New subject: [EXTERN] Decision on Nature Photonics manuscript NPHOT-2024-04-00624

Dec. 16, 2024

      Great news, Impact 32

David will send comments asap

My comments below are only brief

THANKS!

Cheers

Juergen

Von: Kareem Elsayad <kareem.elsayad@meduniwien.ac.at>
Datum: Montag, 16. Dezember 2024 um 04:56
An: "consensus@biobrillouin.org" <consensus@biobrillouin.org>
Betreff: [Consensus Statement] FW: [EXTERN] Decision on Nature Photonics manuscript NPHOT-2024-04-00624

Just got this. Please look through and comment on anything relevant to your particular contribution/expertise (may also poke you individually on this) that we can include in reply. I have no idea who reviewers were but have some guesses. Regardless they are all on our side so should be good: No.1 and No. 2 require a bit of explanation/clarification, and No. 3 is I guess the review you always wished you had 😊.

Things are pretty crazy here at end of year (prob by you too!) so I do not want to try and find a date for meeting or the likes - I want to get this out of the way asap. So please comment to this mailing list on any replies to reviewer comments you would like to make. I will then try to integrate them, and we can send the resubmission by end of year.

I will post an update to the manuscript with the reviewer reply comments to this mailing list before final resubmission.

All the best,

Kareem

From: <giampaolo.pitruzzello@nature.com>
Reply to: <giampaolo.pitruzzello@nature.com>
Date: Monday, 16. December 2024 at 03:57
To: <kareem.elsayad@meduniwien.ac.at>
Subject: [EXTERN] Decision on Nature Photonics manuscript NPHOT-2024-04-00624

15th December 2024

Dear Kareem,

Thank you for your patience. The Consensus statement entitled "Consensus Statement on Brillouin Light Scattering Microscopy of Biological Materials" has now been seen by 3 referees, whose comments are attached at the bottom of this email.
You will see from their comments that all referees agree on the timeliness, value and broader interest of this Consensus statement. Referees #1 and #2 have raised some seemingly minor concerns that require your attention before we can reach a final decision on the piece.
We would accordingly like you to respond to the referees' comments in a revised manuscript, accompanied by a list to explain your revisions.

For review purposes, please resubmit the revised manuscript electronically. When you are ready to submit your revised paper, please use the link below (with the changes clearly marked).

https://mts-nphot.nature.com/cgi-bin/main.plex?el=A2Bp2BIZ1A1BAYD2J5A9ftdLnR...

When revising your manuscript please could you:

- include a point-by-point response to our referees and a list of any changes you have made to the format of the paper;

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- state in a cover note the length of the text, methods (if present) and legends; the number of references; the number and estimated final size of figures and tables.

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Figshare 

Nature Portfolio policies strongly encourage you to share your research data in a public repository (e.g. spreadsheets, text, images). We are partnering with the figshare repository to make this easier; you can use the figshare integration via the ‘Research Data Deposition’ tab when submitting your manuscript. 

Data are stored privately until a manuscript decision is reached. You can edit/withdraw your data up to this point. Your data will be published at the same time as your article.
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We look forward to hearing from you soon.

Yours sincerely,

Dr Giampaolo Pitruzzello
Senior Editor
Nature Photonics

*****************************************************

Reviewers' Comments:
-----------------------------

Reviewer #1 (Remarks to the Author):

In this Consensus Statement, Bouvet et al aimed to synthesize current knowledge and establish the standard practices to improve the comparability of performance across various implementations of Brillouin light scattering (BLS) microscopy in different laboratories. The authors first outlined the physical quantities measured or derivable from BLS microscopy. They then introduced critical parameters that influence the performance of BLS measurements. Finally, technical details regarding different approaches of BLS microscopy and the common artefacts in practice were presented. 

The Statement is both timely and critically important for fostering consistent advancements in BLS microscopy. The content is comprehensive, and the conclusions are precise.

