Terahertz Imaging for Formalin Fixed Malignant Liver Tumors Using Two-Band Beamline at the Accelerator Facility of Nihon University

Round 1

Reviewer 1 Report

The manuscript by Kawashima et al. describes an investigation of malignant liver tumor by transmission imaging in the terahertz and infrared range radiation. The described research deserves the interest of many scientists in the field of medical imaging. The main result of this experimental research is that the free water content, which can be higher in the tumor tissue, can be displayed with the help of THz radiation. Unfortunately, the contrast of these measurements is low for the tumor tissues. However, the fibrous tissue around tumors can be clearly distinguished using THz imaging.

The conclusions of the manuscript are supported with experimental results, although presentation of IR and THz transmission images from the same sample(s) would significantly enhance the comparability of the imaging in these two ranges. In the discussion the problem of the resolution of the THz images was mentioned, which is an inherent specificity of longer wavelength ranges than IR light. The mentioned new research directions are prosperous, that may lead to develop future medical transmission imaging devices based on application of non-ionizing radiation. 

The text needs to be revised in some cases to improve clarity. These are detailed below.

Detailed remarks and questions:

1. Should one consider the optical transmission of formalin for THz and IR radiation? Is the absorption of formalin known for the applied wavelengths / frequencies?
2. It is suggested to revise the abstract and the introduction, so that the reader should clearly understand the difference between the terms of “water molecules” and “free water”. It might be useful to mention that NIR radiation is absorbed by exciting the vibrations of water molecules, while THz radiation interacts more with molecular clusters.
3. The acronym FEL means Free Electron Laser. Therefore, FEL is a device, and it is not the radiation itself. There are quite a few places, where the authors use the term "FEL" as a radiation, which is not appropriate. One of the terms “FEL radiation” or “FEL pulses” or “FEL beam” should be used in lines 39, 59, 62, 65, 98 and 105.
4. Extra hyphen signs appear in the following lines: 26 (hu-man), 56 (im-aging), 60 (undula-tor), 62 (op-tical). Please check the document and remove the unnecessary hyphen signs.
5. Extend the text: “...rate of THz [12]” to “...rate of THz waves [12]” or “...rate of THz radiation [12]”
6. In line 30 the text “... [13,14] such as and localization the THz” should be revised: “... [13,14]. Localization the THz”
7. The sentence in lines 52-54 “This was reflected by a LiTaO3 crystal....” should be simplified, E. g. the temperature control could be at the end of the sentence.
8. The term “dispersion” should be replaced by “fluctuation” or its synonym in line 81.
9. The sentence “To prevent scattered light from injecting the detector, a metal aperture with an opening of 1 × 2 mm2 was placed on the square antenna.” should be revised. “To prevent detector from the scattered light, a metal aperture with an opening of 1 × 2 mm² was placed on the square antenna.” The square symbol must be superscript at mm².
10. In Figure 2 the texts “Sample sandwiched between preparations” do not match with the manuscript description. Both descriptions should be corrected to “Sample sandwiched between quartz slides”
11. Line 116: cm-1: the “-1” should be in superscript.
12. Texts in lines 128-129 “The absorption coefficient α evaluated by the least squares method for the measured transmission data is 52.9 ± 1.0 cm⁻¹. Therefore, we determined the thickness of the samples to be 200 μm in the THz imaging experiments.” is misleading. The following formulation is suggested: “The absorption coefficient α is 52.9 ± 1.0 cm⁻¹, which was calculated from the measured transmission data using the least squares method. Therefore, we found that the appropriate thickness of the samples for the THz imaging experiments is 200 μm.”
13. The sentence in line 136 “... and measured the mean and dispersion of transmission for the tissues” is not correct. Transmission data were measured, from which the mean and standard deviation of the transmission was calculated. Please revise the sentence accordingly.
14. Table caption in lines 145 and 146: “Measured transmissions of the normal tissue and the tumor tissue for each sample. Data are presented as means standard deviation.” should be corrected: “Measured transmissions of the normal tissue and the tumor tissue for each sample. Data are presented as means and standard deviation.” Please give a short description of p values.
15. Please describe in the text, which statistic method was used to get the p values in Table 1.
16. The text “Staining images of the tissues inside…” is misleading. It should be corrected to “Images of the stained tissues inside…”
17. In lines 201-204 transmission data are given for tumor tissue, shown in Figure 4. Transmission value for normal tissue should be given, too.
18. In Figure 6. the adding of the according IR transmission image form sample #4 is suggested. This would help the reader to compare THz and IR images.

