- 學位論文
雙光子激發顯微術於生醫研究之應用
Applications of Two-Photon Excitation Microscopy on Biomedicine
摘要
雙光子顯微術因其優異的特性—包含自發性的光學斷層、較低的光漂白與光損害、較佳的影像對比度以及較長的穿透距離，近年來在生醫領域上，吸引不少學者的高度興趣。在此研究中，藉由觀測以780 nm波長激發、來自巨噬細胞 J774A.1的NAD(P)H自體螢光，得以瞭解螢光強度與細胞的代謝作用之關係。到目前為止，不僅證實了我們所觀測到的訊號的確來自細胞的代謝作用，同時指出LDL 的氧化對細胞所造成的傷害大於ox-LDL。 我們亦觀察了一些較簡單的系統以作為實驗模型，如蛋白質晶體及以YOYO-1染色的DNA纖維。將蛋白質晶體所發出的螢光，以極化分光鏡分為兩垂直方向之光束，並比較它們對雷射極化角度的強度變化，即可判斷我們所觀察的是否為單一晶體形式。此外，亦觀察到來自DNA纖維、因其結構之不對稱性而產生的二倍頻訊號。更進一步地，藉由分析螢光強度對雷射極化之，我們通常能夠分辨DNA纖維是較為延展伸長的，或較為聚集在一起。以上實驗說明了未來以雙光子顯微鏡分析染色質結構的可能性。 實驗結果闡明了雙光子顯微鏡是一有潛力的工具，可用於研究細胞之代謝作用，或可作為評價並量化生物分子之非線性光學反應之儀器。
並列摘要
Two-photon microscopy has attracted high interests on investigation of biomedicine recently, because of its several outstanding characteristics, including intrinsic optical section, lower photo-bleaching and photo damaging, higher contrast and longer penetrating distance. In this research, NAD(P)H autofluorescence of macrophages J774A.1 was observed by 780nm two-photon excitation, to understand the relationship between fluorescence intensity and cells’ metabolisms. So far, we have not only demonstrated that what we observed was from metabolism of cells, but also indicated that LDL oxidation causes more damage than ox-LDL does on macrophages. Simpler systems used as models, such as protein crystals and YOYO-1 stained DNA fibers, were also observed. By separating fluorescence emitted from a protein crystal into two perpendicular polarization directions, and comparing their intensity response dependence to laser polarization, one can tell if what observed is a pure crystalline form. Besides, slight intrinsic Second Harmonic Generation (SHG) signal were collected from DNA fibers, due to the chirality of their structure. Moreover, we can usually tell DNA fibers are more extended or form aggregates, by analyzing the polarization dependence of fluorescence intensity. The above experiments indicate the possibility to analyze chromatin structure using a two-photon microscope in the future. The experimental results bring out that two-photon microscopy is a potential tool to investigate metabolism of cells, and it can also be used as an adequate instrument to evaluate and to quantify the nonlinear responses from bio-molecules.