Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review focuses on the development of fluorescence methods in neuro-oncology. The most commonly used fluorescent dyes are presented. However, a detailed analysis of each dye, as well as a comparison based on statistical data, are practically absent. Unfortunately, the study lacks specific information on the sources and receivers of fluorescence and backscattered light from brain tumors and their immediate surroundings. The wavelengths of excitation, fluorescence, and scattering of fluorophores, as well as which ones are used for fluorescence navigation, are not provided. An analysis of the influence of tumor-associated microglia and macrophages on the fluorescence properties of both the tumor itself and its immediate surroundings is necessary. It would be useful to present the specific fluorescence lifetimes of the dyes used depending on tumor grade. Recommendations for the use of specific technologies for specific tumor types would be desirable.

Author Response

Comment:

This review focuses on the development of fluorescence methods in neuro-oncology. The most commonly used fluorescent dyes are presented. However, a detailed analysis of each dye, as well as a comparison based on statistical data, are practically absent. Unfortunately, the study lacks specific information on the sources and receivers of fluorescence and backscattered light from brain tumors and their immediate surroundings. The wavelengths of excitation, fluorescence, and scattering of fluorophores, as well as which ones are used for fluorescence navigation, are not provided. An analysis of the influence of tumor-associated microglia and macrophages on the fluorescence properties of both the tumor itself and its immediate surroundings is necessary. It would be useful to present the specific fluorescence lifetimes of the dyes used depending on tumor grade. Recommendations for the use of specific technologies for specific tumor types would be desirable.

Response 1

Thank you very much for this valuable and highly constructive comment. We agree that the previous version did not sufficiently discuss the optical and biological principles underlying fluorescence-guided neurosurgery. Accordingly, the manuscript has been substantially revised to provide a more comprehensive overview of the currently available fluorophores and their clinical applications.

Specifically, we have:

• expanded the description of the physical principles of fluorescence imaging by adding the excitation and emission wavelengths of the most commonly used fluorophores (5-ALA/PpIX, fluorescein sodium, and indocyanine green), together with their respective fluorescence navigation systems;
• added a new section describing the role of reflected and backscattered light in optical imaging and its contribution to tissue discrimination;
• included a discussion of the influence of the glioma microenvironment, particularly tumor-associated microglia and macrophages, on fluorophore accumulation, vascular permeability, and fluorescence heterogeneity;
• expanded the discussion of fluorescence lifetime imaging (FLIM), emphasizing its potential role in differentiating tumor tissues based on fluorescence decay characteristics rather than fluorescence intensity alone;
• added information regarding the relationship between fluorescence characteristics and tumor grade, particularly for 5-ALA-induced protoporphyrin IX fluorescence;
• expanded the discussion of the advantages and limitations of each fluorophore and included practical recommendations regarding the preferred use of 5-ALA, fluorescein sodium, and indocyanine green for different neurosurgical indications and tumor types.

These revisions can be found in the revised manuscript in Section 3.1 (Principles of Optical Imaging in Neurosurgery), Section 3.2 (Fluorescence-Guided Surgery in Neurosurgery), and Section 5 (Future Perspectives). All newly added text has been highlighted in the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This review provides a comprehensive overview of the application of fluorescence guided surgery (FGS), optical navigation, augmented reality, and artificial intelligence assisted imaging technologies in neurosurgery. The paper has a clear structure and certain reference value.

1）Most of the references are from 2020-2025, and the latest developments in related fields in the past year can be appropriately added. Early foundational literature can also be supplemented appropriately.

2）The abstract mentioned that the fluorescence specificity is an important controversy, but there seems to be no dedicated review of the relevant solutions later. Section 3.2.1 indicated “5-ALA has important limitations. False-positive and false-negative fluorescence remain significant challenges, particularly in low-grade gliomas and deep-seated lesions.”Here, some relevant progress should also be supplemented with some targeted information.

