二階電化率顯微術於膠原蛋白結構的研究

Abstract

本論文使用二階電化率顯微術來研究膠原蛋白的結構。利用旋轉線性飛秒脈衝激發光，研究病理皮膚，軟骨組織工程與眼角膜。藉由測量影像中每一個點的二階電化率張量，並轉換成影像圖，發現此張量可以用來鑑別病理皮膚，第一型與第二型的膠原纖維。更進一步，建立分子模型，來描述所觀測到的張量變異，並且此模型和已知的高解析x-ray的數據吻合。除此之外，將這顯微術更進一步推廣，測量透明材料的折射率、膠原纖維的相關聯常數和纖維走向的亂度。最後，本研究發現，在眼角膜的正向與背向背頻訊號裡，隱藏著纖維走向有強相關聯的特質。

In the thesis, excitation polarization-resolved second harmonic generation (PSHG) microscopy is utilized to study the most rich mammal protein: collagen. Using PSHG microscopy, second harmonic generation (SHG) signal as a function of the excitation polarization angle was measured for normal and pathological human dermis, engineered cartilage tissue, and cornea. Second-order susceptibility (SOS,χ (2) ) tensor ratios of different collagen types were determined to single-pixel resolution and the results were displayed as images. It was found that χ (2) tensor ratios can be used in the discrimination of normal and pathological skin dermis which normal dermis contain approximately 80% type I collagen and 20% type III collagen. SOS microscopy was found to be useful in distinguish the type I and type II collagen. In particular, χzzz|χzxx = 1.40 ± 0.04 and χxzx|χzxx = 0.53 ± 0.10 are obtained for type I collagen from rat tail tendon, and χzzz|χzxx = 1.14 ± 0.09 and χxzx|χzxx = 0.29 ± 0.11 for type II collagen from rat trachea cartilage. We also extended this methodology to the label-free SOS imaging of engineered cartilage tissue and we found that the presence of type I and II collagen in the tissue can be identified. Next, in an attempt to better explain the SOS results for collagen, we developed a new molecular model which incorporated the SOS contributions of peptide and methylene groups and it was found that the model is consistent with high resolution x-ray diffraction data in collagen-like model peptides. As extensions to the work described above, the methodology described in this thesis was also found to be able to determine fibril orientation. Strong correlation of fibril orientation between forward and backward scattering SHG was found and the described approach has great potential in the application of clinical ophthalmology. Finally, localized birefringence of a material was found to be determinable through depth-resolved SOS microscopy.