本論文主要整合超快雷射三維影像及加工技術，並結合奈米光學理論和生物分子探針技術，來探討SH3GLB2蛋白質的特性。在人體中Spas-1同源SH3GLB2基因已知被過度表現在前列腺癌轉移的細胞中，而在過度表現SH3GLB2基因的非洲綠猴腎細胞株(COS-7 fibroblast)中發現可形成蛋白質聚落。論文主要分三部分，第一部分為超快雷射加工系統的掃描路徑規劃提升，利用Visual Studio C++ 程式開發撰寫DLL動態連結資料庫，將STL (stereo lithography)檔進行轉檔切圖以及向量掃描(vector scan)路徑規劃的演算法，加入到目前以LabVIEW程式控制的超快雷射加工影像系統中。透過以上的整合，可緊密結合從電腦輔助設計端(computer aided design)到雷射加工製造端的流程，再搭配三維雙光子激發影像技術即可提供具影像精確定位和加工多重功能的系統。在第二部分研究，主要利用此飛秒雷射影像加工系統來對於SH3GLB2 蛋白質進行fluorescence recovery after photobleaching實驗，探討SH3GLB2蛋白質聚落在受雷射破壞後恢復的情形。透過單光子計數技術來觀察對於加工前後蛋白質螢光隨時間變化；利用雙光子影像縮時攝影(time lapse)和光流法(optical flow)結合絕對誤差和套合(sum of absolute difference)演算法，追蹤蛋白質聚落的位移以及速率來探討TGF-β1對於SH3GLB2蛋白質聚落活動力的調節和ATP能量代謝的關係，由於生物細胞在基因轉錄或是蛋白質的運送時皆需要能量的參與以進行生物反應。第三部分主要結合奈米光學理論和螢光生命周期影像顯微術(fluorescence lifetime imaging microscopy)來研究SH3GLB2蛋白質聚落在轉染兩種螢光基因表現的COS-7細胞中，其螢光生命周期的變化與蛋白質聚落鍵結的程度關係。

In this thesis, an ultrafast laser system with three-dimensional (3D) molecular imaging and microprocessing has been utilized to investigate SH3GLB2 protein polymerization. In addition, nano-optics theory and bio-molecular probe technique are implemented. In humans, the Spas-1 ortholog SH3GLB2 has been reported to be overexpressed in prostate cancer metastases. It is observed that in the SH3GLB2 overexpressed African green monkey kidney (COS-7 fibroblasts) cell lines the endophilin B2 protein can aggregate to form clusters around the cell nucleus. The research content of this paper can be divided into three parts. The first part is focused on improving the path planning of a developed Ti:sapphire femtosecond laser system. A Visual Studio C++ program having stereo lithography format file transformation and vector scan path allocation has been developed as a DLL (dynamic link library) format and integrated into the LabVIEW platform of the laser system. Through the above integration, the laser microprocessing can be directly manipulated from the computer aid design of 3D microstructures into the laser processing terminal. In the second part, the developed system was utilized to investigate the recovery of SH3GLB2 protein polymerization clusters after photon damage via fluorescence recovery after photobleaching technique based on observing the protein fluorescence variation with time via single photon counting technique. Optical flow method and sum of absolute difference method were combined as a tracking algorithm in two-photon time lapse imaging which evaluates the effective speed of the protein clusters in cytoplasm. The experimental results are used to study and investigate the TGF-beta1-regulated SH3GLB2 protein assembly and the relation to the ATP metabolism since it is always need energy to participate in when the gene express or protein transport. Finally, nano-optics theory and fluorescence lifetime image microscopy are utilized to study the binding affinity of SH3GLB2 expression construct transfected with a green fluorescent protein (GFP)-tagged and red fluorescence protein (DsRed).

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