臺灣博碩士論文加值系統

剪切波影像是一種非侵入式的定量彈性影像技術，可用於重建觀測物之彈性分佈。近年來，光學式剪切波影像系統的發展可實現高靈敏度、空間及時間解析度之彈性成像。此研究結合雷射光斑對比影像技術與剪切波彈性影像，發展了一套三維全域式雷射光斑對比剪切波影像系統。此技術利用因機械擾動造成的局部光斑模糊現象來偵測瞬態剪切波傳遞的狀態。二維 (X–Z) 剪切波彈性影像的仿體測試結果顯示，在剪切模量範圍為1.52 kPa至7.99 kPa的均質仿體中，剪切波速度估算誤差小於6%，而估計標準偏差則小於0.07 kPa。此外，系統之位移靈敏度可偵測到位移移動速度小於0.6 µm/ms。本研究架設及評估兩種三維雷射光斑剪切波彈性影像系統。第一套系統在不同的二維平面上對剪切波傳遞進行成像，利用不同平面的剪切波速度圖重建觀測物之三維彈性分佈。第二套系統採用電腦斷層掃描架構，利用不同角度的剪切波投影來重建剪切波波前之三維空間位置與形狀。因第二套系統架構能利用Time-of-flight演算法於剪切波徑向方向之傳遞路徑估算多方向剪切波傳遞速度，在評估複雜機械特性的觀測物具有優勢。本系統的三維空間解析度皆小於一毫米。然而，由於成像系統的景深以及位移靈敏度（在有靜態散射物質的情況）有限，沿著光軸的方向（Y方向）的有效成像深度大約為6.25 mm。因本系統可量測含有低散射的觀測物之彈性分佈，我們將影像系統應用於量測三維癌細胞培養系統內細胞外基質之空間和時間上的硬度變化並評估測量系統用於力學生物學的可行性。分析結果顯示兩種細胞培養系統（含有4 mg/ml Matrigel以及1mg/ml或2 mg/ml Collagen Type I）中主要的兩種基質纖維密度與量測到的剪切波速度俱有高度相關性，分別為r=0.832，P<0.001和r=0.642，P=0.024。此外，研究中觀察到細胞沿著細胞培養系統中的硬度梯度方向的移動現象，以及因局部基質的硬度所造成不同程度的細胞增生。因本系統可達到無需添加探針式的彈性測量，細胞與細胞微環境之間的雙向機械相互作用及所觀察到的硬度所介導的細胞行為並沒有被影像探針所干涉。此外，接近活體尺寸的細胞微環境之彈性變化資訊有助於提升對在癌症發展過程中所發生的複雜基質重塑機制的理解，進而研發新的癌症治療方法。

The high spatial resolution and motion sensitivity of optical-based shear wave detection have made it an attractive technique in biomechanics studies with potential for improving the capabilities of ultrasound-based shear wave elasticity imaging. In this research, a laser speckle contrast shear wave imaging system was developed. The estimation of shear wave speed is based on the detection of the local blurring of the optical speckle pattern resulting from the mechanical disturbances induced by shear wave propagation. In two-dimensional (2D) imaging, the developed system can estimate the shear wave speed with an error of less than 6% on homogeneous phantoms with shear moduli ranging from 1.52 kPa to 7.99 kPa. The standard deviation of shear modulus estimation was less than 0.07 kPa and motion sensitivity of better than 0.6 µm/ms was demonstrated. In three-dimensional (3D) imaging, two system setups were devised. The first setup performed plane-by-plane 2D reconstruction of the shear wave speed maps at different positions by linearly translating the sample. The second setup, on the other hand, performed tomographic reconstruction of the shear wave wavefront by rotating the ultrasound transducer used for the induction of shear waves. The second setup has the advantage of multi-directional shear wave speed estimation in the radial direction. Thus, it can be beneficial for evaluating the elasticity of an anisotropic sample. The systems exhibit submillimeter spatial resolution in all three dimensions. Due to the limited depth-of-field and motion sensitivity (in the presence of static scatterers), the effective imaging depth along optical axis (Y direction) is approximately 6.25 mm. The applicability of the developed systems for in vitro mechanobiology study with 3D cancer cell culture systems is also explored. The shear wave speed measured for the cell culture sample was found to be strongly correlated with the extracellular matrix fiber density in two types of cell culture system (r=0.832 with P<0.001, and r=0.642 with P=0.024 for cell culture systems containing 4 mg/ml Matrigel with 1 mg/ml and 2 mg/ml collagen type I hydrogel, respectively). Cell migration along the stiffness gradient in the cell culture system as well as an association between cell proliferation and the stiffness of the local extracellular matrix was also observed. As the elasticity measurements can be performed without the need of exogenous probes, the developed system can be used to assist the study on how the microenvironmental stiffness interacts with cancer cell behaviors without possible adverse effects of the exogenous particles. In addition, providing information on the dynamics of the cell microenvironmental stiffness in a scale closer to the in vivo scenario may increase the understanding of the mechanism of the complex matrix remodeling occurs during cancer progression, which may lead to the improvements in cancer treatment strategies.

