此論文的主要目標在於發展一套多功超快雷射系統以完成多光子激發之三維微/奈米加工。另外為搭配掃描式光學顯微鏡架設，使用藉由LabVIEW編譯之現場可編程陣列(field-programmable gate array)資料擷取卡，結合並運用一些重要的光電元件以達成生醫與電漿子應用之功能。研究中發展了一資料格式轉檔程式用以連接多光子加工系統與CAD應用軟體，CAD軟體能協助方便的設計加工所需之三維結構，此外於試片之加工可藉由非線性分子影像作精確定位。
於超快雷射加工研究中，發現孟加拉玫瑰素之雙光子螢光強度在牛血清白蛋白雙光子交聯反應製作結構中會被有效的增強，雙光子交聯反應中孟加拉玫瑰素的作用為光活化劑，其中增強的螢光強度與交聯加工結構中牛血清白蛋白的濃度相關，此結構中材料濃度可藉由控制雷射之功率與脈衝數量而改變，因此三維牛血清白蛋白加工結構可立即的藉由雙光子螢光作觀測，此交聯牛血清白蛋白微結構的合成可作為有不同程度螢光增強之生醫材料。
此外，試著擴張這項技術的應用於電漿子效應方面，將金奈米棒(gold nanorod)加入加工溶液之中並藉由電漿子特性的輔助作結構加工。考慮光活化劑與金奈米棒之交互作用，局部電場強化(local field enhancement)與雙光子冷光(two-photon luminescence)皆對等效雙光子吸收截面(effective absorption cross-section)有所幫助。基於這些特性，金奈米棒在近紅外波段能夠增強非線性光高分子化與光交聯作用。儘管金奈米棒已發現有大範圍的生醫應用，然而目前在三維蛋白質微加工方面的應用還很少。由於嵌入金奈米棒之蛋白質微結構表現出獨特的電學、光學與機械特性，加上光交聯結構中的雙光子螢光增強，應用這些特性於蛋白質微加工中。

The primary goal of this thesis is to develop a multifunctional femtosecond laser system to accommodate two-photon excited three-dimensional micro/nano processing. In addition to adopting a basic laser scanning optical setup, the controller of this system employs a data acquisition board with field-programmable gate array (FPGA) based on LabVIEW programming to integrate and manipulate crucial optoelectronic devices to achieve biomedical and plasmonic functions. A data format converting software has been developed to link the femtosecond laser system and CAD software. CAD software can assistant to conveniently design 3D structures for femtosecond laser processing. It can be applied to process on samples via the positioning assistance of nonlinear molecular imaging.
During femtosecond laser processing, the intensity of two-photon excited fluorescence (TPEF) of xanthene dye, Rose Bengal (RB), was significantly enhanced via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking (TPC) technique. The RB was utilized as the photoactivator in the TPC processing and the enhanced TPEF intensity correlates with the concentration of fabricated crosslinked BSA microstructures via the power control and pulse selection of the employed femtosecond laser. As a result, fabrication of three-dimensional BSA microstructures can be simultaneously monitored by the use of TPEF intensity. The crosslinked BSA microstructures synthesized may be used as an ordered biomaterial for fluorescence enhancement.
Besides, plasmonic effect to assistant the fabrication has been studied. In this aspect, gold nanorods (AuNRs) were added into the fabrication solution and fabricate structures with the assistance of plasmonic phenomena. To consider the interactions of photoactivator and AuNRs, the local field enhancement and two-photon luminescence (TPL) both may aid the improvement of effective two-photon absorption cross-section. Based on these characteristics, AuNRs can enhance nonlinear photopolymerization and photocrosslinking in the NIR region. While AuNRs have found wide biomedical applications, the use of AuNRs in 3D protein microfabrication has been rare. Due to the fact that AuNRs-doped protein microstructures exhibit unique electrical, optical, and mechanical properties, and photocrosslinking enhancement, we explored these properties for protein microfabrication.

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