is banal. The principles of electron-matter interactions are not explained, the volume-resolution dilemma is not even mentioned, the issue of non-isometric voxel is not mentioned as well, and no quantitative metrics are presented in the results, despite the availability of SBF-SEM data to the authors. A novice reader can obtain this level of information by reading Wikipedia or by talking to a technician for 15 minutes.

Detailed comments:

Intro, end of 1st paragraph. This is the place where a passage on the importance of context would fit. The beauty of SBF-SEM microscopy is that a relatively large area can be imaged in every 2D frame, so that the spatial relationships amongst the cells, and between the cells and the extracellular matrix can be analysed in full, guiding the operator towards a higher-resolution method in a correlative manner. Unfortunately, by combining the X-Y voxel of 2 nm with a 50 nm slice thickness the authors generate an inherently astigmatic dataset – in any reconstructed plane (any plane which is not the plane of acquisition) the features would be unresolvable and smeared. And in the plane of acquisition the context is truncated to a tiny square. Such an approach robs the operator of both the context (in the acquisition plane) and of the resolution (in all reconstructed planes).

Overview of the goals: “to provide the best image quality and highest resolution possible”. The first part is trivial – every experimental method strives to provide the best quality, as long as natural limitations permit. The second part is inaccurate – often the largest field of view at a reasonable resolution is the goal. The overall objective of all microscopy methods is rather the harmony between the resolution and the field of view.

Sample preparation section. Specificity of stains – is it your common knowledge, or a more precise account can be provided with a reference to a source (for example, A. Hayat “Electron Microscopy Techniques”)?

TCH and similar binding compounds (glutaraldehyde, tannins) are called mordants.

Where is the osmolarity and pH control mentioned in the preparation sequence?

Cryogenic prep pathway, HPF and FS – this is not only for rare events. Cryogenic preparation serves primarily for delicate preservation of hydrated specimens as close as possible to their native state.

Image acquisition section. What about line- and frame averaging for charging reduction? What about the subsurface interaction volume of the electron beam? Even when the optics are perfect, a beam whose energy is too high will yield BSE/SE from the interaction volume incomparably larger than the desired pixel size, and this is especially germane for biological samples (attributable to their light element composition). Where is your discussion of E2 – the energy at which the incoming and outcoming electrons balance out, and no charging is observed (see Joy and Joy 1996, Low voltage electron microscopy)?

Embedding section. It is unclear how charging results in cutting defects. No conductive resins are mentioned.

Cells fixed in a monolayer – what about compound substrates, such as coated coverslips, or sapphire disks placed in the culture well, for further HPF?

Results. The 2x2x50 voxel makes no sense, but any views different from the plane of acquisition are omitted in the figure. This reviewer observes no difference between the pellet and monolayer cultures. Indeed, to quantify the difference, the volumetric images must be processed and segmented with morphologic metrics provided. How many cells fit in the field of view, what is their morphology, how do their contacts look, what is the distribution of the organelles in the monolayer and pellet specimens? What is the abundance of debris and damaged cells? Is there any evidence of the extracellular matrix elements? Without quantitative analysis this paper can provide very little insight.

Minor comment: numbering pages and lines would be helpful.

Reviewer #2: The manuscript submitted by Noelle V. Antao and colleagues seeks to outline sample preparation and image acquisition for serial block-face scanning electron microscopy imaging of mammalian cell monolayers. The manuscript’s stated purpose is to “highlight the challenges and key steps of sample preparation, and the consideration of imaging variables that will facilitate the acquisition of high quality datasets.” However, in this reviewer’s opinion, the level of detail provided and the depth by which this process is discussed in the primary text is severely lacking. Given the lack of detail and discussion in this manuscript, it is difficult to see its role beyond the methods section provided in the primary author’s most recent publication (DOI: 10.1038/s41467-023-43215-0). The manuscript would benefit from the inclusion of a more in depth discussion of the protocol troubleshooting and optimization methods, expansion of details provided in the main text, and integration of supplemental file “S1_file” with the main text, figures and tables.

