Fluorescent Microscopy of Hot Spots Induced by Laser Heating of Iron Oxide Nanoparticles

Round 1

Reviewer 1 Report

The paper is interesting and the methodology rather well presented.

However, the presentation is not always well organized.

Table 1 is hard to read, please re-organize.

The value of powerlaser is extraordinary high! MW stays for megawatts (note for example the captions of figures 3 and 4), but in general, to my knowledge, powerlaser is normally W/cm2 or less. In other section, the authors mention reasonable values of mW/cm2. Is it only a serious typo?

The authors must mandatory discuss these critical issues before to be publishable.

Author Response

Dear reviewer, thank you very much! Your questions and comments have greatly improved the manuscript.

The paper is interesting and the methodology rather well presented. - Thank you!

However, the presentation is not always well organized. - Thank you for your comments, we have tried to improve the presentation. Therefore, the Introduction, Methods, Results, and Discussion sections have been significantly improved.

Table 1 is hard to read, please re-organize. Table 1 has been reorganized. The description of nanoparticle synthesis has been moved to the Methods section. The Materials subsection has been added to the Methods section. The table itself with the final TEM images and size distribution of each sample has been added to the Results section. Added a supplementary file with data characterizing the synthesized nanoparticles.

The value of powerlaser is extraordinary high! MW stays for megawatts (note for example the captions of figures 3 and 4), but in general, to my knowledge, powerlaser is normally W/cm² or less. In other section, the authors mention reasonable values of mW/cm². Is it only a serious typo? The authors must mandatory discuss these critical issues before to be publishable. - No, this value is correct, we are talking about MW/cm². There was a typo in one place with mW/cm², which has been corrected. Sorry for the inconsistency, everything is corrected now. The fact is that these are the usual power densities for two-photon confocal microscopy, particularly in multi-photon laser scanning microscopy. Typically an ultrashort pulsed infrared emitting laser source provides the high peak power densities needed for the simultaneous absorption of multiple photons by a fluorophore molecule. The Methods section provides a calculation for the power density per scanning laser spot when the laser is focused on an object (Line 221-232). Of course, the irradiation of one point lasts for microseconds, because the scanning speed is quite high. Therefore, the total dose per scan is comparable to an irradiation dose of a hundred mW/cm² and an irradiation time of several tens of minutes. From a physical point of view, the process of NPs heating and heat diffusion from them, of course, differs for cases of "powerful and fast" and "weak and slow". - And this is a very good topic for next investigation.

Reviewer 2 Report

In this manuscript, authors synthesized iron oxide nanoparticles (IONPs) with different shapes and sizes and then studied the hot spots of IONPs with laser heating. This ideal of this study is interested. Experimental results were not enough to support the conclusion, and the content need to reorganize and modify to make readers easy to understand. Thus, I recommend this manuscript could be consider after revision.

Questions and comments for this manuscript are as below.

1. Please provide the full name for all code names when each code name appears at the first time.

2. Same figures use the same format. 

3. Table 1 is not necessary. The content of Table 1 could add to sections of "Meterials and Methods" and "Results".

4. Please systematically conclude all IONPs in experimental results. Current form is disordered.

5. Authors synthesized many nanomaterials, but different experiments selected different IONPs. Why? 

6. A lot of mistakes were in this manuscript. Authors should carefully check again.

7. Did authors consider uptake efficiency when cells were treated with different IONPs? If uptake efficiency is related to hotspot? The shapes and sizes of IONPs will largely affect uptake efficiency. 

8. Conclusion was too extensive. Cannot get the point. What IONP can generate better hotspot?  Were hotspots depended on the shapes and sizes of IONPs. Which one IONP was good to generate hotspot?    

 

        

Suggest authors should reorganize and re-write the content. 

Author Response

Dear reviewer, thank you very much! Your questions and comments have greatly improved the manuscript.

In this manuscript, authors synthesized iron oxide nanoparticles (IONPs) with different shapes and sizes and then studied the hot spots of IONPs with laser heating. This ideal of this study is interested. Experimental results were not enough to support the conclusion, and the content need to reorganize and modify to make readers easy to understand. Thus, I recommend this manuscript could be consider after revision. - Thank you for your comments, we have tried to improve the presentation. Therefore, the Introduction, Methods, Results, and Discussion sections have been significantly improved.

