以非線性顯微術擷取皮膚組織蘇木精-伊紅染整體染色的癌細胞影像：評估莫氏手術邊緣決定的適切性

Nonlinear Microscopy Imaging of Hematoxylin-Eosin Stained Whole-Mount Skin Tissues: Assessment Adequacy of Margins in Mohs Micrographic Surgery

高健庭(Chien-Ting Kao)

指導教授 ： 孫啟光

國立臺灣大學/電機資訊學院/生醫電子與資訊學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703561

摘要

莫氏手術是一種皮膚癌的組織保留手術，在臨床上具有一定的重要性，其方法是透過組織病理學的判讀對手術中切除之組織做邊緣鑑定，在盡可能縮小手術區域的情況下同時達到完全切除腫瘤細胞。目前莫氏手術中，鑑定手術邊緣是否殘留癌細胞的標準流程為冷凍切片病理分析，但這方法仍然相當耗時，且真實的手術邊緣實際上被削除了。在這份研究中，我們利用中心波長1260奈米的鉻貴橄欖石雷射作為非線性顯微鏡的激發光源，以光學虛擬切片的方式擷取蘇木精-伊紅整體染色的皮膚組織影像，其訊號源自三倍頻與三光子螢光、解析度達到次細胞等級，而且同一雷射光源所做出的倍頻顯微鏡系統也能作為手術前邊緣鑑定使用。由於無須經過實際切片及繁雜的染色流程，真實的手術邊緣能被保留，且跟冷凍切片病理分析相比，這套方法可能節省至少三倍以上的時間。染劑及藥劑的使用上，皆為目前標準染色流程所用，若臨床上仍需進一步的冷凍病理分析，將不會有染劑或藥劑不相容的問題。影像判讀方面，光學虛擬切片能即時成像於螢幕上，並後處理成近似傳統的蘇木精-伊紅染色組織學方便病理科醫師判讀。
在擷取蘇木精-伊紅整體染色皮膚組織的光學虛擬切片前，需先確認蘇木精及伊紅的訊號源，也需先確認其非線性影像是否與傳統組織學具有一致性。在非線性頻譜的研究中，顯示了蘇木精會增強三倍頻訊號，而伊紅會釋放三光子螢光；因此受蘇木精所染的細胞核將會呈現於三倍頻影像中，而嗜伊紅結構則會呈現於三光子螢光影像中。而在正常皮膚及皮膚癌組織的薄切片非線性影像中，顯示出非線性影像與傳統明視野影像在對比及病理特徵方面具有相當一致的結果。之後，在厚達一毫米的皮膚組織上，利用本研究中測試出來的穩定染色條件，可以使組織的虛擬切片影像與傳統明視野影像相比擬。為了評估這套技術在莫氏手術邊緣決定的適切性，手術樣本（包括皮脂腺母斑、基底細胞癌、鱗狀細胞癌及乳房外柏哲德氏病）被水平切成若干層以仿效莫氏手術流程，其結果顯示出以非線性顯微術虛擬切片蘇木精-伊紅染整體染色皮膚組織的癌細胞影像與同病人的病理切片有一致的特徵，因此這套技術在莫氏手術中邊緣鑑定的應用上是可行的。這套與現行冷凍切片病理分析有一致性、能更節省時間且保留真實手術邊緣的方法，相信未來能在不失傳統影像的對比及病理特徵的情況下，加速莫氏手術中邊緣鑑定。

關鍵字

莫氏手術 ； 手術中邊緣鑑定 ； 蘇木精-伊紅染色 ； 未切片皮膚組織 ； 非線性顯微術 ； 組織病理學

並列摘要

Intraoperative margin assessment during Mohs micrographic surgery (MMS), a tissue-conserving surgery which minimizes surgical margins by histologically examining the excisions, is clinically critical. Currently, frozen pathology is the gold standard of assessing Mohs excisions for signs of remaining cancerous lesions; however, it is still considered time-consuming and sometimes misses the real margins. In this study, we demonstrate nonlinear microscopy imaging of hematoxylin-eosin (H&E) stained whole-mount skin tissues with a sub-femtoliter resolution through third-harmonic-generation (THG) and three-photon-excited-fluorescence (3PF) by using 1260 nm Cr:forsterite-laser-based nonlinear microscopy, which also enables virtual biopsy for preoperative margin assessment. Without the physical sectioning procedure and with a simplified staining process, real margins were preserved and time before microscopic examination was potentially saved three times shorter than frozen pathology. By exactly stained by H&E, the compatibility allows the adjunction to frozen pathology if further examination is needed. Virtual-sectioning imaging of H&E stained skin tissues is displayed real-time and can be post-processed analogously to conventional H&E histology, facilitating pathologists for diagnosis.
Based on the nonlinear spectroscopy study of H&E, hematoxylin can strongly enhance the THG signals, while eosin can illuminate strong 3PF; hence, hematoxylin stained cell nuclei will appear in THG contrast, and eosinophilic structures will appear in 3PF contrast. In the H&E stained thin section, the nonlinear images of normal and cancerous skin tissues showed fully compatible contrast and features compared with corresponding bright field images. With the robust staining process suggested in this study, virtual-sectioning imaging of non-sectioned skin tissues thicker than 1 mm presented similar contrast to the conventional H&E histology. For assessment adequacy of margins in MMS, surgical tissues including nevus sebaceous, basal cell carcinoma, squamous cell carcinoma and extramammary Paget's disease were horizontally cut into several layers to simulate as process of MMS, and the results indicated the feasibility of margins detection of surgical margins by nonlinear imaging of H&E stained whole-mount skin tissues. This time-saving method is fully compatible with current frozen pathology and preserves real margins, and thus provides a promising histopathological diagnosis technique for rapid margin assessment during MMS without losing the contrast and features of conventional H&E histology.

並列關鍵字

Mohs micrographic surgery ； intraoperative margin assessment ； hematoxylin-eosin stain ； whole-mount skin tissues ； nonlinear microscopy ； histopathology

參考文獻

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