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研究生： 蘇園登
Su, Yuan-Deng
論文名稱： 利用表面電漿子顯微術與微米製作技術於細胞與基質接觸之研究
Study of Cell-substrate Contact via Surface Plasmon Phase Microscopy and Microfabrication
指導教授： 陳顯禎
Chen, Shean-Jen
共同指導教授: 保羅 坎帕尼奧拉
Paul J. Campagnola
學位類別： 博士
Doctor
系所名稱： 工學院 - 工程科學系
Department of Engineering Science
論文出版年： 2010
畢業學年度： 98
語文別： 英文
論文頁數： 113
中文關鍵詞： 表面電漿子 、相位量測 、細胞外基質 、細胞影像
外文關鍵詞： surface plasmon polariton, phase measurement, extracellular matrix, cell images
相關次數： 點閱：100  下載：3 
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    • 表面電漿子(surface plasmon polariton，SPP)相位影像系統不僅可以獲得較高靈敏度的界面之厚度或介電常數變化之相位資訊，而且可以提供大量平行空間影像。本論文中發展出一共光程移相干涉(phase-shift interferometry，PSI)之SPP顯微術來觀測空間平面上之相位資訊，配合五步相位還原演算法來完成相位的重建，量測奈米膜層之相位影像差異，並且討論此干涉方式之長時間相位穩定與空間解析度。此影像系統具有高靈敏度之特性可以量到2x10-7 RIU的折射率變化，並且由於其為一共光程干涉術所以具有相當高之相位穩定，於長時間下之相位穩定可達2.5x10-4π。此系統提供了高解析與高通量之DNA微陣列量測之能力，且不需要對其作額外的螢光標定，不僅如此還可提供量化的依據。而在蛋白質的量測上還結合了微機電製程之微流道系統，實現多通道蛋白質生物分子量測。
    為了觀察細胞之貼附與爬行之情形，本論文中發展了兩套SPP顯微影像系統，並且結合衰逝全反射螢光顯微鏡於細胞貼附之研究。一為傳統菱鏡偶合方式，另一用高數值孔徑之顯微油鏡偶合方式以增進顯微影像之品質，文中比較了此兩系統之優缺點及其限制之外並將其運用於細胞貼附之學習。另外，比較了利用強度與相位量測之實驗結果，相位量測之靈敏度高於強度量測約160倍左右。首先，此SPP相位影像系統用於超過兩個小時之細胞凋亡觀察。細胞與生物基材之接觸影像所相互對應之表面電漿共振(surface plasmon resonance，SPR)角度可以由改變入射角從70到78度掃描之SPP相位量測所獲得。再根據SPR角之影像資訊與多層膜之模擬結果可進一步得知活體黑色素瘤細胞與牛血清蛋白基材之接觸距離資訊。
    最後，利用飛秒(femtosecond)雷射製造出細胞外間質(extracellular matrix，ECM)之交聯結構，此方式可於特定地方製造出二維甚至三維之生物結構，創造了具有線性濃度梯度分佈之纖維蛋白結構，並藉此控制纖維母細胞之爬行與貼附型態之改變。細胞與此結構化之ECM接觸之行為也可透過此SPP相位顯微鏡做進一步的學習與討論。

