PONE-D-19-27146

Near-infrared transillumination imaging combined with aperture photometry for the quantification of melanin in the iris pigment epithelium

PLOS ONE

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Dear Maciej Czepita,

Thank you very much for choosing PLOS ONE journal to submit your research about „Near-infrared transillumination imaging combined with aperture photometry for the quantification of melanin in the iris pigment epithelium „. It is a useful paper since a clinical point of view. Nevertheless, I would like to ask you questions and give you some comments in order to make clear your research:

-In the introduction, there is short description about role of the melanin in Adie’s tonic pupil (lines 85-88). The appropriate reference about it is missing.

-„A total of 4 patiens (7 eyes) were examined. A total of 4 patients (7 eyes) were examined. Three of the patients had pigment dispersion syndrome, while one patient suffered from Adies’ tonic pupil in one eye. Informed consent was obtained from each participant „ (lines 108-109). More patients should be examined. Information about age and sex is missing. The healthy patients also should be examined (as a control).

- „Description of the prototype apparatus” – there should be scheme how the whole system with sample looks like during measurement – not only single parts of the set up (Fig.1).

- How much time takes examination of the one eye? If there is some kind of adaptation to dark conditions before measurement?

- Line 134: „An exemplary near-infrared iris transillumination image can be seen in Fig 2.” Should be indicate A or B.

-Fig.2. – The scale is missing. Better is to superimpose both images.

-„The transmittance τ of the ocular media is estimated to be 0.9 in normal patients” (lines 153-154) – how did you estimate the transmittance?

- „A pupil diameter (de) of 0,7 cm was used as the examination was carried out under scotopic conditions in a dark room.”(lines 154-155) – why exactly 0,7 cm?

-„ The effective focal length of the eye f = 1.7 cm was used according to the model eye of Gulstrand.” (lines 156-157) – the reference is missing

- „By superimposing the dark frame from the images obtained from our patients the exact positions of the hot pixels could be seen (Fig 3).” (lines 173-175) There should be arrows to indicate examples of hot pixels.

- „precision of measurements”, line 195, please explain in the text, what ADU means.

- Table – 1, please explain in the text, what PDS means.

Reviewer #2: The following old and recent papers on transillumination for different applications should be considered in the introduction:

DOI: 10.1109/10.817628

DOI: 10.1364/OL.15.001179

DOI: 10.1109/TBME.2003.812188

DOI: 10.3390/s19040851

DOI: 10.1016/j.biosystemseng.2014.06.014

DOI: 10.1016/j.compag.2019.02.014

Usually transillumination refers to a setup in which light source and detector are one in front of the other. In this case light source and detector are at 90°. Would you be able to comment on the differences? Are you actually detecting side scattered photons? Or is there another way to describe the phenomenon?

Why CCD and not a CMOS? There are CMOS sensors with enhanced responsitivy in the NIR.

Did you need dark room to ensure that the pupil is dilated or to avoid camera saturation with ambient light? In this second case, a narrow optical filter in front of the camera should be sufficient to select only the 940 nm photons

You need to define the acronyom ADU (Analog to Digital Unit) and explain the meaning of it in your experiments.

In many places the authors claim that their measurements enable the quantification of the

melanin. But I do not see any results reporting the exact amount of melanin. Only ADU values are reported but they are not indicating the amount of melanin. So the authors should solve this issue.

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Reviewer #1: No

Reviewer #2: No

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Near-infrared transillumination imaging combined with aperture photometry for the quantification of melanin in the iris pigment epithelium

PONE-D-19-27146R1

Dear Dr. Czepita,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Ireneusz Grulkowski, PhD

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

Reviewer #2: All comments have been addressed

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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: (No Response)

Reviewer #2: (No Response)

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

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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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: (No Response)

Reviewer #2: (No Response)

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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: (No Response)

Reviewer #2: (No Response)

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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)

Reviewer #2: (No Response)

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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

Reviewer #2: No