PONE-D-23-22462Extant Life Detection Using Label-Free Video Microscopy in Analog Aquatic EnvironmentsPLOS ONE

Dear Dr. Snyder,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

Reviewer #3: No

Reviewer #4: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Your work is very interesting and the effort addressing motility as a biosignature makes sense considering our experience in trying to evaluate extant prokaryotes in desert environments.

Some issues:

- How do you differentiate between eukaryotic and prokaryotic cells? Is the size of the "cells" the only difference?

- It is confusing calling the sample "desert spring system" (L 291) considering that, following Lee et al 2018, hypersaline pools are "thermodinamically moderate", and "contains all domains of life and perform complete biogeochemical cycling". Being a hot spring defines it better than being located in a desert.

Specific comments:

Fig 1. L 116 Please change second by thrisd column.

L 253 Figure 3 does not have E and F.

L 329 How do you confirm predation?

L 451 and others Should it says selected trajectory?

Reviewer #2: Summary

The authors present results using holographic microscope in situ from a collection of various environments known to be extreme for life (ice brines, hypersaline pools,etc…), based on prototypes (one of them have been previously published). The authors also present stimuli to interfere with cell motility. Such results could have some useful information for astrobiology to justify to use of holography microscopy on a mission.

However, if they provide a catalog of various results in term of biological environment, taking into account on the difficulties to get such results in situ, still, the number of sample per site is limited which makes difficult to get a statistically strong measurement.

Author provide an overview of biosignature they obtained, but unfortunately many are not quantified even when it is abundant. Such quantification, with a homogenous method and sufficient statistics would really help providing significant biosignature.

Example and evidences

Major issues

Some results have already published : fig 3 was present in Fig 6 in their paper (26) , Fig S6 was Fig1 for instance.

Careful relecture is necessary. For instance, 1) some figures doesn’t correspond: in Figure 3 D to F subpanels are absent. 2) in the text, mismatch between Fig S7 and S8. 3) for Badwater Basin, “motile cells” is used but not clear if it concerns eukaryotes or prokaryotes.

In some movies, we can see global drift. It is not clear how it has been taken into account

For some results, (in different sections, only 2 or 3 cells have been measured), which is really too low. Moreover, it makes difficult to consider a Standard Deviation.

Minor issues

Using time in seconds instead of frame number would help for HMI. For the same reason, adding a timestamp in movies helps to get an idea of the timescale.

Movies contrast is variable, which makes difficult to evaluate. Having all the data with same contrast would be useful

Reviewer #3: This manuscript describes a search for motile microorganisms in various environments, with the aim of understanding how to design astrobiological survey missions. The introduction and discussion are very well written and clear, but the methods/materials section and results section should be revised.

Major points:

1. I was confused about the relationship between HELM, the reconstruction method, and the tracking algorithm. Are the results from the reconstruction sent to the tracking module, and these results sent to HELM? A flowchart would be useful here.

2. The biggest question I have about any detection algorithm is how sensitive the results are to the parameters chosen for the algorithm. There are likely many parameters and detection thresholds in the localization, tracking, and motility detection steps. These should be specified, and there should also be a section that explains and justifies how the parameters/thresholds are chosen. Also there should be some analysis of how sensitive the results (for example, the cell counts and the number of detected motile organisms) are to the choices of parameters and thresholds.

3. The other question in any biological survey is what protocols are used to reduce the risk of contamination. Especially with the samples that show very few microorganisms, or no motility until heating, contamination is a concern. While the authors discuss the use of sterile supplies for sampling, they do not discuss how the imaging and heating environment is kept sterile. Detailed protocols on sterilization and cleaning procedures are needed for others to reproduce the work.

4. The results section was difficult for me to follow. The results are organized by field site, but the methods used are different from site to site. It seemed to me that some sites had no ground truthing experiments performed, or at least none described (Desert Spring System and Ice from Subglacial Void, for example). Some sites had no cell counts. And different detection methods for motility are presented for the sites. It would be more helpful for the reader if these results were organized in a more systematic way. Table 2 is helpful, but the text needs to be revised. Also, ground truth experiments need to be reported for each case.

5. The reported abundance of motile cells (table 2) should be statistically quantified and the uncertainties reported. Uncertainties related to three factors should all be considered: heterogeneity (sample-to-sample variation), sample size (number of detected organisms in field of view), and model uncertainty (variation in number of detected motile organisms with variation in detection thresholds). The total uncertainty is the convolution of these three sources. For these low concentrations, Poisson statistics is likely a good way to estimate uncertainty due to sample size. The model uncertainty should also be straightforward to estimate by varying the detection parameters and thresholds over a reasonable range (see point 2). Uncertainty due to heterogeneity requires quantifying sample-to-sample variation, which may not be available for all sites, but some estimate should be provided here.

