Assessment of the Use of Infrared Laser for Dynamic Laser Speckle (DLS) Technique
, Rolando J. Gonzalez-Peña 3, Lionel X. Dupuy 4,5 and Roberto A. Braga, Jr. 1,*
Round 1
Reviewer 1 Report (Previous Reviewer 1)
The significance of the provided work is still very low with several writing issues, so I don't find the manuscript qualified for publication in Agriculture.
The manuscript has several vague statements, consistency issues or writing Issues. Few examples:
* infra-red sometimes is written as Infra-Red and sometimes infra-red
*line 91: The use of IR laser in dynamic laser speckle (DLS) has been developed recently to monitor the drying of paints [34,35] ==> ref 34 is 2006. It is not recent.
line 96: Others studies IR lasers (780 nm [37], 830 nm [38]) to quantify chlorophyll content in plants/fruits ==> vague statement
Author Response
Reviewer #1
Suggestions in Italics.
The significance of the provided work is still very low with several writing issues, so I don't find the manuscript qualified for publication in Agriculture.
The manuscript has several vague statements, consistency issues or writing Issues.
Answer: We’d imagined that we provided all the suggestions demanded by the Reviewer in the previous round. We will improve all the examples pointed out now, and certainly with the improvements suggested by the other Reviewers we expect to polish the work and make it properly to publish. The text will pass by a peer English review if it is approved in this step, that will also improve the style and writing issues.
Few examples:
* infra-red sometimes is written as Infra-Red and sometimes infra-red
Answer: We adopted the term ‘infrared’, all in lowercase, throughout the text mixing the current suggestion and the suggestion of Reviewer #4. Expect when it begins a paragraph or when the acronym ‘IR’ is used.
*line 91: The use of IR laser in dynamic laser speckle (DLS) has been developed recently to monitor the drying of paints [34,35] ==> ref 34 is 2006. It is not recent.
Answer: You are right. We corrected it.
line 96: Others studies IR lasers (780 nm [37], 830 nm [38]) to quantify chlorophyll content in plants/fruits ==> vague statement
Answer: We improved the reference about the use of IR lasers.
“In apples, the classification using dynamic laser speckle with red laser were better than case of IR laser (780 nm) [37]. In turn, the dynamic laser speckle from IR laser presented lower variability when monitoring tomato ripening than the dynamic laser speckle using the red laser [38].”
Reviewer 2 Report (Previous Reviewer 2)
This is my second review of this paper. The Authors' contribution to the improvement of the manuscript is very noticeable. Nonetheless, I suggest a minor revision of this version. Detailed comments are attached in a pdf file.
Comments for author File: 
Comments.pdf
Author Response
Reviewer # 2
Suggestions in Italics.
This is my second review of this paper. The Authors' contribution to the improvement of the manuscript is very noticeable. Nonetheless, I suggest a minor revision of this version. Detailed comments are attached in a pdf file.
Answer: Thank you for your help in improving the work. We passed by your suggestions thoroughly. The text will pass by a peer English review if it is approved in this step, that will also improve the style and writing issues.
The suggestions in PDF file were all accepted and the text corrected.
A summary of the main corrections are presented here.
In Introduction: We addressed the suggestions, particularly the writing of a clear Objective.
Material and Methods: All the suggestions addressed, particularly in subsection 2.6.
Results and Discussions: All suggestions were addressed. The checks of the sentences for style were carried out. The correction of Fig. 4, its caption, and the information about boxplot. We corrected as well as the wrong designation: “AVD method”.
Reviewer 3 Report (New Reviewer)
The manuscript is interesting as a non-destructive alternative approach to seed differentiation by chemical content.
There are some recommendations for improvement.
1. Please explain the different font color and the presence of a yellow marker in the text.
2. For a more complete demonstration of all the advantages of the experiment, it is desirable to supplement the Introduction section with information on the practical application of the DLS results.
3. To increase reader interest, it is advisable to disclose future research at the end of the Discussion section.
4. It is desirable to include in the keywords the Latin name (Zea mays L.) of the plant whose seeds were used for the study. Also in the text of the manuscript, it is necessary to italicize the epithets of the genus and species in the taxon.
5. It is desirable to specify DOI in the references.
6. As for the ethical aspects related to seeds, a separate section of the type is usually indicated Data Availability Statement: for example, “[latin name of the seed] seeds were used in this study. The seeds were collected in the following provenances (coordinate) in the fall of Year”.
In general, the manuscript can be considered for publication after some adjustments.