Concerns:

1. Introduction mentions the improved resolution of vEM over light microscopy, but spends no time discussing this difference and does not state the resolution limit of light microscopy. In this discussion, it would be necessary to also touch on the resolution limits of super-resolution light microscopic methods that are currently in use.

2. The “target audience” paragraph appears out of place in a methods paper.

3. The authors mention that “the SBF-SEM instrument is comprised of an SEM, an ultramicrotome mounted within the SEM chamber, and the necessary hardware and software to control image acquisition.” This statement is vague and the necessary hardware and software should be covered more thoroughly. If specific hardware or software cannot be stated, the purpose of the hardware/software should be covered.

4. Overview of SBF-SEM: In this section the authors discuss the factors that determine image quality. This appears to be a simplified list, as it does not discuss proper fixation or staining of samples, nor does it mention beam spot size (only pixel size), which is an important factor for image resolution, or the electron beam settings which can determine the focal depth and depth of field.

5. Sample preparation: The authors mention that “a mixture of aldehydes” can be used, but does not provide details on what those mixtures might be or why an individual might choose one over another.

6. Sample preparation: The authors mention that samples can be “trimmed to a shape (truncated pyramid, cube or cuboid) and size that will be amenable to cutting by the ultramicrotome mounted in the SEM chamber” but does not discuss the positives and negatives of various shapes or what a “amenable to cutting” might mean to newcomers to SBF-SEM.

7. Sample preparation: The authors mention that “sample preparation requires[s] optimization at the level of: 1) the combination and concentration of heavy metals, 2) the incubation time of each stain to ensure even stain penetration throughout the sample, 3) the type of embedding resin and 4) the dimensions of the final sample block that is loaded into the SEM chamber.” Nowhere in the manuscript's primary text are methods of optimization discussed, nor are examples given for what this optimization process might entail and how users will know whether their methods are optimized or not. It would strengthen the manuscript considerably if the optimization process was discussed and example images were provided, as is seen in similar publications (e.g., DOI: 10.3791/62045).

8. Image acquisition: “Charging artifacts can be resolved by altering parameters like beam current, dwell time and pixel size during image acquisition.” In what ways should these parameters be altered? What do positive and negative results look like? This section is light on details.

9. Embedding and imaging tissue culture monolayers using SBF-SEM: The authors state that a list of advantages and disadvantages for pellet embedding versus en face embedding are described in the supplementary materials. However, as this is a manuscript focused on vEM of cell monolayers, this information should not be relegated to the supplement and should be expanded upon in the main manuscript body.

10. The section titled “Level of expertise needed to implement this protocol” appears out of place.

11. The materials and methods discussed in the main body of the manuscript are highly specific to a single cell line and experimental conditions conducted in the authors’ laboratory. Given the title of this manuscript, this section should be expanded upon to be much more robust and cater to a much larger range of mammalian cell monolayer systems. I do not see the purpose of this section beyond the methods section provided in the primary author’s most recent publication (DOI: 10.1038/s41467-023-43215-0). The majority of this information is standard in the field and has been covered in a large range of published articles (doi: 10.3390/microorganisms9061194, doi: 10.1242/jcs.188433, doi: 10.3390/v7122940, doi: 10.1016/bs.aivir.2019.07.005, doi: 10.1111/jmi.12667, https://doi.org/10.1016/bs.mcb.2023.01.019).

12. What is the reasoning behind the supplemental file “S1_file” being compiled in the way that it is and why is it relegated to the supplement? It is this reviewer’s opinion that the majority of the information and figures in this file, outside of the step-by-step protocol, should be integrated into the main body text, as these are the details that a large portion of readers will be interested in.

13. The manuscript does not have the required link to the protocol on “protocols.io” in the Materials and Methods section. In addition, Supporting Information file 1 does not contain the required caption, “S1: Step-by-step protocol, also available on protocols.io.”

14. The manuscript mentions a number of embedding resins, but does not provide context in the main text to assist the reader in choosing one.

15. As this process can involve a great deal of optimization and troubleshooting, this manuscript would benefit greatly from a robust discussion on optimization and troubleshooting.