The references of the manuscript were composed with a care.

Author Response

Thank you for your careful peer review.

I wrote ansewers and comments to your questions bellow.

1. Should one consider the optical transmission of formalin for THz and IR radiation? Is the absorption of formalin known for the applied wavelengths / frequencies?

⇒Our manuscript was scoped on the optical transmission of formalin fixed human liver using THz and IR radiation, not for the formalin itself. The absorption of formalin has not investigated in our study.

2. It is suggested to revise the abstract and the introduction, so that the reader should clearly understand the difference between the terms of “water molecules” and “free water”. It might be useful to mention that NIR radiation is absorbed by exciting the vibrations of water molecules, while THz radiation interacts more with molecular clusters.

⇒The terms have been changed.

3. The acronym FEL means Free Electron Laser. Therefore, FEL is a device, and it is not the radiation itself. There are quite a few places, where the authors use the term "FEL" as a radiation, which is not appropriate. One of the terms “FEL radiation” or “FEL pulses” or “FEL beam” should be used in lines 39, 59, 62, 65, 98 and 105.

⇒All of the terms were corrected to “FEL radiation”.

4. Extra hyphen signs appear in the following lines: 26 (hu-man), 56 (im-aging), 60 (undula-tor), 62 (op-tical). Please check the document and remove the unnecessary hyphen signs.

⇒We have removed the hyphen signs.

5. Extend the text: “...rate of THz [12]” to “...rate of THz waves [12]” or “...rate of THz radiation [12]”

⇒We have changed to “...rate of THz waves [12]”.

6. In line 30 the text “... [13,14] such as and localization the THz” should be revised: “... [13,14]. Localization the THz”

⇒We have changed to “[13,14]. The THz”.

7. The sentence in lines 52-54 “This was reflected by a LiTaO3 crystal....” should be simplified, E. g. the temperature control could be at the end of the sentence.

⇒The sentence has been changed to “This was reflected by a LiTaO3 crystal substrate inserted in the FEL beamline, whose refractive index was high in the THz region [30]. It was transported to an experimental room coaxially with the infrared FEL beam at temperature of 297 K.”

8. The term “dispersion” should be replaced by “fluctuation” or its synonym in line 81.

⇒The term has been changed.

9. The sentence “To prevent scattered light from injecting the detector, a metal aperture with an opening of 1 × 2 mm2 was placed on the square antenna.” should be revised. “To prevent detector from the scattered light, a metal aperture with an opening of 1 × 2 mm2 was placed on the square antenna.” The square symbol must be superscript at mm2.

⇒The sentence has been collected.

10. In Figure 2 the texts “Sample sandwiched between preparations” do not match with the manuscript description. Both descriptions should be corrected to “Sample sandwiched between quartz slides”
    ⇒The sentence has been collected to “Marinol fixed sample sandwiched between quartz slides.”
11. Line 116: cm-1: the “-1” should be in superscript.

⇒It has been corrected.

