Dear Dr. Hsiai,

Thank you very much for submitting your manuscript "A Hybrid of Light-Field and Light-Sheet Imaging to Decouple Myocardial Biomechanics from Intracardiac Flow Dynamics" 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 three 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 manuscript would benefit from a better summary of the prior art and its current major limitations. The analysis needs more validation and/or analysis of the variation between specimens. Please also discuss more clearly the major original contributions of the manuscript and their biological significance. Finally, one reviewer noted that the Github repository was not accessible.

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,

Andrew D. McCulloch, Ph.D.

Associate Editor

PLOS Computational Biology

Daniel Beard

Deputy Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: Overall, this is a really interesting and important method to separate and measure the myocardial and hemodynamic kinematics of embryonic heart contractions, something that has typically been explored computationally or at a gross functional level. Understanding how contractility initiation and propagation corresponds with (and drives) local morphogenetic events is very important for understanding mechanisms of maturation and malformation. Although this current paper doesn't address either of those topics, the presented technique sets the stage for such questions to be answered in the zebrafish model to the degree that model system can recapitulate those events.

This being said, the current contributed data does not fully reflect the capability or potential of the measurement technique. The following concerns should be addressed to improve the validation and impact of the contribution:

1) The measurement approach appears to iterate through individual planar slabs of the myocardium. While this can create cubic voxels, it is unclear how accurate/precise the in-plane motions vs. out-of-plane deformations/velocities are within a specimen. Are these known? One simple way would be to re-analyze the same specimen by rotating either the scanning field or the specimen 90 degrees. Obtaining largely identical datasets would establish how locally variable that data is and whether it is biased to the plane of inquiry.

2) Related to #1, it is unclear how the local features measured in either tissue kinematics/velocities are shared in embryos of the same stage or across stages. This paper appears to measure the phenomena in an individual specimen, but does not compare across specimens for consistency across time/space. Shared features across embryos are more likely meaningful, as is an understanding of the variation present locally between embryos.

3) What is the developmental stage range that this technique can be used in the zebrafish? What are the failure modes that are encountered with respect to resolution, field of view, etc.? It doesn't appear that light penetration is the only limitation given the processing algorithms employed, but an understanding of these limitations would improve the proper use and interpretation of data from the approach.

Reviewer #2: This article describes a really nice technique to study the couple interaction of myocardial contraction and blood flow dynamics in the zebrafish heart (3 dpf). The technique combines light sheet microscopy and light field microscopy to simultaneously capture myocardial tissue contraction and red blood motion.

Comments and concerns (minor):

1. Deformable image registration, done to capture the myocardium dynamics, may not capture twisting dynamics of the heart tissue. Overall the image registration is best than nothing, but can be misleading too. Please acknowledge this in the discussion.

2. How was the 'retrospective' synchronization employed checked for accuracy?

3. After reading the description of the blood flow dynamics (results) I had the impression that there was no net flow, given that velocities were negative and negative displacement of red blood cells was about 50% (unless I misunderstood). It is mentioned that the dynamics is inefficient. However, when looking at the movies, the dynamics seem completely different. Blood flow advances and stops, but I cannot see significant backflow. What am I missing? Please describe the dynamics more accurately, or provide better movies.

4. Are there only very few red blood cells inside the ventricle? Is this accurate?

5. Comparing the tubular heart of the zebrafish heart analyzed to the mature heart of humans ('apex twisting') is not appropriate, I think. Heart structure is completely different, and while we can learn from the zebrafish, I feel this is not really a similarity to be drawn upon.

Reviewer #3: The review is uploaded as an attachment

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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: No: Other than code, the data is not available in a repository, it is states it is available upon request.

Reviewer #3: No: Github repository indicated by the authos (https://github.com/aaronzq/cardiac) was not accessible at the time of this review.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: 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.

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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

Attachments

Attachment

Submitted filename: Review_Wang_et_al_2020_PLOSCOMPBIOL.pdf

Dear Dr. Hsiai,

Thank you very much for submitting your manuscript "A Hybrid of Light-Field and Light-Sheet Imaging to Decouple Myocardial Biomechanics from Intracardiac Flow Dynamics" 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 inviting you to modify the manuscript according to the review recommendations. To be acceptable for publication, the English language usage needs to be improved. I recommend using an experienced English language proof reader and editor. The reviewers also expressed concern that not all review comments were fully addressed and that the biomechanical significance of the new methods is difficult to assess.

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,

Andrew D. McCulloch, Ph.D.

Associate Editor

PLOS Computational Biology

Daniel Beard

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: Overall the authors have been responsive to all of the reviewers comments, in particular with the generation of additional data with respect to multiple embryos to elaborate spatial/temporal variation in fluid and myocardial data. In their response to reviewers they acknowledge the challenge in scaling 3D/4D deformation (tissue strain) maps of the embryonic heart, but it is unclear whether those maps are indeed possible to be generated with the technology the authors have developed. It is unclear if their computational approach is capable of rendering fluid or solid biomechanical data as the title indicates. Fluid and tissue velocity profiles are interesting, but not the same as the functionally relevant tissue wall strain and shear stress. These parameters require reference frame decomposition and relative motion measurements between data points in the cloud, while single point velocity tracts (or averaging from a whole segment) does not. It is unclear to what degree of this biomechanically relevant data the authors claim their system is capable to do using the velocity data they currently have. If they indeed have point-to-point information of their solid boundaries and/or complete grids of fluid velocity data (as suggested in their vector maps), it should be straightforward to do. If not, how are their datasets digitized for analysis?

Reviewer #2: Concerns from reviewers 1 & 2 have mainly not been addressed. There is a response - but frequently not to the point and not directly addressing the reviewers concerns or aspects for clarification. As an example, the question of whether the retrospective synchronization was validated (reviewer 2, issue #2) is not addressed. I am guessing the answer is no. To validate you need to acquire images at different angles - including an angle that shows the wave propagation - the retrospective synchronization usually needs to be 'adjusted' with that information. A similar question, I believe, is asked by reviewer 1, (#1 and #2) and also not directly addressed.

Overall, however, answers to reviewer #3 seem to have been addressed. In particular the introduction and discussion, which have been completely re-written, seem to be much more focus on the actual contribution of the paper. These sections are now a really nice description of the methods employed and what is new in this paper - the combination of LSM and LSFM - to both capture myocardium motion and red blood cell velocities.

English needs significant revision - as several typos and grammatical issues can be found in the text. I would suggest that either the authors or the journal use an English Editing service before publication.

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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

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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

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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: Yes

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. Hsiai,

We are pleased to inform you that your manuscript 'A hybrid of light-field and light-sheet imaging to study myocardial function and intracardiac blood flow during zebrafish development' 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,

Andrew D. McCulloch, Ph.D.

Associate Editor

PLOS Computational Biology

Daniel Beard

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 response to comments, textual changes, and additional data address my concerns.

Reviewer #2: Authors have addressed all remaining concerns.

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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: None

Reviewer #2: Yes

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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