本論文以開發多光子激發(multiphoton excitation, MPE)加工與高速三維(three-dimensional, 3D)顯微術系統，以及生醫應用為主，其研究主題大致可分為兩大議題，首先以實驗室所建構之雷射加工直寫(laser direct writing)以及多光子蛋白質交聯(multiphoton protein crosslinking)作用來製作出各式之細胞外基質(extracellular matrix, ECM)之3D微結構，以提供利基(niche)微環境給細胞作3D之生長與爬行，由實驗結果驗證了可透過此系統來應用在建構3D ECM微結構來引導細胞生長，並控制細胞生長之區域在特定3D空間中。
在第二部分主要以建構高速3D顯微多光子影像系統為主，其研究發展階段可大致可分為三部分: 1) 改良傳統之廣視域時域聚焦(temporal focusing, TF)技術，以數位微面鏡裝置(digital micromirror device, DMD)取代傳統閃耀式光柵(blazed grating)，建構廣視域影像激發之TF系統，此新系統為基於DMD之TF系統，並同時兼具數位光調變器(digital light processor, DLP)之結構光照明功能； 2) 在影像接收端部分，以發展可高速擷取動態影像之光場(light field, LF)技術為主，透過單一微透鏡陣列(microlens array, MLA)將各方向之入射光之方向以及位置資訊分別記錄在影像感測器上之不同部位，可透過四維度之矩陣來描述一束光束之行進狀態，並透過傅立葉切片(Fourier slice)技術來重建各個焦平面之影像，透過電腦視覺技術(computer vision)之整合，來將各焦平面影像原始資訊進行計算以重建出三維表面形貌； 3) 整合上述之多光子DMD廣視域TF技術之光學激發技術與LF系統作為高速影像接收顯微系統，並透過調變其傅立葉共軛焦平面上之數值孔徑大小，可控制其3D體積激發在特定空間中，來減少背景之雜訊，並同時可進行動態影像之觀察，在影像收光方面，由於LF只需以單一幀即可記錄下整個三維光學資訊，接著透過3D反褶積(deconvolution)運算即可數位重建出三維之螢光影像，實驗結果以高速布朗運動來進行最終測試與驗證。
最後，透過上述兩大系統主題之開發，可應用於探討生醫微環境在細胞或組織層級交互作用之研究，並可提供一高通量之檢測平台來觀察其生物行為，另外其高速體積影像之優勢亦有潛力應用於神經訊號影像方面之研究。

In this dissertation, the multiphoton excitation (MPE) system, high-speed three-dimensional (3D) microscopy system have been investigated and developed, and applied on the biomedical research. The research topics of this dissertation can be divided into two major issues. First of all, the lab-made laser processing direct writing technique and multiphoton protein crosslinking mechanism are used to fabricate various extracellular matrixes (ECM) micro-scaffolds. The 3D micro-structure provides a 3D growth and migration of cells in the niche microenvironments. From the experimental results, it is verified that the 3D ECM microstructure can be constructed through this system to guide cell growth, and control the growth region of cell in a specific 3D spatial distribution.
In the second part, the main purpose is focused on constructing the high-speed 3D microscopic multiphoton imaging systems. The research and development stage can be broadly divided into three parts: 1) Improvement of the conventional wide-field temporal focusing (TF) technology. A digital micromirror device (DMD) is used to replace the conventional blazed grating, and constructs a TF system for the wide-field image excitation. This new system is the DMD-based TF system, and at the same time, it also act as a digital light processor (DLP) with structured illumination ability; 2) In the image receiving terminal, the light field (LF) technology, that can capture dynamic images at high speed. Through a single microlens array (MLA), the direction and position of incident light can be separated and recorded on the image sensor. The four-dimensional matrix can be used to describe the state of travelling light. Fourier slice theorem is used to reconstruct the images at each focal plane. With the integration of computer vision, each focal plane image can be digitally-refocused. The raw data can be used to calculate and reconstruct the 3D surface morphology; 3) Integrate the MPE technology and LF system of the multi-photon DMD wide-field TF technology as a high-speed image receiving microscopy system. Furthermore, the 3D volume excitation can be controlled by adjusting the numerical aperture in the Fourier conjugate focal plane, and reduce the noise of the background at the same time. The LF can record the entire 3D information in a single frame, and then digitally reconstruct the three-dimensional fluorescence image via 3D deconvolution algorithm. The system is examined and verified by the high-speed Brownian motion experiment.
Finally, through the development of the above two major topic, that can be applied to explore the interaction of the biomedical microenvironments at the cell or tissue level, and provide a high-throughput detection platform to observe the biological behavior via the high-speed volumetric imaging. Moreover, the advantages and the potential of these techniques can be applied to the study of neuronal signal imaging.

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