組織透明化是觀察組織樣本的重要關鍵。而造成組織樣本不透明的主要原因是由於構成生物組織的蛋白質與脂質其折射率較水分子高，造成組織內部分子之間的折射率不均勻，導致光學散射。在光散射的環境下，以顯微鏡觀察厚片組織樣本時(>20 μm)，無法具有良好的光學穿透度與解析度，造成樣本深層組織之影像擷取困難。為了減少組織內的散射，在本實驗中我們製備高折射率水膠，並以高折射率水膠包埋組織，使組織的光學穿透度上升，並應用三維顯微影像技術，觀察透明化之後的消化道組織樣本。
高折射率水膠是由丙烯醯胺單體、甲基丙烯醯胺單體、與交聯劑-三(乙二醇)二甲丙烯酸酯混合而成的水溶液，經由紫外光照射聚合之後所產生的高分子水膠(折射率為1.50 ;水的折射率為1.33)。在本實驗中我們藉由測試高折射率水膠對於水溶液中的蛋白質與脂質(牛奶溶液)，以及動物組織(小鼠大腸與胰臟切片樣本)之光學穿透度，來證明高折射率水膠的組織澄清能力。當以螢光分子標記高折射率水膠組織包埋樣本時，由於組織的透明度增加，在共軛焦顯微鏡下，組織深層的螢光分子能夠被有效地激發並產生螢光訊號，使螢光影像能夠清晰且精確的呈現，得以建構組織之三維影像。
在本實驗中，我們利用高折射率水膠的光學穿透性質以及組織澄清功能與三維顯微影像技術結合，建構消化道組織之三維影像。此外，由於水膠包埋可將檢體固定於水膠立方體之中，使組織樣本有固定的方位與相對座標位置。此一特性，可促成三維共軛焦顯微鏡的光學切面方向，與傳統病理切片的刀切以及觀察方向一致，使我們能在完成非破壞性三維顯微影像擷取之後，以同一組織樣本進行病理組織切片、染色與二維影像擷取，完成三維共軛焦顯微鏡影像與病理二維影像之對位整合。

Background and Aims: Animal tissues are generally opaque. The opacity is derived from the higher refractive indexes of the tissue constituents, mainly the proteins and lipids, than that of the water molecules, causing scattering and creating difficulties in high-resolution deep-tissue microscopy. To reduce tissue scattering, in this research we aim to synthesize a high-refractive-index hydrogel for tissue permeabilization and embedding to prepare transparent gastrointestinal specimens for 3-dimensional (3-D) microscopy.

Methods and Results: An aqueous solution of the monomers acrylamide and methacrylamide with the crosslinker triethylene glycol dimethacrylate is used to synthesize the AMEGA hydrogel with high refractive index (RI at 1.50) through ultraviolet polymerization. The optical-clearing capacity of AMEGA hydrogel is demonstrated by its compatibility with the lipids and proteins in solution (milk) and with tissue (intestinal and pancreatic biopsies), forming transparent gel-milk and gel-tissue complexes after ultraviolet radiation. Importantly, when the intestinal and pancreatic biopsies were labeled with fluorescent probes and examined by confocal microscopy, efficient fluorescence excitation and emission from the gel-tissue complex were achieved, facilitating in-depth 3-D visualization of the tissue microstructure with high definition.

Conclusion: The combination of hydrogel-based optical clearing and 3-D microscopy for penetrative tissue imaging does not require microtome sectioning and provides a useful tool for 3-D presentation and analysis of the gastrointestinal tissues in an integrated fashion.

目錄

中文摘要........i
英文摘要........iii
縮寫對照表......v
圖目錄..........vi
1. 文獻回顧.....1
1.1 組織光學顯微影像: 二維與三維顯微影像之成像技術.....1
1.2 改良腸道與胰臟組織三維顯微影像之光學性質...........2
1.3 水膠...............................................4
1.3.1 水膠的交聯性質...................................5
1.3.1.1 化學性交聯.....................................6
1.3.2 高折射率水膠.....................................7
1.4 研究動機...........................................8
2. 實驗材料與方法......................................9
2.1 高折射率水膠合成...............................9
2.2 高折射率水膠之穿透度測量...........................9
2.2.1 高折射率水膠減少溶液光學散射實驗.................9
2.2.2 高折射率水膠減少組織光學散射實驗.................10
2.3 小鼠消化道組織澄清實驗.............................10
2.4 實驗動物...........................................11
2.5 以染劑灌流小鼠進行血管染色.........................11
2.5.1 染劑配製.........................................11
2.5.2 小鼠灌流.........................................12
2.6 組織染色...........................................13
2.6.1 組織螢光免疫染色.................................13
2.6.2 組織螢光染劑染色.................................13
2.7 封片...............................................14
2.8 共軛焦顯微鏡.......................................14
2.9 影像投影和分析.....................................14
2.10 實驗材料與儀器....................................15
3. 結果與討論..........................................17
3.1 合成高折射率水膠...................................17
3.2 高折射率水膠光學澄清效果之定性與定量分析:
牛奶膠體光學穿透試.................................20
3.3 紫外光照射下，高折射率水膠固化及穩定度之動力學分析.23
3.4 高折射率水膠組織澄清效果: 水膠組織包埋試驗.........26
3.5 高折射率水膠組織澄清技術應用於三維顯微組織學.......31
3.6 三維顯微組織學與二維組織學之整合...................37
4. 結論................................................40
5. 未來工作............................................42
6. 參考文獻............................................43

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