A novel optimized pre-embedding antibody-labelling correlative light electron microscopy technique
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In the intricate environment of a cell, many studies seek to discover the location of specific events or objects of interest. Advances in microscopy in recent years have allowed for high detail views of specific areas of cells of interest using correlative light electron microscopy (CLEM). While this powerful technique allows for the correlation of a specific area of fluorescence on a confocal microscope with that same area in an electron microscope, it is most often used to study tagged proteins of interest. This method adapts the correlative method for use with antibody labelling. We have shown that some cellular structures are more sensitive than others to this process and that this can be a useful technique for laboratories where tagged proteins or viruses, or dedicated CLEM instruments are not available.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology.
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Comments to Author
Dear authors, I have read your manuscript ","Antibody-labelling correlative light electron microscopy" finding it quite interesting. It is clear, it is well written and I liked its approach. The results of several works are not always positive and however any well-conducted study can shed light for other researchers. It is a methodology article to facilitate the location of structures similar to the use of correlative electron microscopy but without requiring the sophisticated instrument. However, one thing that does not convince me is the fact of referring to the results some descriptions that most readers would look for in the material and methods section. Moreover: in Line 104 Please describe the meaning of 6.2. In Line 169 Please revise sentence: and both primary and secondary antibody incubations …
Comments to Author
Dear authors, I have read your manuscript ","Antibody-labelling correlative light electron microscopy" finding it quite interesting. It is clear, it is well written and I liked its approach. The results of several works are not always positive and however any well-conducted study can shed light for other researchers. It is a methodology article to facilitate the location of structures similar to the use of correlative electron microscopy but without requiring the sophisticated instrument. However, one thing that does not convince me is the fact of referring to the results some descriptions that most readers would look for in the material and methods section. Moreover: in Line 104 Please describe the meaning of 6.2. In Line 169 Please revise sentence: and both primary and secondary antibody incubations steps, it was found that a 15 min blocking step followed by a 30 min antibody incubationstep to be perfectly satisfactory
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The reviewers were positive about your article, and are supportive of acceptance for publication following some minor amendments. Please address their comments in full.
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Comments to Author
The manuscript from Doyle et al describes the optimisation of an antibody-labelling correlative light electron microscopy (CLEM) method. While the authors originally aimed to unravel the exact location of RNA synthesis for the infectious bronchitis coronavirus (IBV), the method was not good enough to answer this research question. However, the method they developed will allow non-expert CLEM labs to explore the advantages of CLEM for their own research questions. I only have a few minor suggestions that the authors might want to consider in a revised version of the manuscript: - Either define zER in line 58, or call it zippered ER in line 181. - Figure 1 legend: provide some additional detail in the processing of the samples, e.g. including resin-embedding and sectioning. - Line 82: should read …
Comments to Author
The manuscript from Doyle et al describes the optimisation of an antibody-labelling correlative light electron microscopy (CLEM) method. While the authors originally aimed to unravel the exact location of RNA synthesis for the infectious bronchitis coronavirus (IBV), the method was not good enough to answer this research question. However, the method they developed will allow non-expert CLEM labs to explore the advantages of CLEM for their own research questions. I only have a few minor suggestions that the authors might want to consider in a revised version of the manuscript: - Either define zER in line 58, or call it zippered ER in line 181. - Figure 1 legend: provide some additional detail in the processing of the samples, e.g. including resin-embedding and sectioning. - Line 82: should read "infection of DF1 cells". - Line 135: "low magnification" instead of "low power"? - Section 6.1: I would suggest renaming the title of this section. I would argue that fixation step was the glutaraldehyde incubation (the authors seem to agree with this, as described in the methods (section 5.5) and in the legend of figure 2). So probably this section should be "Optimisation of post-fixation conditions". In any case, the authors should explain why they considered incubating cells in 70% ethanol, which from my point of view is completely counterintuitive. - Line 171: should read "satisfactory" not "perfectly satisfactory". - Lines 174 & 175: why were 3h 25 min and 4 h 50 min tested? I think that fluorescence imaging can be done in a shorter timeframe. - Lines 238-239: does this mean that IBV does not form ROs in LLC-PK1 cells?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