3）Standardization issues are only mentioned in the limitations and current challenges, lacking a summary of standardized clinical operating procedures. 

Author Response

Comment 1

Most of the references are from 2020–2025, and the latest developments in related fields in the past year can be appropriately added. Early foundational literature can also be supplemented appropriately.

Response 1

Thank you for this valuable suggestion. We agree that the manuscript benefits from the inclusion of both the most recent evidence and landmark studies. Accordingly, we have updated the reference list by incorporating several recent publications from 2025–2026 describing advances in fluorescence-guided surgery, quantitative fluorescence imaging, fluorescence lifetime imaging, multimodal optical imaging, and artificial intelligence-assisted intraoperative visualization. In addition, several foundational studies have been included to provide historical context and highlight the evolution of fluorescence-guided neurosurgery. All newly added references and corresponding text have been highlighted in red.

Comment 2

The abstract mentioned that the fluorescence specificity is an important controversy, but there seems to be no dedicated review of the relevant solutions later. Section 3.2.1 indicated “5-ALA has important limitations. False-positive and false-negative fluorescence remain significant challenges, particularly in low-grade gliomas and deep-seated lesions.” Here, some relevant progress should also be supplemented with some targeted information.

Response 2

Thank you for this important comment. We agree that the previous version discussed the limitations of fluorescence specificity without adequately presenting current strategies aimed at overcoming these challenges. Therefore, we have expanded Section 3.2.1 (5-Aminolevulinic Acid) by adding a new paragraph summarizing recent developments in quantitative fluorescence imaging, fluorescence lifetime imaging (FLIM), spectroscopic analysis, artificial intelligence-assisted fluorescence interpretation, and multimodal optical imaging approaches combining fluorescence with Raman spectroscopy and hyperspectral imaging. We also discuss the development of novel tumor-targeted fluorophores as a promising strategy to improve fluorescence specificity. These additions provide a more balanced discussion of both the current limitations and emerging solutions. All revisions have been highlighted in red.

 

Comment 3

Standardization issues are only mentioned in the limitations and current challenges, lacking a summary of standardized clinical operating procedures.

Response 3

Thank you for this constructive suggestion. We have revised the Limitations and Current Challenges section by expanding the discussion on current clinical standardization of fluorescence-guided neurosurgery. The revised manuscript now briefly summarizes commonly adopted clinical operating procedures, including the standard administration protocols for 5-ALA, fluorescein sodium, and indocyanine green, the use of dedicated optical filter systems, correlation of fluorescence findings with preoperative MRI and neuronavigation, and systematic intraoperative interpretation of fluorescence signals. We further discuss ongoing efforts to improve standardization through multicenter studies, quantitative fluorescence analysis, and artificial intelligence-assisted image processing. These additions have been highlighted in red.

Reviewer 3 Report

Comments and Suggestions for Authors

1. How were the studies included in this narrative review identified, selected, and evaluated, and could the authors provide a structured literature search methodology?

2. Can the authors include quantitative clinical outcome data demonstrating the impact of optical navigation and fluorescence-guided surgery on surgical precision and patient outcomes?

3. How do 5-ALA, fluorescein sodium, and indocyanine green compare in terms of sensitivity, specificity, tissue penetration, and clinical indications?

4. What are the major limitations and sources of error associated with fluorescence-guided neurosurgery, and how do they affect intraoperative decision-making?

5. Could the authors expand the discussion on artificial intelligence applications by including recent deep learning and real-time image analysis approaches?

6. How do the costs, infrastructure requirements, and accessibility of advanced optical technologies influence their adoption in different healthcare settings?

7. Can the authors provide a summary table comparing the advantages, disadvantages, and clinical applications of the major optical imaging modalities discussed?

8. What evidence exists regarding the long-term patient outcomes and survival benefits associated with these visualization technologies?

9. How close are emerging technologies such as Raman spectroscopy, holographic navigation, and nerve-specific probes to routine clinical implementation?