#Great

 However, several points need to be addressed before I can recommend this manuscript to the editor for acceptance.

Major comments:
- The idea of minimum reporting table is excellent, particularly given the challenges of identifying key parameters and comparing performance across published studies. However, I suggest that, in addition to parameters for water or phantom beads, the table or figure captions should include the specific parameters (e.g. laser power, single spectrum acquisition time and pixel step size) used for each biological sample. This inclusion is crucial because parameters often differ between biological samples and phantom materials in practice.

#correct, good suggestion

- In section Reporting Consensus, (2) spectral resolution (page 5), for frequency-domain spectrometers, I believe the text is referring to the spontaneous Brillouin spectrometer, as elastic scattering spectra cannot be measured using stimulated Brillouin techniques. Regarding the frequency-domain stimulated Brillouin spectrometer, the spectral resolution is theoretically limited by the laser linewidth. Practically, it can also be affected by the numerical aperture (NA) and the scanning speed of the lock-in-amplifier (LIA) relative to its time constant. A more accurate estimate can be derived by deconvolving a measured water spectrum with data obtained under low NA and slow LIA scanning speeds.

- In section Reporting Consensus, (3) spectral precision (page 6), the suggestion of ‘0.5 % precision’ for both the ν_B and Γ_B may require reconsideration. For pure water at 780 nm, ν_B is ~5GHz, and Γ_B is ~0.3 GHz, making the ‘0.5 % precision’ equivalent to a shift precision of 25 MHz. This is less stringent than the commonly accepted 10 MHz (refs: Nat. Protoc. 16, 1251–1275 (2021); Biomed. Opt. Express 15, 6024-6035 (2024); and as noted in the 3rd paragraph of page 9). Conversely, a ‘0.5 % precision’ for Γ_B (1.5 MHz) may be impractical, as it would demand prohibitively long averaging times. A precision range in the tens of MHz for linewidth at 780 nm may be more realistic. 

- In section Calibration spectra (page 7), calibration is only discussed for spontaneous Brillouin scattering. The authors should also include calibration details for stimulated Brillouin scattering. A frequency counter or an electrical spectrum analyzer can be used to measure the beating frequency between pump and probe beams.

- In section Laser frequency drifting or line-broadening (page 11), unlike frequency drift, temperature fluctuations rarely cause significant line-broadening in single-longitudinal-mode lasers typically used in BLS microscopy. Since frequency drift is more practically relevant, the authors may consider omitting line-broadening from this discussion.

- In Fig. 1E, the large peak Brillouin shift separation observed in the I-SBS results for water and cyclohexane, even exceeding several times the measured linewidth, requires further explanation. The provided reasoning that the I-SBS frequency shift is independent of the refractive index does not sufficiently clarify this observation.

- In Fig. 2C and 2D, the Brillouin shift in the central regions of the beads is lower than peripheral regions. Could the authors provide representative spectra for both the central and peripheral regions of the bead? This additional data would help clarify the observed trend.

Minor points:
- To help reader quickly locate information, specify the section number of the Supplementary Text when referring to it in the main manuscript. 

- In section Mass density (page 4), measurement of BLS gain in accompany with BLS frequency shift and linewidth may not be sufficient to determine mass density without knowledge of the refractive index. This should be clarified. 

- Ref. 42 is incorrectly labeled as "in press", despite having been published over four years ago. Please update this citation. 

Reviewer #2 (Remarks to the Author):

The manuscript aims to standardize parameters and concepts in biomedical Brillouin imaging, offering valuable guidance for experts actively working in the field. While it provides a comprehensive consensus,

#great

 several ambiguities require further clarification and elaboration. Additionally, some sections lack clear definitions for the topics under discussion, and in certain instances, the sequence of explanations is inconsistent. The detailed comments are as follows:

1.The paragraphs after the “abstract” and before the “main text” do not have a title. Is it an introduction?

2.It might be beneficial if the authors added a figure of schematics before showing the spectra of different spectrometer designs or in the supplementary material.

