A Digital Light Microscopic Method for Diatom Surveys Using Embedded Acid-Cleaned Samples

Round 1

Reviewer 1 Report

The topic of the article is interesting and well-structured. Here you can find my comments:

OVERALL: - Please, enlarge and re-arrange all Figures and font sizes to guide the reader properly in all sections. All figures must be composed of HD images. It is mandatory to improve the scientific quality of the whole manuscript. The same for the "supplementary material" file.

-        Please, pay attention to the JOURNAL TEMPLATE in all sections.

INTRODUCTION: Please, consider in the scientific background of your study the use of advanced computer vision-based methods in the management and monitoring of natural resources broadly speaking (i.e.,

Jalonen, J., Järvelä, J., Koivusalo, H., & Hyyppä, H. (2014). Deriving floodplain topography and vegetation characteristics for hydraulic engineering applications by means of terrestrial laser scanning. Journal of Hydraulic Engineering, 140(11), 04014056.

Lama, G.F.C., Errico, A., Pasquino, V., Mirzaei, S., Preti, F., Chirico, G.B. 2022. Velocity uncertainty quantification based on Riparian vegetation indices in open channels colonized by Phragmites australis. J. Ecohydraulics 7(1), 71–76. https://doi.org/10.1080/24705357.2021.1938255)

METHODS: Please, insert a Figure for each cross-section. This will improve the scientific quality of your study, as a great support to all the equations proposed here.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and suggestions for authors

 A digital light microscopic method for diatom surveys

General

This manuscript has a clear methodologic point of view and is in the line of digital transformation of traditional methods. Although the authors stated that the digital workflow proposed can be at least as time consuming (or more) as the traditional analysis by an expert using microscopy, the advantages are also commented particularly at the final section. Reproducibility, ability to develop collaborative international projects, and the promise to be one of the (much) steps to develop future machine learning procedures leading to automated tools assisting identification and analysis of diatoms and, eventually, other organisms.

Main Strenghts

(a)   Team hands-on experience; (b) an up-to-date, detailed and reproducible explanation of workflow that increases usefulness, although an improvement of order and clarity will be acknowledged; (c) it represents one more step in the construction of future automated procedures.

Main Weak points

The graphics are of poor quality and resolution (pixelation and very tiny size) and, particularly some of them, cannot be read or used. They must be improved.

Redaction could be more organized and clearer. 

Additional comments

-Title. The reference to “diatom surveys” is wider and more promising than the actual core of the work. The methodology in general can be applied to diatoms in general, including pelagic both marine and continental species with relevant modifications in sampling and processing samples, but the manuscript is focused on benthic diatoms inhabiting hard substrates, and, hence, slides, biofilm, digestion and many specific, standardized techniques. Perhaps the title (and abstract) should reflect this stress on this category of diatoms, procedures and objectives (water quality and indices) as the potential reader can identify clearly this approach. 

-In the abstract (line 16) they said that 3 replicates of six samples were analysed. This detail (3 replicates) seems to be no clearly stated in the M&M section where just 6 samples are mentioned. Nevertheless, a tenacious reader can discover some reference to these in lines 141-142… So, paragraph lines 98-107 should be improved to clarify the structure of sampling/analysis from sampling to process and results. I think that some figure could help to visualize all the phases (sampling, replicates in the further analysis process, etc) of the whole observation. The river basin is the framework, but you should add the information on replicates in the 6 sampling sites (in the analysis phase) and write more clearly. 6 samples, 3 replicates, 5 cobbles in each site… (3 replicates for each cobble?) and a procedure for each site/cobble difficult to read. Organize better the paragraph or add a graphic scheme. Explain nature of habitat differences if you consider it relevant to explain some results (e.g. fig 2 etc). 

-Some lines could be somehow redundant if considered the whole manuscript as some ideas appear already in the introduction but are repeated in the Results+Discussion section. Review both Introduction and Discussion and try to simplify and reduce the lines of text by either including the ideas mainly in discussion or introduction, or at least shorten some of these paragraphs. 

-Figure 1 is completely unusable due to its small size and pixelation. The figure caption point at a (a) and (b) parts (up and down I assume) but it is impossible to read any word. It must be changed. Figure 2 is large enough but it appears as pixelated and should be changed either by a vector graph or a better resolution bitmap. Figure 4 is almost correct, but text in the axis should be improved due to pixelation and size. Improve also the figures in Supplement (pixelation).