ABSTRACT I
中文摘要 III
CONTENT IV
LIST OF FIGURES VII
LIST OF TABLES XI
CHAPTER 1. INTRODUCTION 1
1.1 ULTRASOUND-BASED SHEAR WAVE ELASTOGRAPHY 1
1.2 PARAMETERS IN DETERMINING THE QUALITY OF SHEAR WAVE ELASTOGRAPHY 3
1.3 OPTICAL-BASED SHEAR WAVE ELASTOGRAPHY 4
1.4 MECHANOBIOLOGY STUDY IN THREE-DIMENSIONAL CELL CULTURE 6
1.5 OBJECTIVES 8
CHAPTER 2. LASER SPECKLE CONTRAST SHEAR WAVE IMAGING: TWO-DIMENSIONAL IMAGING SYSTEM DEVELOPMENT 10
2.1 INTRODUCTION 10
2.2 PRINCIPLES OF LASER SPECKLE CONTRAST IMAGING 10
2.3 MATERIALS AND METHODS 11
2.3.1 System setup 11
2.3.2 Imaging processing 12
2.3.3 Result validation 13
2.3.4 Phantom 14
2.4 RESULTS 15
2.4.1 Experiments with the homogeneous phantoms 15
2.4.2 Experiment with the heterogeneous phantom containing stiff-plate inclusion in the X direction 18
2.4.3 Motion sensitivity 18
2.5 DISCUSSION 19
2.6 SUMMARY 20
CHAPTER 3. MECHANICAL MEASUREMENTS OF THREE-DIMENSIONAL CANCER CELL IN VITRO MODEL 22
3.1 INTRODUCTION 22
3.2 MATERIALS AND METHODS 23
3.2.1 Cell culture sample 23
3.2.2 Experiment setup 24
3.2.3 Imaging and post-imaging process 25
3.2.4 Immunofluorescence, cell count and size distribution analysis 26
3.3 RESULTS 27
3.3.1 Shear wave speed measurements of the 3D cell culture system 27
3.3.2 Immunofluorescence and H&E staining analysis 29
3.4 DISCUSSION 34
3.5 SUMMARY 36
CHAPTER 4. THREE-DIMENSIONAL SHEAR WAVE IMAGING BASED ON FULL-FIELD LASER SPECKLE CONTRAST IMAGING WITH ONE-DIMENSIONAL MECHANICAL SCANNING 37
4.1 INTRODUCTION 37
4.2 MATERIALS AND METHODS 37
4.2.1 System setup and imaging process 37
4.2.2 Phantoms 38
4.3 RESULTS 38
4.3.1 Spatial filtering for reducing the signal from outside of the depth-of-field of the imaging lens 38
4.3.2 Experiment with the heterogeneous phantom containing stiff-plate inclusion in Y direction 39
4.3.3 Three-dimensional shear wave imaging of a heterogeneous phantom with a stiff-cylinder inclusion 40
4.4 DISCUSSION 42
4.5 SUMMARY 42
CHAPTER 5. COMPUTED TOMOGRAPHIC SHEAR WAVE IMAGING 43
5.1 INTRODUCTION 43
5.2 COMPUTED TOMOGRAPHIC SHEAR WAVE IMAGING: PRINCIPLE AND IMAGE RECONSTRUCTION 43
5.3 MATERIALS AND METHODS 44
5.3.1 System setup and imaging acquisition 44
5.3.2 Phantoms 47
5.4 RESULTS 48
5.4.1. Three-dimensional visualization of the shear wave propagation 48
5.4.2. Three-dimensional volumetric reconstruction of the shear wave speed 49
5.5 DISCUSSIONS 52
5.6 SUMMARY 55
CHAPTER 6. CONCLUSIONS AND FUTURE WORKS 56
6.1 CONCLUSIONS 56
6.2 FUTURE WORKS 57
6.2.1 Improving the computed tomographic reconstruction algorithm for shear wave imaging 57
6.2.2 Three-dimensional shear wave imaging for three-dimensional cell culture system 58
REFERENCES 61
PUBLICATION LIST 68

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