Minor points:

6. More details are needed on how the fluorescence-based cell counting was done (lines 236-237).

7. Panels E and F are missing from Figure 3 in the version I received

8. For the "Glaciovolcanic: Glacial Melt and Hot Spring" section, it's not clear to me what ground-truth experiments were done to validate the classification of minerals versus organisms in the DHM analysis.

Reviewer #4: This is a very detailed study of microbial motility detection in a wide range of extreme environments. It is shown that Digital Holographic Microscopy (DHM) can be used in the field to effectively detect motile microorganisms, and thus provide a firm biosignature. This field-based technology is highly relevant for future planetary missions to search for life beyond Earth (particularly Ocean Worlds). Motility itself will of course greatly limit such a search for life, since not all microorganisms evolved to motile forms. However, as the authors point out, in most aquatic environments some fraction of the microbial cells will be motile. Spontaneous, active motility stimulated by e.g. a temperature change, addition of nutrients or a salinity gradient, would provide a very strong biogenicity test. The images produced by DHM were processed using the software package HELM to detect motility and subsequently motion history images (MHI's) were produced. These clearly showed the movement paths of various prokaryotes and eukaryotes. I think this is a valuable study showing a technology that will be of high interest future space missions.

The manuscript is well written and many figures and videos are shown to support the findings. This is good, but also quite confusing for a reader. It is often difficult to follow exactly what is seen in a video in the supplement and how this translates to an observation in a main figures in the paper. Also, it seems that some figures are missing/misplaced, or not referred correctly. Below I list some of these issues. I suggest publication after these minor revisions.

Comments:

line 253 and 264: Fig.3E and 3F are described here but these images are missing in Fig.3.

line 316-319: Reference is made to fluorescence microscopy images here in Fig.S7. Should this be Fig.S8?

line 336-346: In some cases it is difficult for a reader to see how the images link to the motions in the videos. For instance in Fig.5C,D the MHI is shown for motility in video S12. It is really difficult to see what exactly is being tracked. Is it possible to add arrows into the movie to point out what exactly is moving? This is just a suggestion to make it more clear.

line 355: glacial melt samples are presented with reference to Fig.S3. Should this actually be Fig.S4?

line 360-362 '... contained a large amount of silt and microminerals (Fig.6)'. These have elongated and spindle-like shapes. I really wonder what type of silt/minerals have that shape. Can the authors better clarify this? And can it be pointed out in Fig.6A?

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Reviewer #1: Yes:  Cecilia Demergasso

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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PONE-D-23-22462R1Extant life detection using label-free video microscopy in analog aquatic environmentsPLOS ONE

Dear Dr. Snyder,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.  Specifically, the reviewers highlight significant shortcomings concerning the reproducibility and the statistical significance of the experiments, suggesting that the claims made in the manuscript are not supported by the reported data. Unless the technical rigor of the study is improved, it will thus be impossible to recommend publication in PLOS ONE.

Please submit your revised manuscript by Jul 18 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Michael Schubert

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

Reviewer #3: No

Reviewer #4: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I recomend publishing the article in spite of the lack of sufficient statistics, detected by reviewers, based on our experience on extreme environments. I think that the authors have adequately proposed the statistics that can be done in such kind of environments, which is valuable for going ahead.

Reviewer #2: Thanks for the author to the important effort to improve their manuscript which is clearly easier to read.

Based on their feedbacks, I’m still confused about the take-home message and how they provided the sufficiently argued elements.

If I fully understand that the measures on the different sites were done by different teams having their own major goals. However, the variability of the results (image quality , manual/automatic tracking, etc…) could be due to sites or experimentalist. My understanding of providing a biosignature implies a robust and reproducible protocol. For instance L301 : The x, y, t stacks used were usually z projections over multiple planes, but occasionally better SNR was obtained using a single denoised z-plane reconstruction rather than a projection. In Table 2 for the same concentration category, speed is quantified or not among the different sites. In the same idea, claiming an autonomous software package is slightly overselling as authors explain to change of analysis pipeline depending on the SNR of the images.

