以共軛焦顯微鏡技術進行胰小島組織之三維影像擷取

High-resolution, 3-dimensional imaging of mouse pancreatic islet microstructure and tissue networks

呂至軒(Chih-Shen Lu)

指導教授 ： 湯學成

國立清華大學/工學院/化學工程學系/碩士(2010年)

摘要

摘要
胰臟無論是在內、外分泌系統上都扮演重要的角色。其中，位於胰臟組織中的蘭氏小島含有β細胞，負責分泌胰島素以代謝血糖。β細胞因免疫系統攻擊死亡，為第一型糖尿病型成的重要原因。然而造成蘭氏小島功能異常與β細胞死亡的因素很多，其中因蘭氏小島發炎而產生的血管與神經組織結構變化，一直是糖尿病型原因的研究重點。然而，以傳統的二維組織切片方式，進行蘭氏小島的顯微影像擷取，會造成組織變形與切割破壞，無法保持完整的樣本結構。並且，以切片方式所獲得的平面組織影像，無法觀察到血管與神經網路在空間中的完整結構。本實驗以小鼠胰臟組織為樣本，結合螢光標記、染色技術，共軛焦顯微鏡技術，與組織澄清技術，提供了一個不需要使用切片機，即能進行胰臟內蘭氏小島組織觀察的方法，建立觀察蘭氏小島組織的三維顯微影像技術。本研究所發展的立體組織影像獲得方式，可清楚地觀察到蘭氏小島血管與神經網路在空間中的形態。此一影像技術將可提供胰臟與糖尿病領域一項新的研究觀察工具。

關鍵字

生物組織澄清試劑

並列摘要

Abstract
The pancreas is a unique organ where the endocrine microorgans are embedded in the exocrine tissue. To visualize the spatial distribution of pancreatic cells in situ also with three dimensions observation could provide a better understanding of the overall organization of endocrine cells and the relationship between different structures in the pancreas. In this research, we apply an optical-clearing solution, FocusClear, to minimize the random light scattering in the mouse pancreas and show the overall spatial organization of β-cells and islets in the pancreas. This technique, with the advantage than conventional fluorescence microscopy and alleviates technical difficulties such as collection and operating for serial histological sections and avoids structural defects during the sectioning process. Our results thus establish the basis for a new platform which is a noninvasive three-dimensional microscopic imaging method for in situ visualization and assessment of pancreatic islet microstructure.
The three-dimensional images of the fluorescence-labeled structure. Specifically, the islet vasculature and sympathetic nerve fibers were labeled by vessel painting (lipophilic dye labeling of blood vessels) and immunofluorescence staining of neural markers, respectively, for confocal microscopy. Optical sectioning and reconstruction of bulk tissue are simple and rapid. Volume and number of β-cells within a block of tissue can be automatically quantified by freely available software.

並列關鍵字

FocusClearTM

參考文獻

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