The Advances and Applications of Characterization Technique for Exosomes: From Dynamic Light Scattering to Super-Resolution Imaging Technology

Round 1

Reviewer 1 Report

This review mainly focuses on the detection techniques of exosomes, including traditional detection methods such as dynamic light scattering, nanoparticle tracking, scanning electron microscopy, flow cytometry, and emerging detection methods such as fluorescence super-resolution technology. The basic principles of each technology and their applications in exosomes are detailed. There are few literature related to exosome detection and pathological processes such as tumor metastasis. Some references can be added. Overall, the article is rich in content, but there are still certain deficiencies in logic and coherence. In addition, there are also certain issues with the formatting and handling of details in the article. Please revise and organize the article before resubmitting.

Question 1: Format issue. (1) After the complete sentence, there should be a space left between the number and the unit, such as "300 nm" instead of "300nm"; (2) Does the citation format require upper right corner markers; (3) Does the position of Table 1 need to be aligned with the position of the main text. Please carefully revise according to the format requirements of the editorial department.

Question 2: The last sentence of the first paragraph of Section 2.3 mentions that DLS technology cannot measure the concentration of exosomes, which is an unnatural transition from the previous text. Similar structures were not used in the description of other technologies, and it is recommended to coordinate and unify them before and after.

Question 3: In the summary of the entire second part, fluorescence super-resolution technology has already been introduced, while at the beginning of the third part, it starts with traditional fluorescence microscopy, which is not properly connected. In the conclusion of the second part, it is suggested to raise questions and highlight the urgent need for a new technology. This way, starting from traditional fluorescence microscopy later on will not be too abrupt.

Question 4: In the table summarized at the end of Part 2,regarding whether FCM can be measured with multiple parameters, the results contradict the description in section 2.5. Please carefully check and verify.

Question 5: In the last sentence of the first paragraph of section 3.1, "SMLM" is mistakenly written as "SMMLM". Please correct it.

Question 6: The last sentence of the third paragraph in section 3.1.1 suddenly mentions that the authenticity of deep learning needs to be considered, which is quite abrupt and somewhat off topic.

Question 7: In section 3.2, the description of the process of combining STED with UCNPs to achieve EVs observation seems quite confusing. The order of description can be adjusted, and it may be more logical to write according to the idea of improving resolution and ultimately achieving EVs observation.

Question 8：In the third part, the order of introducing super-resolution technology is SMLM, STED, and SIM. Is this order appropriate? According to the development history of fluorescence super-resolution technology, it should be STED, SIM, and SMLM. Moreover, it seems that SMLM technology is the most promising method for detecting exosomes in the future. You can adjust the order of each technology introduction to emphasize your focus.

Question 9: At the end of Part 2, you have a summary table to compare the advantages and disadvantages of traditional exosome detection methods. This will allow for a more intuitive comparison of various techniques. However, in your third section, there is no such summary paragraph, which makes it difficult for us to visually compare the advantages of various super-resolution technologies in exosome detection, and we cannot clearly understand why SMLM technology is more advantageous in exosome detection. It is suggested to add a table similar to the end of the second part to compare the advantages and disadvantages of various techniques in exosome detection.

Question 10: The overall content of the article is very rich, but the wording of each paragraph may make people feel that it is a superposition of many literature, rather than sorting it out according to a certain logic, and the correlation between literature is not enough.

Author Response

Question: This review mainly focuses on the detection techniques of exosomes, including traditional detection methods such as dynamic light scattering, nanoparticle tracking, scanning electron microscopy, flow cytometry, and emerging detection methods such as fluorescence super-resolution technology. The basic principles of each technology and their applications in exosomes are detailed. There are few literature related to exosome detection and pathological processes such as tumor metastasis. Some references can be added. Overall, the article is rich in content, but there are still certain deficiencies in logic and coherence. In addition, there are also certain issues with the formatting and handling of details in the article. Please revise and organize the article before resubmitting.

Answer: Thank the reviewers for their valuable comments on our manuscript. After carefully reading your revision suggestions, we will make replies and explanations one by one, and at the same time give the corresponding changes in the manuscript. The modifications of papers are marked in red.We supplemented the relevant literature on exosomes and the pathological process of tumor metastasis, and the supplementary literature is in 34-38.We have added transitional statements in relevant places to improve the logic and coherence of the article.The details of the article have also been further corrected.

Question 1: Format issue. (1) After the complete sentence, there should be a space left between the number and the unit, such as "300 nm" instead of "300nm"; (2) Does the citation format require upper right corner markers; (3) Does the position of Table 1 need to be aligned with the position of the main text. Please carefully revise according to the format requirements of the editorial department.

Answer 1: Thanks for the reviewer's advice.We have reviewed the content of the review to make sure that there are no errors in the format of the article, and adjusted the format of the article according to the requirements of the editorial department. The modifications of papers are marked in red.

