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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 5  |  Page : 1129-1133

Evaluation of the correlation between infrared thermal imaging-magnetic resonance imaging-pathology of microwave ablation of lesions in rabbit lung tumors


1 Department of Interventional Radiology, First Affiliated Hospital of Fujian Medical University, Fuzhou, China
2 Department of Radiology, Fujian Provincial People's Hospital, Fuzhou, China
3 Department of Radiology, Ningde Mindong Hospital, Fuan City, China
4 Department of Radiology, Quanzhou First Hospital, Quanzhou, China
5 Department of Radiology, Wuping County Hospital, Wuping County, Longyan, China

Date of Submission29-Dec-2019
Date of Decision20-Mar-2020
Date of Acceptance17-Aug-2020
Date of Web Publication29-Sep-2020

Correspondence Address:
Zheng-Yu Lin
Department of Interventional Radiology, First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou 350005
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.296428

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 > Abstract 


Purpose: This study aimed to evaluate the correlation between infrared thermal imaging-magnetic resonance imaging (MRI)-pathology of microwave ablation (MWA) of lesions in rabbit lung tumors.
Materials and Methods: MR-guided MWA was performed in nine VX2 tumor-bearing rabbits. Infrared thermal imaging, postoperative MRI, and pathological presentation were obtained and analyzed. The differences between the infrared thermal imaging-MRI-pathology of MWA were compared.
Results: The center of the ablated lesion exhibited a high signal on T1-Vibe, and an isointense envelope was observed; the center of the ablated lesion exhibited a low signal on fat-suppressed turbo spin-echo T2-weighted imaging (TSE-T2WI-FS) and bands of high signal surrounding it compared with before MWA. No statistically significant difference existed between the maximum diameter of the central low-signal area of the ablation zone on TSE-T2WI-FS after MWA, the high-signal area of the ablation zone on T1-Vibe after MWA, and the maximum diameter of the pathological coagulation necrosis area, as well as between the maximum diameter of the isointense signal area peripheral to the ablation zone on T1-Vibe after MWA, the high-signal area peripheral to the ablation zone on TSE-T2WI-FS, the maximum diameter at the 41°C isothermal zone on infrared thermal imaging, and the maximum diameter of the pathological thermal injury zone.
Conclusions: MWA of malignant lung tumors had specific MRI characteristics that were comparable with postoperative pathology. Infrared thermal imaging combined with MRI can be used to evaluate the extent of thermal damage to lung VX2 tumors.

Keywords: Infrared thermal imaging, magnetic resonance imaging, microwave ablation, pulmonary cancer, VX2 tumors


How to cite this article:
Chen J, Lin XN, Miao XH, Chen J, Lin RX, Su HY, Lin JB, Lin ZY. Evaluation of the correlation between infrared thermal imaging-magnetic resonance imaging-pathology of microwave ablation of lesions in rabbit lung tumors. J Can Res Ther 2020;16:1129-33

How to cite this URL:
Chen J, Lin XN, Miao XH, Chen J, Lin RX, Su HY, Lin JB, Lin ZY. Evaluation of the correlation between infrared thermal imaging-magnetic resonance imaging-pathology of microwave ablation of lesions in rabbit lung tumors. J Can Res Ther [serial online] 2020 [cited 2020 Oct 26];16:1129-33. Available from: https://www.cancerjournal.net/text.asp?2020/16/5/1129/296428




 > Introduction Top


Microwave ablation (MWA) has the advantages of high thermal efficiency, accurate therapeutic effect, accuracy, and minimal trauma and is a common method for thermal ablation of lung tumors.[1] To ensure long-term survival, immediate imaging evaluation of MWA efficacy is essential. Magnetic resonance imaging (MRI) has high tissue resolution and supports multiparameter imaging, making it the best method for immediate evaluation of efficacy.[2] Infrared thermal imaging technology can be used to monitor body surface temperature changes directly, in real time, and in a noninvasive manner [3] and moreover has value for evaluating the extent of thermal damage. In this study, infrared thermal imaging, postoperative MRI, and pathological presentation of rabbit lung VX2 tumors after MWA were compared to analyze the correlation between infrared thermal imaging-MRI-pathology of MWA of lesions in rabbit lung tumors.