16. Supplemental movies – If possible, please include datasets with a greater number of sections.

Reviewer #3: Comments to the author:

The lab protocol article produced by Anteo et al. explores serial block face preparation techniques for the study of in vitro tissue culture cell samples, notably using serial block face SEM (SBF-SEM). The authors share in a comprehensive manner how this technique can be applied, data optimizing methods, and challenges, to facilitate high-quality data acquisition using SBF-SEM which has broad applications in biological communities and beyond. The submitted work aligns well with the aims and scope of this journal, particularly considering its focus on characterization that has implications for interdisciplinary fields, and I certify that the work seems to meet the journal’s criteria for utility, validation, and availability for lab protocol papers. Therefore, I am of the opinion that this work would be eligible to be published in PLoS ONE and I have minor revisions to suggest for strengthening the published work and how it is framed before publication. This reviewer urges the authors to please address the following points for consideration of revision:

1. I thought the introduction was well framed and wanted to suggest when comparing FIB-SEM and SBF-SEM, the authors should provide a numerical comparison range to describe what “larger volumes” are for SBF-SEM to discuss the differences between the techniques. This could involve discussing differences in volume z depth as well as spatially in X-Y for the field of view as applicable. PFIB-SEM could also be mentioned for the completeness of the introductory discussion.

2. It is suggested that the authors frame the introduction more broadly to the audience about these methods and mention how your team is presenting a very specific protocol of how SPF-SEM can be conducted with specific instruments and chemical fixation and embedding methods, but that other instruments and preparation methods may be used.

3. In the introduction for this sentence “More recently this methodology has been adapted to study the ultrastructure of other biological tissue samples e.g. bone tissue, developing unicellular organisms and cell monolayers.” The authors should list references for each of the listed examples for readers to refer to.

4. I note PLoS One describes acceptable lab protocols for publication as follows: “Lab Protocols describing routine methods or extensions and modifications of routine methods that add little value to the published literature will not be considered for publication.” The article is advanced enough, but the introduction frames it as quite routine. I urge the authors to better highlight within their introduction the value of sharing this comprehensive lab protocol article and that limited detailed protocols are widely available within this field to better showcase the impact/importance of the submitted work.

5. In sample preparation, should electron beam sensitivity and softness be better discussed/addressed briefly?

6. In this sentence: “The goal for vEM imaging techniques such as SBF-SEM is to obtain quantitative volumetric information of biological samples with the best image quality and highest resolution possible.” Is the highest resolution possible the goal? Or would it be more appropriate to say something along the lines of being able to achieve the necessary resolution to visualize key biological phenomena to answer your life science questions?

7. In discussing heavy stains used, I suggest also mentioning to readers Uranyless/UAR-EMS Uranyl Acetate Replacement Stains as an emerging safer alternative to uranyl acetate as an additional option to share.

8. In the section overviewing SBF-SEM, for point #2) I recommend rephrasing the section discussing image resolution, which is typically defined by the smallest resolvable feature and related to probe settings and not necessarily pixel size; moreover, many SEMs can push up to 0.5 nm resolution. Reference 17 here seemed ill-fitted here…

9. I’m not sure if this is a stylistic thing pertinent to the style of this article, but I found that the authors use a lot of lists in paragraphs in their writing which made things sometimes hard to follow. E.g.. the paragraph bridging pages 11-12 (start of sample preparation subsection) includes 3 lists within one paragraph!

10. For image acquisition: does pixel size affect imaging in the way you are discussing related to charging artifacts? Or do you mean to discuss that pushing to certain spatial resolutions at higher magnifications can influence imaging due to beam interactions? I wanted to emphasize that pixel size does not equate necessarily to affecting charging, while often they are related; at the end of the day, pixel size has to do with your camera and magnification and can be influenced simply by checking off binning parameters. Something to consider.

11. In the PDFs and supplemental, the text is hard to read in certain figures and quite small. Particularly for figures 1 and 2 in the supplemental. It is suggested that the authors make the text in figures larger accordingly.