-Line 166. I am curious… Why 61 images/layers? The 0,28 µm is related to a real maximum resolution of the 60x 1,42 lens? I have a feeling that is at the optical limit of that lens just with favorable wavelengths. A 0,3 or 0,4 µm step or more… is not enough to reflect the structures? You obtain then a composed layer of some 17 µm covering most of the expected structures. I am thinking of the process/storage capacity of the computers and also in the speed of the procedure perhaps opening a brief discussion on optimization. I have not used this scanner and can also think that is the designer/ engineer who has decided this. If it was not the case, perhaps some very brief justification on this decision would be useful to people using another lens or machine, making the explanation of methods some more universal. 

-And connecting to line 430 and others… When working with undigested and unprepared cells at least a common effect of focus stacking is a blurring of structures, especially with a large amount of layers, when you do not select key layers to enhance when working e.g. with Helicon Focus or similar. In fact, you mention that the loss of depth information is a disadvantage. I somehow feel that this process is quite dependent on the software and method employed as Helicon for example does not provide the same results depending on the algorithm or layers included. As an interested reader, some comment or recommendation of your team regarding to this should be useful in these paragraphs. 

-line 320. A point would improve the sentence: …site M1. Richness values…

-line 321 and ss. Can you explain what you attribute the difference for M1? I have seen after reading the next section a somehow large explanation… perhaps you should calm the reader by commenting that you will talk about it further in the next section.  

Perhaps it can be added in the former sentence (line 321) this: 

This difference was explained by a difference at sampling site M1 (see next section). Richness values obtained

-As mentioned, it appears explained about line 365 as the set analysed by the traditional method was not aggregated in taxonomic complexes? i.e. the digital method accounts for less species just because it aggregated some of them in the complexes? Clarify if my interpretation is correct and write some more clearly this part. 

-Line 523.. Conclusions

The conclusions are written now just as a brief abstract. I would have preferred a synthesis of sections 3.5.1 and 3.5.2 as short telegrammatic sentences with the essence of advantages and disadvantages of the new procedure proposed. 

Actually sections 3.5.1. and 3.5.2 are very relevant to evaluate the method but are long and difficult to read. Select key words (time spent, taxonomic accuracy, reproducibility and so on, and just write a very short conclusion on each advantage/disadvantage of the compared method). 

-The ms has cited 80 references. The authors in the reference list are frequently repeated, which can be explained because it is a very specific topic that only a few teams in the world work on. It is recommended to review those works that, in some way, are already encompassed or present similar ideas as in others from those same teams if possible. The final objective would be a more compact References section.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The article has been considerably improved according to the indications of the reviewer. Just few issues need to be fixed:

FIGURES: Please, enlarge font sizes in the HD figures, this is extremely useful for non-expert readers (i.e., Fiugre 3 and Figure 4a)

INTRODUCTION: Please, consider in the scientific background of your study the importance of accuracy quantification in statistical analysis for eco-engineering (i.e.,

Khan, M.A., Sharma, N., Lama, G.F.C., Hasan, M., Garg, R., Busico, G., Alharbi, R.S. 2022. Three-Dimensional Hole Size (3DHS) Approach for Water Flow Turbulence Analysis over Emerging Sand Bars: Flume-Scale Experiments. Water 14, 1889. https://doi.org/10.3390/w14121889

Xiao, Y., Liu, J., Wang, N., Gualtieri, C., Zhang, T., Liu, J., Fu, J., Zhou, J. 2022. Numerical simulation of overbank hyporheic transport and biogeochemical reactions in a compound channel. Hydrological Processes, e14670. https://doi.org/10.1002/hyp.14670

Lama, G.F.C., Errico, A., Francalanci, S., Solari, L., Chirico, G.B., Preti, F. 2020a. Hydraulic Modeling of Field Experiments in a Drainage Channel Under Different Riparian Vegetation Scenarios. In Innovative Biosystems Engineering for Sustainable Agriculture, Forestry and Food Production; Coppola A., Di Renzo G., Altieri G., D’Antonio P., Eds.; Springer: Cham, Switzerland, 2020; 69–77; doi:10.1007/978-3-030-39299-4_8.

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Author Response

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Author Response File: Author Response.docx