Fossil Plant Remains Diagnostics by Laser-Induced Fluorescence and Raman Spectroscopies

Round 1

Reviewer 1 Report (Previous Reviewer 3)

In the revised manuscript, the authors are focusing more on using spectroscopy to analyze fossil plant and clearly improved the integrity. The spectra details are discussed in-depth, providing more information about how to understand the data. The data are persuasive, showing the value of using spectroscopy for analysis. In conclusion, it is a much-improved manuscript to present the author’s work.
There is also one aspect I need to address. The LIDAR was a important part in the instrumentation and method, however, the new data are proving two points, first, continuous excitation is insufficient to perform fluorescence spectroscopy, second, pulsed excitation is insufficient to perform high-quality Raman spectroscopy. Therefore, it puts the LIDAR in a strange position with one positive point and one negative point in the overall manuscript. It is affecting the logic. I suggest the authors to rewording on associated statement, showing the value of LIDAR, despite the fact that it may not provide good enough Raman spectroscopy. For example, the LIDAR is much more suitable for fieldwork.
There are two technical comments on the figures.
1, For figure 1 panel a, could the author change ‘0’ intensity to a specific color such as blue or black, now its dark red, making the graph hard to read since the top left triangle is showing similar color to data on the right side.
2, Figure 2 and 3 are shown in proximity on the page, it will be better if the curves are in more consistence color and appearance as their sample, therefore simply the reading process.

Author Response

We have marked our answers with the red color.

Referee 1

In the revised manuscript, the authors are focusing more on using spectroscopy to analyze fossil plant and clearly improved the integrity. The spectra details are discussed in-depth, providing more information about how to understand the data. The data are persuasive, showing the value of using spectroscopy for analysis. In conclusion, it is a much-improved manuscript to present the author’s work.

There is also one aspect I need to address. The LIDAR was a important part in the instrumentation and method, however, the new data are proving two points, first, continuous excitation is insufficient to perform fluorescence spectroscopy, second, pulsed excitation is insufficient to perform high-quality Raman spectroscopy. Therefore, it puts the LIDAR in a strange position with one positive point and one negative point in the overall manuscript. It is affecting the logic. I suggest the authors to rewording on associated statement, showing the value of LIDAR, despite the fact that it may not provide good enough Raman spectroscopy. For example, the LIDAR is much more suitable for fieldwork.

Answer: Thank you for careful reading. The pulsed Raman measurements are rather challenging compared to Raman spectroscopy measurements with cw laser excitation due to low efficiency of Raman scattering. With cw laser excitation you can wait for hundreds of seconds or more so good signal-to-noise ratio can be achieved with acquisition during long periods of time. In case of pulsed Raman the number of acquired photons is few orders smaller so the signal-to-noise ratio is poorer.

We agree with the comment on “fieldwork application” so we have modified the manuscript text accordingly.

Abstracts changed:

Perspectives of multiwavelength LIDAR instrument studies under fieldwork conditions are discussed for ancient and geochemically highly altered (graphitic, carbonized) microscopic fossils diagnostics.

Conclusions modified:

A multiwavelength LIDAR instrument has been developed for both laser induced fluorescence and pulsed Raman spectroscopy measurements under field conditions or/and onsite studies. To demonstrate the perspectives for onsite/fieldwork detection of plant fossils …

The method of laser remote sensing can be useful in geological exploration fieldworks in the search for oil and coal-bearing rocks and rocks with a high content of organic matter

There are two technical comments on the figures.

1, For figure 1 panel a, could the author change ‘0’ intensity to a specific color such as blue or black, now its dark red, making the graph hard to read since the top left triangle is showing similar color to data on the right side.

Answer:

We have modified the colors according to the referee’s comment.

Figure 1. (a) - Fluorescence spectra map for isolated plant fossil sample № 4734/67; (b) - fossil sample № 4734/67 and Spectralon® fluorescence spectra comparison with the 305 nm excitation.

2, Figure 2 and 3 are shown in proximity on the page, it will be better if the curves are in more consistence color and appearance as their sample, therefore simply the reading process.

Answer:

We have corrected the figures according to the referee’s comment. We also reordered samples in Table 1 in orger they appear as their spectra are presented.

