董成淵臺灣大學：物理研究所劉源Liu, YuanYuanLiu2007-11-262018-06-282007-11-262018-06-282006http://ntur.lib.ntu.edu.tw//handle/246246/54575多光子顯微術為1990年代初期發展出來的非線性光學技術。多光子顯微術包含多光子螢光與倍頻訊號，其光切片效果及低侵入性，使這項技術非常適合運用於活體觀察。活體與即時的肝臟影像對於了解肝臟的動態過程非常重要。在此我們發展出獨特的肝臟視窗裝置以及手術技巧，以利用多光子顯微術來研究肝臟的動態過程。藉由觀察6-CFDA在肝細胞內被轉換並經由微膽管排出，我們真正看到肝臟的代謝功能。我們並以肝臟的代謝功能為指標，來衡量由膽管結紮所造成膽汁鬱滯的肝功能。相較於正常肝，膽管結紮1天與2天後的肝臟分泌速率較慢。本實驗中，我們展示了運用非侵入式多光子顯術觀察活體肝臟的潛力與可能性。藉由這些技術，我們可以進一步研究肝臟的各種疾病和機制。Multiphoton microscopy is an advanced nonlinear optical technique developed in early 1990s. Including two-photon fluorescence and second harmonic generation, its optical sectioning ability and minimally invasiveness makes it a preferable choice for intravital observation. In vivo and real-time imaging of liver is vital for elucidating hepatic dynamics. Here we develop a novel chronic window device and surgical methods to investigate hepatic dynamics using multiphoton microscopy. The hepatic metabolism is visualized by imaging sequential uptake of 6-carboxyfluorescein diacetate from hepatocytes and its excretion into bile canaliculi. The excretory process is then employed to evaluate the function of liver of cholestasis, which is established by ligating the common bile duct. The liver with bile duct ligated for 1 or 2 days shows a relatively slow rate of excretion compared to the normal liver. In this work, we demonstrate the potential of intravital multiphoton microscopy of hepatic dynamic processes. By these techniques, various liver diseases and mechanisms can be explored in the future.Abstract I 摘要 II Chapter 1 Introduction 1 Chapter 2 Basic Principles 5 2.1 Second Harmonic Generation 5 2.2 Two-Photon Fluorescence 7 2.3 Basic Concepts of Microscopy 12 Chapter 3 Histology of Liver 15 3.1 Liver Structure 15 3.2 Intrahepatic Vascular System and Biliary System 20 3.3 Cells and Elements in Liver 23 Chapter 4 Materials and Methods 26 4.1 Design of the Liver Window 26 4.2 Procedures for the Installation of the Liver Window on Mice 27 4.3 Accommodation of Mice on Microscope Stage 29 4.4 Multiphoton Microscopic Setup 30 4.5 Fluorescent Probes 31 Chapter 5 Results and Discussion 33 5.1 Visualization of Hepatobiliary Excretory Function 33 5.2 Bile Duct Ligation Affects the Metabolism of Liver 37 Chapter 6 Conclusions 46 References 49 Acknowledgements 54 動物實驗許可 IV 誌謝 V11278770 bytesapplication/pdfen-US非侵入式多光子顯微術多光子螢光倍頻訊號活體肝臟代謝膽汁鬱滯膽管結紮intravital multiphoton microscopytwo-photon fluorescencesecond harmonic generationliverin vivohepatic metabolismcholestasisbile duct ligation.thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/54575/1/ntu-95-R93222027-1.pdf