口腔癌為國人癌症死亡的第四大死因，同時也是年輕男性發生率第一的癌症。每年都會有新增將近5400個病例。然而至今，口腔癌的五年存活率仍然很難令人滿意，只有依靠早期診斷，才能真正解決這個問題。臨床上診斷癌症的方式的終極標準是依靠病理切片診斷，但是組織切片常常伴隨著疼痛、流血、創傷甚至是癌細胞擴散的副作用。因此，發展一套非侵入式的檢查方式是一件非常重要的事情。
在這次的實驗中，筆者利用非侵入式倍頻顯微術觀察23個紅斑病與白斑病的口腔癌前病變黏膜樣本。在23個樣品之中，有4個樣品無法取得整層口腔上皮層的影像而排除在接下來的分析。根據病理診斷結果，將這19個病變組織的倍頻影像分成原位癌、重度異生、中度異生、輕度異生與增生性病灶五組並與傳統H&E染色切片病理特徵做比較。為了能夠更清楚的量化不同癌前病變的嚴重程度，筆者更進一步依照病理診斷的準則，分析這19個樣品的上皮層三分之一與三分之二處的細胞密度。在這次的分析結果中，筆者將原位癌與重度異生(以下稱重度異生)合併與其他三組作分析發現，在上皮層的三分之一處重度增生組的細胞密度與其他三組比較發現有明顯的上升(假定質小於0.05)。而在上皮層三分之二處，中度異生的細胞密度也和輕度異生和增生性病灶有顯著的不同(假定質小於0.05)。此結果顯示我們能夠成功辨別重度異生、中度異生與輕度異生以下的病灶。此外，筆者也同時分析了細胞密度隨著上皮層深度的變化，在這分析中發現重度異生的病灶在60微米以內細胞密度就有很顯著的上升。因此，對於無法完全取得口腔上皮層影像的病灶，我們也能夠用此方法來辨別是否為重度異生的病灶。這些結果也指出倍頻顯微術將來非常有機會應用能夠應用在口腔癌前病變的臨床診斷。

Oral cancer is the fourth most common cause of male cancer death in Taiwan and ranks first among the cancers that affect the young male population. About 5,400 new oral cancer cases are reported in Taiwan each year. However, the five-year survival rates of oral cancer patients are still low. The only way to address this problem is early diagnosis. In the oral cancer diagnosis, surgical biopsy followed by pathological exam is the standard operating procedure. But, surgical biopsy is always accompanied by pain, bleeding, trauma, and spreading of cancer cells. Thus, developing a non-invasive diagnosis tool is relatively important.
In this research, we used a noninvasive harmonic generation microscope to observe the ex vivo oral mucosa of 23 precancerous specimens, including leukoplakia and erythroplakia. In all of these specimens, there were 4 specimens excluded in our analysis due to the fact that we could not obtain the whole epithelium imaging of these samples.
According to pathological diagnosis results, we separated 19 samples to carcinoma in situ, severe dysplasia, moderate dysplasia, mild dysplasia, and hyperplasia. We compared the five groups with their H&E stained slides. Moreover, based on the histopathological diagnosis criteria, we further analyzed the cellular density at one-third and two-third of epithelium to quantify the difference between the oral dysplasia lesions. In this analysis, we grouped the carcinoma in situ and severe dysplasia as severe dysplasia. The cellular density of severe dysplasia lesions had a significant increase comparing with other lesions at one third of oral epithelium (P value <0.05). In the two third of epithelium, the cellular density of moderate dysplasia also had statistically difference comparing with mild dysplasia and hyperplasia (P value<0.05).
This result showed that we could distinguish the severe dysplasia and moderate dysplasia from all of precancerous lesions. We then analyzed the changes of cellular density versus depth. Additionally, we also calculated the changes in cellular density versus depth. The results of this analysis indicated that the cellular density of severe dysplasia had a marked increase at first 60μm depth. For these samples, we could not define the one-third and two-third of the epithelium; however, we could still analyze the trend of cellular density versus depth to separate severe/carcinoma in situ lesions from other lesions.
These results indicated that HGM could be a broad potential application to precancerous diagnosis, intraoperative margin, and prognosis.

致謝 I
摘要 IV
ABSTRACT V
LIST OF FIGURES X
LIST OF TABLES XIII
Chapter 1 Introduction 1
1.1 Motivation 1
1.2 Optical imaging in oral cavity over view 2
1.3 Structure of oral epithelium 5
1.3.1 Keratinized epithelium 6
1.3.2 Non-keratinized epithelium 7
1.4 Oral precancerous lesion 7
1.4.1 Leukoplakia 8
1.4.2 Erythroplakia 8
1.5 Thesis aim and organization 9
Chapter 2 Basic principles and properties of harmonic generation microscopy 12
2.1 Conventional optical microscopy 12
2.2 Confocal microscopy 16
2.3 Nonlinear Optical Microscopy 18
2.3.1 Second harmonic generation (SHG) 20
2.3.2 Third harmonic generation (THG) 22
Chapter 3 Experimental Setup 27
3.1 Choosing laser source 27
3.2 HGM system 30
3.3 Sample preparation 32
Chapter 4 HGM Study on oral precancerous lesion 33
4.1 Samples 33
4.2 Ex vivo HGM imaging of dysplasia lesion 34
4.3 Ex vivo HGM imaging of early stage OSCC 47
4.4 Cellular density analysis 50
4.4.1 Analysis method 50
4.4.2 Results of cellular density analysis 52
Chapter 5 Summary 57
Reference 58
Appendix A 62
Appendix B 63
Appendix C 64

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