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研究生:廖愛禾
研究生(外文):Ai-Ho Liao
論文名稱:多模式小動物影像於腫瘤研究之技術開發與應用
論文名稱(外文):Techniques and Applications of Multi-modality Small Animal Imaging in Cancer Research
指導教授:李百祺
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:電機工程學研究所
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:119
中文關鍵詞:高頻超音波影像系統微正子斷層掃瞄小動物影像三維影像定位多模式小動物影像小動物腫瘤高頻超音波對比劑成像經驗模態分解法脈衝反相
外文關鍵詞:High frequency ultrasoundMicroPET3-D reconstructionMulti-modality small animal imagingWF-3 ovary cancerHepatocellular CarcinomaContrast enhanced ultrasoundEmpirical mode decompositionPulse inversion
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近幾年來,小動物模式被廣泛的運用在臨床前人類疾病治療、基因體研究以及藥物開發等生物醫學領域;在這些研究中,想要立即觀察到效果,非侵入式影像系統便顯得重要,而非侵入式的影像系統中,結合結構性以及功能性兩大類的多重影像系統能同時獲得不同的影像資訊,而使研究觀察層面更加深入及徹底。
本論文主要是結合高頻超音波影像系統以及微正子兩套影像系統,並建立兩者結合時的醫學影像定位方式,以壓克力以及瓊膠分別作為製作植有體表腫瘤的小動物定位固定裝置和仿體的材質,然後在相同定位點位置上,放置六個直徑0.43~0.60 mm的玻璃球和0.1 μl [18F]FDG核醫藥物在腫瘤上方,作為能同時成像於兩種影像上的定位點;並使用orthogonal Procrustes algorithm,作為本研究剛體定位的演算法,我們得到的定位點平均誤差為0.31 mm,並進一步呈現融合影像,最後設計測試仿體,將誤差分為定位點誤差以及標的點誤差來評估實驗結果。
結合這兩套系統在小動物腫瘤研究中,我們以非侵入式的高頻超音波影像系統獲得腫瘤活體內的生長曲線,另一方面在腫瘤生長的每個時期,同時進行微正子造影。本研究使用[18F]FDG進行腫瘤功能性微正子造影,所使用之動物腫瘤模式為植有WF-3卵巢癌細胞的C57BL/6J小黑鼠。在獲得兩種影像資料後,接著進行影像區隔(Tumor Segmentation)以及三維影像重建腫瘤影像並運算體積,最後;由腫瘤的生長曲線獲得腫瘤活體內的體積倍增時間為7.46天,此結果我們將與傳統文獻上直接以尺量測腫瘤大小的結果予以比較。而另一方面,我們由腫瘤的微正子造影獲得的核醫藥物放射線比活度曲線獲得腫瘤每週對周遭組織的藥物比率在第五週到達最高,綜合這些資訊,期能提供更多藥物治療癌症時機的資訊。
為了使高頻超音波造影亦具有功能性影像資訊,使兩者影像能不但在結構與功能上互補,亦能比對其功能性的效果,本研究使用自製的高頻超音波對比劑進行肝腫瘤內對比劑的變化週期,以開發高頻超音波影像的功能性影像效果。在這部分,本研究使用本實驗室團隊研發之高頻率超音波影像系統以及研究團隊所合成的高頻超音波對比劑進行對比劑注射B型肝炎基因轉植小鼠超音波造影來診斷肝腫瘤三個血管脈相時期。本研究成功的辨識對比劑注射後腫瘤區塊影像明顯增強的肝腫瘤動脈期以及緊接著門脈期的快速減退,以時間影像強度曲線量化各時期影像強度,並成功的判讀辨識出13隻疑似有肝腫瘤病變的B型肝炎基因轉植小鼠其肝腫瘤類型、良性或惡性,最後以病理組織切片鑑定結果做為標準,計算高頻超音波對比劑成像結果對偵測小鼠肝腫瘤的靈敏度、特異性以及準確度,提供進入臨床前的小動物肝腫瘤研究良好而準確的鑑定造影工具以及技術,並探討微正子影像與高頻超音波影像的結合在腫瘤研究與技術開發之可行性。
最後,本研究使用整合型經驗模態分解法( Ensemble Empirical Mode Decomposition, EEMD )來增強超音波對比劑影像上對血流灌流區的偵測能力,並加入白色噪音(white noise)方式可以產生濾波的效果,解決模態混合的問題得到模態一致的內建模態函數( Intrinsic Mode Function, IMF ),結果發現原始訊號經過整合型經驗模態分解法運算後產生的部分本質模態函數相較於傳統之脈衝反相(pulse inversion)影像,能提升血流灌流區和周遭組織的對比度(contrast-to-tissue ratio)。本研究以模擬組織與對比劑訊號和實驗影像來實現經驗模態分解法在非線性成像增強的效果,實驗影像架構也適當的修正移動雜訊對於分解過程中雜訊的放大的問題,並期此方法能提升超音波對比劑影像之效能。
本研究以結合高頻超音波影像系統以及微正子兩套影像系統為基礎,未來將延伸建構兩套影像系統結合模式,實現各種影像系統(如磁振掃描、光學冷光、光學螢光、自動放射顯影術)影像資訊結合,以目前開發出之超音波對比劑為基礎,研發多模式影像造影劑,實現多模式腫瘤影像之開發。
Small-animal models are used extensively in disease research, genomics research, drug development, and developmental biology. The development of noninvasive small-animal imaging techniques with adequate spatial resolution and sensitivity is therefore of prime importance. In particular, multimodality small-animal imaging can provide complementary information.
This paper presents a method for registering high-frequency ultrasonic (microUS) images with small-animal positron-emission tomography (microPET) images. Registration is performed using six external multimodality markers, each being a glass bead with a diameter of 0.43–0.60 mm, with 0.1 μl of [18F]FDG placed in each marker holder. A small-animal holder is used to transfer mice between the microPET and microUS systems. Multimodality imaging was performed on C57BL/6J black mice bearing WF-3 ovary cancer cells in the second week after tumor implantation, and rigid-body image registration of the six markers was also performed. The average registration error was 0.31 mm when all six markers were used, and increased as the number of markers decreased. After image registration, image segmentation and fusion are performed on the tumor. Our multimodality small-animal imaging method allows structural information from microUS to be combined with functional information from microPET, with the preliminary results showing it to be an effective tool for cancer research.
