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標題: | 倍頻顯微術之淺層疾病診斷臨床應用 Clinical Application of Higher Harmonic Generation Microscopy in Superficial Disease Diagnostics |
作者: | Ming-Rung Tsai 蔡明容 |
指導教授: | 孫啟光(Chi-Kuang Sun) |
關鍵字: | 倍頻顯微術,非侵入式,口腔癌,皮膚癌,病理診斷, Harmonic generation microscopy,noninvasive,oral cancer,skin cancer,histopathological diagnosis, |
出版年 : | 2012 |
學位: | 博士 |
摘要: | 癌症,又稱惡性腫瘤,至今仍然是致死率最高的一種疾病。根據台灣衛生署統計,從民國71年起,連續29年位居十大死因之首。99年死於癌症的人數更是多達4萬多人。因此,早期診斷及早期治療是降低癌症致死率的最佳方法。在醫學以及臨床上,診斷癌症有許多方式,但是活體組織切片是診斷癌症最終極且最準確的方法。此種檢查法雖已被普遍應用,但其過程除了會使病人感覺到疼痛外,產生的傷口更可能會導致流血、形成疤組織及造成癌細胞擴散等副作用。因此發展一個非侵入式的影像工具,並且可提供病理級的資訊,對於癌症的診斷以及選擇治療方式上都是相當關鍵的一環。
相較於活體組織切片,光學虛擬切片術不需要切除組織,不僅可避免或降低活體切片所帶來的疼痛、副作用及診斷上的失誤,更可以減低傳統切片染色法所消耗的時間成本。相較於共軛焦及多光子顯微術,倍頻顯微術已經被證實在活體健康皮膚上具有較深的穿透度、較高的空間解析度和極低的光破壞及光毒性的特性。因此,本論文進一步的利用倍頻顯微術,來探討其臨床應用在口腔癌和皮膚癌的診斷能力。 在口腔研究方面,我們在健康受試者的口腔粘膜得到不同層角質細胞的三倍頻影像以及固有層膠原蛋白的二倍頻影像,並且將這些影像作了相關的分析。由此部份的研究,我們確立了倍頻顯微術應用在活體口腔的影像能力。接著我們更進一步利用手術樣品得到口腔鱗狀細胞癌的倍頻影像,並且使用和病理特徵相同的標準去分辨癌化和正常組織的不同,這些特徵包括麟狀上皮細胞的大小、形狀和其排列,以及分化等等的異常。此外,為了增進三倍頻在口腔粘膜的影像對比度,我們利用醋酸作為三倍頻的對比增強劑塗在受試者的下嘴唇,並且觀察到口腔粘膜的上皮角質細胞之細胞核的確有被增強。 在皮膚研究方面,利用倍頻顯微術我們已經執行了31例活體色素性病變皮膚的臨床試驗研究,包括5例色素性基底細胞癌、1例黑色素細胞癌、17例色素痣及8例脂溢性角化病。依據傳統的病理標準,我們建立了診斷這些疾病的倍頻影像特徵。利用這些影像特徵,我們對手術樣本和臨床實驗總共44個色素性病灶作了敏感性(sensitivity)與特異性(specificity)的統計。在所有條件都列入考慮的狀況下,我們可以得到直接辨別出黑色素細胞癌、基底細胞癌、痣以及脂溢性角化症的總敏感性和特異性分別為92%和97%。若是每種疾病皆呈現兩種具有獨特性的影像特徵的條件下來做評估,得到的總敏感性和特異性為95%和100%。另外,為了增進三倍頻在皮膚的影像功能,我們利用刺青染劑作為活體皮膚的三倍頻對比增強劑。經由細胞、老鼠以及人體皮膚的活體實驗,證實了刺青染劑在的確會增強三倍頻的訊號強度。 由以上口腔和皮膚的研究結果,我們相信此系統在偵測口腔癌和皮膚癌可提供極佳的影像能力,並且具有疾病的監控、早期癌症的檢測與預防的潛力,以降低癌症的死亡率。 Cancer is a major health problem that has significant impacts worldwide. Approximately 12.7 million cancer cases and 7.6 million cancer deaths were reported among U.S. residents in 2008. Thus, early detection and treatment are critical to reduce the mortality of the disease. Cancer can be detected in a number of ways; however, the most definitive diagnoses are usually achieved through biopsy. Biopsy is often uncomfortable and exposes patients to a wide spectrum of potential risks and complications, such as tissue trauma, bleeding, and spread of cancer cells. Therefore, the development of noninvasive imaging tools capable of accurate histopathological diagnosis is of utmost importance. Because it is done without tissue removal, optical virtual biopsy not only avoids or minimizes the inherent disadvantages of conventional biopsies but also reduces the cost and time necessary for traditional pathological processing. Our preliminary in vivo clinical trials of healthy human skin have demonstrated that harmonic generation microscopy (HGM) outperforms confocal and multiphoton microscopy in providing higher penetration depth, higher spatial resolution, and minimized photodamage and phototoxicity. Thus, in this work, we investigate the capability of HGM for oral and skin cancer diagnosis. For the oral study, we perform in vivo HGM on healthy oral mucosa. Third-harmonic generation (THG) provides contrasts in keratinocytes of different epithelial layers and second-harmonic generation (SHG) reveals contrasts in collagen fibers in the lamina propria. We further analyze in vivo HGM images of healthy oral mucosa to obtain relevant information. In vivo observation results confirm the imaging capability of HGM in healthy oral mucosa. We then show contrasts in oral squamous cell carcinoma tissues and establish structural HGM criteria according to histopathological features from ex vivo measurements. The endogenous contrast provided by HGM is adequately high to differentiate cancerous tissues from normal ones in human oral mucosa. Finally, acetic acid is applied to human oral mucosa as an exogenous contrast agent to enhance the THG contrast of nuclei in oral mucosa. For the skin study, we perform in vivo clinical trials on 31 patients with pigmented skin lesions, including 5 patients with basal cell carcinoma, 1 patient with melanoma, 17 patients with melanocytic nevus, and 8 patients with seborrheic keratosis. We also establish HGM imaging diagnostic criteria based on the histopathological features of each type of lesion. To investigate the capability of HGM for differential diagnosis of pigmented skin lesions and determine its sensitivity and specificity, we analyze in vivo and ex vivo images of 44 lesions. Overall evaluation of the specified criteria indicates that HGM presents 92% sensitivity and 97% specificity for diagnostic classification. Utilization of two specific features as diagnostic criteria yields 95% sensitivity and 100% specificity. We further demonstrate the potential of a common tattoo dye as a THG contrast agent for in vivo optical biopsy of human skin. Experiments performed on cultured mouse and human skin cells confirm the THG enhancement effect of the tattoo dye. The results of our oral and skin studies indicate that HGM can provide excellent imaging capability for oral and skin cancer detection. The method has potential applications in early diagnosis of cancer, continuous disease monitoring, clinical disease classification, and determination of appropriate therapeutic guidelines. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63214 |
全文授權: | 有償授權 |
顯示於系所單位: | 光電工程學研究所 |
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