PONE-D-21-10897

Serial imaging of micro-agents and cancer cell spheroids in a microfluidic channel using multicolor fluorescence microscopy

PLOS ONE

Dear Dr. Kaya,

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.

Keeping in mind that novelty is not included in PLOS ONE publication criteria, please review the feedback from reviewers and make any clarifying revisions.  

Please submit your revised manuscript by Jun 13 2021 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: http://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,

Kristen C. Maitland, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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.

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

[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: Partly

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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: No

**********

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: In this manuscript the authors describe and evaluate a fluorescence microscope custom designed for efficient three-channel fluorescence imaging. The microscope design is thoroughly explained and presented by a diagram (Fig. 1). The custom multi-color microscope is demonstrated for imaging microparticles and cancer spheroids within channels and the results of characterization experiments for photobleaching, aberration artifacts, and spectral overlap are presented and discussed. The performance of the multi-color microscope is compared with a brightfield microscope and a traditional fluorescence microscope. Both qualitative and quantitative results are included.

Reviewer #2: Kaya et al presented a widefield microscopy scheme capable of imaging 3 fluorophores with distinct emission profiles. The developed system was demonstrated by imaging labeled tumor spheroid and fluorescent beads loaded in a microfluidic channel stained with a third fluorophore. My main concern is that the novelty of the manuscript is not convincing. The authors applied an existing microscopy technique to new samples, however no new biological findings are reported. Multicolor fluorescence imaging is very common and is extensively used in research labs.

Here are my other concerns/ suggestions:

1. Introduction, page 1, line 6-8. The authors state the advantages of using fluorescence microscopy. However the reasons stated can be true for any light microscopy technique and does not seem specific for fluorescence microscopy.

2. Introduction section, page 2, line 19-20, the authors mention that fluorescence microscopy is used for samples that are stained. This is not true as endogenous fluorophores or fluorescent proteins can imaged using this technique too. Also, rest of the paragraph consists cursory definition of fluorescence an its properties, which seems more appropriate for a tutorial style paper. Moreover, no references have been included.

3. HeLa cell spheroid with culture media does not “create a tumor micro-environment” which would involve other cells like immune cells, cancer associated fibroblast, extracellular matrix , nutrient, oxygen and pH gradients and so on. The authors develop a simplified 3D tumor model. Please consider re-phrasing

4. Please justify the diameters chosen for Hela cell spheroid and polystyrene beads. Are these clinically relevant values?

5. Why was the widefield microscope built with capability of imaging those 3 specific fluorophores? Development an adaptable system that could be used for multiple application would be more useful. Please justify.

6. Maybe I missed it but why was the microfluidic channel loaded with fixed HeLa cell spheroids instead of live cells, since fixed cells has no physiological relevance?

7. From figure 4(d1) it seems like there is bleed through of CellTracker Red CMTPX in the indocyanine green channel

8. Quantification of the data for cross-talk analysis would be more convincing along with the representative images shown.

9. The electrostatic interaction between beads and HeLa spheroids is confusing and does not provide additional data. The data shown are just images showing location of bead attachments on the spheroids. This does not necessarily provide information about the interaction.

10. Advantages over previously published multicolor widefield systems (eg: https://doi.org/10.1364/BOE.7.002285) should be discussed along with disadvantage or shortcomings of the presented technique. Also discuss the advantages of using a ‘round-robin’ detection scheme

**********

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

Reviewer #2: 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.

Serial imaging of micro-agents and cancer cell spheroids in a microfluidic channel using multicolor fluorescence microscopy

PONE-D-21-10897R1

Dear Dr. Kaya,

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,

Kristen C. Maitland, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: