Measuring the Efficiency of Using Raman Photoexcitation to Generate Singlet Oxygen in Distilled Water

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

I have reviewed your article “Measuring the Efficiency of Raman Photoexcitation of Singlet Oxygen in Distilled Water” and expressed my positive feedback regarding your submission, but it should be pointed out more, so I request a major revision.

My comments are as follows:

1. Although the process efficiency is clearly calculated, it would be useful to extend the discussion with a few specific technical suggestions for optimization (eg, choice of wavelength, pulse intensity, cell geometry, or use of optical components for Stokes signal amplification). This supplement can help the reader to see realistic ways to the practical application of the method.
2. At the moment, tests have only been carried out in distilled water. A brief consideration of how the presence of real impurities or ions might affect the Raman generation efficiency of 1O₂ would significantly increase the applicability of the work. This does not have to imply new measurements (if it is not possible), but can be added as a perspective in the discussion or conclusion.
3. Since it is emphasized that the solvent modes are dominant, it would be helpful to include a brief numerical representation of the ratio of the 1O₂ signal intensity to the leading Stokes components of the solvent. Such a table or diagram would help readers quantify the limitations of the method.
4. Water disinfection is mentioned in the conclusion. I recommend that this claim be softened and left as a potential future application.

Best regards

Author Response

Response to Reviewer 1

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in tracked changes in the re-submitted files.

Comment 1. Although the process efficiency is clearly calculated, it would be useful to extend discussion with a few specific technical suggestions for optimization (eg, choice of wavelength, pulse intensity, cell geometry, or use of optical components for Stokes signal amplification). This supplement can help the reader to see realistic ways for practical applications of the method.

Response to comment 1. We chose 410 nm as the pumping wavelength to illustrate the procedure for determining the efficiency of the Raman method. Different pumping wavelengths can be used. Previous publications show that ¹O₂ Raman photogeneration is possible at different pumping wavelengths [see references 14-16]. It would require conducting experiments similar to those discussed in the section of results and the corresponding use of different sets of optical filters. We have added a comment addressing this in lines 112-118.

Table 1 has been added, which provides the main parameters of the experiments, including the pump pulse duration, energy, and intensity, cell pathlength, focusing lens focal length, detector type, and detector rise time (see lines 119-121). The filters used are described on lines 107-111. To detect the pump pulse, we used an interference filter centered at (410±10) nm. To detect the ¹O₂ Stokes signal, we used a long-pass filter with a cut-off at 610 nm. We also used neutral optical filters to avoid detector saturation. The values of the optical density of the filters are provided in lines 157-168. We did not amplify the signals; they were large enough, including the ¹O₂ Stokes signal, to be processed by the oscilloscope for averaging and display.

Comment 2. At the moment, tests have only carried out on distilled water. A brief consideration of how the present of real impurities or ion might affect the Raman generation efficiency of 1o2 would significantly increase the applicability of the work. This does not have to imply new measurements (if it is not possible), but can be added as a perspective in the discussion or conclusion.

Response to comment 2. The presence of impurities or ions in the sample may represent an additional channel for the production of ¹O₂ molecules. However, the efficiency of the Raman method is not expected to be affected, since these other channels do not contribute to the ¹O₂ Stokes response. A brief discussion about this point has been included in the Conclusions section (see lines 288-291).

Comment 3. Since it is emphasized that the solvent molecules are dominant, it would be helpful to include a brief numerical representation of the ratio of the 1O2 signal intensity to the leading Stokes components of the solvent. Such table or diagram would help readers quantify the limitations of the method.

Response to comment 3. A table showing the energy, efficiency, and relative intensity of the Stokes signals from the H₂O vibrational modes and the ¹O₂ Stokes has been provided (see lines 206-207).

Comment 4. Water disinfection is mentioned in the conclusion. I recommend that this claim be softened and left as a potential future application.

Response to comment 4. The comment about the possible application of the Raman method for water disinfection has been “softened” (see lines 291-293).

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors investigate the efficiency of singlet oxygen generated via Raman excitation and calculate the efficiency by dividing the number of generated singlet oxygen molecules. This manuscript is well-designed. I suggest its publication after minor revisions, as indicated below:

1. In Figure 5, why do these absorption peaks undergo a red shift?.
2. Theseimages (Figs. 4 and 5) in the manuscript need further refinement.
3. It is necessary to cite the latest literature in this field, especially those reported in 2025.
4. The manuscript contains an excessive number of short, fragmented sentences, which require further integration and revision to enhance fluency and coherence, such as “However, determining the efficiency of the process is still a task to be completed. The present work eliminates the gap.”
5. There are some errors in expression in the text, such as the improper use of "40 mg/ml," "10 mg/ml," and the incorrect expression in "Figure 3 shows."
6. The paragraphs in the manuscript are highly inconsistent, especially in terms of structure and presentation.