 > Materials and Methods Top


Establishment of rabbit lung VX2 tumor model

Experimental animals

The research protocol was approved by the Institutional Animal Care and Use Committee of Fujian Medical University (Fujian, China). Thirteen healthy adult male New Zealand white rabbits weighing 2.0–3.2 kg (average, 2.82 ± 0.29 kg) were used in the study. The animals were purchased from the Wu Experimental Animal Center (Minhou County, Fujian Province).

Preparation of VX2 tumor strain

General anesthesia was induced in leg tumor-bearing rabbits (gift from the Experimental Animal Center of Fujian Medical University) by intramuscular injection of 1 mL/kg ketamine and 0.5 mL/kg chlorpromazine into the leg muscles. Local anesthesia was achieved using 2% lidocaine in the operation area, and the animal was immobilized on an operating table. Fish-meat-like VX2 tumor tissue with aggressive growth was resected from the thigh margins of the tumor-bearing rabbits and placed in a culture dish containing penicillin and normal saline (1:40). The tumor tissue was cut into pieces, approximately 1 mm × 1 mm × 1 mm in size, using ophthalmic surgical scissors for later use.

Rabbit lung VX2 tumor implantation

Thirteen New Zealand white rabbits were fasted for 6 h before surgery. After general anesthesia, the skin at the site of puncture was prepared according to a routine protocol. The animals were immobilized in the prone position on a 16-slice spiral computed tomography (CT) scanner (Somatom Emotion, Siemens, Erlangen, Germany). After positioning, disinfection, and draping, lung tissue was punctured using a 17-gauge needle (Gallini Srl, Mirandola, Italy) under CT guidance. The needle core was withdrawn, and 3–4 tissue blocks were retrieved using sterile forceps and inserted into the needle sheath. The needle core was used to gently push the tissue blocks into the lung parenchyma. The needle was then withdrawn, and a scan was performed again to determine the presence of postoperative pneumothorax or bleeding. CT of the lungs was performed on postoperative day 20 to observe tumor formation. One tumor-bearing rabbit was randomly selected for euthanization and pathological confirmation of successful implantation of the VX2 tumor in the lung.

Microwave ablation of rabbit lung VX2 tumors

Preoperative magnetic resonance imaging scanning and treatment protocol

Preoperative magnetic resonance imaging scanning

Under general anesthesia, a 1.5 Tesla MRI plain scan (Magnet Espree, Siemens, Germany) of the lungs of the tumor-bearing rabbits was performed using a surface coil. Scanning sequence and parameters using different modalities were as follows:

  1. Fat-suppressed turbo spin-echo T2-weighted imaging (TSE-T2WI-FS): Repetition time (TR), 4000.0 ms; echo time (TE), 80.0 ms; flip angle (FA), 90°; slice thickness (ST), 3.0 mm; slice gap (GAP), 0.6 mm; field of view (FOV), 20 × 15; number of excitations (NEX), 2; and time 130–150 s
  2. T1-weighted three-dimensional volumetric-interpolated breath-hold examination (T1-Vibe): TR, 5.1 ms; TE, 2.4 ms; NEX, 1; ST, 3.0 mm; FOV, 20 × 15; and time, 10–12 s
  3. Diffusion-weighted imaging (DWI): TR, 1500 ms; TE, 94 ms; NEX, 2; ST, 3.0 mm; GAP, 0.6 mm; and b = 50–800 s/mm 3.


Both fat-suppressed T2WI and DWI sequences were performed using a diaphragm trigger. The needle insertion point on the skin was determined, the distance between the intended needle insertion point and the body surface Vitamin E capsule was measured, and body surface was positioned and labeled using a colored pen. In addition, the penetration depth and angle were measured.