Author Response File: Author Response.docx

Reviewer 2 Report (Previous Reviewer 1)

The authors have made a strong effort to answer my comments from the first review. One of the major issues I had was concerning the unsubstantiated claim of lidar studies. The change of thrust (title and claims) of the paper has obviated this primary concern, and most of the smaller items and addition of references have also been addressed. Therefore, the paper will be publishable after the minor modifications below are made:

(1) in abstract: "... chlorophyll derivatives rather than highly altered fossilized graphitic material can be studied by carbon D and G bands" would read much better as "... chlorophyll derivatives, but resolves highly altered fossilized graphitic material via the carbon D and G bands"

(2) There are other places that the language could be improved throughout the paper, although it is largely understandable.

Line 54 and throughout " perspective"; line 60 " on samples"; line 80 "suck", for example.

(3) line 283, the broad bands at 300-700 cm-1 could easily be unresolved broad Raman peaks from disorganized material. Work out the wavelengths involved and you will find it narrow for fluorescence. The quantity of disorganized material in fossils is large, and it does show up in Raman spectra due to its quantity even if the cross section is small. I would at least put it in as an option.

(4) My comment number 12, accepted and corrected at that place by the authors, is still present on lines 314-315.

(5) The authors might tie back to the introduction in the conclusion to comment on how this study might impact or provide guidance to data processing of the data generated by the missions described in the introduction.

Author Response

We have marked our answers with the red color.

Referee 2

The authors have made a strong effort to answer my comments from the first review. One of the major issues I had was concerning the unsubstantiated claim of lidar studies. The change of thrust (title and claims) of the paper has obviated this primary concern, and most of the smaller items and addition of references have also been addressed. Therefore, the paper will be publishable after the minor modifications below are made:

(1) in abstract: "... chlorophyll derivatives rather than highly altered fossilized graphitic material can be studied by carbon D and G bands" would read much better as "... chlorophyll derivatives, but resolves highly altered fossilized graphitic material via the carbon D and G bands"

Answer: Thank you for suggestion. Referee’s variant is better so we changed the abstract accordingly.

(2) There are other places that the language could be improved throughout the paper, although it is largely understandable.

Line 54 and throughout " perspective"; line 60 " on samples"; line 80 "suck", for example.

Answer:

Thank you for careful reading. We have corrected the text to improve English.

Changed:

“Consequently, detection of such species by laser remote sensing is very promising.”

“Another promising application of laser remote sensing technology is a paleobotany.”

“In order to estimate the capabilities of onsite laser induced fluorescence spectroscopy measurements…”

“Furthermore, Raman spectroscopy became a powerful instrument for …”

“This study opens new trends for soil pigment composition…”

“Recently, alternative analytical techniques were utilized for paleontological sample studies to extract more information on chemical structure rather than morphology of the sample.”

“Plant fossilized materials, as a rule, mainly contain a cuticle, inside which … “

(3) line 283, the broad bands at 300-700 cm-1 could easily be unresolved broad Raman peaks from disorganized material. Work out the wavelengths involved and you will find it narrow for fluorescence. The quantity of disorganized material in fossils is large, and it does show up in Raman spectra due to its quantity even if the cross section is small. I would at least put it in as an option.

Answer:

We agree with the referee’s concern that Raman bands can be also presented in 300-700 cm-1 peaks. The text have been corrected accordingly:

A broad band at 300-700 cm-1 contained broad bands which can be attributed to the fluorescence but Raman scattering peaks can be also presented.

(4) My comment number 12, accepted and corrected at that place by the authors, is still present on lines 314-315.

Answer:

Sorry for that. We have corrected the Conclusions according to referee’s 12 comment in the first revision.

Corrected:

Spectra of fossilized materials can be distinguished compared to the rock fluorescence in the spectral ranges of 550-580 and 600-750 nm, so fossil plants can be detected at rock surface.

(5) The authors might tie back to the introduction in the conclusion to comment on how this study might impact or provide guidance to data processing of the data generated by the missions described in the introduction.

Answer:

We have added the discussion in the Conclusions.

Added:

The combination of laser induced fluorescence and Raman spectroscopies is very beneficial in extraterrestrial missions focusing on life traces detection.

Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

·         Abstract is too long. It should highlight the significant findings, research purpose, or importance of the work, and the main outcomes

·         Schematic diagram of he multiwavelength lidar system should be added in the experimental part.

·         Multiwavelength lidar

·         The English language of the manuscript needs to be revised.