10. What future research directions and technological developments are most likely to overcome the current challenges of standardization, fluorescence specificity, and interoperability?

11. The authors are encouraged to strengthen the discussion on emerging optical sensing, graphene-enabled imaging platforms, and AI-assisted biomedical technologies by citing relevant recent studies, including:

    1. Design of Split Ring Resonator Graphene Metasurface Sensor for Efficient Detection of Brain Tumor (2024).
       

Author Response

1. How were the studies included in this narrative review identified, selected, and evaluated, and could the authors provide a structured literature search methodology?

Response

Thank you for this valuable comment. To improve the transparency and reproducibility of the review, we have expanded the Materials and Methods section by providing a more structured description of the literature search strategy. In addition to the searched databases, search terms, inclusion and exclusion criteria, we now describe the criteria used to evaluate the retrieved studies, including their methodological quality, level of clinical evidence, and scientific relevance. We also clarify that, owing to the narrative nature of the review, no formal risk-of-bias assessment or meta-analysis was performed. These revisions have been incorporated into the Materials and Methods section and highlighted in blue.

2. Can the authors include quantitative clinical outcome data demonstrating the impact of optical navigation and fluorescence-guided surgery on surgical precision and patient outcomes?

Response

We thank the Reviewer for this excellent recommendation. We agree that the inclusion of quantitative clinical outcome data enhances the clinical relevance of the review. Accordingly, we have added a new comparative table (Table 1) summarizing the principal fluorophores currently used in fluorescence-guided neurosurgery, including their optical characteristics, mechanisms of action, clinical indications, representative quantitative clinical outcomes, major limitations, and key references. The table includes landmark evidence such as the improved gross total resection and progression-free survival achieved with 5-aminolevulinic acid (5-ALA), representative gross total resection rates reported for fluorescein sodium, and quantitative intraoperative vascular assessment outcomes obtained with indocyanine green (ICG). In addition, the table incorporates recent literature (2025–2026) to provide an updated overview of current clinical evidence. We believe this new summary substantially improves the readability of the manuscript and provides readers with a concise evidence-based comparison of the most widely used fluorophores in contemporary neurosurgical practice. All additions have been highlighted in blue.

3. How do 5-ALA, fluorescein sodium, and indocyanine green compare in terms of sensitivity, specificity, tissue penetration, and clinical indications?

Response

To facilitate comparison between the principal fluorophores currently used in fluorescence-guided neurosurgery, we have expanded Table 1 to include their relative sensitivity, specificity, tissue penetration characteristics, and principal clinical indications, in addition to representative quantitative clinical outcomes and major limitations. We believe these additions provide a clearer and more comprehensive comparison of the currently available fluorescence agents.

4. What are the major limitations and sources of error associated with fluorescence-guided neurosurgery, and how do they affect intraoperative decision-making?

Response

The major limitations and potential sources of error associated with the principal fluorophores have now been summarized in the newly added Table 1, including factors affecting fluorescence specificity, tissue penetration, and false-positive or false-negative findings. In addition, we have included a brief statement emphasizing that fluorescence findings should always be interpreted in conjunction with neuronavigation, preoperative imaging, anatomical landmarks, and surgical experience to support intraoperative decision-making. All modifications have been highlighted in blue.

5. Could the authors expand the discussion on artificial intelligence applications by including recent deep learning and real-time image analysis approaches?

Response

In response, we have expanded Section 3.4 (Artificial Intelligence and Computational Optical Neurosurgery) by incorporating a more comprehensive discussion of recent advances in deep learning and real-time intraoperative image analysis. Specifically, we have included the emerging role of convolutional neural networks (CNNs) and transformer-based architectures for automated tissue segmentation, fluorescence signal quantification, tumour boundary delineation, and identification of infiltrative tumour margins. We further describe the integration of artificial intelligence with fluorescence imaging, neuronavigation, operative microscopy, preoperative magnetic resonance imaging, and spectroscopic data to support real-time intraoperative image interpretation and surgical decision-making. Finally, we emphasize that these technologies are intended to complement rather than replace the surgeon's expertise and discuss their current stage of clinical validation and future prospects for routine neurosurgical practice. All modifications have been highlighted in blue.