Questions and comments for this manuscript are as below.

1. Please provide the full name for all code names when each code name appears at the first time. - Thank you for your comment, we have added a full description for all abbreviations in the first place used.
2. Same figures use the same format. - Thank you for your comment, we have corrected the figures to the same formats.
3. Table 1 is not necessary. The content of Table 1 could add to sections of "Materials and Methods" and "Results". - Table 1 has been reorganized. The description of nanoparticle synthesis has been moved to the Methods section. The Materials subsection has been added to the Methods section. The table itself with the final TEM images and size distribution of each sample has been added to the Results section. Added a supplementary file with data characterizing the synthesized nanoparticles.
4. Please systematically conclude all IONPs in experimental results. Current form is disordered. - Thanks for the note; we have added the results of the experiments for all IONPs.
5. Authors synthesized many nanomaterials, but different experiments selected different IONPs. Why? - Thanks for the note; we have added the results of the experiments for all IONPs.
6. A lot of mistakes were in this manuscript. Authors should carefully check again. - Sorry for the inconsistency, everything has been corrected now. The English language of the manuscript was carefully reviewed and improved.
7. Did authors consider uptake efficiency when cells were treated with different IONPs? If uptake efficiency is related to hotspot? The shapes and sizes of IONPs will largely affect uptake efficiency. - Thank you for your question. This is a very interesting point. Unfortunately, this work did not include TEM of cells incubated with NPs to assess uptake. However, we relied on literature data supporting the accumulation of NPs in lysosomes in the form of ensembles [13 (https://doi.org/10.1002/adfm.201803660), 15 (https://doi.org/10.1021/acsnano.5b07249), 18 (doi:10.3390/nano10081548) ], which we used as a basis for modeling.
8. Conclusion was too extensive. Cannot get the point. What IONP can generate better hotspot? Were hotspots depended on the shapes and sizes of IONPs. Which one IONP was good to generate hotspot? - Thanks for this comment; it helped us to structure the output. We have revised the conclusion. In particular, it was added:

"As a summary of modeling, we can say about four regularities for heating IONPs with light: 1) large NPs heat up better than small ones; 2) NPs ensembles are heated better than single NPs; 3) Fe₂O₃ is better than Fe₃O₄; 3) efficiency grows in a row of rods / flakes / spheres / cubes. However, the results with water IONPs colloids did not confirm such regularities. On the contrary, large spherical and cubic NPs heated up worse than small ones, and about the same as rod-like NPs. This distribution is probably due to different properties of the hydrophilic shells of NPs in relation to accessibility for RhB, -carboxymaltose and PEI for nanorods, DOPAC for cubic and spherical NPs.

In the experiment on cells, RhB bound to the mitochondrial membrane, and the IONPs uptaked in lysosomes; therefore, the thermometer and the IONPs ensemble were separated in space, and the NPs surface should not have affected the RhB fluorescence lifetime. In the experiment on cells, IONPs were heated more efficiently in the series: hexagonal (modeled with a flakes), spherical, cubic, rod-like, octopod-likes (modeled with a cubes). The regularities are also observed that large NPs of the same shape heat better than small ones and Fe₂O₃ is better than Fe₃O₄. Possibly, the incomplete compliance with the regularities derived from the modeling was due to a different cellular uptake of NPs, which we did not study in this work."

Comments on the Quality of English Language

Suggest authors should reorganize and re-write the content. - The English language of the text has been carefully checked and improved.

Reviewer 3 Report

1. Authors have reported the methods used for the synthesis of various forms of iron oxide nanoparticles. Did the authors verify the formation of the synthesis by any method? Authors need perform at least one physicochemical analysis for the formation and purity. XRD data may be provided. otherwise, the authors need to use commercially available materials for the study. 

2. Authors need to provide a sub-section for instruments with details of make and model as well as their purpose of study in the materials and method section.

3. Authors need to provide a sub-section for materials with details of make used in their purpose of study in the materials and method section.