Magnetic resonance imaging-guided microwave ablation of rabbit lung VX2 tumors

Routine disinfection and draping were performed before the operation. A small skin incision was made at the puncture point using a sharp blade. The intraoperative MRI-guided puncture sequence was TSE-T2WI-FS, T1-Vibe, using the same parameters described above. During the puncture process, TSE-T2WI-FS scanning was performed several times to ensure that the needle was inserted in the correct direction and that blood vessels and the trachea in the needle track were avoided as much as possible. The scanning direction was oblique sagittal or oblique transverse axial parallel to the MRI-compatible microwave antenna (14G, 15 cm, VISION Medical Equipment Co., Nanjing, China) to show the full length of the microwave antenna. The microwave antenna penetrated along the center of the tumor and extended 0.5 cm beyond its distal end. TSE-T2WI-FS and T1-Vibe scans were performed again to confirm the relationship between the needle tip, the tumor, and important surrounding tissue structures. After confirming that the needle position was satisfactory, water-cooling circulation was connected to the coaxial cable, the microwave ablation parameters were set, and ablation was started. At the same time, the distance and angle of the infrared thermal imager (Hangzhou Yuanzhou Medical, Co., Ltd., China) were adjusted, the centerline of the lens was perpendicular to the body surface of the ablation zone, the distance between the lens and the body surface was maintained at 130 cm, and infrared thermal imaging was monitored in real time. The MWA output power was set to 80 W, ablation was performed for 4 min, and temperature field change data were collected.

Magnetic resonance imaging scanning after microwave ablation and immediate evaluation of efficacy

An MRI scan was performed immediately after MWA to observe the ablated lesions. Tumor-bearing rabbits were euthanized, and lung specimens were harvested and cut along the MWA antenna needle path for histopathological examination. Postoperative maximum diameter of the central high-signal tumor on T1-Vibe (DT1), maximum diameter of the central low-signal tumor on TSE-T2WI-FS (DT2), pathological lung tissue coagulation and necrosis (D coagulation), maximum diameter of the signal area around the ablation zone on T1-Vibe (DT1Z), ablation margin of high-signal zone on TSE-T2WI-FS (DT2Z), maximum diameter of the 41°C isothermal zone on infrared thermal imaging (DIR), and maximum diameter of the pathologically thermally damaged zone (D injury) were measured.

Statistical analysis

SPSS version 22.0 (IBM Corporation, Armonk, NY, USA) was used for statistical analysis. The paired t-test was performed on all data, which are expressed as mean ± standard deviation (x ± s); differences with P < 0.05 were considered to be statistically significant.


 > Results Top


Tumor formation and magnetic resonance imaging examination

Isolated tumors were found in the lungs of 9 of the 13 experimental rabbits (4 in the left lower lobe and 5 in the right lower lobe). The maximum diameter was 1.02–1.41 cm, with an average of 1.17 ± 0.14 cm. Extensive metastasis to the pleura was observed in 1 rabbit, and no clear tumors were found in 2 rabbits. One tumor-bearing rabbit was euthanized and confirmed to have a VX2 tumor. MRI examination revealed a solitary nodule in the lung with a shallow lobular change at the edge of the lesion and clear margins. Compared with the signal from the chest wall muscle layer, the lesion exhibited a slightly higher signal on the TSE-T2WI-FS sequence [Figure 1]a and clear margins and an isointense signal on the T1-Vibe sequence [Figure 1]b.
Figure 1: (a) The lesion exhibited a slightly higher signal on the fat-suppressed turbo spin-echo T2-weighted imaging sequence. (b) The lesion exhibited an isointense signal on the T1-Vibe sequence

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Microwave ablation intraoperative infrared imaging examination

The average surface temperature at the operation site was approximately 30.5°C before MWA was started and gradually increased thereafter. The thermal field was roughly concentric and radiated outward from the center, with the highest temperature in the center [Figure 2]. The temperature reached a peak when ablation was completed. The maximum mean surface temperature at the operation site was 45.51 ± 0.63°C.
Figure 2: (a) Different colors represent different temperature zones in infrared thermal imaging: the white zone has the highest temperature, and temperature decreases in the order of red, yellow, green, and blue. After microwave ablation was started, the high-temperature zone gradually expanded at 30 s. (b) After microwave ablation was started, the high-temperature zone gradually expanded at 120 s. (c) After microwave ablation was started, the high-temperature zone gradually expanded at 240 s

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Microwave ablation postoperative imaging examination

