Dear Dr. Vesna Bacheva,

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.

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Fentahun Adane Nigat, MSc., PhD

Academic Editor

PLOS ONE

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“National Science Foundation STC Center for Research on Programmable Plant System under grant number DBI-2019674.

V. Bacheva was supported by Schmidt Science Fellows, SNF Postdoc.Mobility (grant number 214477), and KIC Postdoctoral Fellowship”

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“National Science Foundation STC Center for Research on Programmable Plant System under grant number DBI-2019674.

V. Bacheva was supported by Schmidt Science Fellows, SNF Postdoc.Mobility (grant number 214477), and KIC Postdoctoral Fellowship”

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."

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“This project was supported by the National Science Foundation STC Center for Research on Programmable Plant System under grant number DBI-2019674. V. Bacheva was supported by Schmidt Science Fellows, SNF Postdoc.Mobility (grant number 214477), and KIC Postdoctoral Fellowship”

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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:

“National Science Foundation STC Center for Research on Programmable Plant System under grant number DBI-2019674.

V. Bacheva was supported by Schmidt Science Fellows, SNF Postdoc.Mobility (grant number 214477), and KIC Postdoctoral Fellowship”

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

6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comments (if provided):

Thank you for submitting your manuscript entitled “Low-cost, DIY programmable tissue processor for solvent exchange in biological sample preparation” to PLOS ONE. The reviewers and I recognize the novelty and practical relevance of your work, which presents an innovative and potentially impactful approach to affordable, open-source laboratory automation. However, several critical issues must be addressed before the manuscript can be reconsidered for publication.

Manuscript structure and organization

The current version does not fully adhere to the journal’s required structure. Please revise the manuscript to include clearly defined sections—Introduction, Materials and Methods, Results, Discussion, and Conclusion—with logical flow and sufficient methodological detail. Ensure that limitations are clearly discussed.

Title and scope

Revise the title to better reflect the evaluative and experimental nature of the study. For example, a title such as “Evaluation of a Low-Cost, DIY Programmable Tissue Processor for Solvent Exchange in Biological Sample Preparation” would more accurately convey the focus on device performance.

Validation of the software and hardware

Provide experimental validation data for both the control software and hardware system. This should include:

Software reliability: accuracy of timing and sequencing, error-handling capacity, and reproducibility.

Hardware performance: comparison with a commercial or standard tissue processor in terms of tissue quality, staining uniformity, and solvent exchange efficiency.

Quantitative results and appropriate statistical analyses should be presented to support the device’s claimed performance.

Quantitative performance assessment

Include clear performance metrics such as processing time, solvent exchange rate, and reproducibility across multiple runs. Comparative data will strengthen the technical credibility of the device.

Statistical and experimental details

Please specify the number of replicates (n), the statistical tests used, and any measures of variability (e.g., mean ± SD). These additions are necessary to validate reproducibility and reliability.

Safety, scalability, and reproducibility

Add a section discussing safety considerations (e.g., solvent vapor management) and the device’s scalability, including its ability to process multiple samples simultaneously. Mention expected lifespan and maintenance requirements for key components.

Context and literature

Expand the Introduction to include comparative context with other open-source automation tools (e.g., OpenTrons, microfluidic systems). Clarify the unique innovation of your design (e.g., whether it automates both dehydration and rehydration steps).

Figures, tables, and data presentation

Ensure all figures have complete legends, proper scale bars, and consistent numbering. Tables should include total cost estimates and availability of components. Verify that all figures are cited sequentially in the text.

Language, formatting, and references

Conduct a thorough grammatical and editorial revision to improve clarity and professional tone. Ensure consistent formatting of units, symbols, and reference style, and include DOIs for all references where possible.

Additional scientific context

As noted by one reviewer, please mention in the Introduction that imaging techniques are used not only for examining cell growth and responses but also for assessing cell death, to provide a more comprehensive context for the use of tissue imaging.

Once these revisions are completed, please provide:

A revised manuscript with all corrections clearly highlighted or tracked, and

A detailed point-by-point response addressing each reviewer comment and editorial note.

We believe that, with substantial revision and additional validation, this study could make a meaningful contribution to the field of open-source biomedical instrumentation.

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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?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: No

Reviewer #3: 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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Manuscript title: Low-cost, DIY programmable tissue processor for solvent exchange in biological sample preparation

General comments: Thank you for the opportunity to review this manuscript, which presents a novel and potentially valuable low-cost, DIY programmable tissue processor. The concept is highly relevant, as accessible and affordable instrumentation can significantly advance biological and medical researches. However, in its current form, the manuscript requires major revision before it can be considered for publication. The main drawbacks are lack of structural rigor and insufficient validation data. The following major flaws must be addressed to meet the scientific standards expected by this journal.

