Clinical Application of Higher Harmonic Generation Microscopy in Superficial Disease Diagnostics

• Main Authors: Ming-Rung Tsai, 蔡明容
• Other Authors: Chi-Kuang Sun
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/80129009179592453382

博士 === 國立臺灣大學 === 光電工程學研究所 === 101 === 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.