利用Ti:Sapphire飛秒雷射作為生物顯微術的光源，由於其具備了近紅外光光長波段，對於生物組織擁有較好的穿透深度，同時擁有極短脈衝寬度，因此可以在極低的平均輸出功率下，在雷射聚焦點處獲得強大的峰值功率，如此瞬間強大的能量與物質產生交互作用時，得以引發局部非線性光學效應，進而發展出全新的加工方法，特別是應用在生物細胞組織上。
本論文首先透過顯微物鏡將雷射光束聚焦在組織樣本上，經過光學非線性效應激發出雙光子螢光(two-photon excited fluorescence)或二倍頻光(second-harmonic generation)，這些訊號可以建構出三維分子影像。論文主軸是應用飛秒雷射對於如膠原蛋白凝膠與細胞等生物組織進行三維加工。由於生物組織對紅外光是相當透明的材料，就如同上述分子影像技術，所以我們可以利用高數值孔徑(numerical aperture)的顯微物鏡，使得雷射光束可以在不影響上下層部份組織的情況之下，順利穿透材料並精確地聚焦在其內部進行加工。將系統及雷射參數加以調整，同時配合三維掃描系統之各個元件，如XY震鏡掃描器、聲光調變器與壓電載物移動平台，以及三維模型建立，目前已經成功利用自行研發之飛秒雷射加工系統，於膠原蛋白凝膠中完成二、三維高解析度之微米加工結構。
關鍵字︰雙光子螢光，二倍頻，膠原蛋白凝膠

Ti:Sapphire femtosecond laser is adopted as a powerful light source in biological microscopy due to its near-IR wavelength with deeper penetration in tissue. Also, its short pulse duration indicates that a very high peak power in a low average energy is confined in the focal point. The laser interacts with materials through the great peak power which may induce nonlinear optical effects in the local focal point; hence a brand new three-dimensional micromachining processing has been developed in biological tissue specially.
In this thesis, the femtosecond laser beam is focused in the bulk of materials through a microscope objective, and then 3D molecular images are constructed by utilizing two-photon excited fluorescence and second-harmonic generation signals. The main aim in this thesis is to use the femtosecond laser to create 3D machining patterns in biomaterials, including collagen gel and living cells which are near transparent in the near-IR region. With the help of the molecular image microscopy, we can use the same high numerical aperture objective to focus the light within the biomaterials without affecting other undesired machining regions. The femtosecond laser machining system has been successfully developed with optimal machining parameters such as controlling laser power, XY galvo mirror, acousto-optic modulator, PZT actuator, and 3D CAD model. In summary, the high resolution 2D and 3D microstructures in collagen gel without destroying adjacent parts have been developed.
Keywords︰two-photon excited fluorescence, second-harmonic generation, collagen gel

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