- 學位論文
病毒感染細胞螢光蛋白光譜分布之動態量測
Dynamic Measurement of Fluorescent Proteins Spectral Distribution on Virus Infected Cells
摘要
近幾年來，使用螢光蛋白於細胞分析的應用已經逐漸增加。這項發展主要隨著螢光偵測儀器的進步而發展，如顯微影像系統和影像分析及資料彙整技術，其不但增加了螢光蛋白在一些特殊應用的彈性及使用性，且已經廣泛的應用於各種生物醫療方面如腫瘤的轉移監控和癌細胞的遷移觀察。結合改進的螢光成影裝置，加上高複雜的影像分析邏輯系統的建立，螢光蛋白技術將會在更精密且高容量以細胞為基礎的掃描技術中扮演越來越重要的角色。我們建立了一套動態光譜量測系統，可以在一次的量測中同步地量測多螢光團的光譜強度及其分布。我們利用此系統監控秋行軍蟲卵巢細胞株（Sf21）被有基因重組功能的桿狀病毒感染的分布情況，病毒中包含了讓細胞顯現紅螢光蛋白和綠螢光蛋白生產機制的基因，正常未被感染的細胞並不會因雷射的激發而發出螢光，而相對於被感染的細胞，基因的改變使其開始生產出螢光蛋白，經過適當波長和強度的雷射激發後，將會有螢光的效應出現，藉此我們可以利用這種特性去觀察病毒感染細胞的情況和分布。系統是由倍頻雷射、掃瞄系統和光譜儀所組成。我們另外也用了一些演算法和訊號補償技術來消去相對於訊號的背景雜訊和色相補償。
並列摘要
We constructed a dynamic spectroscopy system that can simultaneously measure the intensity and spectral distributions of samples with multi-fluorophores in a single scan. The system was used to monitor the fluorescence distribution of cells infected by the virus, which is constructed by a recombinant baculoviruses, vAcD-Rhir-E, containing the red and green fluorescent protein gene that can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. The system was composed of an excitation light source, a scanning system and a spectrometer. We also developed an algorithm and fitting process to analyze the pattern of fluorescence distribution of the dual fluorescence produced in the recombinant virus-infected cells. All the algorithm and calculation are automatically processed in a visualized scanning program and can monitor the specific region of sample by calculating its intensity distribution. The spectral measurement of each pixel was performed at millisecond range and the two dimensional distribution of full spectrum was recorded within several seconds. We have constructed a dynamic spectroscopy system to monitor the process of virus-infection of cells. The distributions of the dual fluorescence were simultaneously measured at micrometer resolution.