High-Performance Microcomputing Tomography of Chick Embryo in the Early Stages of Embryogenesis

Round 1

Reviewer 1 Report

 

 

The paper describes a comparison of different staining approaches for microtomography analysis applied to chicken embryos. Compared with similar articles in the scientific literature, this contribution adds a systematic approach considering different reagents in various concentration, and gives an interpretation of the results obtained with the single combinations. The conclusions are appropriate and particularly useful for the scientific community.

Nevertheless, even if the experimental setup taken into consideration (a commercial desktop microtomograph) is ideal for the everyday use in a laboratory, the authors should at least mention the phase-contrast approach using a synchrotron radiation source [Dullin 2017]. This imaging technique can increase the contrast-to-noise ratio up to a factor of 200. It is also clear, on the other hand, that while phase-contrast CT can thus be employed even for unstained samples, reaching incredible high contrast-to-noise ratios and a spatial resolution which can reach sub-micron values, it is practically only feasible at synchrotron light sources and systems using fine focus x-ray tubes.

C. Dullin, R. Ufartes, E. Larsson, S. Martin, 
M. Lazzarini, G. Tromba, J. Missbach-Guentner, D. Pinkert- Leetsch, D.M. Katschinski, F. Alves, “μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry”, PLoS ONE 12(2): e0170597 (2017).

Minor editing of English language required

Author Response

We thank the Reviewer 1 for his/her time and valuable recommendations that helped to improve our manuscript. All comments were considered. Corrections in the text are marked by red. Responses to comments and recommendations are provided below.

 

The paper describes a comparison of different staining approaches for microtomography analysis applied to chicken embryos. Compared with similar articles in the scientific literature, this contribution adds a systematic approach considering different reagents in various concentration, and gives an interpretation of the results obtained with the single combinations. The conclusions are appropriate and particularly useful for the scientific community.

Thank you very much for so kind evaluation of our work.

Nevertheless, even if the experimental setup taken into consideration (a commercial desktop microtomograph) is ideal for the everyday use in a laboratory, the authors should at least mention the phase-contrast approach using a synchrotron radiation source [Dullin 2017]. This imaging technique can increase the contrast-to-noise ratio up to a factor of 200. It is also clear, on the other hand, that while phase-contrast CT can thus be employed even for unstained samples, reaching incredible high contrast-to-noise ratios and a spatial resolution which can reach sub-micron values, it is practically only feasible at synchrotron light sources and systems using fine focus x-ray tubes.

1. Dullin, R. Ufartes, E. Larsson, S. Martin, 
M. Lazzarini, G. Tromba, J. Missbach-Guentner, D. Pinkert- Leetsch, D.M. Katschinski, F. Alves, “μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry”, PLoS ONE 12(2): e0170597 (2017).

Thank you for recommendation. This aspect was added and discussed in the Introduction.The suggested reference was useful, thank you.

Minor editing of English language required

The text was checked and revised by native English speaker.

Reviewer 2 Report

In this study, the use of silver proteinate, eosin, Lugol's solution, and phosphotungstic acid in various concentrations during the CE contrast at the early period of embryogenesis (HH22-HH34 embryonic stages) were investigated to obtain micro-CT results providing reliable micro structural analysis. It would be meaningful in practical research to evaluate the image contrast obtained by measuring actual samples under varying measurement conditions, as was done in this study. On the other hand, the following points should be noted in the evaluation of the obtained images. After the following modifications appropriately made, this study may be worthy of publication.

 

1.     How many samples were used for each measurement condition? How did the authors evaluate individual differences in the samples used to obtain their results?

2.     In Table 2, it is shown that each measurement condition, such as thickness of absorber and X-ray intensity, is different. How did the author optimize these parameters for optimal measurements and analysis?

3.     Each tomographic image in Fig.1 and 2 are too small for the assessment. It is recommended to show clearer.  

Non.

Author Response

We thank the Reviewer 2 for his/her time and valuable recommendations that helped to improve our manuscript. All comments were considered. Corrections in the text are marked by red. Responses to comments and recommendations are provided below.

 

1. How many samples were used for each measurement condition? How did the authors evaluate individual differences in the samples used to obtain their results?

Thank you for your comment. To work out various methods of contrast and features of microCT, as well as measurement of visualized volume relative to no stain (mm3 (%)), we took 10 embryos (please see Section 2. Materials and Methods, Subsection 2.3. Stain, Table 1.). To determine the X-ray density (HU) of embryos and selected organs, we took 5 embryos (please see Section 3. Results, Table 3). For the experiment, we used embryos (HH25-HH27 embryonic stage) as close as possible in anthropometric characteristics.

 

2. In Table 2, it is shown that each measurement condition, such as thickness of absorber and X-ray intensity, is different. How did the author optimize these parameters for optimal measurements and analysis?

Thank you for the comment. We used the methodological recommendations of the microCT manufacturer to select the necessary scanning and reconstruction parameters for each specific level and features of the radiopacity of the object. The passing level of radiation in most planes of the CE should be in the range of 30-50%. When reconstructing the histogram (grey value) of all images was included in the contrast region (minimum and maximum gray values), so as to most effectively highlight the general and differentiated contrast of parts of the object. A wider filter indicated an increase in the overall level of object contrast and helped to highlight the embryo in relation to the surrounding space.

