董成淵臺灣大學：物理研究所孫梓凌Sun, Tzu-LinTzu-LinSun2007-11-262018-06-282007-11-262018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/54573肝癌長久以來一直是國人揮之不去夢魘，而國內對於肝癌的研究也是相當重視。但是醫師對於癌症判斷，卻僅止於經驗法則，雖然此法已行之有年，但主觀上判讀仍是存在精確度的疑慮。若有更客觀的方式，去建立一套分析的準則，不僅可以提高癌症級數判讀的準確度，同樣也是輔助醫師的一項利器。 而本實驗室便是利用雙光子螢光與二倍頻顯微術(以下簡稱顯微術)來觀測肝細胞癌(HCC)切片，藉此達到上述之目的。藉著本實驗室顯微術系統，掃描各級數是切片樣本，在擷取其螢光與二倍頻影像，再進一步分析。 將取得之影像對其雙光子螢光或是二倍頻訊號強度，與各種參數分析，試著去找出其中的對應關係與趨勢。而利用這些結果去建立一套較客觀的準則，藉此輔助或是精進癌症程度的判斷。 而目前所得結果顯示，就自發螢光而言，非癌症樣品較癌症樣品高，但仍需較多的樣本數來驗證。並試著藉此結論建立分級標準。Hepatic disease has always been a major health problem in Taiwan, with hepatocellular carcinoma (HCC) being among the leading causes of death. Since the diagnosis of the degree of liver carcinoma is done according to the doctor’s clinical experience, there is a need for a more objective determination of the extent of this cancer. In this work, we attempt to develop an objective method to help improve the accuracy of HCC classification which will be a useful aid for clinical investigations. In this study, we make use of two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy in imaging hepatocellular carcinoma (HCC) specimens. By scanning thin sections of different HCC specimens, we attempt to correlate the change in the sample’s autofluorescence with parameters such as tumor size. We hope that this approach can lead to a more accurate classification of HCC. Our preliminary results show that the autoflurescence intensity of non-tumor sample is higher than that of tumor sample. Our approach demonstrates the possibility of using tissue autofluroescence for tumor classification and the potential of this methodology for clinical diagnosis.Content 口試委員審定書 I 致謝 II 摘要 IV Abstract V Content VI Figure Catalog VII Chart Catalog VIII Table Catalog VIII Chapter 1 Introduction 1 Chapter2 Basic Principles 4 2.1 Second Harmonic Generation 4 2.2 Two-Photon Fluorescence 7 Chapter 3 Histology of Liver 13 3.1 Liver Structure and Function 13 3.2 Liver Circulation System 18 3.3 Cells of Liver 20 Chapter 4 Experimental Setup, Materials and Method 23 4.1 Experimental Setup 23 4.2 Experimental Materials 25 4.3 Method 26 Chapter 5 Results and Analysis 32 5.1 Introduction of TPF and SHG Imaging 32 5.2 Morphological Analysis 36 5.3 Autofluorescence Intensity Ratio Analysis 48 5.4 Optical Biopsy of Liver Fibrosis 49 Chapter 6 Conclusion 56 Reference 58 Figure Catalog Fig.2.1.1 Second Harmonic Generation 5 Fig.2.2.1 Level k is spread into a density of states. 9 Fig.2.2.2 Two-Photon Fluorescence 11 Fig.2.2.3 Limited excitation volume of TPE 12 Fig.3.1.1 Liver anatomy 13 Fig.3.1.2 Structure of classical liver lobules 14 Fig.3.1.3 Three zones of liver acinus 15 Fig.3.1.4 The arrangement of hepatocytes and sinusoids 17 Fig.3.2.1 The circulation process of intrahepatic vascular system 18 Fig.3.2.2 The process of biliary circulation 19 Fig.3.3.1 The H&E stained paraffin fixation section of liver of B6 mou.e.. 21 Fig.4.1.1 Experimental setup 24 Fig.4.3.1 Multiphoton imaging of non-tumor specimens 28 Fig.4.3.2 Autofluorescence imaging of non-tumor specimens 28 Fig.4.3.3 Autofluorescence intensity inversion imaging 29 Fig.4.3.4 Nucleoli label 29 Fig.4.3.5 Result of nucleus counting 30 Fig.5.1.1 TPF and SHG microscopy imaging of non-tumor tissue (A), and histological comparison of non-tumor tissue (a). 33 Fig.5.1.2 TPF and SHG microscopy imaging of HCC tissue (A), and histological comparison (a). 34 Fig.5.1.3 Magnified view of selected areas in Fig.5.1.1 and Fig. 5.1.2 of TPF and SHG image and the corresponding histological section image. 35 Fig.5.2.1-1 TPF and SHG microscopy imaging of HCC grade1 (a), and histological comparison (b). 37 Fig.5.2.1-2 Magnified view of selected region in Fig.5.2.1-1. 38 Fig.5.2.2-1 (a) TPF and SHG imaging of HCC grade2, and (b) histological comparison. 39 Fig.5.2.2-2 Magnified view of selected regions in Fig.5.2.2-1. 40 Fig.5.2.3-1 (a) TPF and SHG microscopy imaging of HCC grade3, and (b) histological comparison. 41 Fig.5.2.3-2 Magnified view of selected regions of Fig. 5.2.3-1. 42 Fig.5.2.4-1 (a) TPF and SHG micrpscopy imaging of HCC grade4, and (b) histological comparison. 43 Fig.5.2.4-2 Magnified view selected regions of Fig. 5.2.4-1. 44 Fig.5.4.1 (a) TPF and SHG microscopy imaging of tissues with METAVIR grade0, and (b) the histological comparison. 50 Fig.5.4.2 (a) TPF and SHG microscopy imaging of tissues with METAVIR grade1, and (b) the histological comparisons. 51 Fig.5.4.3 (a) TPF and SHG microscopy imaging of tissues with METAVIR grade2, and (b) the histological comparisons. 52 Fig.5.4.4 TPF and SHG microscopy imaging of tissues with METAVIR grade3, and (b) the histological comparisons 53 Fig.5.4.5 (a) TPF and SHG microscopy imaging of tissues with METAVIR grade4, and (b) the histological comparisons. 54 Chart Catalog Chart 4.3.1 Saturation test of non-tumor section 27 Chart 5.2.1 (a) The nuclear number per 100×100 μm2 area of non-tumor and tumor specimens for different grade of cancer. (b) The nuclear number per 100×100 μm2 area for non-tumor and tumor specimens 45 Chart 5.2.2 (a) NC ratio for different grades of cancer. (b) NC ratio for non-tumor and tumor specimens. 46 Chart 5.3.1 Autofluorescence intensity ratio for different grades of cancer. 48 Table Catalog Table 4.3.1 Saturation test of non-tumor section 26 Table 5.4.1 METAVIR grading system 495577870 bytesapplication/pdfen-US雙光子螢光二倍頻肝細胞癌two-photon fluorescence microscopy (TPFM)second harmonic generation (SHG)hepatocellular carcinoma (HCC)[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/54573/1/ntu-96-R94222002-1.pdf