Author Response
Suggestions in Italics.
Reviewer #3
The manuscript is interesting as a non-destructive alternative approach to seed differentiation by chemical content.
There are some recommendations for improvement.
Answer: Thank you for your help in improving the work.
- Please explain the different font color and the presence of a yellow marker in the text.
Answer: The color (red) was linked to new text in the former review. The improvements are now using the Track Changes function. The yellow mark was a mistake, it was dismissed.
- For a more complete demonstration of all the advantages of the experiment, it is desirable to supplement the Introduction section with information on the practical application of the DLS results.
Answer: We improved the Introduction with information on the practical application and advantages of the DLS (in red), after the list of examples. We limited a bit the size of new information, since one of the Reviewers complained about a large Introduction section.
“In biology, DLS has been used to measure superficial blood flow rate in human patients and rats in laboratory experiments [13, 14] or to monitor a broad range of biological samples in agriculture [15, 16]. Applications in agriculture include the assessment of the viability of seeds [17, 18], the fungal contamination of seeds [19], the identification of meristematic regions in roots [20], the monitoring of fruit maturation [21] or water activity in leaves [22], as well as to develop diagnostic tools for the early detection of the bruising in fruits [23]. The technique has also been used in animal and human science to assess the aging of meat [24] and to detect the presence of parasites in blood [25]. Those are some examples of the feasibility using DLS as an alternative tool to follow tiny changes in biological material with the advantage to offer objective judgment, possibility to automation, in some cases with low cost and being a non-contact and non-destructive (NDT) technique.”
- To increase reader interest, it is advisable to disclose future research at the end of the Discussion section.
Answer: We increased the text with the suggested information.
“The results lead to a potential use of IR laser in DLS technique with some advantages with relation to the visible lasers traditionally adopted. In summary, the reduction of the variability of the results, the non-inlfuence of the light in the biological tissue, and the reduction of the influence of the pigments of the surface in the DLS results, such as the case of the chlorophyl. We believe that new studies particularly linked to each application should be carried out to bias the use, and check if the IR laser adoption presents the advantage claimed. For example, if you need to follow the water activity, the use of IR laser in DLS can compromise the application.”
- It is desirable to include in the keywords the Latin name (Zea mays L.) of the plant whose seeds were used for the study. Also in the text of the manuscript, it is necessary to italicize the epithets of the genus and species in the taxon.
Answer: We have included in the keywords the Latin name (Zea mays L.) and we did italicize the genus and species in the taxon in the whole text.
- It is desirable to specify DOI in the references.
Answer: We have included the DOI where it lacks.
- As for the ethical aspects related to seeds, a separate section of the type is usually indicated Data Availability Statement: for example, “[latin name of the seed] seeds were used in this study. The seeds were collected in the following provenances (coordinate) in the fall of Year”.
Answer: We have included it.
“Zea mays L. seeds were used in the study, and the crop was located at latitude 21º 14' S, longitude 45º 00' W Gr. and 918 m above sea level, in Lavras MG Brazil, Köppen climate classification, Cwa, temperate rainy. The harvesting was in the fall of 2018 when the seeds were used in the assays. The seedlings (Zea mays L.) were illuminated …” The text will pass by a peer English review if it is approved in this step, that will also improve the style and writing issues.
Reviewer 4 Report (New Reviewer)
In the manuscript Assessment of the use of infra-red laser for Dynamic Laser Speckle (DLS) technique authors tested whether infrared lasers improve the robustness of the DLS analysis in seed germination applications. The dynamical properties of the laser speckles obtained from living biological samples were compared when illuminated by red and IR lasers.
The subject is quite interesting as authors performed improvements in the current model. However, this paper requires corrections. Here are some comments for the authors to improve their work:
1. I suggest in the title change infra-red with infrared- Line 2
2. The introduction section is quite long, with only a few details about laser systems used in agriculture, and does not highlight the advantages and limitations of DLS compared to these methods.
3. The writing must be ordered. There are many unnecessary spaces, smaller and larger fonts, different line spacing, different paragraphs
Line 30- introduce Tab
Line 38- introduce Tab
Line 41 – delete space before Dynamics….
Line 82 – delete space before a function
Line 87 – delete one TAB
Materials and methods – different line spacing from Introduction
Line 203 -Seed imaging system- delete bold
Line 242 ec. (1) and line 256 ec. (2)
2.5- different font size and line spacing…..etc
4. Compared to Results section, conclusions are too short
Please arrange References
Author Response
Reviewer #4
Suggestions in Italics.
