Peer Review History
Original SubmissionSeptember 29, 2020 |
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PONE-D-20-30666 Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging PLOS ONE Dear Silvio, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor points raised by one of the reviewer. Espacially, the methods section needs to be better developed as it lacks some information. Please submit your revised manuscript by december 15. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:
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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ********** 3. Have the authors made all data underlying the findings in their manuscript 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: Yes Reviewer #2: Yes ********** 4. Is the manuscript 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 note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 5. 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 Authors are presenting a protocol that enables the correlation between three imaging techniques. Starting from a fully established CLEM protocol based on sequential imaging with Light Microscopy (LM) and Electron Microscopy (EM) of thin resin sections of vitrified/freeze substituted resin embedded material, the Authors succeeded in analyzing the same sections with secondary ion mass spectroscopy (SIMS). The resulting method, CLEM-SIMS, offer the possibility to overlay to the EM ultrastructure the compositional information gained by nanoSIMS having also the chance to target specific cells or organelles due to the correlation with LM. As proof of principle the Authors imaged in a correlative manner cultured cells stained with MitoTracker or mito-mCitrine transfected. CLEM-SIMS approach showed that non-functional mitochondria (i.e. MitoTracker negative) despite having a similar morphology, have a molecular composition different from functional ones. This clearly shows the advantage of correlating analytical information with a CLEM approach over an independent correlation between SIMS and LM or EM alone. I agree with the authors that their protocol will be extremely useful to correlating SIMS signal to specific cellular structure and potentially to study turnover of proteins and molecule. I have only few technical concerns. 1)Uranyl acetate fluorescence The Authors claimed that the fluorescent signal observed in the DAPI channel is the signal from uranyl acetate ( see lines 122-123; 130 and 435). In a recent publication Tuijtel, M. W. et al. (DOI: 10.1038/s41598-017-10905-x) used indeed uranyl acetate (UA) fluorescence in order to correlate CLEM images. Instead of what the Authors are claiming Tuijtel, M. W. et al. reported that the fluorescent signal coming from UA is visible using filter set for GFP (excitation 470/40, emission 525/50) at cryogenic temperature, and far less intense at 21°C. Moreover the fluorescent signal arising from UA revealed the outline of the cells, nucleoli and mitochondria. In figure 2 of this manuscript instead the DAPI channel is highlighting heterochromatic regions. Could the authors comment on these discrepancies? 2)Material and Methods The authors should reported the excitation and emission parameter of the fluorescent filter sets and the imaging parameter used. Information of the type of transmission electron microscope used, camera and pixel sizes are missing. Image registration In the image registration chapter, the author should explain in more detail the procedure of alignment and ideally give an estimation of the alignment accuracy over the 3 imaging modalities. As suggestion the Authors could draw a line on structures visible in the 3 modalities (i.e mitochondria) calculate the full-with-half maximum of the intensity over the line for EM, LM and nanoSIMS in order to obtain an estimation of the alignment accuracy. Such images with their respective plots calculated from the signal of the 3 images modalities could be added as supplementary data. Minor comments. High pressure freezing/freeze substitution (HPF/FS). The authors correctly decide to avoid chemical fixation and process their sample with HPF/FS as they explained in the Discussion chapter. Nevertheless, in order to process the samples in HPF planchettes, the cells were detached using trypsin. In my opinion for further studies the Author should consider to use sapphire disc in order to vitrify cultured cells without the need of tripisinization. NanoSIMS compare to other analytical methods. Other analytical methods have been recently developed in the contest of a correlative approach. The author did a good effort in highlighting potentiality and pitfall of the technology they developed. For a reader would be useful also to compare this new methods with other correlative approached based on other analytical techniques (i.e. energy dispersive X-ray (EDX) or electron energy loss spectroscopy (EELS)). Reviewer #2: This is a very interesting and useful manuscript in which the authors present a novel and insightful methodology to correlate the ultrastructure of cells with the molecular composition of cell compartments. The technique is based in previous methodological advances achieved by the group of Dr. Silvio Rizzoli who leads this study. The novelty is the combination of electron microscopy and fluorescence microscopy with secondary ion mass spectrometry (SIMS) to visualize the localization of spots enriched in different types of natural isotopes, which is useful to, for example, detect specific organelles (such as mitochnodria o difference in chromatin organization). The group is pionneering here the implementation of high pressure freezing which is an advantage to investigate cellular ultrastructure under conditions that are better to preserve structures compared to conventional fixation methods. This is a methodological and useful study in which the conclusions are supported by high quality data. The authors explain and discuss the methods in detail. The paper is very nicely and clearly written and opens new perspectives to investigate specific aspects of the biology of the cell such as the aging of organelles. ********** 6. 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 [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. 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Revision 1 |
Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging PONE-D-20-30666R1 Dear Silvio, 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, Stephane Gasman Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ********** 4. Have the authors made all data underlying the findings in their manuscript 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: Yes Reviewer #2: Yes ********** 5. Is the manuscript 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 note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 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: (No Response) Reviewer #2: (No Response) ********** 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 |
Formally Accepted |
PONE-D-20-30666R1 Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging Dear Dr. Rizzoli: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Stephane Gasman Academic Editor PLOS ONE |
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