Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy

Jason Seth Rothman; Carolina Borges-Merjane; Noemi Holderith; Peter Jonas; R. Angus Silver

doi:10.1371/journal.pone.0277148

Peer Review History

Original SubmissionOctober 19, 2022
3 Jan 2023 Decision Letter - Shaokoon Cheng, Editor PONE-D-22-28847 Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy PLOS ONE Dear Dr. Rothman, 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 points raised during the review process. Please submit your revised manuscript by Feb 17 2023 11:59PM. 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. 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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 https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 3. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 study presents the results of original research. In the manuscript, the author has made great efforts to validate the stereological method with TEM. Both introduction and conclusions are well-written with sufficient and appropriate information provided to the scientific community. The validation of innovative technique is supported by using relevant assays and mathematical model, and the data is presented in a comprehensive manner using different figures and tables with appropriate statistical analysis performed. Reviewer #2: The manuscript evaluates the application of a pre-existing method for particles size measurement. The manuscript is well-written, however, it may require some amendments as explained below to improve before being accepted by the journal. 1. The manuscript requires a Conclusion section. Given that the authors have already mentioned about the limitations and assumptions of the particles size measurement methods focused in this research, they should discuss any further work needed to be done in the future to overcome such limitations. Or is there a need for a more conclusive method, which is not limited in terms of the particles' transparency, etc. 2. The manuscript may not be ‘validating’ the particles size measurement method, but rather ‘evaluating’ its applicability/ accuracy for certain particles studied in this work. The title and text may need to be revised from this aspect. 3. The abstract should be re-written and the main findings of the work need to be highlighted including the findings about “estimating the size and density of somata, nuclei, and vesicles in rodent cerebella”. 4. The introduction needs some amendments. Some terms such as ‘cap’, ‘section angle’, etc. could be briefly defined in the text to make the manuscript readable for readers of different backgrounds. Plus, the authors should include an explanation why they have selected certain particles for their study in the introduction. 5. The sentence in the lines 42-44 is incomplete and has to be re-written. The text should be thoroughly checked to avoid such issues. ****** 6. 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Revision 1
28 Feb 2023 Author Response 1. The manuscript requires a Conclusion section. Given that the authors have already mentioned about the limitations and assumptions of the particles size measurement methods focused in this research, they should discuss any further work needed to be done in the future to overcome such limitations. Or is there a need for a more conclusive method, which is not limited in terms of the particles' transparency, etc. We agree with Reviewer #2 and have added a Conclusions section to our manuscript (lines 1317-1337) including limitations of our methods, how the limitations can be overcome with technological developments and future directions for research. 2. The manuscript may not be ‘validating’ the particles size measurement method, but rather ‘evaluating’ its applicability/accuracy for certain particles studied in this work. The title and text may need to be revised from this aspect. We chose the word validation rather than evaluation as the former conveys the message that the method is valid, while the latter just specifies that a test was done without information on the outcome. We believe validation is justified given that we test the method both against 3D EM experimental data (now extended to include nuclei as well as vesicles; S9 Fig in S1 File; Table 3) and more generic Monte Carlo simulations. We believe the most novel and newsworthy aspect of our analysis is the model validation, which is why we highlighted it in the title. The words ‘validate’ and ‘verify’ have similar meaning and have been used extensively to describe the ‘verification’ of stereological methods, as cited in our manuscript on lines 160-161 [22,24,28,51]: [22] Saper 1996: “Stereological methods have since been validated and extended by many other workers”. [24] Geuna 2000: “Those who use a model-based approach usually have to provide a calibration study to verify the robustness of the model itself and the appropriate application of the method.” [28] Bartheld 2001: “We need a rigorous comparison and validation of 2-D and 3-D counting and its direct comparison with counts derived from 3-D reconstruction of serial sections.” [51] Coggeshall 1992: “Verification by serial-section methods: How can an investigator be sure estimates of neuronal or synaptic numbers using any particular method are unbiased? In my opinion, the only way is to verify the counts, which implies comparison against a standard. The obvious standard is numbers obtained from serial-section reconstructions”. 3. The abstract should be re-written and the main findings of the work need to be highlighted including the findings about “estimating the size and density of somata, nuclei, and vesicles in rodent cerebella”. We agree that the main findings of the work were not described sufficiently in our original abstract. To address this we have restructured and reworded the abstract. We have also included the definition of ‘cap angle’ in the abstract, which was originally referred to as ‘section angle’ and included a justification of its use. 4a. The introduction needs some amendments. Some terms such as ‘cap’, ‘section angle’, etc. could be briefly defined in the text to make the manuscript readable for readers of different backgrounds. The word ‘cap’ is defined on lines 115-117: “These latter particles whose north and south poles appear on the bottom and top of the section are known as ‘caps’.” The word is also defined in the legend of Fig 1 which is referenced in the same paragraph, in which case Fig 1 should be embedded within the Introduction of the final manuscript. We removed ‘section angle’ from the Abstract since this phrase is not used anywhere else in the manuscript. Instead, we have used ‘cap angle’. We updated our definition of ‘cap angle’ (θ) as follows (lines 134-135): “the half angle subtended by a particle’s cap from the particle’s center (Fig 1).” The cap angle is also defined in Fig 1 (legend and illustrations A and B) and the section “Definition of Key Terms”. Other key terms such as section thickness (T), mean particle diameter (μD) and section z-depth (ζ) are defined in the Introduction and Fig 1, in the definition of Eqs 1-3 and in “Definition of Key Terms”. 4b. Plus, the authors should include an explanation why they have selected certain particles for their study in the introduction. As requested, we added the following sentence to the last paragraph of the Introduction (lines 166-169): “Synaptic vesicles in MFTs and the nuclei and somata of GCs were chosen for the analysis since they contain a wide range of particle sizes and have high densities that are problematic for design-based stereological methods.” 5. The sentence in the lines 42-44 is incomplete and has to be re-written. The text should be thoroughly checked to avoid such issues. We believe this sentence is complete: it begins with the conjunction “Moreover”, linking this sentence with the previous sentence. We have completed a thorough proof-read of our manuscript for any other grammatical errors. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0277148.r002
2 Mar 2023 Decision Letter - Shaokoon Cheng, Editor Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy PONE-D-22-28847R1 Dear Dr. Rothman, 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, Shaokoon Cheng Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277148.r003
Formally Accepted
7 Mar 2023 Acceptance Letter - Shaokoon Cheng, Editor PONE-D-22-28847R1 Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy Dear Dr. Rothman: 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. Shaokoon Cheng Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277148.r004

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