Thermoacoustic Imaging Using Single-Channel Data Acquisition System for Non-Invasive Assessment of Liver Microwave Ablation: A Feasibility Study

Comments 1: In Lines 144, the expression “different depths…as shown in Figures 2(d)-(g)” should be “different depths…as shown in Figures 3(a)-(d)”.

Response 1: Agree. We have changed "Figures 2(d)-(g)" to "Figures 3(a)-(d)" as you requested. The changes are shown on page 4, line 223.

Comments 2: In Page 5, the order of the Figure 2 is incorrect. Please correct it.

Response 2: Agree. We corrected the serial number of the picture. Change the figure 2 to Figure 3. The changes are shown on page 5, line 247.

Comments 3: In Paragraph 1 on Page 5, the description of the first two sentences is somewhat hard to comprehend. It is recommended to first describe the results at 0 mm and then at 80 mm. This will help the reader better understand the imaging depth of the TAI system. Additionally, the Figure 2 in these sentences should be Figure 3. Please correct it.

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have made corresponding changes in the first paragraph of page 5. Added description of the analysis of experimental results at 0mm - " From Figure 3(d), it can be observed that when no porcine liver is placed above the soy sauce tube (0 mm), the outline of the soy sauce tube is the most distinct.". In addition, the serial numbers of the pictures in this paragraph have been all modified according to your requirements. The changes are shown on page 5, lines 249-252.

Comments 4: From Figure 4, it can be observed that as the imaging depth increases, the amplitude of the thermoacoustic signals gradually decreases, but the contrast seems to not change significantly, indicating that the imaging depth of this TAI system is quite considerable. Please revise or improve the description at the end of the first paragraph on the fifth page.

Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have added this description in the first paragraph of page 5 - “The relative microwave absorption amplitude of the soy sauce tube showed a slight downward trend, but the contrast seemed to change little.”. The changes are shown on page 5, lines 258-259.

Comments 5: In lines 188-189, the Figure 3 should be Figure 5. Please correct it.

Response 5: Agree. We corrected the serial number of the picture. Change the Figure 3 to Figure 5. The changes are shown on page 7, lines 316-321.

Comments 6: Please improve the language format of the article, for example: line 38, "it" should not be bold font; Line 40, extra space before "and"; Line 67, an extra ", "; In line 115, there should be a space after "'r"; In line 178, change "a" to "an".

Response 6: Agree. We have corrected each of the above errors. On the first page, line 38, "it" has been de-bolded. On the first page, line 40, the space before "and" was removed. On page 2, line 67, the superfluous ", "has been deleted. On page 3, line 115, we added a space. On page 6, line 178, the "a" is changed to "an".

Comments 1: While not serious, there are minute issues in writing. For example, as the authors know ‘in vivo’ and ‘ex vivo’ are usually written in Italics. ‘charactristics of ~~ efficient’ (should be ‘efficiency’) on line 15 is not correct. Also, the verb tense in the abstract would not be future (e.g., ‘will’ on line 25). Please proofread the manuscript again.

Response 1: Thank you for pointing this out. We have corrected each of the above errors. All the words' in vivo 'and' ex vivo 'are in italics. The word "efficient" has been changed to "efficiency" in line 15. At the same time, I have corrected the future tense of lines 22 and 25 in the abstract.

Comments 2: I checked the iThenticate report. Many sentences are indicated as sentences from other sources (especially, the second and third paragraphs in the Introduction, the first paragraph of Section 2.1). Please thoroughly rewrite them since it may be able to cause the issue of plagiarism. Please note that this is an extremely critical issue if not resolved.

Response 2: We have rewritten the second and third paragraphs of the introduction and added the corresponding references. Changes are shown in the first and second paragraphs of the second page. Regarding the second paragraph, we need to explain the high repetition rate of the content. The experimental system used in this study is mostly similar to the previous study published by our group. To address this issue, we have cited the appropriate references in this section. The changes are shown on page 2, line 95, and page 3, line 163.

Comments 3: With a 50 Hz repetition frequency and 50 times average in addition to the time required for the movement of the rotator, the total experimental duration should be longer than 2 minutes with 360 Deg. and 220 Deg. measurement. By the way, how long does it take for image reconstruction using DAS?