3. Each tomographic image in Fig.1 and 2 are too small for the assessment. It is recommended to show clearer.  

Thank you for recommendation. The main idea of figure 1 was to show all samples in one place to make it possible to carry out a comprehensive analysis. Initially each image is in high resolution. As Applied Sciences publishes articles in electronic (online) version, the reader can zoom the figure to investigate its’ separate parts. However, we added separate parts of Figure 1 in Supplementary to make it possible for readers to study all parts in higher resolution. Figure 2 was expanded in the text to improve its’ readability.

 

Minor editing of English language required

The text was checked and revised by native English speaker

Reviewer 3 Report

The manuscript is worth sharing with the scientific community and will significantly help in reproductive and developmental toxicology. However, i have some suggestions to improve the quality of the work.

1. The grammar needs to improve.

2. Line 32: Chicken Embryo (CE) should first be written in full with the abbreviation in parenthesis. Subsequently, the abbreviation CE may be used.

3. High-resolution images should be used.

 

There are a lot of grammatical errors. Thus, the English language needs to be improved.

Author Response

We thank the Reviewer 3 for his/her time and valuable recommendations that helped to improve our manuscript. All comments were considered. Corrections in the text are marked by red. Responses to comments and recommendations are provided below.

The manuscript is worth sharing with the scientific community and will significantly help in reproductive and developmental toxicology. However, i have some suggestions to improve the quality of the work.

1. The grammar needs to improve.

Thank you for the comment. The text was checked and revised by native English speaker

2. Line 32: Chicken Embryo (CE) should first be written in full with the abbreviation in parenthesis. Subsequently, the abbreviation CE may be used.

Thank you for your attentiveness. We decided not to introduce the CE abbreviation in Abstract and used full spelling of chick embryo. CE abbreviation is introduced in Introduction.  

3. High-resolution images should be used.

Thank you for recommendation. The main idea of figure 1 was to show all samples in one place to make it possible to carry out a comprehensive analysis. Initially each image is in high resolution. As Applied Sciences publishes articles in electronic (online) version, the reader can zoom the figure to investigate its’ separate parts. However, we added separate parts of Figure 1 in Supplementary to make it possible for readers to study all parts in higher resolution. Figure 2 was expanded in the text to improve its’ readability.

There are a lot of grammatical errors. Thus, the English language needs to be improved.

Thank you for the comment. The text was checked and revised by native English speaker

Round 2

Reviewer 2 Report

In response to my review comments, other than the size of Figures 1 and 2, it is unclear how the author addressed them. I recommend that these points be improved.

See below.

1.     How many samples were used for each measurement condition? How did the authors evaluate individual differences in the samples used to obtain their results?

2.     In Table 2, it is shown that each measurement condition, such as thickness of absorber and X-ray intensity, is different. How did the author optimize these parameters for optimal measurements and analysis?

 

Author Response

Dear Reviewer 2, we are sorry for insufficient convincing processing of your comments and recommendations at the previous stage. We have studied your comments again and tried to give better response and to revise the main text where was needed. All changes in the text are marked by red. Please find response to the comments bellow.

1. How many samples were used for each measurement condition? How did the authors evaluate individual differences in the samples used to obtain their results?

The following information was added to new subsection 2.6.

For each contrast method indicated in Table 1, 10 samples were used. For visualization of 2D and 3D structures, as well as for visualization of radiopacity profiles, the most characteristic representative materials were used. Segmentation and quantification were carried out according to the recommendations of Kim et al. [26].

The assessment of individual differences in the samples was carried out using statistical analysis using ANOVA followed by post hoc testing using P < 0.05 as a significance threshold

 

2. In Table 2, it is shown that each measurement condition, such as thickness of absorber and X-ray intensity, is different. How did the author optimize these parameters for optimal measurements and analysis?

Table 2 shows the optimized parameters of separate microtomography using the Skyscan 1176 Control software (Bruker, Kontich, Belgium) and reconstruction of the tomographed object using the NRecon 1.7.4.2 software program (Bruker, Kontich, Belgium).

The main variable factor set in the Skyscan 1176 Control software (Bruker, Kontich, Belgium) was the thickness of the absorber (filter), which depends on the level of X-ray contrast and the thickness of the object. On the majority of CE flights, the passing radiation level should be between 30 and 50%. In this case, the intensity of X-ray radiation was determined automatically depending on the chosen filter

To obtain the best image quality with minimal scanning time, the voxel size and scans averaged parameters were constant for all samples.

The reconstruction parameters: ring artifact correction, beam hardening correction and minimum and maximum for CS to image conversion were selected depending on the output image and the histogram of distribution of gray values of all images. When reconstructing the histogram (grey value) of all images was included in the contrast region (minimum and maximum gray values), so as to most effectively highlight the general and differentiated contrast of parts of the object.

The above facts led to the formation of the various scanning and reconstruction conditions indicated in Table 2 with different contrast techniques.

Described information was added to subsection 2.4.