ACMI-D-23-00242 Antibody-labelling correlative light electron microscopy I wish to commend the authors for envisaging a new optimized pre-embedding protocol, which in turn, holds the potential to novel discoveries in the intricate localization of specific events with the spatiotemporal revelations, utilizing the revolutionary CLEM tool. Perhaps, authors have also highlighted that their protocol can be used in a laboratory workflow, where a dedicated CLEM is not available. Albeit, authors have revealed that their protocol was unsuitable for correlating sites of IBV RNA synthesis but had a greater potential for other cellular ultrastructures where a membrane-bound structure was not the focus of investigation. The manuscript takes up an interesting topic and the presented results are interesting and …
Comments to Author
ACMI-D-23-00242 Antibody-labelling correlative light electron microscopy I wish to commend the authors for envisaging a new optimized pre-embedding protocol, which in turn, holds the potential to novel discoveries in the intricate localization of specific events with the spatiotemporal revelations, utilizing the revolutionary CLEM tool. Perhaps, authors have also highlighted that their protocol can be used in a laboratory workflow, where a dedicated CLEM is not available. Albeit, authors have revealed that their protocol was unsuitable for correlating sites of IBV RNA synthesis but had a greater potential for other cellular ultrastructures where a membrane-bound structure was not the focus of investigation. The manuscript takes up an interesting topic and the presented results are interesting and pragmatic. Furthermore, the manuscript explores a new avenue in antibody-labeling with CLEM and presents their findings in a lucid manner giving appropriate discussion along with a greater scope for further improvisation of the protocol. A few minor clarifications can further improve the submission. 1. Whether there is a scope for revision of the title? As to include "a novel optimized pre-embedding protocol" or a high-yield technique" in antibody CLEM? 2. Furthermore, can the authors add a note in comparison of this new protocol (easily attainable) from the advanced hardware of an "Integrated CLEM platform like SECOM", which can further speed up the entire laborious process by minimizing the contamination of handling the specimen of interest in two different interfaces (FM & TEM)? Secondly, what were the initiatives taken in handling the specimens to minimize the contamination aspects of this optimized antibody labeling and imaging protocol? 3. Authors are suggested to write briefly in their discussion on the region of interest (ROI) and its user-friendly nature in grid workup, block trimming (for both confocal and EM) (Leica TCS SP8 or Leica Stellaris 5), or any improvisation they adapted in their CLEM facility. 4. As correctly pointed out in the membrane permeabilization step, "Antibody target of interest may influence further optimization", it would be prudent if the authors have some data/experience to support this. What were the antibody targets considered in this novel protocol other than BrU labeling of nascent viral RNA (unsuitable for authors' objectives as it limits visualizing BrU antibody immunofluorescence signal) Weather authors find any other finding (apart from their research question) which includes any other ultrastructure which has advantages over the above findings? 5. Authors have adapted "overnight OsO4 incubation combined with a UA incubation step" which indeed revealed great images. In Figure 2b and Figure 4 arrowheads (lipid droplet?) were labeled as "V" for white arrowhead, kindly amend the same. The improvised "15 min blocking step followed by a 30 min antibody incubation" i.e., rapid IF labeling and the minimum incubation indeed improved in preservation of cellular structure and yielded good images. Albeit, they couldn't reach a conclusion in their research question but with LLC-PK1 cells were infected with PDCoV and processed using the optimized protocol gave good results in ultrastructure (except a drawback of ROs was not being observed, which limited their comparison with both cell types) adding a finding that optimized protocol can have improved results based on the cell type utilized in CLEM, which expands the scope and utility of this protocol. Wishing the best! Dr. Vanam
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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