食道癌是全球十大致命的癌症之一，過去幾十年來，雖然衛生環境的改善確實讓胃癌的威脅大幅降低，但飲食習慣的改變卻讓食道癌風險不減反增，其中在亞洲又以食道鱗狀細胞癌的發病率最高。目前主要的內視鏡及切片檢查要在早期發現癌變非常困難，因此食道癌常在晚期才被確診。
為了提高早期食道癌診斷的準確性和預防食道癌的發生，我們的研究專注於偏振解析雙光子顯微術在食道鱗狀細胞癌的應用，由於食道的鱗狀上皮充滿著豐富的膠原蛋白，而過去的研究表明，膠原蛋白分子其三股螺旋結構的異變與癌細胞侵襲程度具有高度的相關性。因此，研究膠原蛋白與食道癌組織在不同階段的結構關係具有其重要意義，其中癌變組織所產生的非線性光學訊號與膠原蛋白分子的二階非線性極化張量為我們主要的研究重點。
除此之外，使用偏振解析雙光子顯微術，不僅在膠原蛋白的結構組織具有優秀的空間解析度，而且透過二倍頻(SHG)與雙光子激發螢光(TPEF)可以分析膠原蛋白在癌細胞浸潤下的變化。由於膠原蛋白分子的二倍頻訊號強度會隨著入射光的偏振狀態而改變，因此我們透過旋轉半波片和四分之一波片調控入射光的偏振狀態來分別求得膠原蛋白分子的二階非線性極化張量X22/X16，以及由分子排列所產生的二倍頻圓二色效應(SHG-CD)，在上述結果的幫助下，我們成功地將食道癌分為四個不同階段，這與傳統組織病理學所做的階段劃分完全一致。因此，我們證明了偏振解析二倍頻顯微術除了可以在影像上提供強度與樣本的型貌之外，更可透過張量的計算得到受到癌細胞侵襲後，受損分子的結構資訊。使此研究方法不僅具更精確的食道癌分期鑑別能力，更具有生醫應用上的潛力。

Esophageal cancer has fatally attacked human health over the past few decades. It is often diagnosed at a late stage due to how difficult detecting early cancerous changes when using conventional endoscopy and/or biopsy.
In order to improve the accuracy of diagnosis and prevent the occurrence of esophageal cancer in the early phase, a quantitative analytical method used to discriminate among the various types of cancerous esophagus tissues is demanding to provide sufficient accuracy in cancer staging. Previous studies indicate that the structural change in collagen ascribes to misfolding of the triple helices that is highly associated with cancer cell invasion. Thus, it is of great significance to study the structural correlation between collagen fibers and esophageal cancer tissues at varying stages. Here we used polarization-resolved two-photon microscopy, not only the structural organization of collagen fibrils in squamous cell carcinoma was revealed at high spatial resolution but also the ratios of second harmonic generation (SHG) / two-photon excited fluorescence (TPEF), tensor elements of second-order nonlinear susceptibility, X22/X16, and SHG-circular dichroism (SHG-CD) were obtained.
With the aid of the above results, we successfully classified esophageal cancers at four different stages, which complied well with the stage differentiation made by histopathology. This research indicated a reliable and highly accurate inspection of various types of esophageal cancer tissues, showing great potentials in cancer research.

論文審定書 i
誌謝 ii
摘要 iv
Abstract v
目錄 vi
圖目錄 viii
表目錄 x
第一章 緒論 1
1.1研究背景 1
1.2食道癌簡介 2
1.3食道癌的診斷標準 3
1.4食道癌的診斷工具 6
1.4.1內視鏡技術 7
1.4.2光學顯微技術 9
1.5近期研究 11
1.6研究動機與目的 14
第二章 理論基礎 15
2.1 非線性光學與二倍頻簡介 15
2.2 膠原蛋白的結構與排列 17
2.3 偏振解析二倍頻顯微術 21
2.3.1 偏振原理 21
2.3.2 膠原蛋白的二階非線性極化率 23
2.3.3 SHG偏振分析理論 25
2.3.4 二倍頻圓二色性 27
2.4 雙光子螢光影像 31
第三章 樣本製備與實驗方法 33
3.1 偏振解析雙光子顯微鏡系統 33
3.2 食道鱗狀細胞癌樣本製備 34
3.3 SHG/TPEF強度比值分析 36
3.4 二階非線性極化率分析 38
3.5 二倍頻圓二色性分析 41
第四章 結果與討論 43
4.1 H&E染色與雙光子影像 43
4.1.1 癌細胞的浸潤範圍與膠原蛋白的型態變化 45
4.1.2 SHG/TPEF強度比值分析 48
4.2膠原蛋白之二階非線性極化率 50
4.3二倍頻圓二色性與膠原蛋白的分子導向 53
第五章 結論與未來展望 55
參考文獻 56

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