光學定位之超解析度螢光顯微術利用定位演算法、多色螢光偵測及軸向定位技術已證實可實 現10-20nm 側向解析度和30-50nm 軸向解析度之三維多色螢光影像造影，甚至是單分子造影， 並取得於細胞生物、神經生物及其他生物醫學研究中的標的分子的重要空間分布情形與生物 物理參數等資訊。然而，光學定位之超解析度螢光顯微術於厚生物樣本的應用仍然是一大問 題。因傳統的超解析度螢光定位影像技術的螢光激發機制是以單光子激發，此激發方式的缺 點是造成其他未取像層之螢光分子的光漂白和具散射樣本內較淺造影深度。因此，本計畫中 提出將建構以時域聚焦多光子螢光像散成像之三維雙色超解析度多光子激發螢光定位顯微 術。藉由快速時域聚焦多光子螢光激發顯微鏡的光學切片激發方式和雙通道同步數據擷取方 式來取得二種螢光分子的影像進而定位出其位置數據。此三維雙色超解析度多光子激發螢光 定位顯微術具有數奈米等級解析度、單分子偵測、三維造影、低光漂白、較深穿透深度等優 點，將可應用於活體厚生物樣本內分子作用的動態研究。本計畫中、此造影系統將用於研究 二項重要生醫議題。一項為探討在腫瘤相關纖維母細胞提取之三維基質或膠原蛋白三維基質 環境內的受體寡聚體/叢聚體形成和細胞內吞調節作用下非小細胞肺癌的上皮細胞生長因子受 體活化機制。三維雙色超解析度定位影像將可提供腫瘤生成時，受體寡聚體/叢聚體形成和受 體招入網格蛋白被膜對於癌症相關的變異上皮細胞生長因子受體活化之重要的數據。另一項 為探討神經退化性疾病的大腦之神經突觸中特定蛋白(如Aβ、tau、α-syn 等其他蛋白)的突觸 間傳遞機制。此病理蛋白聚集的三維超解析度定位影像也將可提供神經退化性疾病中突觸間 傳遞機制是否促進病理蛋白聚集於神經元間傳遞。這些研究數據與知識將可於開發異常之上 皮細胞生長因子受體活化相關疾病之藥物標靶步驟和神經退化性疾病之更有效治療協助開啟 新的方向。 ;The localization-based super-resolution fluorescence microscopy using the localization algorithm, the multi-fluorescence detections, and the axial positioning techniques has been demonstrated to achieve three dimensional (3D) multi-color fluorescence imaging with a lateral and axial resolution of 10-20 nm and 30-50 nm to extract the significant spatial distribution and biophysical factors of target molecules in cell biology, neuronal, and other biomedical researches. However, the localization-based 3D super-resolution microscopy still has a serious drawback for the applications of thick biological specimens. Because the fluorescence excitation of conventional super-resolution fluorescence localization imaging is based on single photon excitation, the drawbacks of this approach is photoblaching of fluorophores at other layers and the less imaging depth within the scattering specimens. Therefore, 3D dual-color super-resolution multiphoton excitation fluorescence localization microscopy using the temporal focusing multiphoton fluorescence astigmatic imaging is proposed in this proposal. Images of two fluorophores are obtained to locate their positions by optical sectioning excitation using a fast temporal focusing multiphoton excitation microscope and by the simultaneous collection of data in two channels. The proposed 3D dual-color super-resolution multiphoton excitation fluorescence localization microscopy is useful for the dynamic study of molecular interactions in living thick biological specimens with a nanoscale resolution, single molecule detection, 3D imaging, nominal photobleaching, and superior penetration depth. The proposed 3D dual-color super-resolution multiphoton excitation fluorescence localization microscopy will used to explore two significant biomedical subjects. One is the epidermal growth factor receptor (EGFR) activation of non-small cell lung cancer (NSCLC) with formation of receptor oligomers/clusters and the endocytosis modulation in the tumor associated fibroblast-derived 3D matrices or the 3D collagen matrices. 3D dual-color super-resolution localization imaging can show the significant evidence about the formation of receptor oligomers/clusters and the recruitment of receptor to CCPs for its activation of cancer related EGFR mutants in tumorigenesis. The other one is the trans-synaptic propagation of neurodegenerative disease-specific proteins, which including Aβ, tau, α-syn, and others, in the neuronal synapse of brain. 3D super-resolution localization image of the pathological aggregates will also provide the evidence whether interneuronal transfer of pathological aggregates is facilitated by the trans-synaptic propagation in the neurodegenerative disease. These results will help to open a new direction to develop drug target steps in disease related aberrant EGFR activation and the effective therapies of neurodegenerative diseases.