12. One last comment, I felt that post-processing was not discussed in the protocol in great detail. I think readers would benefit from an expansion of how Fiji was used for instance in this work, even if just by a couple sentences and leading readers elsewhere to learn more.

Reviewer #4: This paper begins with a simple introductory opening for the non-expert in SBF and FIB-SEM but then the paper never dives deeper into the reasons for selecting certain parts of the protocol ie the resin or stain. It essentially presents standard methods for cell embedding with little reference to prior work or placing the protocol in context. Therefore, this paper could benefit from substantial expansion to the references and discussion. There’s nothing referenced on the types of resins or stains used, or why. These are critical in the workflow for preparing a sample.

Please reference this sentence, readers will like to have access to these works too : More recently this methodology has been adapted to study the ultrastructure of other biological tissue

samples e.g. bone tissue, developing unicellular organisms and cell monolayers.

Similarly – do you have references to these stains used in SBF or FIB-SEM, it would be helpful for readers to access protocols: These heavy metal salts include: osmium

tetroxide, which preferentially interacts with unsaturated lipids; thiocarbohydrazide, which

binds to osmium in the tissue and acts as a bridging agent to which more osmium can

bind, thereby enhancing the contrast of lipid components in the cell; uranyl acetate, which

stains nucleic acids and proteins an provides general contrast to biological samples; lead

aspartate, which interacts with membranes and proteins.

Some discussion on the type of resin used for embedding (Durcupan, Embed 812, Epon, PMMA, etc) would be particularly useful – there are several with varying hardness, could you refer readers to more information on this (given it wasn’t the subject of this investigation)? Are any particularly well suited to cells?

Fig 2: FCC – define in caption

No references are provided in the “Image Acquisition” section – there are papers to highlight the effect of these settings on imaging – it would be worthwhile to reference some. These seem like novice tips on basic SEM operation.

CLEM is mentioned in the intro and abstract, but nothing is really presented on this in the paper. Either include more or remove reference to it being demonstrated.

What was different between the samples prepared in different ways? Since different imaging parameters were used, it's also hard to make any conclusions – can you comment on any pros/cons of either method – as you observed in what's shown in Fig 3 (not just in general)?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Sample preparation and data collection for Serial Block Face Scanning Electron Microscopy of Mammalian Cell Monolayers

PONE-D-23-34295R1

Dear Dr. Bhabha,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Furqan A. Shah

Academic Editor

PLOS ONE

Additional Editor Comments:

My decision of Accept was based on the assessment that concerns raised by Reviewer #1 were pertaining to theoretical aspects of electron optics and imaging, and were mostly beyond the scope of a Lab Protocol manuscript. Yes, as Reviewer #1 has pointed out that the authors have not shown any 3D visualisation, that is true. However, 2D visualisation of sequential slice-and-view methodology is still a key starting point.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has marginally improved, but yet contains numerous errors re electron-substrate interaction. For example, the spot size depends on the electron source, electron optics, and electron current (limited by the aperture) and not by the voltage. The unfortunate effect of charging besides the degradation of the substrate, degrades the image quality and masks/distorts the features of interest. Fixative and mordants are different things. Neutral charging is an unusual expression. Overall, the manuscript still contains factual errors (obviously, a bad thing) and no 3D quantitative data on the substrate (a weakness undermining the very purpose of 3D imaging) and as such will do a disservice to the readers. The authors should consult a qualified electron microscopist or physicist to bring the paper to the publishable quality.

Reviewer #2: This manuscript resubmission by Noelle Antao and colleagues, titled "Sample preparation and data collection for Serial Block Face Scanning Electron Microscopy of Mammalian Cell Monolayers" adequately addresses the concerns of this reviewer through extensive revision of the text.

Reviewer #3: I thank the authors for their comprehensive response to my revision points and the revision points of the other reviewers, which were quite extensive! I believe the authors went above and beyond in their revision for a protocol paper considering the guidelines provided by PLOS ONE in my opinion, particularly after surveying other similar-in-style protocols published. I strongly suggest accepting the article for publication now that it is greatly improved.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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