In this study, we used a microUS system that we developed in-house as an alternative method for tumor growth calipers. In addition, microUS was combined with small-animal positron-emission tomography (microPET) for tumor metastatic assessment. MicroUS provides anatomical information that can be used for tumor volume measurements while microPET is a functional imaging method with positron-emitting radiophamaceuticals, such as 18F-labeled deoxyglucose, [18F]FDG. In this study, microUS and microPET were performed in a mouse tumor longitudinal study (2-8 weeks), both with 3D tumor segmentation and volume measurements. The average tumor volume doubling time as determined during the exponential phase was 7.46 days by microUS. MicroUS and microPET are complementary to each other as microUS has superior spatial resolution and microPET provides functional information such as hypoxia or necrosis in the progression of the tumor. With image registration and fusion, the combination can be a valuable tool for cancer research.
To investigate the feasibility of the functional information which provided from microUS, we used the contrast enhanced ultrasound (CEUS) techniques to characterize liver focal lesions and detect three vascular contrast phases in Hepatitis B virus X (HBx) transgenic mice. Specifically, high-frequency ultrasound liver imaging with albumin-shelled microbubbles was employed to detect three vascular contrast phases and characterize focal liver lesions that developed in thirteen HBx transgenic mice at around 14 to 16 months of age. In the thirteen mice, the arterial phase ranges from 2 to 60 seconds post contrast injection. The time period from 10 to 30 minutes post contrast injection was defined as the parenchyma phase in this study. Comparing the imaging and the pathology results, the sensitivity, specificity and accuracy of CEUS for the detection of malignant focal liver lesion in HBx transgenic mice were 91%, 100% and 92%. To characterize the features of the focal liver lesion and detect the three vascular contrast phases of malignant focal liver lesions, the results were arranged according to the guidelines of European Federation of Societies for Ultrasound in Medicne and Biology. Histopathology investigations confirmed the development of the lesion in these thirteen mice.
Finally, we propose to use a novel technique, called the ensemble empirical mode decomposition (EEMD) for contrast nonlinear imaging, to improve the contrast in CEUS imaging. Compared with the results based on the traditional nonlinear imaging technique, the new approach obtains improved performance for tissue components removal from the mixed signals effectively and objectively, and provides us with more accurate contrast nonlinear signals.
These results demonstrated that high-frequency CEUS imaging is potentially for characterizing malignant focal liver lesions in mice and is valuable to provide functional information for preclinical study. The CEUS technique can combine with microPET imaging in the future. The combing methods of microUS and microPET multimodality imaging systems could be extended and other imaging modalities (ex: MRI, in vivo bioluminescent imaging, in vivo fluorescent imaging, autoradiography) integrate into these new techniques. The homemade microbubbles could be constructed as a multimodality contrast agent. A multiplicity of ligands may be coupled to microbubbles directly via covalent bonds or indirectly through avidin-biotin interactions. Ultrasonically reflective particles can be complexed to paramagnetics for MR or radionuclide for nuclear or D-luciferin for bioluminescent or fluorescence for microscope multimodal imaging. The new technique provides an alternative method for cancer research in small animal.
CONTENTS