MRI imaging performed immediately after MWA revealed that the center of the ablated lesion exhibited a low signal on TSE-T2WI-FS, with blurred margins, bands of high signal surrounding it, and high-signal membrane edema and thickening in the adjacent chest [Figure 3]a. The center of the ablated lesion exhibited a high signal on T1-Vibe, the peripheral margins were unclear, and an isointense envelope was observed [Figure 3]b.
Figure 3: (a) The center of the ablated lesion exhibited low signal on fat-suppressed turbo spin-echo T2-weighted imaging, with blurred margins, bands of high signal surrounding it. (b) The center of the ablated lesion exhibited a high signal on T1-Vibe, and an isointense envelope was observed

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Microwave ablation postoperative pathological examination

Gross morphology after microwave ablation

After MWA, with the needle track at the center, morphology from the inside to outside appeared as follows: carbonized area (the area around the needle track, black, hard, and brittle); tumor coagulation necrosis area (peripheral to the carbonized area, gray-white); lung tissue coagulation necrosis area (peripheral to the tumor coagulation necrosis area, reddish-brown); and the peripheral thermal injury area (a band of normal lung tissue coagulation necrosis with congestion and edema, light red) [Figure 4]a. Under light microscopy, rabbit lung VX2 tumor after MWA (hematoxylin and eosin staining) exhibited large areas of tumor cell necrosis and complete destruction of nuclear structure, with only the outlines of cells remaining. The lung coagulation necrosis area exhibited destruction of nuclear structure and outlines of the remaining lung tissue cells. A large amount of exudate was found in the alveoli of the heat-damaged area, and some of the alveoli were completely filled. The exudate contained fragmented red blood cells and various inflammatory cells, and the boundaries with normal lung tissue were unclear [Figure 4]b.
Figure 4: (a) After microwave ablation, with the needle track at the center, morphology from the inside to outside: carbonized area; tumor coagulation necrosis area; lung tissue coagulation necrosis area; and the peripheral thermal injury area. (b) Light microscope from inside to outside divided into tumor coagulation necrosis zone (I), coagulation necrosis area (II), pulmonary congestion and edema (III), and inflammatory cell infiltration zone (IV)

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The results are summarized as follows:

  1. Pairwise comparison of the central low-signal area of the ablation zone on TSE-T2WI-FS after MWA, the high-signal area of the ablation zone on T1-Vibe after MWA, and the maximum diameter of the pathological coagulation necrosis area revealed no statistical differences (i.e., P = 0.865)
  2. Pairwise comparison of the isointense signal area peripheral to the ablation zone on T1-Vibe after MWA, the high-signal area peripheral to the ablation zone on TSE-T2WI-FS, and the maximum diameter of the pathological thermal injury zone revealed no statistical differences (i.e., P = 0.934)
  3. Comparison of the maximum diameter at the 41°C isothermal zone on infrared thermal imaging and the maximum diameter of the pathological thermal injury zone demonstrated no statistical differences (i.e., P = 0.579).



 > Discussion Top


MWA is widely used in the treatment of lung cancer and has demonstrated good curative effects.[4],[5],[6] CT is currently the most commonly used method for guiding and evaluating the efficacy of lung tumor MWA. However, CT scanning produces ionizing radiation, cannot be used in real time to guide puncture(s), and only has a single imaging parameter. Ground-glass opacities surrounding the lung tumor often obscure the original tumor after ablation, and its extension by 5–10 mm beyond the surrounding area is considered to be a sign of successful tumor ablation.[7] However, because mechanical lung puncture can easily cause intrapulmonary hemorrhage, confounding between intrapulmonary hemorrhage and ground-glass opacities after ablation can sometimes affect the evaluation of efficacy, resulting in inaccurate assessment of efficacy. Therefore, CT has some shortcomings, despite being the most commonly used modality for guidance of lung tumor thermal ablation. Ultrasound is widely used to guide MWA due to its simple operation and real-time guidance for puncture. However, because gasses have greater effects on ultrasound, ultrasound is rarely used to guide lung ablation except for lesions close to the chest wall. The water vapor generated by the high temperatures during MWA is hyperechoic on ultrasound, which can easily be confused with the hyperechogenicity of the lesion, making it difficult to evaluate the immediate efficacy of ablation treatment.[8] MRI produces no ionizing radiation damage and has high soft-tissue resolution. It can be arbitrarily oriented and supports multiparameter imaging, can provide information about blood vessels around the lesion without contrast agent, does not produce artifacts from metal puncture needles or bone, and can better depict injury to the pleura, soft tissues of the chest wall, and adjacent bones, nerves, and other tissues after MWA than CT or ultrasound.[8],[9],[10] Therefore, MRI-guided MWA has unparalleled advantages over CT and ultrasound.

