雙光子激發螢光相干顯微術於人類胚胎腎臟293細胞質中綠螢光蛋白之研究

Abstract

本論文研究為利用雙光子螢光相干顯微光譜(Two-photon-based fluorescence correlation spectroscopy )對綠螢光蛋白於磷酸鹽緩衝溶液、細胞質內綠營光蛋白濃度的量測，而細胞內綠營光蛋白表現量在轉染後6-27小時內會隨著時間增加而增加所以需量測出最適合的量測時間還有我們的FCS系統可量測到的最高很對低濃度。本研究的目的是為了建立一個可長時間、低生物傷害的細胞研究平台，以利於研究細胞骨架以及胞內物質運輸等等用途，可進一步了解有關細胞移動和癌細胞轉移等等重要特性。一般對於細胞內蛋白的運輸，例如細胞骨架或細胞膜流動速率等等使用的方式是螢光漂白後復原(Fluorescence Recovery After Photobleaching, FRAP)，使用的是平均能量較高的光子造成螢光物質漂白再計算由他處螢光物質補充回來的時間來推算出細胞膜等物質的擴散速率，由於漂白後的螢光物是無法再表現螢光且用平均能量較高的雷射光長時間照射細胞會有細胞傷害的風險，例如突變或死亡或是因為蛋白質變性改變細胞的訊息傳遞等等。而且，雙光子具有較深的穿透深度更適合用於胞內的研究，所以選用雙光子激發的光源是讓細胞更接近原本的環境且又可以得到細胞訊息的方式。螢光相干顯微光譜則是跟螢光漂白後復原一樣計算帶有螢光物標誌物質的擴散係數，但不同的是此方法為一個空間內計算螢光分子進出的時間，不需要先將螢光物質漂白，可以不受制於只能是固定態的物質，例如細胞膜，才能夠做測量，細胞骨架為3D的結構在胞內，不似螢光漂白後復原或是電子顯微鏡需佐以影像。綠螢光蛋白(Green Fluorescence Protein, GFP) 可以形成融合蛋白(fused protein) 當作標的蛋白的標示且不易引起其他免疫反應或造成細胞死亡的指示物。本實驗目的是為了未來用綠螢光蛋白於細胞內研究而所作的基本參數量測。由於螢光相干光譜有其濃度限制，在數個奈米莫耳濃度到數個微米莫耳濃度會是最佳產生相干濃度。於是在轉染後細胞表現的綠螢光蛋白濃度變會影響螢光相干量測結果，最佳濃度量測的時間為轉染後6到30的小時內。這也包含細胞存活和背景雜訊干擾的考量而得到最是當的量測時間。少於6小時綠螢光蛋白表現量不夠且細胞處在不穩定的狀態，而大於30小時則是綠螢光蛋白數量過多且因為細胞死亡而吐出的綠螢光蛋白增加背景訊號。

In this thesis, we use the two-photon based fluorescence correlation spectroscopy system to measure the dynamic parameter of green fluorescence protein in PBS, and in cytosol. The concentration of GFP was increased with time during the period of the time, 6-27 hours after transfection. According the correlation we should know our system limitation of the concentration GFP. Our purpose is to set up a platform which is low bio-damage and for long-term observation for the study of the cell biology such as cytoskeleton and transportation in cell which are related to the metastasis of cancer cell or cell movement. In general, the studies of cytotskeleton or membrane transfusion rate are use Fluorescence recovery after photobleaching (FRAP) as a tool to study. The laser of FRAP was one photon excitation. Compare to the one photon excitation two photon excitation had less photobleaching, less cell damage and longer penetration depth. Not only for the immobile phase such as membrane, but also the protein in cytosol can be measured the parameter in our system – Two photon excitation fluorescence correlation spectroscopy (TPE-FCS). As for these reasons that two photon excitation was the suitable tool to develop a platform for long period of time in observation of cell biology. As a given excitation volume, the diffusion coefficient of molecules due to the Brownian motion into or out of the excitation volume is determined. In our system, the limit concentration of measurement is between sub-nanomolar to sub-micromolar. This is associated to the transfection. After tansfection, the HEK 293 cell started express the green fluorescence protein. However, less than 6 hour after transfection, the cells were at an unstable situation and there are less GFP in the cell. On the other hands, more than 30 hours that too much GFP were expressed in cell even secrete out to the medium. Thus became more background noise to the FCS measurement.