PONE-D-23-14581

Comprehensive Tool for a Phase Compensation Reconstruction Method in Digital Holographic Microscopy Operating in Non-Telecentric Regime

PLOS ONE

Dear Dr. DOBLAS,

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Ireneusz Grulkowski, PhD

Academic Editor

PLOS ONE

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   "Funding. This research was partially funded by C. Trujillo’s and A. Doblas’ funding resources. C. Trujillo acknowledges the support provided by Vicerrectoría de Ciencia, Tecnología e Innovación from Universidad EAFIT. A. Doblas acknowledges the support provided by National Science Foundation (NSF) through her NSF CAREER grant (grant number 2042563)."

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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: Yes

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

Reviewer #1: N/A

Reviewer #2: N/A

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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: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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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: In the submitted manuscript, the authors present a computational method to process the phase images acquired with a non-telecentric, digital-holographic microscope. The proposed computational method is thoroughly described. Here are some suggestions.

In digital holographic microscopy, the phase modulation not induced by the sample can be easily and accurately subtracted using a background image, which is acquired for an empty field of view. The same background image can be used repeatedly for multiple sample images acquired afterward; thus, the process does not increase the data acquisition time much. The computational method proposed by the authors would be useful if it provides the same resolution and phase accuracy as the background subtraction method. That said, I’d strongly recommend the authors to perform a more thorough characterization of the proposed method.

- The resolution measurement has not been performed. This could be done by analyzing the edge of the star-target image (Figure 6).

- More important, the phase measurement accuracy needs to be shown using samples of known refractive index values (e.g., polymer microspheres in index-matching liquid).

- The low-frequency phase distribution in the background region (i.e., non-sample region) would be useful to confirm the effectiveness of the proposed method.

Reviewer #2: The authors report about a method for compensation of phase aberrations in quantitative phase imaging (QPI) with non-telecentric off-axis digital holographic microscopy (DHM). After an explanation of the underlying principles and characterization of the method by utilizing a phase test chart the application on a technical sample and red blood cells is illustrated.

In general, the manuscript is motivated, organized, an includes adequate references. The experimental investigations appear to be accurately performed. The results are plausible. The authors address an important topic in QPI with DHM: The compensation of spherical phase aberrations which may be of interest for the field of DHM and the interdisciplinary areas of QPI and label-free biomedical imaging. In summary, the content of the manuscript appears to be suitable for the journal PLOS one.

However, the authors should consider revisions:

1. Abstract: From the abstract the novelty aspects of the proposed phase compensation concept with respect to reference 35 becomes not fully clear. The authors should clarify the abstract concerning this topic.

2. Introduction:

a. To complete the description of the-state-of-the-art the authors may consider adding that non-telecentric arrangements can simplify the combination of DHM with commercial optical microscopes as, for example, reported in Drug Deliv. and Transl. Res. 12, 2207–2224 (2022).

b. In the last paragraph of the introduction the novelty aspects/extensions of the proposed phase compensation concept with respect to reference 35 become not fully clear. The authors should consider adding further clarifying details.

3. Section 2 “Off-axis Digital Holographic Microscopy operation in non-telecentric mode”:

a. The authors may consider removing “operation” from the section title.

b. In general, the explanations in this section include many details and are partly difficult to understand. The author may consider shortening and clarifying the text and to further emphasize the most important statements/topics.

c. Fig. 1: The authors show the sketch of a specific experimental arrangement in which sample illumination and reference wave are plane waves. However, in practice, e.g., a sample illumination via a condenser lens (for illustration see, e.g., ref. 35 of the manuscript or Drug Deliv. and Transl. Res. 12, 2207–2224 (2022)) or utilization of a spherical reference wave can also result in a spatial frequency spectrum as illustrated in Fig. 2 of the manuscript. The authors thus may consider adding a discussion concerning the possible transfer of their approach to a more general regime.

4. Section 3:

a. Figs. 3 and 5 (major point): From the explanations it becomes not fully clear how the spherical phase aberrations are compensated and what are the differences in the procedure reported in reference 35 of the manuscript. For, example: Is equation 10 multiplied or subtracted from phase images like shown in Fig. 5a? The authors should add substantial clarifying information concerning this topic (and perhaps may extend Fig. 4 for an additional illustrating/clarifying sub figure?).

b. Figs. 7 and 8: The investigated sample “wedding cakes” should be explained with more details. The authors may consider adding cross-section plots through the phase images in Figs. 8c and 9a.

c. 2nd paragraph below Fig. 7: The description concerning the minimization algorithms should be supported by data, perhaps, in a figure or table.

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Reviewer #1: No

Reviewer #2: No

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Comprehensive Tool for a Phase Compensation Reconstruction Method in Digital Holographic Microscopy Operating in Non-Telecentric Regime

PONE-D-23-14581R1

Dear Dr. DOBLAS,

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.

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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 #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 #2: Yes

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

Reviewer #2: N/A

**********

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 #2: Yes

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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 #2: Yes

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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 #2: (No Response)

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Reviewer #2: No

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