超音波對比劑微氣泡已被證實經靜脈注射後於活體內具有多種重要醫療用途，例如吸收超音波能量後可誘發其體積發生脹縮變化，進而對週遭血管內皮細胞產生物理性刺激並提升血管通透性；或是可作為藥物/基因載體，將藥物釋放至靠近病灶處之血管壁，達成局部藥物控制遞送。但實際在血管中流動時，微氣泡主要是沿著血管中心線流動，僅有少量的微氣泡靠近血管壁，此現象將大幅降低其相關應用之成效。為解決此困境，本研究將提出一非侵入式方式，藉由超音波產生之聲輻射力操控微氣泡於血管中的空間分佈，使流動之微氣泡可大量累積至特定區域。此外，目前市面上仍缺乏能夠同時針對微氣泡相關之治療與診斷並兼具良好空間/時間解析度的非侵入性成像工具。藉此，本研究提出結合超音波漩渦式聲鉗與平面波成像實現以同一顆探頭操控微氣泡於血管中之空間分布並可同時監控的成效。實驗採用 5-MHz 二維陣列式超音波探頭 (11 × 11 陣元，3 個週期，聲壓為 2000 kPa) 與自製微氣泡 (平均粒徑約 1.2 μm)，於流速 20 mm/s 的流場環境進行實驗。漩渦式聲鉗之波型是藉由將發射脈衝重複頻率設計為非對稱 (流入端：4 kHz；流出端：8 kHz) 而形成。為了達成以同一顆探頭同時捕捉微氣泡並進行造影，漩渦式聲鉗脈衝訊號與超音波影像脈衝訊號以交錯串接的方式連接，影像由 9 個角度 (-7° 至 7°) 的平面波脈衝進行複合組成複合超音波平面波影像 (幀速率：200 fps；橫向解析度：0.65 mm)。微氣泡捕捉之成效首先於直徑 200-μm 纖維管中利用螢光顯微影像驗證，再由 3-D 複合超音波平面波影像於直徑 3-mm 管狀仿體進行聲學觀測。由顯微鏡觀測結果顯示微氣泡確實可在流場中被漩渦式聲鉗聚集而形成微氣泡團簇 (直徑約為 71 μm)，並能抵抗流速持續於定點位置約 22 秒。在漩渦式聲鉗脈衝訊號與超音波影像脈衝訊號交錯發射 1 秒後，超音波 3D 影像中顯示微氣泡團簇相對於背景區域之訊號強度提高了約 13 dB，此結果表明我們提出之方式確實可僅透過單一探頭就將流動之微氣泡於特定位置大量累積，並可同步觀察此現象，未來將可透過此工具提高微氣泡應用相關之成效，並應用於多種疾病模型治療。

Ultrasonic contrast agent microbubbles (MBs) have been proven to have a variety of important medical applications in vivo following intravenous injection. For example, ultrasonic energy can induce volumetric change of MBs, which provides a physical force to the surrounding vascular endothelial cells, enhancing the permeability of blood vessels; or MBs can be used as a drug/gene carrier for release drugs to the blood vessel wall near the lesion to achieve local drug delivery. However, when flowing in a blood vessel, MBs frequently flow along the centerline of the blood vessel, and only a small amount of MBs are close to the blood vessel wall. This phenomenon will greatly reduce the outcome of MBs-related applications. In addition, there is still lack of non-invasive imaging tools for therapeutic and diagnostic applications of MBs with good spatial/temporal resolution. Therefore, this research aims to combine acoustic vortex tweezers (AVT) and ultrasonic plane wave imaging to achieve the effect of controlling the spatial distribution of MBs in blood vessels and simultaneously monitoring this process via the same probe. The experiments were conducted by a 5-MHz 2D array ultrasonic probe (11 × 11 array elements, 3 cycles, acoustic pressure of 2000 kPa) and self-made MBs (mean size: 1.2 μm) with a flow rate of 20 mm/s. The waveform of the AVT is formed by modulating the repetition frequency of the transmitted pulse to be asymmetric (inflow end: 4 kHz; outflow end: 8 kHz). In order to simultaneously capture MBs and perform imaging with the same probe, the AVT pulse signal and the ultrasonic image pulse signal are connected in a staggered series, and the imaging is performed by plane wave pulses at 9 angles (-7° to 7°) compounded plane wave imaging (CPWI, frame rate: 200 fps; lateral resolution: 0.65 mm). The effect of capturing MBs was first verified by fluorescence microscopy in a 200-μm fiber tube, and then acoustically observed on a 3-mm tubular analogue from 3-D CPWI. Microscopic observations showed that MBs could indeed be gathered by the AVT in the flow field to form MBs clusters (about 71 μm in diameter), and could resist the flow and stay at a fixed point for about 22 s. After the AVT signal and the ultrasonic image pulse signal turns on for 1 s, the ultrasonic 3D image showed that the signal intensity of the MBs clusters increased 13 dB comparing with the background area. This result showed that the proposed strategy could accumulate flowing MBs in a desired location and to observe this phenomenon simultaneously with a single probe. In the future, this tool can be used to improve the outcomes of MBs-related applications for treating various disease models.