Question 2: The last sentence of the first paragraph of Section 2.3 mentions that DLS technology cannot measure the concentration of exosomes, which is an unnatural transition from the previous text. Similar structures were not used in the description of other technologies, and it is recommended to coordinate and unify them before and after.

Answer 2: Thank the reviewers for their valuable comments.In response to your comments, we have revised the end of section 2.3 to make it structurally consistent with the description of other technologies. The modifications of papers are marked in red.

Question 3: In the summary of the entire second part, fluorescence super-resolution technology has already been introduced, while at the beginning of the third part, it starts with traditional fluorescence microscopy, which is not properly connected. In the conclusion of the second part, it is suggested to raise questions and highlight the urgent need for a new technology. This way, starting from traditional fluorescence microscopy later on will not be too abrupt.

Answer 3: Thanks for the expert's suggestion! The modifications of papers are marked in red.At the end of this second part, we have improved the imaging advantages of optical microscopy by comparing traditional characterization techniques, furthermore, how to break through the imaging resolution of optical microscope in order to reach the imaging requirement of exosome is put forward, which forms a transition with the research result of breaking through the diffraction limit of the superresolution imaging technology at the beginning of the third part.

Question 4: In the table summarized at the end of Part 2,regarding whether FCM can be measured with multiple parameters, the results contradict the description in section 2.5. Please carefully check and verify.

Answer 4: Thank the reviewers for their valuable comments. The modifications of papers are marked in red.According to the suggestions of reviewers, we re-read this part and unified the conclusions of FCM multi-parameter measurement.

Question 5 : In the last sentence of the first paragraph of section 3.1, "SMLM" is mistakenly written as "SMMLM". Please correct it.

Answer 5: Thank the reviewers for their valuable comments. The modifications of papers are marked in red.We have made corrections according to the recommendations of the reviewers.

Question 6: The last sentence of the third paragraph in section 3.1.1 suddenly mentions that the authenticity of deep learning needs to be considered, which is quite abrupt and somewhat off topic.

Answer 6: Thank the reviewers for their valuable comments. The modifications of papers are marked in red. According to the suggestions of reviewers, we have modified this sentence, and combined with the previous article, we have made a concluding discourse on the application and development of algorithms in SMLM technology.

Question 7: In section 3.2, the description of the process of combining STED with UCNPs to achieve EVs observation seems quite confusing. The order of description can be adjusted, and it may be more logical to write according to the idea of improving resolution and ultimately achieving EVs observation.

Answer 7: Thanks for the reviewer's advice! The modifications of papers are marked in red. According to your comments, we reorganized the description of STED combined with UCNPs in Section 3.2, and re-described the application of this method in EVs observation according to the improvement of resolution, so as to make the content more logical.

Question 8：In the third part, the order of introducing super-resolution technology is SMLM, STED, and SIM. Is this order appropriate? According to the development history of fluorescence super-resolution technology, it should be STED, SIM, and SMLM. Moreover, it seems that SMLM technology is the most promising method for detecting exosomes in the future. You can adjust the order of each technology introduction to emphasize your focus.

Answer 8: Thanks for the reviewer's advice! The modifications of papers are marked in red. We have read this part carefully, and the order you proposed is indeed more logical in the history of the development of super-resolution imaging technology, but after consideration, we think that SMLM should still highlight the dominant position of exosome imaging research, so we retain a narrative order of SMLM technology before STED and SIM technology.

Question 9: At the end of Part 2, you have a summary table to compare the advantages and disadvantages of traditional exosome detection methods. This will allow for a more intuitive comparison of various techniques. However, in your third section, there is no such summary paragraph, which makes it difficult for us to visually compare the advantages of various super-resolution technologies in exosome detection, and we cannot clearly understand why SMLM technology is more advantageous in exosome detection. It is suggested to add a table similar to the end of the second part to compare the advantages and disadvantages of various techniques in exosome detection.

Answer 9:Thanks for the valuable suggestions given by the reviewers. The modifications of papers are marked in red. According to your comments, we added a summary in the third part of the paper to analyze the advantages and disadvantages of SMLM, STED and SIM techniques in exosome imaging studies based on  several key points，such as the requirements of fluorescent probes, imaging spatio-temporal resolution and live cell imaging. Highlighting the advantages of SMLM in exosome imaging studies.The shortcomings of STED and SIM were pointed out, and the optimization direction of both exosome imaging was summarized.

Question 10: The overall content of the article is very rich, but the wording of each paragraph may make people feel that it is a superposition of many literature, rather than sorting it out according to a certain logic, and the correlation between literature is not enough.

Answer 10: Thank the reviewers for their valuable comments. The modifications of papers are marked in red. According to the suggestions of the reviewers, we added transitional words in the relevant paragraphs of the article to highlight the conclusions in the literature, explain the logical relationship between various parts, strengthen the connection between the contents of various parts, and improve the logical relationship of the content of the article.

