Dear Dr. Lagache,

Thank you very much for submitting your manuscript "Robust tracking of single neuron calcium dynamics in behaving Hydra" 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.

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,

Boris S. Gutkin

Associate Editor

PLOS Computational Biology

Lyle Graham

Deputy Editor

PLOS Computational Biology

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

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The review is uploaded as a PDF attachment.

Reviewer #2: Lagache and colleagues present a complete user-friendly software package for tracking neurons during short calcium recordings in moving and deforming hydra. They demonstrate that their method can track neurons even when those neurons are inactive, without the need for an additional fluorescent marker. The development of effective algorithms that are integrated into user-friendly and open source software packages like Icy is very valuable for the community.

There are two substantive concerns.

1) It is unclear how the method performs on recordings longer than 25 seconds.

I could not find any description of the effective recording length for the validation performed against synthetic data with empirically derived noise, so I don't know if that was greater than 25s. But it is important to show that the method can work over longer recordings because calcium recordings are often many minutes long, and the sequential nature of this method would seem to be particularly susceptible to accumulating errors over time. For example, I might reasonably expect to accumulate errors in stitching tracks together such that after a few minutes a significant fraction of neurons may now be assigned different labels at the end of the recording compared to the beginning. One way the authors could address this concern is by showing how performance degrades over recording length when validated against realistic synthetic data.

2) Authors should clarify novelty and correct description of a previous method.

A key selling point of the method is that it uses deformation defined by tracked neurons to infer the location of missing neurons during gaps. The text highlights this method extensively, as if it were novel. But method [9], Ngueyn et al, explicitly performs the same step. Like this method, [9] uses thin plate splines of tracked neurons to infer the position of missing neurons, see main text accompanying Fig 5 in that reference [9].

The authors make a special point to explicitly compare to reference [9] in detail (for example on p. 7) and they also compare to many other methods in Table 1 and throughout the manuscript, all of which is commendable. However, they incorrectly claim that [9] is " designed to link two sets of particle detections in consecutive time frames." But this is incorrect. The method in [9] is time independent, in the sense that all the recorded volumes could be shuffled in time without changing tracking performance. Similarly, Table 1 incorrectly states that ref [9] is not robust to long gaps. On the contrary, because it is time independent, ref [9] is robust to exceptionally long gaps.

The authors should clarify novelty and correct descriptions of the previous method.

Additional technical concern:

There seems to be an inconsistency with Figure 4. It shows calcium activity lasting 1,400 s or 23 mins, whereas the main text and Table 3 suggests that recordings lasted less than 2 mins (1000 frames @ 10hz). Please resolve this inconsistency.

**********

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: None

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: review_PCB_Lagache_2020.pdf

Dear Dr. Lagache,

Thank you very much for submitting your manuscript "EMC2: A versatile algorithm for robust tracking of calcium dynamics from individual neurons in behaving animals" 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 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.

In particular, it is necessary that you address the concerns of Reviewer #2 regarding the relevant time scale for the method, which is a critical factor for it's experimental application.

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,

Boris Gutkin

Associate Editor

PLOS Computational Biology

Lyle Graham

Deputy Editor

PLOS Computational Biology

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

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 more than adequately adressed all my relevant comments. The addition of a second test case and the exploration of the performances of EMC2 with respect to the fraction of stable cells, cell density and time of recording both help in demonstrating the versatility of the method. I believe the paper will acquire greater visibility and applicability as it now stands. I also appreciated the clarifications the authors made on the stitching procedure both in the Methods section and in modifying Fig1. Finally, quantification of performances of the different methods in terms of reconstructed tracks is very instructive and allows for a rational comparison. I strongly support publication of this paper.

Reviewer #2: In this revision Lagache and colleagues have made important revisions and clarifications. It remains unclear, however, whether the method can track neurons in hydra over a sufficiently long timescale to be useful.

The authors specifically motivate their work by the need to "achieve robust and automatic tracking of individual cells over long time-lapse sequences." Most calcium activity experiments are for at least a few minutes, and recent published work by this group recorded calcium activity in moving hydra for 2 hours (Yamamoto and Yuste 2020, see Fig 3). It is therefore imperative that the authors show what accuracy to expect on the 5 min timescale and beyond.

With synthetic data, the authors now show that their method's accuracy drops from 97% for 250 frames (presumably 25s) to an accuracy of ~80% for 1000 frames (presumably 100s). It seems possible that accuracy will continue to drop dramatically as the timing approaches 5 mins.

Related: synthetic data is reported in terms of frames, but the authors should instead report the equivalent time in seconds, based on the timescale of the motion in the synthetic data. The authors should make this easy to find in the text and figure or caption.

If the method is unable to achieve reasonable accuracy on the multiple minutes timescale, the authors should scale back their claims, and it may also reduce the significance of the work.

