本論文主要是利用多光子交聯(multiphoton crosslinking)機制來進行細胞外基質結構(extracellular matrix，ECM)之三維(three-dimensional，3D)微製作，並藉由多光子顯微術做細胞與ECM交互作用探討，其中包含四個研究主題：1）經由一個液晶模組調製之偏極光二倍頻(second harmonic generation，SHG)顯微鏡來改善其取影像之能力與提出其分析法進行解析；2）使具形態分布的生物組織影像外，更能提供分子或超分子(supramolecular)結構等級之成分資訊的能力；3）以及多光子激發微製作技術之發展有具金奈米柱(gold nanorods，AuNRs)之3D微米polyacrylamide和牛血清蛋白(bovine serum albumin，BSA)結構；4）並實現以多光子激發所獲取的高解析度之生物組織灰階影像可直接進行3D仿生骨架製作的逆向工程系統。
實驗結果顯示利用史托克向量(Stokes vector)偏振分析法可檢測液晶模組調製之偏光二倍頻顯微鏡並達成無機械式之具180度線性偏振激發，其中橢圓偏振殘留小於0.08 (ellipticity < 0.08)。藉由圓柱對稱的染色(Di-8-ANEPPS)之單層巨型微胞(giant unilamellar vesicles，GUV)顯微影像展現偏振激發影像的正確性並符合理論依據。在SHG影像上，以代表性的肌腱與肌肉組織進行量測，其偏振反應與分析取得之組織特性(helical pitch angles)皆吻合文獻結果。更進一步地結合先前分析法進而發展以單一像素進行分析的新偏振解析法，並針對混型的膠原蛋白膠體結構之等效螺旋角(α-helix pitch angle)進行研究，其實驗結果顯示此方法可分辨在不同比例的I型與III型混合的膠原蛋白膠體並與肽鏈模型之結果一致。另外，本文也成功地製作出3D具AuNRs之acrylamide與BSA微米結構，也證實其具有表面電漿子(surface plasmons)之特性。最後，為了提升此技術的應用性以及製作仿生骨架之能力，結合生物組織灰階影像並以此轉換模型直接進行製作，此法證實可利用ECM精準的複製出切片的老鼠心臟組織結構，並具有很大的潛力在細胞與組織研究的應用。

In this thesis, multiphoton excitation microscopy (MPEM) and fabrication of three-dimensional (3D) extracellular matrices (ECM) at submicron scale have been investigated. The primary works focus on: 1) improving the polarization-resolved second harmonic generation (SHG) imaging via a liquid crystal modulator located in the infinity space; 2) providing its analysis method with great potential to use in tissue characterization, as extracting molecular and supramolecular structural details; 3) developing the multiphoton fabrication capability, including to make a 3D polymerized polyacrylamide or crosslinked bovine serum albumin (BSA) microstructures containing gold nanorods (AuNRs); and, 4) providing a powerful 3D fabrication scaffold tool based on directly map the greyscale image data from the high resolution MPEM.
First, the performance in terms of polarization purity is validated using Stokes vector polarimetry, and then found to have minimal residual polarization ellipticity. We also demonstrated the performance by imaging cylindrically symmetric giant unilamellar vesicles with Di-8-ANEPPS stained dye. The experimental data is well fit to the theoretical prediction. The SHG intensity depended on the linear polarization angle is measured for the well-characterized cases of chicken tendon and skeletal muscle, and the extracted helical pitch angles agree with previous results. Furthermore, the net collagen α-helix pitch angle within the gel mixtures was investigated by using a new pixel based polarization resolved approach by combining previous analyses. The extracted pitch angles are consistent with those of peptide models and the method has sufficient sensitivity to differentiate collagen type I from the collagen type I/ collagen type III mixtures. Furthermore, AuNRs in designated positions of the fabricated 3D polyacrylamide microstructures has been successfully achieved. The AuNRs-doped BSA microstructures can be applied in biomedical scaffolds with surface plasmonic properties. Finally, a fabricated approach with modulated raster scanning has been developed for precisely recreating a single section of mouse heart. Also, this method should be useful in the cell-matrix studies with the fabricated artificial scaffolds.

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