1. The current title is descriptive but does not accurately reflect the evaluative nature of the work that is required for a scientific publication. It should be revised to emphasize the performance assessment of the device. Consider a title such as ‘Evaluation of a Low-Cost, DIY Programmable Tissue Processor for Solvent Exchange in Biological Sample Preparation’.

2. The manuscript does not adhere to the standard structure (e.g., Introduction, Methods, Results, Discussion, Conclusion) required by the journal. The absence of clearly defined sections for Methods, Results, and a Discussion that without the study limitations makes it difficult to assess the validity and scope of the work. The manuscript must be thoroughly reorganized to meet these fundamental publication standards.

3. A critical omission is the validation of the custom-developed software. The manuscript should include data demonstrating the software's reliability, such as Accuracy- Does the software execute the programmed timing and sequence steps correctly and consistently? Error Handling - How does the software manage user input errors or hardware communication failures? Reproducibility- Are the programmed protocols reproducible across multiple runs and by different users?

4. The study lacks a quantitative performance evaluation of the hardware itself. To establish the device's utility, it is essential to compare its performance against a commercial or standard benchmark. This analysis should include: - Sensitivity- can the device consistently and effectively process a range of tissue types and sizes to a high-quality standard? Specificity/Robustness- are the results free from artifacts introduced by the device? Data comparing tissue morphology, staining quality, and processing consistency (e.g., against a commercial processor) is necessary. Performance Metrics- include data on processing time, solvent exchange efficiency, and consistency of results across multiple batches.

5. The authors are strongly encouraged to perform a comprehensive revision of the manuscript, ensuring it fully aligns with all specific formatting and content guidelines for the journal (PLOS ONE). This includes the incorporation of all standard scientific manuscript sections, a clear description of limitations, and the critical validation data outlined above.

Reviewer #2: Comments to the authors:

Title: “Low-cost, DIY programmable tissue processor for solvent exchange in biological sample preparation”

Manuscript Number: (PONE-D-25-48079)

Dear the editor of PLOS ONE Journal

I would like to express my sincere gratitude for the opportunity to review this manuscript and contribute my expertise to the advancement of scientific knowledge. I also wish to extend my appreciation to the authors for their valuable contribution in exploring an innovative and time-efficient technology for tissue processing. Their work addresses a significant need in histological research and diagnostics by proposing a method that has the potential to reduce the time, labor, and resource demands traditionally associated with conventional tissue processing procedures.

1. Title: “Low-cost, DIY programmable tissue processor for solvent exchange in biological sample preparation” (PONE-D-25-48079):

• Comment: The title is concise, descriptive, and captures the core innovation of the work “low-cost,” “DIY,” and “programmable” are strong keywords that attract interest in open-source hardware and biological sample preparation.

• Suggestions:

o Consider specifying the type of tissues or applications (e.g., “for histological and fluorescence microscopy sample preparation”) to make the scope clearer.

o Describe the “DIY” first and make formal contexts since journals like PLOS ONE prefer academic tone.

2. Abstract

• Comment: The abstract clearly summarizes the motivation, approach, design, and findings. It effectively emphasizes accessibility and cost reduction.

• Suggestions:

o The problem statement could briefly include quantitative efficiency (e.g., percentage reduction in manual time).

o Add a brief mention of validation results (e.g., comparable fluorescence intensity to manual staining).

3. Introduction

• Comment: The introduction is well structured, providing a logical flow from biological imaging needs to limitations of existing systems and the motivation for a low-cost alternative. References (1–16) are appropriate and current.

• Suggestions:

o Include a comparative statement with similar open-source automation tools (e.g., OpenTrons or other microfluidic DIY devices).

o Add a clear hypothesis or research question at the end of the section (e.g., “We hypothesize that a low-cost programmable tissue processor can achieve staining results equivalent to commercial systems”).

o Clarify the novelty, does the processor uniquely automate both dehydration and rehydration steps or simply adapt existing robotics principles?

o Check citation consistency (e.g., space before parentheses: “subsequent tissue clearing(6)” → “subsequent tissue clearing (6)”).

4. Design of the Automated Tissue Processor

• Comment: This section provides excellent technical clarity. Figures 1–2 support understanding.

• Suggestions:

o Include a brief cost breakdown within this section, not only in Table 1, to strengthen the claim of affordability.

o A risk assessment or safety note (e.g., ethanol vapor management) could improve completeness.

o Clarify scalability, can this handle multiple samples simultaneously?

o Add comparison with existing low-cost automation platforms for context (e.g., Arduino-controlled microfluidic systems).