摘要 i
Abstract iii
誌謝 v
目錄 vi
圖目錄 ix
表目錄 xii
第一章 緒論 1
1.1 超音波結合微氣泡用於藥物遞送 1
1.1.1 靜脈注射藥物遞送途徑之困境 1
1.1.2 超音波對比劑微氣泡用於藥物遞送 1
1.1.3 超音波聲輻射力增強微氣泡於血流中之遞送效率與其限制 2
1.2 超音波聲鉗 3
1.2.1 超音波聲鉗的種類與其應用限制 3
1.2.2 超音波漩渦式聲鉗 5
1.2.3 整合式醫學影像導引超音波微氣泡藥物遞送 6
1.3 超音波成像 7
1.3.1 傳統超音波成像 7
1.3.2 平面波超快速成像 8
1.3.2.1 平面波成像方式 8
1.3.2.2 平面波影像之應用 10
1.3.2.3 平面波影像結合超音波對比劑微氣泡之應用 11
1.3.3 四維平面波體積影像 12
1.4 研究目的及論文架構 14
第二章 實驗材料與方法 16
2.1 概論 16
2.2 微氣泡之製備與特性量測 16
2.2.1 DiI-loaded MBs (DiI-MBs) 之薄膜製備 17
2.2.2 DiI-MBs之製備 17
2.2.3 微氣泡之光學定性 18
2.2.4 微氣泡之濃度與粒徑分布量測 18
2.2.5 微氣泡之穩定性測量 19
2.3 超音波相關實驗之硬體設備 20
2.3.1 Verasonics開放式超音波陣列研究平台 20
2.3.2 二維陣列式超音波探頭 21
2.4 發射波型設置 22
2.4.1 探頭陣元對位 22
2.4.2 聚焦波型 23
2.4.3 漩渦聲場波型 23
2.4.4 流場應用修正後之漩渦聲場波型 24
2.5 聲場模擬與量測 25
2.5.1 聲場模擬方法 25
2.5.2 實際發射聲場量測 25
2.6 螢光顯微系統觀測實驗 26
2.6.1 光學實驗系統架構 26
2.6.2 光學顯微影像分析 27
2.7 三維平面波影像實現方式 28
2.7.1 三維複合平面波影像 28
2.7.2 探頭陣元靈敏度設置 29
2.8 聲學影像解析度測試 30
2.8.1 金屬線仿體製作 30
2.8.2 聲學影像解析度測試之實驗架構 30
2.9 聲學影像觀測實驗 32
2.9.1 仿體製作 32
2.9.2 聲學實驗系統架構 32
2.9.3 漩渦聲場波型結合平面波脈衝之發射序列設計 34
2.10 統計分析 35
第三章 實驗結果與討論 36
3.1 微氣泡之物化性質 36
3.1.1 微氣泡之光學定性 36
3.1.2 微氣泡之濃度與粒徑分析 36
3.1.3 微氣泡之穩定性測量 37
3.2 聲場模擬與量測 38
3.2.1 探頭陣元對位 38
3.2.2 聚焦波型 39
3.2.3 漩渦聲場波型 40
3.2.4 漩渦聲場波型之發射相位優化 41
3.2.5 流場應用修正後之漩渦聲場波型 43
3.3 螢光顯微系統觀測實驗 44
3.3.1 靜場之微氣泡捕獲現象 44
3.3.2 流場中不同發射波型對微氣泡之影響效果比較 46
3.3.3 流場中漩渦式聲鉗之實驗參數比較 48
3.3.4 漩渦式聲鉗之流速極限測試 49
3.3.5 漩渦式聲鉗之捕獲累積過程 50
3.3.6 漩渦式聲鉗結合複合平面波之可行性驗證 51
3.4 微氣泡團簇於聲學影像的解析情形 52
3.5 漩渦式聲場串接複合平面波之聲學影像觀測實驗 54
3.5.1 微氣泡團簇於直徑3-mm中空管仿體之空間解析情形 54
3.5.2 靜場之微氣泡捕獲現象 54
3.5.3 流場之微氣泡捕獲累積現象 56
第四章 結論與未來展望 60
4.1 結論 60
4.2 未來應用與發展 60
參考文獻 61
附錄Ⅰ、5-MHz 二維陣列探頭之平面波影像測試 64
附錄Ⅱ、18-MHz 線型陣列探頭輔助觀測結果 69
附錄Ⅲ、Turnitin檢測結果 73