Author Response File: Author Response.docx

Reviewer 2 Report

 See my review.

The manuscript can be accepted for publication after making some changes: 1) check spelling by observing the spacing between the words; 2) the figures are very small, if possible, zoom in on the fugures; 3) it is necessary to formate references  correctly.

Comments for author File: Comments.pdf

Author Response

The manuscript can be accepted for publication after making some changes: 1) check spelling by observing the spacing between the words; 2) the figures are very small, if possible, zoom in on the fugures; 3) it is necessary to formate references correctly.

Answer : Thank the reviewers for their detailed comments and suggestions on our manuscript.We appreciate you pointing out the problem with the manuscript.We will carefully modify the feedback. The modifications of papers are marked in red.

• We went over the whole article and corrected the spelling mistakes and missing s
• As for the problem with the picture of the article you pointed out, we have re-checked the picture according to the requirements of the editorial department and inserted a new corresponding illustration.

3) According to your comments, we used the professional software (Endnote) to revise the literature part of the article.

Author Response File: Author Response.doc

Reviewer 3 Report

The review paper written by Shijia. et al gives a comprehensive review on the state-of-art exosome characterization techniques, especially those optical methods. The angle of the authors looking into the analysis of exosomes is novel, but some modifications are suggested before it is considered publishable. My comments are provided below:

1. the general English writing can be improved. Many sentences are hard to follow. I suggest the authors rewrite their abstract and modify their introduction. For example, on line 15, what is the meaning of 'the advances of its application...'? What is this 'its' referring to?

2. on line 35 to 37, I believe some of the mentioned surface proteins are universal, but not tumor-specific. The authors may want to specify how they can be used for tumor identification.

3. line 58, what is 'paied'?

4. some of the spaces are missing after a period. The author should correct all the typos.

5. I believe many publications have used DLS to characterize the concentration of exosomes, for example, Palmieri, V., Lucchetti, D., Gatto, I. et al. Dynamic light scattering for the characterization and counting of extracellular vesicles: a powerful noninvasive tool. J Nanopart Res 16, 2583 (2014). https://doi.org/10.1007/s11051-014-2583-z. The author may want to provide stronger evidence on why DLS can not detect concentration of exosomes.

6. line 83, 'setttled' is a typo.

7. In my point of view, many of the characterization methods are significant to understand the nature of exosomes but may not be catagorized as a detection method. The author may want to specify the definition of detection they used in the manuscript. 

8. The connection between the super-resolution part and the front parts is not tight. It is hard to follow why the comparison between super-resolution and other techniques, such as DLS or EM should be made. 

Too many typos need to be corrected and addressed. Too many grammar mistakes. 

Author Response

The review paper written by Shijia. et al gives a comprehensive review on the state-of-art exosome characterization techniques, especially those optical methods. The angle of the authors looking into the analysis of exosomes is novel, but some modifications are suggested before it is considered publishable. My comments are provided below:

1. The general English writing can be improved. Many sentences are hard to follow. I suggest the authors rewrite their abstract and modify their introduction. For example, on line 15, what is the meaning of 'the advances of its applicatio..'? What is this 'its' referring to?

Answer 1: Thank the reviewers for their detailed comments and suggestions on our manuscript.We have revised the summary and introduction as suggested. What we want to express in line 15 is to describe the application of every characterization techniques mentioned above one by one, which has been revised here.

2. on line 35 to 37, I believe some of the mentioned surface proteins are universal, but not tumor-specific. The authors may want to specify how they can be used for tumor identification.

Answer 2: Thanks for the reviewer's advice!Here we add a description of the link between exosome surface protein detection and tumor identification as you suggested. Modify as follows:" The surface proteins of cancer exosomes are often different in different stages, which indicates that these proteins are closely related to the process of cancer. Similarly, the surface proteins of cancer exosomes from different sources are also different, this can be used for early diagnosis of cancer. Research has proven that ADAM10, metalloprotease, CD9, Annexin‐1, and HSP70 are enriched in exosomes isolated from the pleural effusion or serum of breast cancer patients. "

3. line 58, what is 'paied'?

Answer 3: Thanks for the comments of the reviewers, The author has modified this section by suggestion. The modification of papers are marked in red.

4. some of the spaces are missing after a period. The author should correct all the typos.

Answer 4: Thanks for the reviewer's advice!We went over the whole article and filled in the missing Spaces.

5. I believe many publications have used DLS to characterize the concentration of exosomes, for example, Palmieri, V., Lucchetti, D., Gatto, I. et al. Dynamic light scattering for the characterization and counting of extracellular vesicles: a powerful noninvasive tool. J Nanopart Res 16, 2583 (2014). https://doi.org/10.1007/s11051-014-2583-z. The author may want to provide stronger evidence on why DLS can not detect concentration of exosomes.