Active motion is presented as a key element. However, L311 it is mentioned Brownian motion analysis but we don’t understand 1) where are used / shown the results, 2) how they split between directional vs Brownian movement (as for instance NanoTrackJ is done exclusively for diffusive objects). Another question is still how authors split between eukaryotes and prokaryotes only based on their size. Some eukaryotes could be as small as 5µm (Ostreoccucus & others), so for a reader, it could induce erroneous information.

Again, I really appreciate the effort of the authors to improve the text, but several weaknesses in the data analysis seem to me too important to publish under these conditions.

Reviewer #3: I thank the authors for their revisions to the text in response to the reviewers' comments. Unfortunately I cannot recommend the manuscript for publication because my comments about analysis and statistical significance (raised also by reviewer 2) have not been addressed. As I understand, PLOS ONE aims for a high degree of technical rigor, and in its current state the manuscript does not rigorously support its claims.

In particular I remain concerned about the analysis approach, the very small number of samples, and the lack of ground truthing for several sites. The authors note in the revised manuscript that 'Cells were identified as either motile or non-motile via manual observation of the x, y, t stacks. The concentration of observed motile microbes was very low at some of the sites, with a total of only a few organisms identified over many recordings.' They further note that 'The parameters/thresholds used to process the images to increase their trackability are unique for each recording and are chosen through an iterative process of comparing identified localization of particles via the automated tracking algorithms to manual observations of the recordings at each image processing step.'

This approach has several major problems: (1) Relying on manual observation to identify motilility makes the results subject to the particular observer and can introduce bias -- especially considering that the image processing parameters/thresholds were determined on the basis of manual observations. False positive identifications can easily result from such a procedure. (2) The small number of observed motile microbes and the absence of ground truth for some of the data sets means that there is no way to assess what the false positive rate is, and thus no way to verify the claims. (3) Because the criteria for processing the images differ from recording to recording and depend on the intervention of an observer, it would be very difficult to replicate the results in future, independent studies.

I do appreciate the amount of field work and analysis that went into this study, and the aims of the study are well-motivated. But these features alone are not sufficient for publication in PLOS ONE. The technical rigor must be improved.

A minor comment: the figures are overcompressed, and figure 3 in particular is unreadable in my copy of the manuscript.

Reviewer #4: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: Yes:  Cecilia Dermergasso

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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<div>PONE-D-23-22462R2Extant life detection using label-free video microscopy in analog aquatic environmentsPLOS ONE

Dear Dr. Snyder,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the remaining points raised during the review process.

Please submit your revised manuscript by Feb 09 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Michael Schubert

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Partly

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: I Don't Know

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: I apologize to the authors for the delay in getting them this review. The authors have made many changes in response to my and the other reviewers' comments. In response to my comments, they have now noted why they rely on manual identification. I think the revised justification is fine for the manuscript, which is trying to establish some baseline for future analyses. However, the authors note in Line 256 that "Trained experts manually identified and classified active motion, morphology, and optical properties as biosignatures through inspection of...". In the interests of reproducibility, it will be important for the authors to specify who the experts are and, more importantly, how they were trained. After the authors add this information, I would favor acceptance. I do not need to see the manuscript again.

Reviewer #4: Based on the earlier reviews and revisions, it is clear that there are some shortcomings in the statistics of the observed motion-based biosignatures (small number of observed motile microbes, absence of ground truthing, identifying motility manually instead of automated tracking for verification).

However, I think the authors have now very well explained 1) the exact purpose of this study, 2) the limits of getting good statistics in the extreme environments that were studied, 3) the need for such studies for future space missions, in which similar lack of statistics will likely be a reality.

In the Introduction of the paper (line 105-107) the authors write: “The hypothesis of this work is that while not every microbe will exhibit detectable motion, morphology, or optical property biosignatures, within most environments – no matter how extreme – some fraction of microbes will be identifiable by these biosignatures”. I think this is the important message, and makes this study relevant for future space missions.

Overall, the authors made every effort to provide ground truthing in the form of fluorescence microscopy and in some cases gene sequencing. They presented many DHM images, and videos. Applying the method to in situ samples in extreme environments, rather than ideal lab culture samples, is in my view a very important test. In that sense it forms a more realistic basis for future space missions.

So overall, although I recognize the shortcomings pointed out by the other reviewers (particularly with respect to statistical rigor), I think this study warrants publication. It is a very valuable basis for future studies that can add similar data and thereby increasing the statistics of this method.

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Reviewer #3: No

Reviewer #4: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Extant life detection using label-free video microscopy in analog aquatic environments

PONE-D-23-22462R3

Dear Dr. Snyder,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michael Schubert

Academic Editor

PLOS ONE