Dear Dr. Giovannucci,

Thank you very much for submitting your manuscript "VolPy: automated and scalable analysis pipelines for voltage imaging datasets" for consideration at PLOS Computational Biology.

As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Both reviewers agree on the need for analysis tools for voltage-imaging data. Due to the nature of the underlying signals and the reporters, these data do pose a significant challenge for analysis, to detect real signals, disambiguate the cellular sources etc. This is obviously an evolving field since breakthroughs in reporters will change the requirements for analysis, hopefully making life easier. Both reviewers agree that the approaches taken in this article are reasonable given the data sets that they consider. The proposed pipeline is a collection of previously described approaches and techniques, not new techniques.

Both reviewers agree that the paper would have far higher impact if it included comparisons to existing methods, some of which have been tested on voltage imaging data. I agree and comparisons should be made to the tools mentioned by reviewer 1. Claims of validity of methods were based on a small number of annotators, so again, it is crucial to check claims and expected behavior against other methods and revise either the methods or claims if and when unexpected results appear.

There are also a large number of specific questions and concerns from reviewer 1, which must be addressed point by point.

Please also answer the following questions from the editor: 1. Were surrogate data sets used or can they be used to test validity of the methods? 2. Were model data sets with known ground truth spike times, cell identities, etc. constructed or used or can they be used to test the validity of methods?

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Corey Acker

Guest Editor

PLOS Computational Biology

Kim Blackwell

Deputy Editor

PLOS Computational Biology

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Both reviewers agree on the need for analysis tools for voltage-imaging data. Due to the nature of the underlying signals and the reporters, these data do pose a significant challenge for analysis, to detect real signals, disambiguate the cellular sources etc. This is obviously an evolving field since breakthroughs in reporters will change the requirements for analysis, hopefully making life easier. Both reviewers agree that the approaches taken in this article are reasonable given the data sets that they consider. The proposed pipeline is a collection of previously described approaches and techniques, not new techniques.

Both reviewers agree that the paper would have far higher impact if it included comparisons to existing methods, some of which have been tested on voltage imaging data. I agree and comparisons should be made to the tools mentioned by reviewer 1. Claims of validity of methods were based on a small number of annotators, so again, it is crucial to check claims and expected behavior against other methods and revise either the methods or claims if and when unexpected results appear.

There are also a large number of specific questions and concerns from reviewer 1, which must be addressed point by point.

Please also answer the following questions from the editor: 1. Were surrogate data sets used or can they be used to test validity of the methods? 2. Were model data sets with known ground truth spike times, cell identities, etc. constructed or used or can they be used to test the validity of methods?

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: attached

Reviewer #2: Voltage imaging with genetically encoded indicators is a powerful emerging technique for measuring neural activity. In this manuscript, Cai et al. describe a suite of computational tools – VolPy – for extracting time series proportional to voltage changes from voltage imaging datasets. VolPy builds upon past algorithmic developments from these authors to provide a scalable pipeline for motion correction, ROI segementation, spike detection, and denoising. This tool efficiently handles large voltage imaging datasets and should be extremely useful for labs establishing voltage imaging as an experimental technique. The authors do a commendable job benchmarking VolPy on several existing voltage imaging datasets and demonstrate generalization to new datasets differing qualitatively from those used for training.

Major comments:

1) In the most likely use case, labs implementing VolPy will annotate additional training data and re-train the Mask R-CNN for best results. While the authors include a brief description of this process in the Discussion, the manuscript would benefit from some additional description of the tools included in VolPy for this purpose in the materials and methods and results. I suggest the authors include some quantification of how precision, recall, etc. change with increasing amounts of training data from new datasets differing qualitatively from those used for initial training. This would help give prospective users a better idea of the time investment that would be needed for adoption.

2) The manuscript appears to lack analysis of how spike identification is improved by the modified Spike Pursuit algorithm compared to other simpler approaches.

Minor comments:

Line 198: “More in details” appears to be a typo and should be omitted or otherwise corrected.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions, please see http://journals.plos.org/compbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Giovannucci,

Thank you very much for submitting your manuscript "VolPy: automated and scalable analysis pipelines for voltage imaging datasets" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Corey Acker

Guest Editor

PLOS Computational Biology

Kim Blackwell

Deputy Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have done an impressive job addressing my comments and questions, improving GT annotations and implementing comparisons to competing methods, highlighting both computational scalability, as well as better performance in extracting spatial footprints and spikes. I recommend the paper be accepted for publication.

Small comment regarding Figure 3 – using red and green contours is probably not an accessible choice for color-blind readers.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

**********

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

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Suggested addition from the editor: Excellent job addressing the concerns of the reviewer and upgrading the paper and the tools. If possible, please add at least a discussion point to make sure readers are aware of fundamental differences between calcium and voltage imaging. Even though in practice it might not be that important, useful signal from voltage sensors are only from the cell membrane, not cytosol like most calcium sensors, and depending on details of the voltage sensor and imaging system (ex. resolution), this may or may not play an important role in how data are extracted/analyzed. I will leave the reviewer's point about figure line colors up to your discretion.

Dear Dr. Giovannucci,

We are pleased to inform you that your manuscript 'VolPy: automated and scalable analysis pipelines for voltage imaging datasets' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Corey Acker

Guest Editor

PLOS Computational Biology

Kim Blackwell

Deputy Editor

PLOS Computational Biology

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