Answer 5: Thank the reviewers for their valuable suggestions!we have readed and referenced this paper. According to your comments, we added that the reason why DLS cannot detect exosome concentration is that DLS technology detects all particles in the sample at the same time, so this technology can only be used for particle diameter distribution detection, and cannot determine the concentration or number of a certain particle detection. Modify as follows:" As DLS analyzes all particles in a sample simultaneously and therefore cannot provide information on particle number or concentration. For example, DLS provides a clear range of the diameter of EVs derived from ovarian cancer cells，but the concentration was difficult to analyze. Therefore, DLS technology is often combined with other technologies to complete the characterization of EVs. For example，Tajik T combines DLS technology with electron microscopy,showed cannabis-derived EVs (CDEVs) can be considered as exosome-like nanovesicles. And highlight that CDEVs can be an ideal natural vehicle for bioactive phytocannabinoids,which promotes the research of EVs in cancer diagnosis. "

6. line 83, 'setttled' is a typo.

Answer 6: Thank the reviewers for their valuable comments on our manuscript. The author has modified this section by suggestion. The modifications of papers are marked in red.

7. In my point of view, many of the characterization methods are significant to understand the nature of exosomes but may not be catagorized as a detection method. The author may want to specify the definition of detection they used in the manuscript.

Answer 7: Thanks to the reviewers for their suggestions.According to your comments, the detection of exosomes that we emphasized in this review includes the detection of basic characteristics such as exosome concentration, particle size, morphology and particle tracking, etc. In the introduction part of the article, we added the relevant definition description. Modify as follows:" In this review, we propose that these characterization techniques for obtaining this information are exosome detection techniques. "

8. The connection between the super-resolution part and the front parts is not tight. It is hard to follow why the comparison between super-resolution and other techniques, such as DLS or EM should be made.

Answer 8: Thank the reviewers for their valuable comments!In response to your comments, we have revised the end of the second part of the article to make it more closely aligned with the super-resolution imaging part. Modify as follows:" Therefore, there is an urgent need for an exosome detection technique that can preserve the fluorescence specificity and achieve high-throughput sample single-molecule detection.In recent years, researchers pay more attention to fluorescence microscopy, fluorescence microscope has the advantage of live cell imaging and specific labeling, however， the resolution ranges from 200 nm to 500 nm can not be applied to detection studies of exosomes. Therefore, how to improve the imaging resolution of optical microscopy and apply it to the imaging observation of subcellular structures such as exosomes has become a research hotspot in recent years."

Author Response File: Author Response.doc

Round 2

Reviewer 3 Report

The authors have carefully addressed my comments, and no other problems have been detected. I only have two more suggestions to the authors before publishing this work:

1. the authors may want to build up the relation between other techniques and super-resolution in the early part of the introduction or abstract but not leave it until section 2.6. 

2. some of the references have a spacing in front of them, but some of them do not. The author may want to keep the format consistent. 

Author Response

1. the authors may want to build up the relation between other techniques and super-resolution in the early part of the introduction or abstract but not leave it until section 2.6.

Answer: Thanks for the reviewer's advice. We have reviewed the content of the review. We are in the third paragraph of the introductory section to build up the relation between other techniques and super-resolution. And retain a summary of traditional characterization techniques in section 2.6. Modify as follows:" Conventional characterization methods can achieve basic characterization of exosomes, yet they are subject to certain limitations. DLS can only obtain the size distribution of exosomes, but cannot detect concentration. Although flow cytometry (FCM) realizes multi-parameter detection, the analysis results are based on high-throughput samples and cannot obtain the morphological characteristics of a single EV. EM can visually observe the morphological characteristics of a single EV, but it is not suitable for living cells, and the direction is limited in biological research. Therefore, there is an urgent need for an exosome detection technique that can preserve the fluorescence specificity and achieve high-throughput sample single-molecule detection. In recent years, researchers have paid more attention tofluorescence microscopy. The fluorescence microscope has the advantage of live cell imaging and specific labeling; however, its resolution ranges from 200 nm to 500 nm, which can not be applied to detect exosomes. Therefore, how to improve the imaging resolution of optical microscopy and apply it to the imaging observation of subcellular structures such as exosomes has become a research hotspot in recent years. With the development of fluorescence microscopy, super-resolution imaging technology breaks through the diffraction limitation, which is conducive to the field of exosomes. Compared with others, it has the advantages of high resolution, specific detection, and live-cell imaging to realize the qualitative and quantitative analysis of proteins of living or fixed cells and track the mechanism of exosomes."

2. some of the references have a spacing in front of them, but some of them do not. The author may want to keep the format consistent.

Answer: Thanks for the expert's suggestion! We carefully checked the references in the review and kept the format consistent.