 

6. How do the costs, infrastructure requirements, and accessibility of advanced optical technologies influence their adoption in different healthcare settings?

Response

We have expanded the Limitations and Current Challenges section by discussing the economic, infrastructural, and organizational factors that influence the implementation of advanced optical technologies in clinical practice. The revised manuscript now addresses the impact of equipment acquisition and maintenance costs, the need for specialized imaging platforms, computational infrastructure, multidisciplinary training, and institutional expertise, as well as the differences in accessibility between tertiary referral centres and resource-constrained healthcare settings. We also highlight that continued technological simplification, cost reduction, and standardized imaging platforms are expected to facilitate broader clinical adoption.

7. Can the authors provide a summary table comparing the advantages, disadvantages, and clinical applications of the major optical imaging modalities discussed?

 

Response

To improve the readability of the manuscript and facilitate comparison between the principal optical imaging modalities, we have added a new summary table (Table 2). The table compares the major optical imaging techniques discussed in this review with respect to their underlying principles, principal advantages, limitations, and current clinical applications. We believe this overview provides readers with a concise and practical summary of the strengths and weaknesses of the currently available optical imaging technologies.

8. What evidence exists regarding the long-term patient outcomes and survival benefits associated with these visualization technologies?

Response

The revised manuscript emphasizes that the strongest evidence is available for fluorescence-guided resection of high-grade gliomas, where improved extent of resection and progression-free survival have been demonstrated, whereas definitive independent overall survival benefits remain to be confirmed. We also note that, for other emerging optical imaging modalities, current evidence primarily supports improvements in diagnostic accuracy and surgical precision, while robust long-term survival data are still limited.

 

9. How close are emerging technologies such as Raman spectroscopy, holographic navigation, and nerve-specific probes to routine clinical implementation?

Response

Bevore table 2. we have added a concise paragraph discussing the current stage of clinical translation of emerging optical technologies. The revised manuscript highlights that Raman spectroscopy is the closest to routine clinical implementation, supported by increasing clinical evidence and the development of clinically applicable imaging platforms, whereas holographic navigation systems and nerve-specific fluorescent probes remain promising technologies that require further prospective validation, workflow standardization, and broader regulatory approval before widespread adoption. These additions have been highlighted in blue.

 

10. What future research directions and technological developments are most likely to overcome the current challenges of standardization, fluorescence specificity, and interoperability?

Response

Rather than introducing a separate discussion, we have revised the opening of the Future Perspectives section to explicitly describe how emerging technological developments—including multimodal optical imaging, artificial intelligence, improved interoperability, and next-generation imaging platforms—are expected to address the current challenges of standardization, fluorescence specificity, and clinical integration. These revisions have been highlighted in blue.

11. The authors are encouraged to strengthen the discussion on emerging optical sensing, graphene-enabled imaging platforms, and AI-assisted biomedical technologies by citing relevant recent studies, including:Design of Split Ring Resonator Graphene Metasurface Sensor for Efficient Detection of Brain Tumor(2024).

Response

We have expanded the Future Perspectives section by briefly discussing emerging graphene-based biosensing technologies as a promising complement to future optical imaging approaches. We have also incorporated the recommended 2024 study and highlighted the potential role of graphene-enabled metasurface sensors and AI-assisted biomedical technologies in advancing next-generation image-guided neurosurgical diagnostics. These additions have been highlighted in blue.

 

 

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have substantially revised the article and responded fully to comments and suggestions. In this form, the article will be useful to readers practicing neurosurgery. It is eligible for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed all my comments and the manuscript now can be accepted for publication

Comments on the Quality of English Language

Accept in present form