5. Software Design and Operation

• Comment: Comprehensive description of the user interface and operational logic. The inclusion of a flowchart (Fig. 3) is excellent.

• Suggestions:

• Indicate whether the software includes error handling or alerts (e.g., empty solvent reservoirs).

• Consider including code versioning or repository DOI earlier for transparency.

• A short subsection on usability testing (who tested it, user feedback) could strengthen reproducibility claims.

6. Performance of the Tissue Processor

• Comment: Validation using propidium iodide (PI) staining on multiple plant species is appropriate and convincing. Quantitative fluorescence comparison (signal-to-noise ratio ~80) supports equivalency.

• Suggestions:

o Include statistical analysis (e.g., t-test comparing mean intensity between manual and automated samples).

o Provide replicate numbers (n) for reproducibility.

o Add discussion on long-term performance or maintenance stability (e.g., after repeated use).

o Minor: specify microscope model consistently (e.g., “Zeiss LSM 880 confocal microscope” should appear consistently, not “Zeiss u880”).

7. Design Considerations

• Comment: Good rationale for material and component selection.

• Suggestions:

o Add a table of solvent compatibility for quick reference.

o Include expected lifespan or maintenance recommendations for pumps and valves.

o Clarify whether temperature or humidity control is needed for reliable operation.

o Mention possible future modifications (e.g., incorporation of sensors or remote data logging).

8. Conclusion

• Comment: The conclusion effectively summarizes the study’s achievements and potential applications.

• Suggestions:

o Add a quantitative summary (e.g., cost reduction, processing time, and staining reproducibility).

o End with a forward-looking statement about potential expansion to animal tissues or integration with automated imaging systems. Example: “Future work could extend this platform to integrate real-time imaging or temperature-controlled sample handling.”

9. Materials and Methods

• Comment: Extremely detailed and well written, especially for reproducibility. The use of open-source files and a GitHub repository aligns with PLOS ONE’s data policy.

• Suggestions:

o For clarity, include subheadings under “Assembly Procedure” (e.g., “Control Box,” “Pump Module”).

o Add a short note on ethical or biosafety considerations if using plant materials.

o Ensure units and symbols follow consistent formatting (e.g., “°C,” “μm”).

o In “Electrical wiring,” specify any safety isolation between power and control circuits.

o Table 1 could include total estimated cost and country availability of parts.

10. Figures and Tables

• Comment: Figures are clear and informative. Table 1–3 provide high transparency.

• Suggestions:

o Ensure all figures include scale bars for microscopy images.

o Figures 1 and 7: add legends with abbreviations for clarity.

o Combine Fig. 2 (pump calibration) and a small chart comparing manual vs. automated results for visual impact.

o Verify all figure numbers are cited in the text sequentially.

11. References

• Comment: The reference list is recent, relevant, and includes both foundational and contemporary studies (e.g., PLOS ONE 2024 papers).

• Suggestions:

o Ensure reference consistency (some journal names italicized, others not).

o Add DOIs for all references where possible.

o A few references (e.g., “Tissue Processing Systems Market...”) are non-peer-reviewed and should be supported by primary sources if available. The status of the paper is ongoing.

o Include one or two recent citations (2023–2025) on open-source lab automation to contextualize innovation.

Overall Assessment

• Strengths:

o High reproducibility and transparency (open-source design, detailed methods).

o Excellent integration of engineering and biological applications.

o Figures and methods are well structured and clear.

• Weaknesses & Improvements:

o Limited statistical validation and quantitative comparison.

o Some redundancy across sections (especially Abstract vs. Conclusion).

o “DIY” phrasing may reduce perceived technical rigor.

o Lack of discussion on safety, error handling, and scalability.

Reviewer #3: Imaging techniques are not only used to examine the cell growth and responses but also to examine cell death. Therefore, please add this information under the introduction part. Also work on the grammatical and editorial corrections.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Dr. Hussen Abdu Muhidin

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Comments to the authors.docx

Design and evaluation of a low-cost, DIY programmable tissue processor for solvent exchange in biological sample preparation

PONE-D-25-48079R1

Dear Dr. Bacheva and co-authors,

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.

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Kind regards,

Fentahun Adane Nigat, MSc., PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: I thank the authors for their rigorous revision. My comments were fully addressed. I think the revised manuscript was ready for publication.

Reviewer #2: I would like to thank the authors for their commitment to address all of the comments I have given to them.

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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: Yes: Dr. Hussen Abdu Muhidin

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