Author Response

Response to Reviewer 2

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in tracked changes in the re-submitted files.

Comment 1. In Figure 5, why do these absorption peaks undergo a red shift?

Response to comment 1. The red shift observed in Figure 5(b) is small (≈1 nm) compared to the full-width-half-maximum (FWHM) of 25 nm. This is within the standard deviation of the curve. The red shift in Figure 5(a) is close to zero.

Comment 2. These images (Fig. 4 and 5) in the manuscript need further refinement.

Response to comment 2. The figures were updated and evaluated by the editing services provided by MPDI.

Comment 3. It is necessary to cite the latest literature in this field, specially those reported in 2025.

Response to comment 3. The reference list has been updated to include some of the latest publications in the field.

Comment 4. The manuscript contains an excessive number of short, fragmented sentences, which requires further integration and revision to enhance fluency and coherence, such as “However, determining the efficiency of the process is still a task to be completed. The present work eliminates the gap.”

Response to comment 4.  We have revised the style of the entire manuscript avoiding fragmented sentences. The manuscript was also evaluated for fluency and consistency by MPDI’s editing services.

Comment 5. There are some errors in expression in the text, such as the improper use of “40 mg/l,” “10 mg/ml”, and the incorrect expression in “Figure 3 shows.”

Response to comment 5. The correct unit (mg/L) has been added to the text and Figure 3. Thank you for noticing this.

Comment 6. The paragraphs in the manuscript are highly inconsistent especially in terms of structure and presentation.

Response to comment 6. The style has been revised for consistency and clarity. The style and grammar were checked using the services provided by MPDI.

Response to Reviewer 2

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in tracked changes in the re-submitted files.

Comment 1. In Figure 5, why do these absorption peaks undergo a red shift?

Response to comment 1. The red shift observed in Figure 5(b) is small (≈1 nm) compared to the full-width-half-maximum (FWHM) of 25 nm. This is within the standard deviation of the curve. The red shift in Figure 5(a) is close to zero.

Comment 2. These images (Fig. 4 and 5) in the manuscript need further refinement.

Response to comment 2. The figures were updated and evaluated by the editing services provided by MPDI.

Comment 3. It is necessary to cite the latest literature in this field, specially those reported in 2025.

Response to comment 3. The reference list has been updated to include some of the latest publications in the field.

Comment 4. The manuscript contains an excessive number of short, fragmented sentences, which requires further integration and revision to enhance fluency and coherence, such as “However, determining the efficiency of the process is still a task to be completed. The present work eliminates the gap.”

Response to comment 4.  We have revised the style of the entire manuscript avoiding fragmented sentences. The manuscript was also evaluated for fluency and consistency by MPDI’s editing services.

Comment 5. There are some errors in expression in the text, such as the improper use of “40 mg/l,” “10 mg/ml”, and the incorrect expression in “Figure 3 shows.”

Response to comment 5. The correct unit (mg/L) has been added to the text and Figure 3. Thank you for noticing this.

Comment 6. The paragraphs in the manuscript are highly inconsistent especially in terms of structure and presentation.

Response to comment 6. The style has been revised for consistency and clarity. The style and grammar were checked using the services provided by MPDI.

Reviewer 3 Report

Comments and Suggestions for Authors

This study presents a novel photosensitizer-free approach for generating singlet oxygen (¹O₂) through Raman excitation, with the first quantitative measurement of its quantum yield (Φ_R = (8±2)×10⁻⁹). Comparative analysis with Rose Bengal photosensitization demonstrates superior long-term stability of the Raman method. The work addresses a significant gap in photochemical ¹O₂ generation methodologies and shows potential for biomedical/environmental applications. While conceptually innovative, methodological details require clarification to fully support the claims.

Key Concerns Requiring Clarification

1. Quantum Yield Determination
   • Detail the experimental methodology for quantifying "Raman-generated ¹O₂ molecules":

   • Is 1270 nm phosphorescence intensity the sole metric? Provide calibration protocol.

   • How were pump photon losses (scattering/reflection) accounted for in total photon count?
     • Specify error propagation analysis for the reported Φ_R = (8±2)×10⁻⁹.

2. Experimental Parameters
   • Define laser specifications: pulse duration, repetition rate, beam profile/area, and sample path length.
   • Clarify trapping assay conditions: Uric acid concentration, reaction kinetics model, and temporal resolution of ¹O₂ quantification.

3. Comparative Fairness
   • Provide initial quantum yields for Rose Bengal (0–10 min) to contextualize long-term stability claims.
   • Confirm identical irradiance conditions (W/cm²) and sample geometry for both methods.