·         The aim should be clearly addressed

·         Why wide broadband emissions without no peaks were observed at 532 nm. Authors should elaborate more on that.

·         Did the author compare their results to results obtained by LIBS technique. LIBS has been used remotely to check rocks before.

·         Conclusion is also too lengthy. It is more than a page. Authors should concentrate on the main findings without repeating what was mentioned in the manuscript.

Author Response

We have marked our answers with the red color.

Referee 3

Abstract is too long. It should highlight the significant findings, research purpose, or importance of the work, and the main outcomes

Answer: We have slightly reduced the abstracts according to the referee’s comment.

• Schematic diagram of he multiwavelength lidar system should be added in the experimental part.

Answer: We have presented in the lidar’s scheme in our previous paper [Lednev, V.N.; Bunkin, A.F.; Pershin, S.M.; Grishin, M.Y.; Artemova, D.G.; Zavozin, V.A.; Sdvizhenskii, P.A.; Nunes, R.A. Remote Laser Induced Fluorescence of Soils and Rocks. Photonics 2021, 8, 411] so we decided to skip the scheme and provide the reference.

• Multiwavelength lidar

Answer: We haven’t understood this comment.

• The English language of the manuscript needs to be revised.

Answer: We have carefully read the manuscript trying to improve English.

• The aim should be clearly addressed

Answer: We cannot fully agree with the referee’s comment. The purpose of the study has been discussed in abstract, introduction section and in conclusions: to demonstrate the prospective of remote laser sensing for fossilized materials by laser induced fluorescence spectroscopy and Raman spectroscopy. Is it possible to distinguish the fossilized material at rock matrix with pulsed fluorescence and Raman scattering measurements?

• Why wide broadband emissions without no peaks were observed at 532 nm. Authors should elaborate more on that.

Answer: The referee have not specified the spectrum (or figure) so we will suppose that referee asked on the laser induced fluorescence spectra of the fossilized materials.

The third harmonic (355 nm) was filter out by color glass filters so no fundamental (1064 nm) or second (532 nm) harmonic wavelength can be detected at LIDAR output. Laser induced fluorescence spectrum of the plants and fossilized plant materials is a sum of the fluorescence bands for different molecules like humic substances (peaks at 560-580 nm), aromatic hydrocarbons (peaks at 420-460 nm) or kerogen (600-700 nm). We have not observed fluoresce at 532 nm in our experiments but we also studied through literature but we haven’t find out any 532 nm fluorescence bands for fossilized substances.

• Did the author compare their results to results obtained by LIBS technique. LIBS has been used remotely to check rocks before.

Answer: Authors have a huge experience with the LIBS technique. However, LIBS sampling will damage the valuable targets. The fossil leaf plant remains are rather thin (<50 um) and LIBS technique will ablate sample material for a few to few tenths of microns. Such damage cannot be accepted for valuable samples which were available for us.

• Conclusion is also too lengthy. It is more than a page. Authors should concentrate on the main findings without repeating what was mentioned in the manuscript.

Answer: We agree with the referee’s comment and have compacted the conclusions.

Author Response File: Author Response.docx

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 describes fluorescence and Raman measurements of fossils. The authors purport to make remote Lidar measurements of these materials, but do not show such results and it is not clear that the results presented here are relevant to true remote sensing of fossils for several reasons, including a strong chemical treatment of the samples (definitely not remote), the fact that the wavelengths used will not penetrate rock but are susceptible to surface contamination measurements, the lack of evidence that the results are of endogenous rather than contamination, contradictory fluorescent and Raman conclusions, lidar is not used except to reduce background light via timing (not lidar), and lack of sample damage discussions. Thus, the paper is not suitable for publication at this time. Particular comments follow in no particular order other than roughly as they appear.

(1) lines 2-3: title is misleading (above)

(2) throughout text: the term 'compressions' or 'plant compressions' is understandable in context, but 'fossil' or another term would be more descriptive. Also, 'chlorine' is an atomic species and used where 'chlorin(s)' or 'chlorophyll derivatives' might be more appropriate.

(3) line 42: reference 11 refers to a very different type of lidar (point cloud) than is relevant for spectroscopic studies.

(4) references are needed for the statements in lines 77-78, 80, 82-83.

(5) paragraph starting on line 90: gating to a surface is not really lidar, but in any case, how is the surface located, what are the size of the range bins and how many are chosen for the gate.