Response 3: We apologize for the calculation error. Upon review, we believe the experiment time should be within 4 minutes. The correction is shown on page 3, line 166. Additionally, the DAS reconstruction time is under 1 second.

Comments 4: Please provide the product number and manufacturer of the data acquisition card.

Response 4: The changes are shown on page 3, line 163.

Comments 5: Please provide the conditions for mimicking MWA using the soldering iron (e.g., time and temperature).

Response 5: Thank you for pointing this out. The soldering iron rises to about 70℃ and burns about 60s. The changes are shown on page 4, line 193.

Comments 6: I have several questions and suggestions for Fig. 2. Question: (a) and (b) do not match well. Could you provide any explanation for it? Suggestions: 1) Please add a color bar for (b); 2) Please indicate the lesion; 3) Could you consider showing photos and TAI images before and after inducing the lesion for better representation? 1)

Response 6: Thank you for pointing this out. We have decided to make some changes to Figure 2. We got rid of the red dotted line. We apologize for the misunderstanding caused by the red dotted line we drew earlier. We have used red arrows to indicate the boundaries of normal tissue and yellow arrows to indicate the boundaries of damaged tissue. Besides, I'm sorry we didn't leave a picture the photos and TAI images before and after inducing the lesion. The revision is presented on pages 4, lines 195-206.

We are sorry that we did not retain the ultrasound and TAI images before MWA. At that time, we believed that the images showing both normal tissue and ablated tissue were sufficient to meet the comparison requirements, and thus did not keep the pre-MWA images. This was an oversight on our part.

Comments 9: Where is the transducer located in Fig. 3 (you mentioned Fig. 3 as Figure 2 which should be modified)?

Response 9: The sensors are distributed in a circular arrangement around the soy sauce pipe. For example, in Figure 3, if the Marker (soy sauce pipe) is taken as the X-axis and the yellow dashed line in the image represents the Y-axis, then the sensors are arranged in a circle around the soy sauce pipe, with the pipe serving as the horizontal plane and the distribution occurring around the Y-axis.

Comments 10: Could you designate where MWA was performed in Fig.5(a) and (b)?

Response 10: Thank you for pointing this out. The blue arrows we have used in Fig.5(a) and (b) indicate the ablation areas.

Comments 11: I would like to recommend the authors consider showing ultrasound and TAI images before and after needle insertion and MWA in Fig. 5. It is not really clear from the figures in the current form.

Response 11: We are sorry that we did not retain the ultrasound and TAI images before MWA. At that time, we believed that the images showing both normal tissue and ablated tissue were sufficient to meet the comparison requirements, and thus did not keep the pre-MWA images. This was an oversight on our part.

Comments 1: In Fig 2, the boundary of ablated tissue is not clearly seen without the red dashed lines, and does not match well with the photograph. What is the expected spatial resolution of the system? Soldering iron would damage only the contacting surface, but the TAI might be imaging the inside of the liver sample. Please comment on this.

Response 1: Thank you for pointing this out. In response to your question, we have decided to make some changes to Figure 2. We got rid of the red dotted line. You can indeed see clear organizational boundaries in Figure 2 (b). We apologize for the misunderstanding caused by the red dotted line we drew earlier. We have used red arrows to indicate the boundaries of normal tissue and yellow arrows to indicate the boundaries of damaged tissue. In addition, it should be noted that because the imaging system is a rotating acquisition signal, certain deformation may occur in the final synthesized image. But the TAI is roughly consistent with the photo.

And the spatial resolution of TAI in this study is 0.9mm, which was analyzed in our previous study（Huang, L.; Li, T.; Jiang, H. Technical Note: Thermoacoustic Imaging of Hemorrhagic Stroke: A Feasibility Study with a Human Skull. Med. Phys. 2017, 44, 1494–1499, doi:10.1002/mp.12138.）. It should be noted that the ex vivo porcine liver sample we used was thin slice. Due to the insufficient vertical resolution of the imaging system, its ability to image normal liver tissue beneath the damaged tissue layer is limited. Additionally, the purpose of this study was to preliminarily assess whether TAI can be used to observe normal and damaged tissue. This constitutes a feasibility study. Naturally, there are areas in the experimental design that require improvement.