4. Statistical analysis section should be added.

 

5. Statistical analysis between groups and compared to control is unclear for figures (figs 7, 9, 12, 13, 14). Meaningful levels should be indicated by an asterisk (p ≤ 0.001 ***, p ≤ 0.01:**, p ≤ 0.05: *). 

6. The interoperation of results is not clear and should be improved.

7. There are many grammatical and sentence errors in the article, and the language organization needs to be improved.

 

 

There are many grammatical and sentence errors in the article, and the language organization needs to be improved.

Author Response

Dear reviewer, thank you very much! Your questions and comments have greatly improved the manuscript.

1. Authors have reported the methods used for the synthesis of various forms of iron oxide nanoparticles. Did the authors verify the formation of the synthesis by any method? Authors need perform at least one physicochemical analysis for the formation and purity. XRD data may be provided. otherwise, the authors need to use commercially available materials for the study. - Thank you for your comment. The description of nanoparticle synthesis has been moved to the Methods section. The Materials subsection has been added to the Methods section. The table itself with the final TEM images and size distribution of each sample has been added to the Results section. Added a supplementary file with data characterizing the synthesized nanoparticles - XRD analysis and ⁵⁷Fe Mössbauer spectroscopy.
2. Authors need to provide a sub-section for instruments with details of make and model as well as their purpose of study in the materials and method section. - Subsection IO NPs synthesis with careful description of their characterization have been added to the Methods section.
3. Authors need to provide a sub-section for materials with details of make used in their purpose of study in the materials and method section. - The Materials subsection has been added to the Methods section.
4. Statistical analysis section should be added. - We apologize, but perhaps this question was not intended for this manuscript.
5. Statistical analysis between groups and compared to control is unclear for figures (figs 7, 9, 12, 13, 14). Meaningful levels should be indicated by an asterisk (p ≤ 0.001 ***, p ≤ 0.01:**, p ≤ 0.05: *). - We apologize, but perhaps this question was not intended for this manuscript.
6. The interoperation of results is not clear and should be improved. - Thank you for your comments, we have tried to improve the presentation. Therefore, the Introduction, Methods, Results, and Discussion sections have been significantly improved.
7. There are many grammatical and sentence errors in the article, and the language organization needs to be improved. - The English language of the text has been carefully checked and improved.

Comments on the Quality of English Language

There are many grammatical and sentence errors in the article, and the language organization needs to be improved. -- The English language of the text has been carefully checked and improved.

Round 2

Reviewer 1 Report

The authors addressed all the critical issues evidenced after first revision stage.

The paper can be published in the revised form.

Author Response

- Thank you! Thanks to your comments, we were able to significantly improve the presentation of the work.

Reviewer 2 Report

Authors had addressed most of questions in revised manuscript. In addition to Fe₃O₄ and Fe₂O₃ and the sized difference. Different compounds are required to discuss. CoFe₂O₄ NPs, β-FeOOH, and iron carboxymaltose were also synthesized, but the conclusion just discussed Fe₃O₄ and Fe₂O₃ and the sized difference. I recommend this manuscript could be accepted after minor revision.

It is fine in current form.

Author Response

- Thank you for your comment, we have tried to answer it. The following conclusions were added to the results description (end of 3.3. Assessing the temperature in cells):

«"Hot spots" for β-FeOOH 14 nm rods - (95.7655 ± 30.3)°C coincide with "hot spots" for Fe₃O₄ 14 nm octopods - (95.6931 ± 41.3)°C. For slightly larger iron carboxymaltose rods, 20 nm, with Fe(OH)₃ nuclei, "hot spots" of (119.036 ± 46.0)°C coincide with "hot spots" of CoFe₂O₄ 31 nm octopods - (120.614 ± 48.6)°C. …

We did not find refractive indexes and extinction coefficients for β-FeOOH, Fe(OH)₃ and CoFe₂O₄ materials in the databases. However, there are separate publications with measurements that allow us to conclude that β-FeOOH is close to the Fe3O4 oxide in terms of optical properties [52], and the CoFe₂O₄ is greater to the Fe₂O₃ [53]. Here, in cellular experiments, we can only observe the most obvious patterns related to the temperature of the "hot spots", as we have not studied the uptake of synthesised NPs by TEM and we cannot reliably assess the number of NPs in the ensemble inside lysosomes. »