中文摘要 I
ABSTRACT II
CONTENTS III
LIST OF FIGURES IV
LIST OF TABLES V
CH 1. INTRODUCTION 1
1.1 Multimodality Small Animal Imaging 4
1.2 High Frequency Ultrasound Imaging System 5
1.3 MicroPET Imaging System 7
1.4 Imaging Registration for MicroUS/MicroPET 9
1.5 Basics of Ultrasound Contrast Agents 9
1.6 Contrast-Enhanced Ultrasound of HCC 10
1.7 Contrast Improvement in Ultrasound Nonlinear Imaging Using EEMD 11
1.8 Scope and Organization of the Dissertation 12

CH 2. A THREE-DIMENSIONAL REGISTRATION METHOD FOR MICROUS/MICROPET MULTIMODALITY SMALL ANIMAL IMAGING 14
2.1 Small-Animal Model 15
2.2 Registration Phantom and Animal Holder Designed 15
2.3 Experimental Setup for MicroPET and MicroUS 18
2.4 MicroUS/microPET Data Processing and 3D Reconstruction 19
2.5 Test Phantom Design and Estimation 23
2.6 Rigid-Body Transformation and Error Estimation 24
2.7 Discussions 27

CH 3. NONINVASIVE TUMOR IMAGING WITH HIGH FREQUENCY ULTRASOUND AND MICROPET IN SMALL ANIMALS 29
3.1 Small-Animal Model 31
3.2 Small-Animal High-Frequency Ultrasound Imaging 31
3.3 Small-Animal MicroPET Imaging 33
3.4 MicroUS/microPET Data Acquisition, 3D Tumor Reconstruction, and Volume Measurement 34
3.5 In Vivo Growth Curve 40
3.6 Discussions 42

CH 4. CHARACTERIZATION OF MALIGNANT FOCAL LIVER LESIONS WITH CONTRAST-ENHANCED 40 MHZ ULTRASOUND IMAGING IN HEPATITIS B VIRUS X TRANSGENIC MICE: A FEASIBILITY STUDY 46
4.1 Hepatocellular Carcinomas in Human 46
4.1.1 Pathology and Hepatocarcinogenesis 46
4.1.2 Unenhanced Sonography 48
4.1.3 Contrast Enhanced Sonography 48
4.2 Transgenic Mouse Model 50
4.3 Microbubbles for High-Frequency Ultrasound Imaging 51
4.4 Small-Animal High-Frequency Ultrasound Imaging 53
4.5 Contrast-Enhanced Ultrasound Imaging at 40 MHz 54
4.6 Time-Intensity Curves 62
4.7 Histopathology 64
4.8 Characterization of Focal Liver Lesion 64
4.9 Discussions 65

CH 5. CONTRAST IMPROVEMENT IN ULTRASOUND NONLINEAR IMAGING USING EEMD 68
5.1 Empirical Mode Decomposition 71
5.2 Ensemble Empirical Mode Decomposition 72
5.3 Simulation 73
5.4 Experiments 77
5.5 Denoising 82
5.6 Discussions 83

CH 6. CONCLUSIONS AND FUTURE WORKS 90
6.1 MicroUS/MicroPET Multimodality Small Animal Imaging 90
6.2 Feasibility of Using Contrast-Enhanced 40 MHz Ultrasound Imaging to Characterize Hepatocellular Carcinomas in Small Animal 91
6.3 Future Works 92
6.3.1 Applications of the Three-Dimensional Registration Method for MicroUS/MicroPET Multimodality Small Animal Imaging 92
6.3.2 Microbubbles for Therapeutic Application in Hepatocellular Carcinomas 92
6.3.2.1 Production of Anticancer Microbubbles 94
6.3.2.2 Potential of Multimodality Microbubbles 94
6.3.3 Evaluation Blood Flow Changes with Contrast Enhanced 40 MHz Ultrasound in HBx Transgenic Mice 95

REFERENCES 97

APPENDIX 110
Positron Emission and Annihilation 110
MicroPET Image Processing 111
A-1 Ordered Subsets Expectation Maximization (OS-EM) 111
A-2 Filtered Back Projection (FBP) 112
Histology of Liver Focal Lesion in HBx Transgenic Mice 114

PUBLICATION LIST 115
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