High Resolution STEM Images of the Human Tooth Enamel Crystals
Round 1
Reviewer 1 Report
Look at the attached file
Comments for author File: 
Comments.docx
Author Response
Referee Comment. “Figure 2 shows the HRTEM image of the human tooth enamel crystals as they begin to dissolve: it starts at the center of the crystal”. Herein, Authors are referring to the sample treated with orthophosphoric acid for simulating acid attacks. Please, comments better by shortly commenting the idea at line 62.
Answer: Thank you very much for this comment. It is true, Figure 2 shows a HRTEM image of a sample previously treated with orthophosphoric acid to simulate the acid attack, which is also observed during the caries process. We have included this comment on page 2 and in the caption of Figure 2.
Referee Comment. “Modern STEM microscopes are equipped with aberration correction systems and the low and high angle scattered electron annular detectors provide bright field (BF), low angle annular dark field (ADF) and dark field high-angle (HAADF) images with sub-angstrom resolution [10]. STEM-BF images include the transmitted beam and the 79 small-angle scattered beams produced by Rutherford elastic scattering, plasmons, and 80 phonons [11].“ Since Authors are focusing on structural investigation of the human tooth enamel and not on the interesting basic principles of the characterization techniques, the reported comments are nice for Materials and Method Section.
Answer: We are very sorry, but here we differ a bit with this comment. Our idea is to introduce the basic principles of the STEM microscope images in this section so the reader has an idea of the technique that we use in this work to analyze the structure of human tooth enamel. It is an important part of the introduction. Therefore, we consider that this paragraph is best placed where it is, in the first paragraph on page 3.
Referee Comment. Authors well describe the different meaning of the dark and bright contracts of CDL by using three different imaging techniques in Figure 3 and 4. But properly comment for Figure 3c needs clarify the implication or why Authors show HAADF-STEM image in terms of the structural properties of the enamel and not on the interesting basic principles of the characterization techniques. Furthermore, the proposed comments should be compared to the reported for Figure 3a and 3B and I suggest to the Authors to provide a single display items containing both Figures 3 and 4 since the present description jump from Figure 3 to 4 and back without follow the scientific story for describing the different microscopy images.
Answer:Thank you very much for this comment since it has greatly improved this section, now in page 3. Figures 3 and 4 have been unified in the new Figure 3. Therefore, we have discussed the implication in terms of the structural properties of enamel as well as the basic principles of STEM techniques. The first paragraph of page 3 has been modified accordingly and including the caption of figure 3.
Referee Comment. Please indicate on which nanometric region of the presented TEM images the EDP has been probed since the Authors before well discussed the interesting CDL atomic defect by direct electron imaging and after present a common Electron Diffraction Pattern (EDP) of the human tooth enamel crystal, wherein the CDL not clear visible in the reciprocal space. Please, show (indicate) and comments better where the atomic defect is representative in the EDPs (probably nano-diffraction will show better CDL).
Answer: No, this is not the idea. So far, because it size, the CDL is not visible in the reciprocal space, at least not yet by the available EDP techniques. The EDP technique used is the nanodiffraction (nano-EDF). So, the diffraction patterns (EDP) come from the nanometric region of the human tooth enamel crystal, that is, it comes from the whole crystal. The idea behind the EDPs shown in Figure 4 is to introduce the electron beam directions for the STEM images that we present in this work and lead the reader to have an idea of the relative orientation between the different directions and images presented. We have slightly modified the last paragraph on page 3 to include this idea more clearly.
Referee Comment. Figure 6, the unit measure Angstrom is not properly write.
Answer: It is right, thank you very much for this indication. We have correctly written the Angstrom unit of measure in Figure 6.
Referee Comment. Sincerely, I can well observe the crystalline periodicity, but I do not find any planar defect or CDL in Figure 7a. Maybe a FFT processes of Figure 7a could easily show the presence of the linear defect covered by yellow line capable of hiding atomic defects or dislocation in the real space imaging.
Answer: Yes, it is true, but this is exactly the result we are reporting from the STEM images: the crystalline periodicity can be clearly observed in human tooth enamel crystals and no defects are observed in the CDL region. Perhaps, a detailed FFT-type analysis of these images could show something additional about the presence of a linear defect, we have already done so, but so far we have not detected any defect.
The yellow line on the CDL was reduced in thickness to a minimum for a better observation of the CDL contrast and not to hide any type of atomic defects or dislocations in the image. All this has been done in Figures 6, 7 and 8 (previously Figures 7, 8 and 9).
Referee Comment. "Figure 7D shows a magnification of the region " please specify that it is a magnification of the simple crystallographic image that it is already well visible in Figure 7b. Furthermore, Authors is not clear if Authors performed an atomic simulation for the Figure 7a or it is a simple overlapping images.
These comments are also referred to the Figure 8 and 9.
Answer: Figure D in Figures 6, 7 and 8 (previously Figures 7, 8 and 9) shows an enlargement of the region in and around the CDL for clarity of the presented crystal arrangement in the CDL area. Yes, it is true, this is just an enlargement of the simple crystallographic image of Figure 7b since so far we have not done an atomic simulation and it is only a simple superposition of images of the unit cell of hydroxyapatite in the direction mentioned. The overlapping produced is a result of the deformation fields present in the atomic arrangement of the crystals. In a short future, we will find a way to carry out this atomic simulation because it is necessary.
Referee Comment. Authors presented high resolution images at different orientation of the zone axes in Figure 7, 8, and 9 without commenting the useful difference among them in terms of atomic species, position, lenght and angle bond defects, etc.. Please, comments and discussion are requested.
Answer: True, in Figures 6, 7 and 8 (previously Figures 7, 8 and 9) we are presenting high resolution STEM-HAADF images in three different orientations (corresponding to the zone axes of the Figure 4). Since the ordering of the atomic positions is different in different directions of the hydroxyapatite unit cell, the idea is to observe the contrasts of the CDL in these three directions and to register some possible defect (if any) in the atomic positions, bond defects of length and angle, etc. ., which could provide some extra information on the structure of the CDL. However, and this is what we report in this work, no structural defects were observed in these directions except the existence of a deformation field.
Reviewer 2 Report
The paper titled "High Resolution STEM Images of the Human Tooth Enamel Crystals" could be of interest to the readers. The methods research finding have been adequately explained. Minor language revisions and better image for the Fig 4 would be helpful.
Author Response
Referee Comment. Better image for Fig 4 would be helpful.
Answer: Thank you very much for this comment. Figure 3d (previously figure 4) was slightly modified and included in figure 3.
Referee Comment. Minor language revision
Answer: One of us (Dr. Etienne Brès) is of English native. He has done the handwriting review of the manuscript. We hope this correctly answers this comment. However, and if it is still considered necessary, we are willing to submit the manuscript to an additional language review.