1. Shewach, D.S. and R.D. Kuchta, Introduction to cancer chemotherapeutics. Chem Rev, 2009. 109(7): p. 2859-61.
2. Chang, E.I., et al., Vascular complications and microvascular free flap salvage: the role of thrombolytic agents. Microsurgery, 2011. 31(7): p. 505-9.
3. Patra, J.K., et al., Nano based drug delivery systems: recent developments and future prospects. J Nanobiotechnology, 2018. 16(1): p. 71.
4. Li, C., et al., Recent progress in drug delivery. Acta Pharm Sin B, 2019. 9(6): p. 1145-1162.
5. Malam, Y., M. Loizidou, and A.M. Seifalian, Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer. Trends Pharmacol Sci, 2009. 30(11): p. 592-9.
6. Huang, T., N. Li, and J. Gao, Recent strategies on targeted delivery of thrombolytics. Asian J Pharm Sci, 2019. 14(3): p. 233-247.
7. Liu, Y., H. Miyoshi, and M. Nakamura, Encapsulated ultrasound microbubbles: therapeutic application in drug/gene delivery. J Control Release, 2006. 114(1): p. 89-99.
8. Gong, Q., et al., Drug-Loaded Microbubbles Combined with Ultrasound for Thrombolysis and Malignant Tumor Therapy. Biomed Res Int, 2019. 2019: p. 6792465.
9. Chandan, R., S. Mehta, and R. Banerjee, Ultrasound-Responsive Carriers for Therapeutic Applications. ACS Biomaterials Science & Engineering, 2020. 6(9): p. 4731-4747.
10. Gorick, C.M., et al., Sonoselective transfection of cerebral vasculature without blood-brain barrier disruption. Proc Natl Acad Sci U S A, 2020. 117(11): p. 5644-5654.
11. Tzu-Yin, W., et al., Ultrasound and microbubble guided drug delivery: mechanistic understanding and clinical implications. Curr Pharm Biotechnol, 2013. 14(8): p. 743-52.
12. Monteith, S., et al., Potential intracranial applications of magnetic resonance-guided focused ultrasound surgery. J Neurosurg, 2013. 118(2): p. 215-21.
13. Yalcin, O., et al., Plasma expander viscosity effects on red cell-free layer thickness after moderate hemodilution. Biorheology, 2011. 48(5): p. 277-91.
14. Poelma, C., et al., Accurate blood flow measurements: are artificial tracers necessary? PLoS One, 2012. 7(9): p. e45247.
15. Wilhelm, S., et al., Analysis of nanoparticle delivery to tumours. Nature Reviews Materials, 2016. 1(5): p. 16014.
16. Dayton, P., et al., Acoustic radiation force in vivo: a mechanism to assist targeting of microbubbles. Ultrasound Med Biol, 1999. 25(8): p. 1195-201.
17. Rychak, J.J., A.L. Klibanov, and J.A. Hossack, Acoustic radiation force enhances targeted delivery of ultrasound contrast microbubbles: in vitro verification. IEEE Trans Ultrason Ferroelectr Freq Control, 2005. 52(3): p. 421-33.