In the manuscript Assessment of the use of infra-red laser for Dynamic Laser Speckle (DLS) technique authors tested whether infrared lasers improve the robustness of the DLS analysis in seed germination applications. The dynamical properties of the laser speckles obtained from living biological samples were compared when illuminated by red and IR lasers.
The subject is quite interesting as authors performed improvements in the current model. However, this paper requires corrections. Here are some comments for the authors to improve their work:
- I suggest in the title change infra-red with infrared- Line 2
Answer: We adopted it.
The introduction section is quite long, with only a few details about laser systems used in agriculture, and does not highlight the advantages and limitations of DLS compared to these methods.
Answer: We believe that theme is complex and the association of agriculture contents with physics always demands additional information. Since the Introduction is quite long, we included some comments in the Introduction as well as in the Discussion.
“(…)Those are some examples of the feasibility using DLS as an alternative tool to follow tiny changes in biological material with the advantage to offer objective judgment, possibility to automation, in some cases with low cost and being a non-contact and non-destructive (NDT) technique.”
“The results lead to a potential use of IR laser in DLS technique with some advantages with relation to the visible lasers traditionally adopted. In summary, the reduction of the variability of the results, the non-inlfuence of the light in the biological tissue, and the reduction of the influence of the pigments of the surface in the DLS results, such as the case of the chlorophyl. We believe that new studies particularly linked to each application should be carried out to bias the use, and check if the IR laser adoption presents the advantage claimed. For example, if you need to follow the water activity, the use of IR laser in DLS can compromise the application.”
- The writing must be ordered. There are many unnecessary spaces, smaller and larger fonts, different line spacing, different paragraphs
Line 30- introduce Tab
Line 38- introduce Tab
Line 41 – delete space before Dynamics….
Line 82 – delete space before a function
Line 87 – delete one TAB
Materials and methods – different line spacing from Introduction
Line 203 -Seed imaging system- delete bold
Line 242 ec. (1) and line 256 ec. (2)
2.5- different font size and line spacing…..etc
Answer: All the suggestions were accepted and changed in the text.
- Compared to Results section, conclusions are too short
Please arrange References
Answer: We improved the section “Conclusion” and arranged the section References.
The text will pass by a peer English review if it is approved in this step, that will also improve the style and writing issues.
Round 2
Reviewer 1 Report (Previous Reviewer 1)
NA
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript discussed the assessment of dynamic speckle techniques using infrared laser. I evaluate the paper needs major revision.
The authors should indicate the clear contribution of this work and how the science or industry can benefit from the provided findings.
Detailed comments are attached in a pdf file.
Comments for author File: Comments.pdf
Reviewer 2 Report
Dear Authors
As the reader who is not a phisilogist or biologists but is interested in spectral methods I missed a description or explanation of few aspects of your experiment.
1. Please justify in the paper a practical application of the gel with a black dye (where or why it is used, why black dye not the others)
2. Please explain (preferably in Materials and methods) the choice of red and green pigment for drying experiment.
3. Please highliht stronger the novelty of research presented in this paper.
4. Please improve your discussion since in present form it is rather raw Results. For example: the Authors did not refer to the large dispersion of mean intensities obtained for the red laser. Lack of analysis of intensity distribution within ROI puts into question whether mean is a good measure of central tendency, especially if the distribution is strongly skewed, which in case of biological objects is a frequent phenomenon. The same observation concerns results obtained from enamel drying test. Results for red and IR laser showed a higher dispertion, exluding experiment with green enamel drying.
Moreover I found few things that should be improved before publication and I'm kindly encourage you to do that.
1) Please consider changing the abbreviations of BSL to LBS (laser biospeckles) and DSL to DLS (dynamic laser speckles) because in some cases the former ones makes the sentence illogical, ex: (line 11) Biospeckle laser (BSL) is a phenomenon ... (in this sentence the laser seems to be the phenomenon while in fact the biospeckles are). Despite of using BSL and DSL abbrev. in other papers they are still confusing and make it difficult to understand the content, especially by readers who are just begginers in exploring of speckles phenomenon.
2) Please provide information on resolution of images acquired by both cameras, preferably in the chapter 'Materials and methods'.
3) Please rewrite the sentence in lines 78-80 (Also, the use of IR lasers...) - there is too much information in one sentence, which makes it incomprehensible. Please consider dividing the sentence into simpler ones, for example: 'Also, the use of IR lasers requires more expensive instruments. Additionally, such laser light is not visible, inducing additional difficulties during the set-up of instruments. These are some challenges that have been faced so far considering using IR lasers for LBS analyses' or similar.