In this study, conventional MRI scans were performed before MWA. Compared with the muscles of the chest wall, tumors exhibited shadows with slightly higher signal on T2WI, while some had nonuniform signals. Tumors exhibited isointense signals and clear boundaries on T1-Vibe. After MWA, there was a low signal in the center of the ablation zone on TSE-T2WI-FS that covered the original high-signal lesion area, and a ring-shaped signal was found in the surrounding area on TSE-T2WI-FS. Here, a high signal in the center of the ablation zone on T1-Vibe and a ring-shaped signal were found in the surrounding area on T1-Vibe. The peripheral isointense T1-Vibe signal and the high-signal thermal damage reactive zone on TSE-T2WI-FS extended beyond the original lesion by 5–10 mm. After thermal ablation, the coagulated necrosis area included both coagulated necrotic tumor tissue and coagulated necrotic lung tissue.[11] Coagulation necrosis presents as decreased water content in tissues and cells on pathology, and higher T1-Vibe signal and lower TSE-T2WI-FS signal in the tumor on MRI compared with before MWA, and the boundary between the coagulation necrosis zone signal and the tumor is unclear, with both having high signal on T1-Vibe and low signal on TSE-T2WI-FS. MWA surgery can also cause thermal damage, congestion, and edema in the surrounding lung tissue, which presents as increased tissue water content on pathology, and patchy areas of low T1-Vibe signal surrounding the ablation zone and high TSE-T2WI-FS signal on MRI. The present study statistically confirmed that the coagulation necrosis area and the surrounding thermal injury area after MWA were consistent with the changes in MRI signals. These results are consistent with those reported by Oyama et al.,[12] demonstrating that MRI presentation is strongly correlated with histopathological changes. Therefore, conventional plain MRI scan after ablation can be used for immediate and accurate evaluation of the efficacy of lung tumor ablation.

Postoperative MWA injury is caused primarily by increased temperatures; therefore, thermal field distribution in the lung can be used to predict the effect of ablation and the extent of thermal injury. MR temperature measurement techniques can be used to measure the temperature of the region of interest within the scan field but also has certain limitations. MR temperature measurement techniques require specialized software and are very susceptible to artifacts caused by breathing, heartbeat, and/or movement. Lung tissue contains a large amount of gas, which also affects the accuracy of temperature measurement. In addition, it has some time delay and is not a form of real-time monitoring; as such, it is not completely suitable for real-time monitoring of temperature during thermal ablation treatment of lung tumors.[13] During MWA, internal temperature changes in the body are transmitted to the body surface through blood circulation and heat conduction among tissues. Infrared scanning devices can collect heat energy radiating from the body in real time. Computer postprocessing software can represent different temperatures using various color levels and integrate them into an infrared thermal imaging map that reflects the surface temperature of the body. In this study, the maximum diameter of the 41°C isotherm did not differ significantly from that of the pathological heat injury zone. In monitoring the extent of thermal injury to normal lung tissue during the operation, MR examination indirectly reflects the extent of thermal injury through alveolar hemorrhage or exudation after thermal injury to lung tissue, whereas infrared thermal imaging technology can be used to monitor changes in body surface temperature noninvasively, in real time, and directly. Therefore, infrared thermal imaging combined with MRI can be used to evaluate the extent of lung VX2 tumor thermal damage more accurately.

Limitations

There were limitations to the present study, the first of which was its small sample size. Furthermore, MRI-compatible microwave antenna artifacts were small, and sometimes, the antenna needle tip was not clearly evident in the lung. Postoperative enhanced MRI and CT scans were not used to further evaluate the efficacy of tumor ablation, and were only used to observe acute thermal injury without dynamic observation of the evolution of the ablation zone on imaging and pathology, and must be addressed in future studies.