4. Mechanistic Rigor
   • Justify pump wavelength selection (410 nm): Is this resonant with specific vibrational modes of H₂O/O₂?
   • Discuss alternative pathways (e.g., two-photon absorption) and their exclusion.
   • Analyze the physical origins of low Φ_R (e.g., Raman cross-section limitations).

Author Response

Response to Reviewer 3

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in tracked changes in the re-submitted files.

Comment 1. Quantum Yield Determination - Detail the experimental methodology for quantifying “Raman generated ¹O₂ molecules.” Is 1270 nm phosphorescence intensity the sole metric? Provide calibration protocol.

Response to comment 1. The number of Raman-generated ¹O₂ molecules is quantified by determining the energy of the Stokes peak centered around 610 nm when pumping at 410 nm. Figure 3 shows this peak for different concentrations of oxygen. When the oxygen concentration nears zero, the signal also disappears. The phosphorescence signal is weak, several orders of magnitude smaller than the Stokes signal shown in Figure 3(a). Sensitive photon-counting detection methods are generally applied for this purpose. On the contrary, the phosphorescence method measures the number of ¹O₂ molecules independently from the excitation mechanism. In the present study, the Stokes signal detected can only be due to Raman excitation. Measuring the Stokes signal produced by ¹O₂ photoexcitation is a more direct and precise method that does not require complex calibration procedures. We used a calibrated detector to measure the energy of the ¹O₂ Stokes signal. We used the same detector to measure the incoming pumping energy. A division of both energies provides the efficiency reported. We have provided further explanation in the text regarding these points (see lines 172-188).

Comment 2. Quantum Yield Determination – How were pump photon losses (scattering/reflection) accounted for in the total count? Specify error propagation analysis for the reported F_D=(8±2) 10^-5.

Response to comment 2. We defined the efficiency of ¹O₂ Raman photoexcitation as the ratio between the number of ¹O₂ molecules generated over the number of incoming pump photons. We use the term “efficiency” instead of “quantum yield”; because of this definition, there is no need to estimate the losses due to reflection, scattering or other processes. The reported F_D standard deviation corresponds to ten different experiments completed under the same conditions. The error propagation analysis is based on the measurements of the energy of the ¹O₂ Stokes peak and the measurement of the pumping energy. We have provided additional explanations in the text to improve clarity (see lines 212-216)

Comment 3. Experimental Parameters – Define laser specifications; pulse duration, repetition rate, beam profile area, and sample path length.

Response to comment 3. We have added Table 1 (lines 119-120), which show the main experimental parameters.

Comment 4. Experimental Parameters – Clarify trapping assay conditions: Uric acid concentration, reaction kinetic model, and temporal resolution of ¹O₂ quantification

Response to comment 4. The uric acid concentration is indicated in line 223. Uric acid has been used as a quantitative probe for ¹O₂ despite its limitations (see the newly added reference 25). Parabanic acid has been identified as a ¹O₂-specific oxidation product of uric acid (see the newly added reference 26). Comments about these points have been added on lines 130-136. The temporal resolution of ¹O₂ quantification is indicated in lines 243-244.

Comment 5. Comparative Fairness. Provide the initial quantum yield for Rose Bengal (0-10 min) to contextualized long-term stability claims.

Response to comment 5. We included the Rose Bengal quantum yield ¹O₂ photoproduction from other authors (see lines 69-70 and references 22-23). In our experiments we did not measure the Rose Bengal quantum yield since it was not the main purpose of the work.

Comment 6. Comparative Fairness. Confirm identical irradiance conditions (W/cm2) and sample geometry for both methods.

Response to comment 6. Both experiments were conducted under the same conditions. A statement confirming that was added on lines 237-238.

Comment 7. Mechanistic Rigor. Justify pump wavelength selection (410 nm): Is this resonant with vibrational modes of H₂O/O₂.

Response to comment 7. We chose 410 nm as the pumping wavelength to illustrate the procedure for determining the efficiency of the Raman method. Different pumping wavelengths can be used. Previous publications show that ¹O₂ Raman photogeneration is possible at different pumping wavelengths [see references 14-16]. It would require conducting experiments similar to those discussed in the Results section and the corresponding use of different sets of optical filters. We have provided a brief discussion about the choice of 410 nm in lines 112-118.

At 410 nm the pumping beam is not resonant with the vibrational modes of H₂O or O₂. This is a Raman transition that does not require resonance at this particular wavelength. However, resonance can significantly increase the Raman response. We have added a comment about this possibility in the Conclusions (see lines 284-288).

Comment 8. Mechanistic Rigor. Discuss alternative pathways (e.g. two photon-absorption and their exclusions).