3.The introduction lacks a brief discussion about scattering (elastic and inelastic): these terms are used several times later.

4.In “Hypersonic acoustic speed (V)”, what are the acoustic modes?

5.The authors define “Longitudinal Loss Tangent (tan 𝛿)” as a measure of attenuation independent of ⍴. What attenuation? 

6.The authors use different notations for shear modulus, G and G´.

7.It might be beneficial to add the definition of acoustic and optical phonon in the introduction as it is used several times later.

8.In “Landau-Placzek Ratio”, “For simple homogeneous samples it is equal to the difference between the specific heat at constant pressure and that at constant volume, relative to that at constant volume.” is hard to understand.

9.In “Resolution,” it would be beneficial to define the spectral resolution before discussing how to improve it by deconvolution or fitting. 

10.In “Reporting Consensus”:
a.Spectrometer: What is the sampling step size? Is it BS/pixel or something else?
b.Spectral resolution: “In the frequency domain, it can be obtained from measuring the elastic scattering peak width from a highly scattering sample like a mirror.”
How does this measurement represent the spectral resolution? BS/pixel?
c.Spectral resolution: “While the spectral resolution is not a direct indication of the precision with which the key parameters can be extracted if the shape of the peak is known,” known or unknown? Is it a typo?
d.Spectral precision: Is this definition applicable to time-varying or heterogeneous samples? It is a systemic parameter that should be characterized for the system, not for samples.

11.The last paragraph of the “Calibration spectra” is a little vague.

12.In the “Fitting of BLS spectra” second paragraph, what variance are the authors referring to?

13.In “Uncertainties,” it is better to define the uncertainty first.

Reviewer #3 (Remarks to the Author):

Summary and Overall Recommendation:
I am pleased to provide a highly positive recommendation for this manuscript. As a generalist in this field, I can appreciate the broader significance of this contribution to the photonics and life sciences community. 

#great

The manuscript addresses a pressing need for standardization in Brillouin Light Scattering (BLS) spectroscopy by proposing a consensus statement for reporting protocols and identifying common artifacts. 

#great

This is a valuable endeavor, given the increasing utilization of BLS in life sciences and the variability in experimental approaches across studies. The participation of leading experts as authors further underscores the importance and reliability of this work.

Significance:
The field has experienced considerable progress in recent years, but this has been accompanied by challenges, particularly disagreements in the interpretation of measurements and variability in reported results due to differences in apparatus and experimental conditions. By providing clear recommendations for standardized reporting, this consensus statement has the potential to serve as a foundational document that enables reproducibility and consistency in future BLS studies. Moreover, it will help facilitate meaningful comparisons across studies and promote the broader adoption of this technique in biological research.

Key Strengths:

Timeliness: The manuscript is particularly timely, as BLS studies in biological contexts are still in their nascent stages, often employing custom-built spectrometers. 

#correct, still research projects also on instrumentation can be got, since further improvements are necessary

Establishing agreed-upon standards now will undoubtedly streamline future advancements.

Authorship: The inclusion of many leading researchers in this field lends substantial credibility to the work and ensures that the recommendations reflect a broad consensus.

#great

Practical Utility: The reporting recommendations and detailed discussion of common artifacts are of immense practical value to both new and experienced researchers in this area.

#great

Clarity: The abstract and overall objectives of the manuscript are clearly articulated, making the motivation and contributions of the study easy to understand.

Conclusion:
This manuscript makes a highly valuable contribution 

#great

to the field of photonics and its applications in the life sciences. By addressing a long-standing need for standardization in BLS spectroscopy, it is poised to have a significant and lasting impact. I strongly recommend the acceptance of this work without further revisions.

#great

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Re: FW: [EXTERN] Decision on Nature Photonics manuscript NPHOT-2024-04-00624

Czarske, Juergen

Robert Prevedel

Palombo, Francesca

Yakovlev, Vladislav V

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