**********

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: None

**********

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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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Lagache,

Thank you very much for submitting your manuscript "EMC2: A versatile algorithm for robust tracking of calcium dynamics from individual neurons in behaving animals" 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. In particular the authors must address the issues brought up by reviewer 1 on the limitations of the method for extended recording times that had come up after the last revision. Indeed, in order to meet the editorial requirements of PLoS CB it is necessary to show that the method presents a significant advance on existing 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,

Boris S. Gutkin

Associate Editor

PLOS Computational Biology

Lyle Graham

Deputy Editor

PLOS Computational Biology

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

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: I still support the publication of this paper.

Reviewer #2: With further simulation studies, the authors now show that their method's performance degrades as recording duration increases. For Hydra, the method is only suitable for very short recordings (<5 min timescale). This contradicts claims made in the abstract and throughout the text related to "long time lapse sequences" or "long-term" monitoring. Claims about closing potentially "long" gaps also lack evidence. In short there is a disconnect between many of the authors' claims and the evidence.

Evidence does not support the authors claim that that this method "greatly supersedes existing approaches." In contrast, other methods (NeRVE from Ngueyn et al. 2017; Conditional Random Fields from Chaudhary et al. elife 2021) are fundamentally time-independent and thus maintain the same performance regardless of recording duration. The authors need to revise their comparisons to other methods with a frank assessment of this limitation, which seems to arise from the sequential time-dependent nature of the algorithm. For example, it would be relevant to report in Table 1 whether each algorithm is time-dependent or not.

Crucially, the authors need to better justify why this method is meritorious despite being limited to only very short recordings. To be compelling the authors should provide specific use cases, and also include a discussion of why and how errors accumulate with time and what fundamentally about the algorithm imposes this limitation.

Minor, but important: it is imprecise to claim that accuracy "remained above ~70% for all animals" when Table 3 reports accuracy of 66.7% and 67.4% for two of the three animals.

**********

Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code 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 and code 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 or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: None

**********

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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: Yes: Olivier Cochet-Escartin

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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Lagache,

Thank you very much for submitting your manuscript "EMC2: A versatile algorithm for robust tracking of calcium dynamics from individual neurons in behaving animals" 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 follow the suggestions laid out by the reviewer 2.

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,

Boris S. Gutkin

Associate Editor

PLOS Computational Biology

Lyle Graham

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: I thank the authors for the clarifications of time-dependent and time-independent methods following Reviewer 2's comments. I feel these clarifications now clearly define both the advantages and limitations of the EMC2 method in comparison with previously developped solutions. By clearly discussing in which situations, such as Hydra neuronal tracking, EMC2 provides improvement from existing methods, the authors now clearly provide the reader with both a novel tracking method and rational arguments for using it, or not, depending on her/his own experimental data.

Reviewer #2: The authors have taken important steps to bring their claims more into alignment with the evidence they present, but some problematic language remains.

• The phrase “Long time lapse sequences” still appears on p. 4 This should be removed because it implies that the method will address the “long time lapse” case, when it does not.

• The placement of new language about “…premature ending of a track due to missing detection(s), can lead to important error propagation” on p. 8 is problematic. That text appears in subsection “2 - Limitations of Standard SPT Algorithms,” but the authors claim incorrectly that EMC^2 overcomes those limitations of standard SPT algorithms in subsection “3 – EMC^2 Algorithm”: “To overcome these limitations, we have developed EMC2…” The authors need to revise this framing so that they do not incorrectly claim that they have overcome the limitations on accumulation of errors over time.

• The claim (p.16) that EMC^2 is robust “independent of the type and complexity of particle motion” is not supported by the data. Indeed if this were true it would be extraordinary. I suspect that the performance of EMC^2, like all time-dependent tracking algorithms before it, is dependent on the amount and type of motion per frame, and with enough motion per frame its performance would degrade precipitously. Please qualify this claim or provide evidence to support it.

Minor, but important:

• This statement on p. 7 incorrectly implies that the sparse detectability of neurons is uniquely problematic for Hydra and not C. elegans or other organisms, when in fact this depends only on whether the animal is expressing a reference fluorophore like RFP, and is not an inherent property of the organism. Please reword and clarify.

“while the aforementioned mapping methods used in C. Elegans can accommodate moderate changes in the total number of neurons between frames due to missing or spurious detections (counting noise), the intermittent and sparse detectability of neurons in calcium imaging definitely hinders the applicability of mapping methods to Hydra [7]”

• Subsection “4 - Validation of EMC^2”: “We used the first 250 frames” p.13 The authors should specify in the main text the duration in seconds of these 250 frames (Perhaps 25 seconds?) This information is important for the reader to interpret the method’s performance.

• In the abstract the authors write: “Our results prove…”. The statement of proof is unnecessarily, and in my opinion, inappropriately strong. Best usage would be to reserve proofs for mathematical proofs. Please revise.

**********

Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code 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 and code 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 or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: 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

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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

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.

Dear Dr. Lagache,

We are pleased to inform you that your manuscript 'Tracking calcium dynamics from individual neurons in behaving animals' 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,

Boris S. Gutkin

Associate Editor

PLOS Computational Biology

Lyle Graham

Deputy Editor

PLOS Computational Biology

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

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #2: The revisions have adressed my concerns. Thank you.

**********

Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code 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 and code 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 or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: None

**********

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Reviewer #2: No