    Surface plasmon polariton (SPP) phase image system can not only obtain high sensitivity phase information of thickness or dielectric constant change but also provide high-throughput spatial information. A common-path phase-shift interferometry (PSI) SPP image system has been developed to measure the nano-layer at sensing surface. The system uses five-step phase reconstruct algorithm to obtain the spatial phase information. It has very high sensitivity and the limitation of measurement can achieve 2x10-7 reflect index unit (RIU). Moreover, the phase stability can achieve 2.5x10-4 π in long-term measurement due to the common-path setup. The imaging system offers high resolution and high-throughput screening capabilities for microarray DNA image without the need for additional labeling, and provides valuable quantitative information. It also combines with a microfluidic system which is fabricated by MEMS to achieve the measurement of multichannel protein chip.
    To study the behavior of cell-biosubstrate contacts, two kinds of system configuration for SPP phase microscopy are developed. One is based on prism coupling and the other one is high numerical aperture objective-based coupling. The sensitivity comparison of intensity and phase demonstrates that the sensitivity of the phase measurement is 160-fold greater than that of the intensity measurement. Also, a more than 2-hour cell apoptosis observation via the SPP phase microscopy is presented. To implement the incident angle from 70° to 78°, cell-biosubstrate contact images corresponding to surface plasmon resonance (SPR) angles are obtained by utilizing the SPP phase measurement. According to the information of the corresponding SPR angle images and a multilayer simulation, the contact distances between a living melanoma cell and a bovine serum albumin substrate at four different locations have been estimated.
    Finally, a femtosecond laser is utilized to fabricate a cross-linked extracellular matrix (ECM) with arbitrary shape and concentration. It can form two-dimensional or even three-dimensional biometrical specificities. Fibronectin with a linear gradient concentration is fabricated and it is utilized to control the cell migration, adhesion, and differentiation of fibroblast cells. The behaviors of cell contacts with the ECM structures have been studied via the SPP phase microscopy.

    Abstract I 摘要 III Acknowledgements IV Contents V List of Figures IX List of Tables XV Abbreviation XVI Chapter 1 Introduction 1 1.1 Introduction 1 1.2 Literature review 2 1.3 Motivation 5 1.4 Outline 6 Chapter 2 Theory of Surface Plasmon Resonance 9 2.1 Surface plasmon wave and dispersion relationship equation 9 2.1.1 TE modes 10 2.1.2 TM modes 12 2.2 Excitation of surface plasmons 15 2.2.1 Coupling methods of surface plasmons 15 2.2.2 Penetration depth of surface plasmons 17 2.2.3 Propagation length of surface plasmons 18 2.3 Multilayer simulation for surface plasmon excitation 19 2.4 Phase jump 22 2.5 The properties of surface plasmons 23 2.5.1 Different thickness of the metal film 24 2.5.2 Different thickness and dielectric constant of the dielectric layer 26 Chapter 3 Surface Plasmon Polariton Phase Image System 29 3.1 Phase-shift interferometry 29 3.1.1 Phase reconstruction by five-step phase-shifting algorithm 29 3.1.2 Phase-shift calibration 30 3.1.3 Phase jump and unwrapping 31 3.2 Common-path phase-shift interferometry SPP image system 33 3.2.1 SPR sensor design and manufacture 34 3.2.2 Optical system setup 34 3.2.3 Control system 36 3.3 Phase stability and sensitivity 38 3.4 Self-assembled monolayer 40 3.5 DNA microarray 41 3.5.1 Sample preparation 41 3.5.2 ssDNA phase image 42 3.5.3 DNA hybridization 43 3.6 Protein interaction with the microfluidic chip 44 Chapter 4 Surface Plasmon Polariton Microscopy for Cell Membrane Observation 49 4.1 Prism couple based SPP phase microscope 50 4.1.1 Optical system setup 50 4.1.2 Cell culture protocol and sensor coating 52 4.2 Cell images via prism-based SPP microscope 53 4.3 SPP excitation via a high numerical aperture objective 57 4.4 Objective-coupled SPP microscope 63 4.4.1 Optical setup 63 4.4.2 Surface modification 65 4.4.3 Cell lines and cell culture 66 4.5 Lateral spatial resolution testing 66 4.6 SPP cell images with objective-based system 68 4.6.1 Long-term live cell membrane imaging 68 4.6.2 Cell-biosubstrate contact distance 70 Chapter 5 Extracellular Matrix Microfabrication 77 5.1.1 Photochemistry 77 5.1.2 Fabrication instrumentation setup 78 5.1.3 Surface modification and reagent 79 5.1.4 Gradient design and characterization 80 5.1.5 Cell culture 81 5.1.6 Dual staining for focal adhesions and F-actin. 81 5.1.7 Statistic analysis 82 5.2 Experimental results 83 5.2.1 Gradient characterization 83 5.2.2 Cell response to the gradient 84 5.3 Observation of cell adhesion on ECM via SPP phase microscopy 91 5.4 Discussions 94 Chapter 6 Conclusions 97 References 99 Curriculum Vitae 111

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