PONE-D-24-10924zIncubascope: long-term quantitative imaging of multi-cellular assemblies inside an incubatorPLOS ONE

Dear Dr. Badon,

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 Aug 24 2024 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Florian Rehfeldt

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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. Thank you for stating the following financial disclosure: 

"This project was supported by grants from the French National Agency for Research (ANR-22-CE42-0019, ANR-21-CE18-0038, ANR-21-CE19-0029) and the Institut National du Cancer ( PLBIO 20-135 ). The authors also acknowledge the financial support from the Grand Research Program LIGHT Idex University of Bordeaux, the Graduate program EUR Light S & T PIA3 ANR-17-EURE-0027 and GdR ImaBio."

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"We thank all the other members of the BiOf (Bioimaging and Optofluidic) team, in particular Camille Douillet, Aur´elien Richard, Laetitia Andrique, L´eon Rembotte, Fernanda Lopez-Garcia, for beta testing the device and suggesting improvements. We are also grateful to all the colleagues that encouraged us to develop a user-friendly 

version of the zIncubascope by expressing enthusiastically their need for such an instrument. This project was supported by grants from the French National Agency for Research (ANR-22-CE42-0019, ANR-21-CE18-0038, ANR-21-CE19-0029) and the Institut National du Cancer ( PLBIO 20-135 ). The authors also acknowledge the

financial support from the Grand Research Program LIGHT Idex University of Bordeaux, the Graduate program EUR Light S & T PIA3 ANR-17-EURE-0027 and GdR ImaBio."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"This project was supported by grants from the French National Agency for Research (ANR-22-CE42-0019, ANR-21-CE18-0038, ANR-21-CE19-0029) and the Institut National du Cancer ( PLBIO 20-135 ). The authors also acknowledge the financial support from the Grand Research Program LIGHT Idex University of Bordeaux, the Graduate program EUR Light S & T PIA3 ANR-17-EURE-0027 and GdR ImaBio."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. Please upload a copy of Supporting Information Figure/Table/etc. S1-S5 Video which you refer to in your text on page 14/17.

[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

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

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

**********

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

**********

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 manuscript presents an open-source and low-budget compact bright-field microscope built with off-the-shelf parts, which easily fits in a standard cell culture incubator and is suitable for several days-long time lapse recording of live specimens. The authors describe the setup and subsequently showcase applications with encapsulated cells, cysts, and yeast.

The element of novelty appears to be not really high, as the authors already published a very similar setup. The major improvement in this work is the integration of a motorized z-axis to record image stacks in bright field. I would suggest to consider the publication of the manuscript upon the accomplishment of major changes.

The positive aspects of this work are:

- Compact system built with off-the-shelf parts, it could be assembled and tested in few days provided that all the needed information for part purchase and assembly is given (which is not the case in the present version).

- Apparently stable over time and able to record sufficiently long time-lapse data in bright-field.

- Any open-source, low-cost solution to perform basic time-lapse live imaging experiments with bright-field illumination is positive fort he scientific community, especially for low-budget laboratories.

Criticisms/recommendations

- With one microscope only one condition can be examined. This limits its applicability to specific cases, so it’s not as universal as claimed and the authors should discuss it more critically.

- A part list for the building of the microscope is missing. A person skilled in opto-mechanics and microscopy will be able to identify the off-the-shelf parts immediately. However, users with less experience in these fields, who are an important target of the paper, will find very useful a list of parts with catalogue number and price, and the authors should add it.

- The GitHub repository contains the code, CAD data, and experimental data (e.g., time-lapse data). However, there is no explanation whatsoever, tips and tricks, troubleshooting, etc. Inserting these details in the repository will make the adoption of the system, particularly by inexperienced users, much easier. I suggest to revise the repository adding a comprehensive explanation to accompany the material. Without this, the repository is not useful.

- It would be recommendable to give hints on how to upgrade the system, e.g., with a motorized stage. In fact, a motorized xy-stage would be important for the community of users, and make the device more „universal “. The authors may want to comment on this.

- The system seems to be quite incremental with respect to the previous publication by the same authors. All the professional objective lenses have the same parfocal length and tube lens distance, so the previous system could already be used with other objective lenses, I suppose. The authors should clarify this point.

**********

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

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

zIncubascope: long-term quantitative imaging of multi-cellular assemblies inside an incubator

PONE-D-24-10924R1

Dear Dr. Badon,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Florian Rehfeldt

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

**********

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

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

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

**********

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

**********

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)

**********

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

**********