12. Texts in lines 128-129 “The absorption coefficient α evaluated by the least squares method for the measured transmission data is 52.9 ± 1.0 cm−1. Therefore, we determined the thickness of the samples to be 200 μm in the THz imaging experiments.” is misleading. The following formulation is suggested: “The absorption coefficient α is 52.9 ± 1.0 cm−1, which was calculated from the measured transmission data using the least squares method. Therefore, we found that the appropriate thickness of the samples for the THz imaging experiments is 200 μm.”
    ⇒The text has been corrected.
13. The sentence in line 136 “... and measured the mean and dispersion of transmission for the tissues” is not correct. Transmission data were measured, from which the mean and standard deviation of the transmission was calculated. Please revise the sentence accordingly.
    ⇒The sentence has been corrected.
14. Table caption in lines 145 and 146: “Measured transmissions of the normal tissue and the tumor tissue for each sample. Data are presented as means standard deviation.” should be corrected: “Measured transmissions of the normal tissue and the tumor tissue for each sample. Data are presented as means and standard deviation.” Please give a short description of p values.
    ⇒The p values were “Significant probability when we hypothesized that the permeability of tumor tissue is equal to the permeability of normal tissue.”.
15. Please describe in the text, which statistic method was used to get the p values in Table 1.
    ⇒I have added “We have calculated the p values when we hypothesized that the permeability of tumor tissue is equal to that of normal tissue.” in the manuscript.
16. The text “Staining images of the tissues inside…” is misleading. It should be corrected to “Images of the stained tissues inside…”
    ⇒The text has been corrected.
17. In lines 201-204 transmission data are given for tumor tissue, shown in Figure 4. Transmission value for normal tissue should be given, too.
    ⇒I believe you mentioned about the sample #4 in table 1. The transmission value for the normal tissue was 33.06±07％.
18. In Figure 6. the adding of the according IR transmission image form sample #4 is suggested. This would help the reader to compare THz and IR images.
    ⇒We have not take the image IR transmission from sample #4. Therefore we can’t add the image.
19. The references has been changed to Applied Sciences' style.

Reviewer 2 Report

The manuscript under consideration presents an experimental research on terahertz imaging system for tumor detection. The authors apply THz and NIR transmission characteristics of formalin fixed human liver tumor samples. The difference in NIR transmission for normal and tumor tissue was not detected. However, it was determined that THz transmission (about 0.1THz) for tumor tissue was lower than for normal tissue. The research revealed heterogeneity in THz transmission of tumor samples. Some tumor samples demonstrated even higher THz transmission than normal tissue. The reason of this was found in extended content of fibrosis, which has better transmission due to lower water content. The authors proposed to use higher THz frequencies to avoid problems with heterogeneous sample response in future studies.

To my opinion, the manuscript presents novel interesting approach for biomedical society and can be accepted for publication.

I have some minor comments:

1) References 14 and 37 are duplicates, one of them must be removed.
2) Misprint at line 237 (Debye relaxation band).

Author Response

Thank you for your careful peer review.

I wrote answers to your comments bellow.

1) References 14 and 37 are duplicates, one of them must be removed.
⇒reference 37 was removed.

2) Misprint at line 237 (Debye relaxation band).
⇒Misprint has been corrected.

Reviewer 3 Report

I would like to get answers to two main questions in your further research:
1. How applicable are the results of the article in practical medicine for diagnosing tumors in living patients.
2. Is it possible to replace the proposed methods of traditional methods of diagnosis - digital radiography, x-ray computed tomography, dual-energy x-ray computed tomography and magnetic resonance imaging. 

Author Response

Thank you for the comments.   I wrote answers to your questions.   1. How applicable are the results of the article in practical medicine for diagnosing tumors in living patients. ⇒Thank you for the comment. I believe that it will take some time to applicable for diagnosing tumors in living patients. Because the size of the facility is too big for the hospital. We need to reduce the size of the facility as big as a computed tomography or a MRI.
2. Is it possible to replace the proposed methods of traditional methods of diagnosis - digital radiography, x-ray computed tomography, dual-energy x-ray computed tomography and magnetic resonance imaging. ⇒Thank you for the comment. I believe it is possible. However, further research with more samples is necessary.