4) line 82: please change 'One author showed....' to 'Perez et al. showed'
5) lines 95 - 100, The aim of the study should be formulated more clearly (it is about the style not the merit). For example (please keep in mind that I'm not obligue the Autors to formulate the goal in this particular way):
6) This work aimed at comparison of properties of dynamic patterns (known as biospeckles) obtained for living biological samples with IR and red laser, commonly used in LBS (LDS) applications. We started with an approach consisting direct illumination of samples with laser light, nonetheless we focused our study on the effect of pigment on penetration depth and sensitivity of IR laser in comparison with the red laser.
7) lines 103, 104: please check the sentence: what was actually collected on acquisition set-up? - not laser but overtime changing images of speckles/biospeckles formed by IR and visible red laser light scattered back from the same part of the sample with keeping its orientation.
8) How was the ambient light limited during the experiment? - please provide that information in Materials and methods.
9) Considering lines 114-115, the sentence in line 103 should not include the term 'simultaneously'.
10) line 129: please change '...paper opened...' to '...paper was opened...'
11) line 146-147: please consider a change: '...in biospeckle laser applications in root growing...' to ...in application of laser biospeckles in root growing...'
12) line 205: what were the ROI dimensions? Were the intensities averaged spatially and temporarily to get the mean for ROI?
13) line 206: please change biospeckle to biospeckles, since speckle means one spot and analysis considers whole pattern
14) line 246: Shouldn't there be mean and avd value instead of histogram and mean? Thus the caption on the Y-axis of the boxplot A in Figure 5 should be corrected. Histogram is a kind of plot (or vector), rather than variable which values are presented on the plot. I suppose that boxplot A presents means, SDs and extremas of mean intensities of red and IR laser light, obtained from their histograms. Consequently Figure 5 caption should be also corrected.
15) lines 258-262: This part should be corrected regarding the style.
16) line 266: To maintain the naming style of the chapters I suggest to change the sub-chapter title into more informative, ex: The effect of pigments on DLS obtained by IR and red laser or similar
17) lines 268-269: please check the correctness of the statement, in present form, its context is such that scattering ... determines the intensity and the dynamics of the scattering.... Aren't intensity and dynamics concern the drying process? Moreover, it is rather obvious that the surface reflects the visible light of the same colour while absorbing the radiation that complements white light. In this experiment the visible light was monochromatic (only one wavelength of 635nm) thus was fully reflected by red coating and almost fully absorbed by the green coating thus scatter effect occured to be weak in the latter case. Similarly for near infrared laser light.
Reviewer 3 Report
This work aims at assessing the use of a 780 nm – laser compared to a 635 nm – laser for biospeckle techniques. The hypothesis is that the IR laser would improve the results of BSL techniques by reducing the several drawbacks, such as interaction with samples, external light, influence of colors ( ??), improved penetration depth.
Improving the BSL measurements is an interesting matter, however the conclusions of the article go way beyond the results presented.
First of all, the optical setup presented is questionable. The use of 2 distinct cameras for 635nm and 780nm is not justified, and what is more both references of the cameras used are specified by the supplier to have an IR cut-off filter > 650 nm. The setup of the magnification and/or illumination level is unclear and seems wrong (l110 – l116-120).
The discussion about the water activity in the paragraph 3.1, l226-227 is too weak. The cited reference [19] (Cardoso 2011) does not give any more answer, this work presenting the frequency decomposition of BSL images to argue about water in seeds, and not about wavelength decomposition. The reference to water absorption in IR (l228-229) is erroneous, as water absorbance is very low at 780nm.
The discussion in paragraph 3.2 is unclear (l250-265). Again the conclusions are not justified (l253-255 and l260-262). The penetration depth in the sample, which would have been an interesting information to study, is not explored.
The last paragraph 3.3, much longer than the other two, aimed at justifying the choice of the laser wavelength to obtain consistent BSL results. However, it seems obvious that using a laser with a wavelength strongly absorbed by the sample would lead to imprecise BSL results.
The vocabulary is not precise enough regarding « color » (we generally talk about reflectance/absorbance, or we can plot a spectrum for instance) or the expression « the first moment of the drying process » l275, 284. The scattering of a sample depends on its granularity and not on its « color » (l269-270).
The relevance of the figures 9 and 10 is questionable and the justification in l 322-324 is unclear.
Finally, the conclusion is very short and is not related to the experiments presented.