 > Conclusions Top


Through MWA experiments in rabbit lung VX2 tumors, we found that MWA of malignant lung tumors had specific MRI characteristics that were comparable with postoperative pathology. Infrared thermal imaging combined with MRI can be used to evaluate the extent of thermal damage to lung VX2 tumors.

Acknowledgments

This work was supported by The Start-up Fund for Scientific Research, Fujian Medical University (Grant number: 2017XQ1094), and The Research and Develop Program of Focus Field of Guangdong Province, PRC (Grant number: 2019B110233001).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Zheng A, Ye X, Yang X, Huang G, Gai Y. Local Efficacy and Survival after Microwave Ablation of Lung Tumors: A Retrospective Study in 183 Patients. J Vasc Interv Radiol 2016;27:1806-14.  Back to cited text no. 1
    
2.
Chen J, Lin Z, Chen J, Lin Q, Chen J, Yan Y. Magnetic resonance imaging-guided transperineal prostate biopsy. J Cancer Res Ther 2019;15:394-7.  Back to cited text no. 2
    
3.
de Jesus Guirro RR, Oliveira Lima Leite Vaz MM, das Neves LM, Dibai-Filho AV, Carrara HH, de Oliveira Guirro EC. Accuracy and reliability of infrared thermography in assessment of the breasts of women affected by cancer. J Med Syst 2017;41:87.  Back to cited text no. 3
    
4.
Healey TT, March BT, Baird G, Dupuy DE. Microwave ablation for lung neoplasms: A retrospective analysis of long-term results. J Vasc Interv Radiol 2017;28:206-11.  Back to cited text no. 4
    
5.
Li L, Wu K, Lai H, Zhang B. Clinical application of CT-guided percutaneous microwave ablation for the treatment of lung metastasis from colorectal cancer. Gastroenterol Res Pract 2017;2017:1-9.  Back to cited text no. 5
    
6.
Ierardi AM, Coppola A, Lucchina N, Carrafiello G. Treatment of lung tumours with high-energy microwave ablation: A single-centre experience. Med Oncol 2017;34:5.  Back to cited text no. 6
    
7.
Liu JJ, Wu ZY, Huang W, Ding XY, Wang ZM. Clinical application of CT-guided coaxial tumor biopsy combined with microwave ablation for lung tumors. J Interv Radiol 2018;27:141-6.  Back to cited text no. 7
    
8.
Li H, Lin Z, Chen J, Guo R. Evaluation of the MR and pathology characteristics immediately following percutaneous MR-guided microwave ablation in a rabbit kidney VX2 tumor implantation model. Int J Hyperthermia 2019;36:1197-206.  Back to cited text no. 8
    
9.
Lin Z, Chen J, Yan Y, Chen J, Li Y. Microwave ablation of hepatic malignant tumors using 1.5T MRI guidance and monitoring: Feasibility and preliminary clinical experience. Int J Hyperthermia 2019;36:1216-22.  Back to cited text no. 9
    
10.
Yan Y, Lin ZY, Chen J. Analysis of imaging-guided thermal ablation puncture routes for tumors of the hepatic caudate lobe. J Cancer Res Ther 2020;16:258-62.  Back to cited text no. 10
    
11.
Iguchi T, Hiraki T, Gobara H, Fujiwara H, Matsui Y, Soh J, et al. Percutaneous radiofrequency ablation of lung cancer presenting as ground Glass opacity. Cardiovasc Intervent Radiol 2015;38:409-15.  Back to cited text no. 11
    
12.
Oyama Y, Nakamura K, Matsuoka T, Toyoshima M, Yamamoto A, Okuma T, et al. Radiofrequency ablated lesion in the normal porcine lung: Long-term follow-up with MRI and pathology. Cardiovasc Intervent Radiol 2005;28:346-53.  Back to cited text no. 12
    
13.
Ertürk MA, Sathyanarayana Hegde S, Bottomley PA. Radiofrequency ablation, MR thermometry, and high-spatial Resolution MR parametric imaging with a single, minimally invasive device. Radiology 2016;281:927-32.  Back to cited text no. 13
    


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