Response to comment 8. Alternative pathways for the generation of singlet oxygen without photosensitizers are possible. For example, Bregnhoj and Ogilby studied photosensitizer-free ¹O₂ production through two-photon excitation of oxygen-solvent charge-transfer (CT) states [see reference 18]. A complex of two oxygen molecules (dimols) may absorb visible light and then relax to two ¹O₂ molecules in vibration excited states without the use of a photosensitizer. Finally, solvent impurities may act as photosensitizers, producing small but detectable amounts of ¹O₂. These different mechanisms may compete in the process of visible light ¹O₂ photoexcitation. However, only the Raman effect generates Stokes components directly associated with ¹O₂ excitation. We discussed these possibilities in our previous publication (see reference 16). None of these alternative paths generate the ¹O₂ singlet signal around 610 nm, as shown in Figure 3.

Comment 9. Mechanistic Rigor. Analyze the physical origin of low F_D (e.g. Raman cross section-limitations).

Response to comment 9. The main reason for the low F_D value is that the number of oxygen molecules dissolved in water is much smaller than the number of water molecules. The concentration of molecular oxygen in water is around 8 mg/L, which correspond to 25 10¹⁵ molecules per mL. In the same mL, there are 62 10²⁰ molecules of water. Raman cross-sections need to be evaluated, but this is not within the scope of the present work. We have added a comment about this point in the Conclusions section (see lines 269-272).

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript presents a Raman excitation-based technique to experimentally determine the efficiency of singlet oxygen (¹O₂) generation in distilled water using a photosensitizer-free Raman excitation method. The work fills a relevant gap by quantifying Φ_Δ and comparing the results with those obtained using conventional photosensitisation with Rose Bengal. The authors show that there is no photobleaching and that ¹O₂ generation can be maintained over long periods of time.

The manuscript is well written; the experimental design is appropriate and well documented and includes the appropriate control experiments. The figures are clearly structured and the quantitative values, e.g. the conversion efficiencies for each Raman component, are provided with uncertainties. The results are clearly presented, and the discussion is adequate and well justified. It is emphasised that no photobleaching occurs, which is a practical advantage, and possible applications are also suggested.

The major issues identified with this manuscript are the following:

The reported Φ_Δ differs between sections: 8×10⁻⁵ in the abstract and results, and 8×10⁻⁴ in the conclusions. This must be clarified.

The choice of 410 nm pumping should be justified beyond availability of the OPO source.

A concise table of experimental parameters (pump wavelength, pulse width, energy, focal length, cuvette path length, detector specifications) should be included.

The statement that 4×10^{11 1}O₂ molecules are generated per pulse is interesting, but readers would benefit from having also this expressed as a concentration (µM or nM) in the experimental volume to better assess application feasibility.

While water disinfection is mentioned, potential limitations in real-world conditions (e.g., scattering, absorption, presence of quenchers) should also be acknowledged.

The formatting consistency for DOIs and journal titles in the reference list should be checked.

Author Response

Response to Reviewer 4

Thank you very much for taking the time to review this manuscript. Please find our detailed responses below and the corresponding revisions in tracked changes in the re-submitted files.

Comment 1 - The reported F_D differs between sections: 8 10^-5 in the abstract and results, and 8 10^-4 in the conclusions. This must be clarified.

Response to comment 1. The correct F_D value is 8 10^-5. We have corrected the misprint in the Conclusions section; thank you for noticing this.

Comment 2 – The choice of 410 nm pumping should be justified beyond availability of the OPO source.

Response to comment 2. We chose 410 nm as the pumping wavelength to illustrate the procedure for determining the efficiency of the Raman method. Different pumping wavelengths can be used. Previous publications show that ¹O₂ Raman photogeneration is possible at different pumping wavelengths [see references 14-16]. It would require conducting experiments similar to those discussed in the Result section and the corresponding use of different sets of optical filters. A comment about this point has been added in the description of the experimental section (see lines 112-117).

Comment 3 – A concise table of experimental parameters (pump wavelength, pulse width, energy, focal length, cuvette pathlength, detector specifications) should be included

Response to comment 3. The suggested table has been included in the experimental section (see lines 118-122)

Comment 4 – The statement that 4 10^{11 1}O₂ molecules are generated per pulse is interesting, readers would benefit from having also this expressed as a concentration (mM or nM) in the experimental volume to better assess application feasibility.

Response to comment 4. We have estimated a concentration of 3.6 nM of ¹O₂ molecules excited within the volume defined by the pulse excitation inside the water sample. This estimation has been provided in lines 217-219.

Comment 5 – While water disinfection is mentioned, potential limitations in real world conditions (e.g. scattering, absorption, presence of quenchers) should be acknowledged.

Response to comment 5. A comment about this point has been provided in lines 292-293.

Comment 6 – The formatting consistency for DOIs and journal titles in the reference list should be checked.

Response to comment 6. The reference format has been revised for consistency and correctness.