The revision is presented on pages 4, lines 195-206.

Comments 2: In the imaging depth measurement, which part of the body (or specifically liver) does the soy sauce tube represent? In 0 mm depth result in Fig 3, the soy sauce tube generates much higher intensity than the surrounding liver tissue, which does not seem common in real tissues. However, as you mentioned in Fig 2, the ablated region would look as blank, not strong signal. Thus, the soy sauce imaging cannot demonstrated the imaging depth of ablated tissue.

Response 2: Thank you for your questions. We think there are some misunderstandings that need to be clarified. In imaging depth experiments, soy sauce tubes are not used to represent organs of the body. The main purpose of this part of the experiment is to discuss microwave attenuation. In this experiment, we gradually increased the thickness of liver tissue in the area from the antenna to the target tissue (soy sauce tube), in order to explore the effect of microwave attenuation generated by the system under the increase of liver thickness on TAS. Because of the high contrast between the soy sauce tube and the transformer oil and the clearly observable boundary, we chose the soy sauce tube as the target tissue. In addition, the thermoacoustic signal generated by pulsed microwave radiation is the ultrasonic signal with a frequency of less than 2MHz (Xu, Y.; Wang, L.V. Signal Processing in Scanning Thermoacoustic Tomography in Biological Tissues. Med. Phys. 2001, 28, 1519–1524, doi:10.1118/1.1380436.). And the attenuation of ultrasound signals in the liver at low frequency is relatively low (Parker, K.J.; Asztely, M.S.; Lerner, R.M.; Schenk, E.A.; Waag, R.C. In-Vivo Measurements of Ultrasound Attenuation in Normal or Diseased Liver. Ultrasound Med. Biol. 1988, 14, 127–136, doi:10.1016/0301-5629(88)90180-9.). Therefore, the imaging depth of the TAI imaging system in liver tissue can be approximated by discussing the changes in the signal-to-noise ratio and TAS caused by microwave attenuation caused by different thickness of liver. Of course, in the subsequent system optimization, real-time imaging by placing the antenna and ultrasonic probe on the same level will further promote the clinical application of TAI technology. In addition, we can see from Figure 2 that the damaged tissue is not a completely blank area (yellow arrow). The signal of damaged tissue is weaker than that of normal tissue, which may be related to the design of TAI system. I hope my explanation can answer your questions. Thank you very much!

Comments 3: In Fig 5, it is mentioned that the diameter of the ablated lesion was 22 mm, but it looks similar to the scale bar length. The margin does not seem clearer than the ultrasound image.

Response 3: We apologize for the typographical error. We confused the diameter of the ablated tissue with the subcutaneous distance. The correct description should be: “We observed from TAI image that the ablated lesion was 10 mm in diameter (red circle), and located 22 mm beneath the rabbit's skin (yellow arrow), which corresponds well with the ultrasound images.”. We sincerely apologize once again for our mistake. Furthermore, the reason the ultrasound images appear visually clearer is that many bubbles form in the liver tissue after ablation, leading to significant changes in the ultrasound. However, this does not represent the actual ablation boundary. The revision is presented on pages 7, lines 321-322.

Comments 1: While I understand that the authors did not collect the data that I requested, the authors will be able to consider collecting data from normal or non-treated tissue for comparison.

Response 1: Thank you for pointing this out. Our initial idea for designing the experiment was to control the complex influencing factors by displaying both normal and damaged tissues within a single experimental image. That can make a rough comparison. Your suggestion is very useful, and we will incorporate this idea into our new experimental designs in the future. Thank you once again for your proposal.

Comments 2: "MWA needs to minimize damage to healthy tissue" can be "Damage to healthy tissue should be minimized in MWA" or something like that. Meanwhile, the authors may be able to proofread the manuscript once more.

Response 2: Agree. The changes are shown on page 2, line 46. In addition, we also checked the contents of the manuscript again and made modifications to address similar errors.

Comments 3: "60s" can be "60 s".

Response 3: Thank you for pointing this out. The changes are shown on page 4, line 126.