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標題: | 非線性光學顯微術於組織工程上的觀測 Monitoring Tissue Engineering Using Nonlinear Optical Microscopy |
作者: | Te-Hsuen Chen 陳德軒 |
指導教授: | 黃義侑(Yi-You Huang) |
關鍵字: | 非線性光學顯微術,組織工程,二倍頻,偏振二倍頻,三倍頻, nonlinear optical microscopy (NLOM),tissue enginnering (TE),second harmonic generation (SHG),polarization-second haemonic generation (P-SHG),third harmonic generation (THG), |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22770 |
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顯示於系所單位: | 醫學工程學研究所 |
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