18. Ozcelik, A., et al., Acoustic tweezers for the life sciences. Nat Methods, 2018. 15(12): p. 1021-1028.
19. Meng, L., et al., Acoustic tweezers. Journal of Physics D-Applied Physics, 2019. 52(27).
20. Hwang, J.Y., et al., Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells. Ultrasonics, 2015. 63: p. 94-101.
21. Ding, X.Y., et al., On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(28): p. 11105-11109.
22. Kang, S.T. and C.K. Yeh, Potential-well model in acoustic tweezers. IEEE Trans Ultrason Ferroelectr Freq Control, 2010. 57(6): p. 1451-9.
23. Lamsam, L., et al., A review of potential applications of MR-guided focused ultrasound for targeting brain tumor therapy. Neurosurg Focus, 2018. 44(2): p. E10.
24. Izadifar, Z., et al., An Introduction to High Intensity Focused Ultrasound: Systematic Review on Principles, Devices, and Clinical Applications. J Clin Med, 2020. 9(2).
25. Hsiao, Y.H., et al., Clinical Application of High-intensity Focused Ultrasound in Cancer Therapy. J Cancer, 2016. 7(3): p. 225-31.
26. Elhelf, I.A.S., et al., High intensity focused ultrasound: The fundamentals, clinical applications and research trends. Diagn Interv Imaging, 2018. 99(6): p. 349-359.
27. Kinoshita, M., et al., Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption. Proc Natl Acad Sci U S A, 2006. 103(31): p. 11719-23.
28. Mikhail, A.S., et al., Magnetic Resonance-Guided Drug Delivery. Magn Reson Imaging Clin N Am, 2015. 23(4): p. 643-55.
29. Luan, Y., et al., Lipid shedding from single oscillating microbubbles. Ultrasound Med Biol, 2014. 40(8): p. 1834-46.
30. Peng, H., et al., Ultrafast ultrasound imaging in acoustic microbubble trapping. Applied Physics Letters, 2019. 115(20).
31. Bercoff, J., Ultrafast ultrasound imaging, in Ultrasound imaging-Medical applications. 2011, InTech.
32. Tanter, M. and M. Fink, Ultrafast imaging in biomedical ultrasound. IEEE Trans Ultrason Ferroelectr Freq Control, 2014. 61(1): p. 102-19.
33. Montaldo, G., et al., Coherent plane-wave compounding for very high frame rate ultrasonography and transient elastography. 2009. 56(3): p. 489-506.
34. Bercoff, J., M. Tanter, and M. Fink, Supersonic shear imaging: a new technique for soft tissue elasticity mapping. IEEE Trans Ultrason Ferroelectr Freq Control, 2004. 51(4): p. 396-409.
35. Bercoff, J., et al., Ultrafast compound Doppler imaging: providing full blood flow characterization. IEEE Trans Ultrason Ferroelectr Freq Control, 2011. 58(1): p. 134-47.
36. Jensen, J.A., et al., Ultrasound Vector Flow Imaging-Part I: Sequential Systems. IEEE Trans Ultrason Ferroelectr Freq Control, 2016. 63(11): p. 1704-1721.
37. Jensen, J.A., et al., Ultrasound Vector Flow Imaging-Part II: Parallel Systems. IEEE Trans Ultrason Ferroelectr Freq Control, 2016. 63(11): p. 1722-1732.
38. Couture, O., et al., Ultrasound contrast plane wave imaging. 2012. 59(12): p. 2676-2683.
39. Couture, O., et al., Tumor Delivery of Ultrasound Contrast Agents Using Shiga Toxin B Subunit. Molecular Imaging, 2011. 10(2).
40. Errico, C., et al., Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging. Nature, 2015. 527(7579): p. 499-502.
41. Provost, J., et al., 3D ultrafast ultrasound imaging in vivo. Phys Med Biol, 2014. 59(19): p. L1-L13.
42. Correia, M., et al., 4D ultrafast ultrasound flow imaging: in vivo quantification of arterial volumetric flow rate in a single heartbeat. Phys Med Biol, 2016. 61(23): p. L48-L61.
43. Rabut, C., et al., 4D functional ultrasound imaging of whole-brain activity in rodents. Nat Methods, 2019. 16(10): p. 994-997.
44. Sauvage, J., et al., 4D Functional Imaging of the Rat Brain Using a Large Aperture Row-Column Array. IEEE Trans Med Imaging, 2020. 39(6): p. 1884-1893.
45. Ng, A. and J. Swanevelder, Resolution in ultrasound imaging. Continuing Education in Anaesthesia Critical Care & Pain, 2011. 11(5): p. 186-192.