臺灣博碩士論文加值系統

非線性光學顯微術藉由多光子激發搭配其它的非線性光學對比機制，已發展成為研究生物樣本的三維結構之利器。雙光子搭配高頻倍頻顯微術可以同時擷取自發螢光、二倍頻訊號和三倍頻訊號，即時觀測生物樣本。無需繁複的樣本製備和染色，因此多模態非線性光顯微術適合研究複雜的生物系統。
在本論文中的第一部分，我們先量測不同的生物支架材料的非線性光學特性，得知在1230 nm激發下，幾丁聚醣具有自發螢光、二倍頻訊號和三倍頻訊號；聚己內酯(PCL)具有二倍頻和三倍頻訊號；聚乳酸(PDLLA)有很強的三倍頻。第二部分為該技術在脂肪組織工程和神經組織工程的應用。在脂肪組織工程的部分，利用脂肪油滴的三倍頻訊號、膠原蛋白的二倍頻訊號和細胞的自發螢光訊號，觀察到誘導分化條件下，脂肪前驅細胞培養在膠原蛋白水膠中，分化成成熟的脂肪細胞，而不需繁複的樣本處理步驟即可監測脂肪前驅細胞的分化過程。而在神經組織工程的應用，藉由偏振二倍頻技術區別在大鼠脊髓冷凍切片樣本中的二倍頻訊號，且可細分其訊號來源為星狀膠質纖維或膠原蛋白纖維。最後將非線性光學技術應用在活體即時皮膚凍傷觀測的研究，藉由分析細胞自發螢光和膠原蛋白二倍頻訊號，評估皮膚受到冷凍破壞後細胞和膠原蛋白的變化過程。

Nonlinear optical microscopy has been developed as a powerful tool to investigate biological samples in 3-Dimensional scale, utilizing multiphoton excitation in conjunction with other nonlinear contrast mechanism. Two-photon together with high harmonic microscopy can simultaneously observe the autofluorescence, second and third harmonic signals for real time sampling. The advantage of such technique provides hassle-free sample preparation and staining, and also can be applied for multi-mode nonlinear microscopy for a complex biological system.
In this research investigation, we measured the nonlinear optical characteristics of different biodegradable materials for tissue engineering application. Under the near-infrarad excitation of 1230nm, chitosan was observed with strong autofluorescnece, as well as second and third harmonic signals. In contrast, polycaprolactone (PCL) exhibited only second and third harmonic signals, and the Poly(dl-lactic acid) (PDLLA) possessed only strong third harmonic signal.
Multi-modal microscopy techniques were also applied for in vitro adipose tissue engineering and neural tissue engineering. The third harmonic signals of adipocyte lipid droplet, collagen second harmonic signals, and cellular autofluorescence allows clear observation of the process where pre-adipocyte differentiate to mature adipocyte cultured in collagen gel. In addition, polarization second harmonic signals can also apply to neural tissue engineering by differentiating the astrogliar filament and the collagen fibrils.
For in vivo application, nonlinear optical microscopy can analyze in real-time of the nonlinear optical signalsresulting from cellular autofluoescence and collagen fibrils to monitor the cells and collagen after cryoinjury.

中文摘要 I
英文摘要 II
目錄 IV
圖目錄 VII
表目錄 XI
第一章 緒論 1
第二章 文獻回顧 3
2.1 非線性光學簡介 3
2.2 雙光子顯微術原理 5
2.3 二倍頻顯微術原理 13
2.4 三倍頻顯微術原理 15
2.5 非線性光學於生物醫學之應用 17
雙光子螢光(Two Photon Fluorescence, TPF)顯微鏡 17
二倍頻顯微鏡(SHG microscopy) 19
三倍頻顯微鏡(THG microscopy) 20
同調反司托克拉曼散射(Coherent anti-Stoke Raman Scattering, CARS) 20
多模態顯微鏡 21
第三章 實驗儀器裝置 22
3.1 正立式顯微鏡系統 22
3.2 倒立式顯微鏡系統 24
第四章 生醫材料之非線性光學特性 26
4.1 簡介 26
4.2 實驗材料與方法 28
4.2.1 實驗材料和方法 28
4.2.2 顯微系統 29
4.3 實驗結果與討論 30
4.3.1 幾丁聚醣之非線性光學特性 30
4.3.2 聚乳酸(PDLLA)非線性光學特性 31
4.3.3 聚己內酯(PCL)非線性光學特性 33
4.4 結論 36
第五章 非線性光學於脂肪組織工程之應用 37
5.1 簡介 37
5.2 實驗材料與方法 38
5.2.1 動物來源 38
5.2.2 脂肪前驅細胞初代培養 38
5.2.3 膠原蛋白膠體製備 40
5.2.4 微球體支架製備 41
5.2.5 非線性光學影像系統 42
5.3 實驗結果與討論 43
5.3.1 膠原蛋白膠體影像 43
5.3.2 微球體支架影像 43
5.3.3 大鼠皮下組織影像 45
5.3.4 小鼠白色脂肪組織 45
5.3.5 In vitro 脂肪細胞油滴 46
5.4 結論 50
第六章 非線性光學於神經組織工程之應用 51
6.1 簡介 51
6.2 實驗材料與方法 52
6.2.1 建立大鼠脊髓損傷模型 52
6.2.2 影像系統 53
6.2.3 影像處理和分析 53
6.3 實驗結果與討論 56
6.3.1 脊髓損傷模型非線性光學影像 56
6.3.2 偏振二倍頻(PSHG)實驗 57
6.4 結論 68
第七章 非線性光學於皮膚凍傷之研究 69
7.1 簡介 69
7.2 實驗材料與方法 72
7.2.1 體外(ex vivo)和活體實驗 72
7.2.2 動物實驗 72
7.2.3 活體(in vivo)觀測視窗夾具(window chamber)手術 72
7.3 實驗結果與討論 75
7.3.1 體外(ex vivo)測試結果 75
7.3.2 活體觀測裸鼠正常皮膚 76
7.3.3 正常與凍傷背部皮膚的活體觀測 77
7.3.4 連續活體觀察的數據分析 78
7.4 結論 82
第八章 總結 83
參考文獻…………… 84

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