(6) lines 111-112: a calculation is needed to show that the sizes/height claimed are reasonable, and experimental results shown that support the calculation.

(7) line 113 starts: why are the samples chemically treated? How are they pre-cleaned to remove non-endogenous substances from the surface? Why does the sample have to be strongly oxidized? It seems that a strong oxidation only hinders the measurement and results in loss of sample information. Explain why this is not the case.

(8) line 125: several sample pictures are missing. The sample names are not helpful unless they stand for something we are not being told.

(9) lines 138-140: after such a strong treatment for both, and lack of ruling out contamination, there is no proof here.

(10) page 5: it would be useful to present data from pure minerals corresponding to minerals in the rocks, as a reference.

(11) figure 2 has poorly rendered text in the legend -- avoid jpeg.

(12) lines 148-149: this statement assumes that preservation is indicated by a difference. Usually it is indicated by species present and by the sharpness of peaks. A difference only indicates that something different is there, not what it is or was.

(13) lines 159-161: references needed for these statements.

(14) line 206: Fig. 5 is missing, and is the 'most interesting'

(15) line 213: an indicator that non-endogenous materials are being analyzed is given here, but without comment.

(16) lines 241-4, figure 6: It seems that the remote Raman system is not giving any real information. The D and G bands are observed in all kinds of materials, from carbon black to carbon nanotubes to graphite minerals. Seeing their presence or lack is not scientifically interesting. The data used to indicate rock type (or at least a component) is in a lab-based system. This relates to the comments above about irrelevance for lidar and sample damage.

(17) There is a note about sample damage on line 252, but how that was determined and for what specimen is not described (it will depend on the sample material).

(18) Line 258: calcite is observed. Were other minerals? They ought to be discernable with Raman.

(19) line 259: The 300-700 cm-1 feature is narrow by fluorescence standards, and is typical for Raman measurements of complex biological materials, due to overlap of contributions from many materials, each broadened by disorder.

(20) line 265: this is a big problem to just mention without testing on reference samples or quantitative estimates.

(21) line 279: remote sensing has not been carried out here

(22) lines 290-3: see comment 12. I also add that no measurements have been shown on non-chemically-treated rock surfaces that demonstrate signal and indicate endogenous material.

(23) lines 301-2: can we really rule out other possibilities not mentioned as the source of these emission lines? A list of possible contaminants and their spectra must be compiled and given to substantiate this claim.

(24) lines 314-315: fluorescence and Raman did not clearly show the same thing and D and G bands are well-known, so 'new insights' are not supported either.

(25) line 320: 'ancient life' not proven, but some counter-indication is given (comment 15)

(26) line 325: 'under field conditions' would include no sample preparation and outside, random orientation, etc. Need to demonstrate this in an undisturbed fossil field.

(27) lines 326 - 332: these capabilities may exist, but are not what the paper is about nor are they shown in the paper.

In summary, the claims of the paper are often not substantiated and the choices made in the sample conditions are unclear and perhaps not relevant to the claims. The paper and measurements would need major revisions to make a coherent case for publication.

Reviewer 2 Report

Manuscript ID: photonics-1941410

Review of “Fossil plant remains diagnostics by combined fluorescence and Raman LIDAR”

In the manuscript the authors report the application of the LIDAR technology to plant fossil study, combining laser induced fluorescence and pulsed Raman spectroscopy measurements.

The reported study is very interesting and it could be relevant in different geological and biological applications. Therefore, in my opinion the work should be published in the photonics journal. I suggest to improve the introduction adding more recent papers related to lidar applications. 

 Minor comment:

1.    Introduction: please report more appropriate and recent papers using lidar for atmospheric study.

2.    Lines 132 and 145: Figure 5 is indicated in the text but it is missing

3.    The Figures number must be consecutive.

4.    The text in the figures is too small.

Reviewer 3 Report

The authors presented application of a previously reported home built LIDAR system to the investigation of chemical composition in fossil plants. They reported fluorescence and Raman spectra of both control samples and fossil plant samples. From the presented spectra, differences are visible. However, lacking further analysis is limiting the value of the work. The manuscript needs to be polished in language to make it concise and coherent. Additionally, the manuscript preparation was poor. It is apparent that proofreading was missing, as Figure 5 was cited in the main text, but was not shown in the manuscript, besides some typos. I would suggest reorganizing and remaking figures, and rewriting the manuscript.